U.S. patent application number 10/930194 was filed with the patent office on 2005-04-21 for mekk1 molecules and uses thereof.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Liao, Sha-Mei, Palombella, Vito J..
Application Number | 20050084884 10/930194 |
Document ID | / |
Family ID | 33418890 |
Filed Date | 2005-04-21 |
United States Patent
Application |
20050084884 |
Kind Code |
A1 |
Palombella, Vito J. ; et
al. |
April 21, 2005 |
MEKK1 molecules and uses thereof
Abstract
The invention provides full-length, human isolated nucleic acids
molecules, designated MEKK1 nucleic acid molecules, which encode a
MEKK family member. The invention also provides antisense nucleic
acid molecules, recombinant expression vectors containing MEKK1
nucleic acid molecules, host cells into which the expression
vectors have been introduced, and nonhuman transgenic animals in
which a MEKK1 gene has been introduced or disrupted. The invention
still further provides isolated MEKK1 proteins, fusion proteins,
antigenic peptides and anti-MEKK1 antibodies. Diagnostic methods
utilizing compositions of the invention are also provided.
Inventors: |
Palombella, Vito J.;
(Needham, MA) ; Liao, Sha-Mei; (Lexington,
MA) |
Correspondence
Address: |
MILLENNIUM PHARMACEUTICALS, INC.
40 Landsdowne Street
CAMBRIDGE
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
33418890 |
Appl. No.: |
10/930194 |
Filed: |
August 31, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10930194 |
Aug 31, 2004 |
|
|
|
09697898 |
Oct 27, 2000 |
|
|
|
6818427 |
|
|
|
|
Current U.S.
Class: |
435/6.12 ;
435/194; 435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C07H 21/04 20130101;
C12N 9/1205 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/194; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/12 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule selected from the group
consisting of: a) a nucleic acid molecule comprising the nucleotide
sequence which is at least 90% identical to the nucleotide sequence
of SEQ ID NO:1 or the cDNA insert of the plasmid deposited with the
ATCC as Accession Number PTA-1836; and b) a nucleic acid molecule
which encodes a polypeptide comprising the amino acid sequence of
SEQ ID NO:2, or the amino acid sequence encoded by the cDNA insert
of the plasmid deposited with the ATCC as Accession Number
PTA-1836.
2. The nucleic acid molecule of claim 1 further comprising vector
nucleic acid sequences.
3. The nucleic acid molecule of claim 1 further comprising nucleic
acid sequences encoding a heterologous polypeptide.
4. A non-human host cell containing the nucleic acid molecule of
claim 1.
5. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence which is encoded
by a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1
or nucleotides 7-4545 of SEQ ID NO:1; b) a polypeptide comprising
an amino acid sequence which is encoded by a nucleic acid
comprising the nucleotide sequence of the cDNA insert of the
plasmid deposited with the ATCC as Accession Number PTA-1836 or a
portion thereof, comprising the coding region; c) a fragment of a
polypeptide comprising the amino acid sequence of SEQ ID NO:2,
wherein said fragment comprises at least 500 contiguous amino acids
of SEQ ID NO:2 and has MEKK1 activity; d) a polypeptide comprising
an amino acid sequence which is encoded by a nucleic acid which has
at least 90% nucleotide sequence identity with the entire length of
the nucleotide sequence of SEQ ID NO:1, wherein said nucleic acid
comprises nucleotide residues 1 to 64 of SEQ ID NO:1 and wherein
said polypeptide has MEKK1 activity; e) a polypeptide comprising an
amino acid sequence which is encoded by a nucleic acid which has at
least 90% nucleotide sequence identity with the entire length of
the nucleotide sequence of the insert of the plasmid deposited with
the ATCC as Accession Number PTA-1836, wherein said nucleic acid
comprises nucleotide residues 1 to 64 of SEQ ID NO:1 and wherein
said polypeptide has MEKK1 activity; f) a polypeptide comprising
the amino acid sequence of SEQ ID NO:2 or the amino acid sequence
encoded by the cDNA insert of the plasmid deposited with ATCC as
Accession Number PTA-1836; g) a polypeptide comprising a MEKK1
variant, wherein said MEKK1 variant comprises an amino acid
sequence has at least 90% amino acid sequence identity with the
entire length of SEQ ID NO:2, wherein said MEKK1 variant has amino
acid residues 1 to 20 of SEQ ID NO:2, and wherein said MEKK1
variant has MEKK1 activity; and h) a polypeptide comprising a MEKK1
variant, wherein said MEKK1 variant comprises an amino acid
sequence having at least 98% amino acid sequence identity with the
entire length of SEQ ID NO:2 and wherein said variant has MEKK1
activity.
6. The isolated polypeptide of claim 5 selected from the group
consisting of: a) a polypeptide comprising the amino acid sequence
of SEQ ID NO:2; and b) a polypeptide comprising the amino acid
sequence encoded by the cDNA insert of the plasmid deposited with
ATCC as Accession Number PTA-1836.
7. The polypeptide of claim 5 further comprising heterologous amino
acid sequences.
8. The polypeptide of claim 5 further comprising a detectable
label.
9. An antibody which selectively binds to a polypeptide of claim
5.
10. A method for producing a polypeptide comprising the amino acid
sequence of SEQ ID NO:2 or the amino acid sequence encoded by the
cDNA insert of the plasmid deposited with the ATCC as Accession
Number PTA-1836; comprising culturing the host cell of claim 4
under conditions in which the nucleic acid molecule is
expressed.
11. A method for detecting the presence of a polypeptide of claim 5
in a sample, comprising: a) contacting the sample with a compound
which selectively binds to a polypeptide of claim 5; and b)
determining whether the compound binds to the polypeptide in the
sample.
12. The method of claim 11, wherein the compound which binds to the
polypeptide is an antibody.
13. A kit comprising a compound which selectively binds to a
polypeptide of claim 5 and instructions for use.
14. A method for detecting the presence of a nucleic acid molecule
of claim 1 in a sample, comprising the steps of: a) contacting the
sample with a nucleic acid probe or primer which selectively
hybridizes to the nucleic acid molecule; and b) determining whether
the nucleic acid probe or primer binds to a nucleic acid molecule
in the sample.
15. A kit comprising a compound which selectively hybridizes to a
nucleic acid molecule of claim 1 and instructions for use.
16. A method for identifying a compound which binds to a
polypeptide of claim 5 comprising the steps of: a) contacting a
polypeptide, or a cell expressing a polypeptide of claim 5 with a
test compound; and b) determining whether the polypeptide binds to
the test compound.
17. The method of claim 16, wherein the binding of the test
compound to the polypeptide is detected by a method selected from
the group consisting of: a) detection of binding by direct
detecting of test compound/polypeptide binding; b) detection of
binding using a competition binding assay; c) detection of binding
using an assay for MEKK1-mediated signal transduction.
18. A method for modulating the activity of a polypeptide of claim
5 comprising contacting a polypeptide or a cell expressing a
polypeptide of claim 5 with a compound which binds to the
polypeptide in a sufficient concentration to modulate the activity
of the polypeptide.
19. A method for identifying a compound which modulates the
activity of a polypeptide of claim 5, comprising: a) contacting a
polypeptide of claim 5 with a test compound; and b) determining the
effect of the test compound on the activity of the polypeptide to
thereby identify a compound which modulates the activity of the
polypeptide.
20. An isolated polypeptide selected from the group consisting of:
a) a polypeptide consisting of the amino acid sequence of SEQ ID
NO:2; and b) a polypeptide consisting of the amino acid sequence
encoded by the nucleotide sequence of the insert of the plasmid
deposited with the ATCC as Accession Number PTA-1836.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the identification of isolated,
full-length human MEKK1 nucleic acid molecules encoding MEKK1
proteins, and methods of using the nucleic acid molecules and
proteins.
BACKGROUND OF THE INVENTION
[0002] Controlling the state of phosphorylation is an important
mechanism by which signaling molecules regulate the activity of
other proteins. One common theme in such molecular signaling is the
so-called kinase cascade, in which a linear series of kinases is
activated by phosphorylation by upstream kinases. The
mitogen-activated protein kinase (MAPK) pathway is one such example
of a kinase cascade.
[0003] MAPKs are activated and phosphorylated by MAPK kinases
(MKKs). The MKKs, in turn, are activated and phosphorylated by
serine/threonine kinases (MKKKs), which themselves may be activated
and phosphorylated by MKKK kinases (MKKKKs). There are currently
over ten different groups of kinases covering more than twenty-two
different genes that act upstream of one another and regulate the
MKKs. One family, the MAPK/ERK kinase kinases (MEKKs) directly
activate and phosphorylate specific MKKs. (Schlesinger et al.,
Front Biosci. 3:d1181-1186 (1998).)
[0004] MEKKs are activated by a number of diverse extracellular
stimuli, indicating that not only can these molecules affect a wide
variety of downstream activity, but they can also react to a
diverse array of extracellular stimuli. For example, both EGF
receptor stimulation and TNF .alpha. lead to an increase in MEKK1
activity. (Lange-Carter et al., Science 265:1458-1461(1994);
Winston et al., Proc. Natl. Acad. Sci., 92:1614-1618 (1995);
Ishizuka et al., J. Biol. Chem. 271:12762-12766 (1996); Kaga et
al., J. Immunol. 160:4182-4189 (1998).) MEKK1 is also activated in
response to DNA damaging stresses such as UV irradiation,
etoposide, cisplatin, and mitomycin C. (Widmann et al., Mol Cell.
Biol. 18:2416-2429 (1998); Cardone et al., Cell 90:315-323
(1997).)
[0005] To date, six different MEKK genes have been at least
partially cloned in mammalian cells. (Lange-Carter et al., Science
260:315-319 (1993); Blank et al., J. Biol. Chem. 271:5361-5368
(1996); Gerwins et al., J. Biol. Chem. 272:8288-8295 (1997); Wang
et al., J. Biol. Chem 271:31607-31611 (1996); Wang et al., Biochem.
Biophys. Res. Commun. 253:33-37 (1998).) There are a number of
functional motifs found within the N-terminal regulatory domains
of, at least, MEKK1 and MEKK4. Both molecules contain putative
pleckstrin homology domains. Pleckstrin domains associate with
polyphosphoinositides and mediate localization to specific regions
of the plasma membrane. MEKK1 and MEKK4 also contain proline rich
regions at the N-terminus, which may be of functional significance.
Proline rich regions have been shown to be important for binding
proteins that contain Src homology 3 (SH3) domains. Moreover, with
regard to MEKK1, 14-3-3 proteins bind at the N-terminal regulatory
domain. Although 14-3-3 association does not appear to dramatically
affect MEKK activity, 14-3-3 proteins are important for MEKK
regulation by mediating interactions with other regulatory proteins
and for controlling subcellular localization of these kinases.
[0006] MEKK1 is important in regulating cell survival and
apoptosis. MEKK1 is a substrate for caspases, a family of proteases
required for apoptosis. The apoptotic signaling appears to be
dependent on cleavage of MEKK1 and subsequent caspase activation,
as cleavage resistant mutants do not induce apoptosis. Thus, MEKK1
plays a critical role in regulating cell survival and death, acting
as a molecular switch when cleaved by caspases. Cleavage by a
caspase changes MEKK1 from a survival promoting kinase to an
effector of cell death.
[0007] MEKK1 activates both the Activator Protein-1 (AP-1) stress
response pathway and the NF.kappa.B pathway. The transcription
factor AP-1 is a critical regulator of T-cell activation, cytokine
production, including IL-2, IL-3, and GM-CSF, and the production of
metalloproteinases. (Gottschalf et al., J. Exp. Med. 178:1681
(1993); Want et al., J. Mol. Cell. Biol. 14:11153 (1994); Rao,
Immunol. Today 15:274 (1994); Angel et al., Biochem. Biophys. Acta.
1072:129 (1991).) With regard to cytokine regulation, AP-1 mediates
positive transactivation independently or in association with NF-AT
(Nolan, Cell 77:795 (1994)). AP-1 activity is induced by many
stimuli, including the phorbol ester tumor promoter
12-0-tetradecanoylphorbol-13-acetate (TPA), growth factors,
cytokines, T-cell activators, neurotransmitters, and UV
irradiation. AP-1 is composed of dimers of different members of the
Fos and Jun family of proteins. AP-1 activity is regulated at the
level of both C-Jun and C-Fos transcription and by
post-translational modification of their protein products by
phosphorylation and dephosphorylation.
[0008] Moreover, independent of its MAPK activity, MEKK1 also plays
another role in regulating transcription factor NF.kappa.B, which
is a dimer maintained in the cytoplasm via an inhibitory regulatory
subunit, I.kappa.B. (Lee et al., Cell 88:213 (1997).) Upon
stimulation with specific cytokines or environmental stresses,
I.kappa.B is phosphorylated by I.kappa.B kinase which induces
proteolytic degradation of I.kappa.B, thereby releasing NF.kappa.B
to translocate to the nucleus effecting changes in transcription.
(PCT Publication No. WO 97/35014.) Overexpression of MEKK1
activates NF.kappa.B.
[0009] The two transcription factors, NF.kappa.B and AP-1, have
been shown to regulate the production of many proinflammatory
cytokines and related proteins that are elevated in
immunoinflammatory diseases. These transcription factors regulate
IL-1, IL-2, TNF .alpha., IL6, and IL-8 levels in a variety of cell
types. NF.kappa.B and other related transcription factor complexes
are involved in the rapid induction of genes whose products
function in protective and proliferative responses upon exposure of
cells to external stimuli. Similarly, AP-1 has a significant role
in the regulation of IL-2 transcription during T-cell activation.
Thus, the role of NF.kappa.B and AP-1 is to act as a transducer of
certain stimuli that lead to immune, inflammatory, and acute phase
responses and, when overactivated, can lead to a disease state
(Suto et al., Current Pharm. Design 3:515-528 (1997). No known
antiinflammatory or autoimmune drugs have been specifically
developed clinically as inhibitors of NF.kappa.B or AP-1.
Therefore, a critical need exists for new therapies to treat
immunoinflammatory and autoimmune disorders.
[0010] Prior to the instant invention, cloning efforts to obtain
the full-length human MEKK1 gene have been unsuccessful for several
reasons. Earlier efforts by others failed to identify the
full-length cDNA. In some instances, the libraries that were
screened were 5'-stressed and would not have expressed a
full-length coding region. Specifically, the 5' region of the gene
proved difficult to clone for reasons that were, until now,
unclear. Other attempts using reverse PCR methodology were also
unsuccessful.
[0011] Nevertheless, in order to completely understand MEKK1
biology and to develop potential regulators or modulators of MEKK1,
the full-length MEKK1 nucleic acid molecule had to be cloned and
isolated. There is a need, therefore, to identify and clone the
full-length MEKK1 nucleic acid to further understand and exploit
the role of MEKK1.
SUMMARY OF THE INVENTION
[0012] The present invention is based, in part, on the discovery of
a novel full-length human gene referred to herein as "MEKK1". The
polynucleotide sequence of a cDNA encoding MEKK1 polypeptide is
shown in SEQ ID NO:1, and the amino acid sequence of a MEKK1
polypeptide is shown in SEQ ID NO:2. In addition, the
polynucleotide sequence of the coding region is depicted in SEQ ID
NO:3.
[0013] Accordingly, in a first aspect, the invention features a
full-length nucleic acid molecule which encodes a MEKK1 protein or
polypeptide, e.g., a biologically active portion of the MEKK1
protein. In a preferred embodiment the isolated nucleic acid
molecule encodes a polypeptide having the amino acid sequence of
SEQ ID NO:2. In other embodiments, the invention provides isolated
MEKK1 nucleic acid molecules having the polynucleotide sequence
shown in SEQ ID NO:1, SEQ ID NO:3, or the sequence of the DNA
insert of the plasmid deposited with ATCC Accession Number
PTA-1836. In other embodiments, the invention provides a nucleic
acid molecule which hybridizes under stringent hybridization
conditions to a nucleic acid molecule comprising the polynucleotide
sequence of SEQ ID NO:1 or the sequence of the DNA insert of the
plasmid deposited with ATCC Accession Number PTA-1836, wherein the
nucleic acid encodes a full-length MEKK1 protein.
[0014] In a related embodiment of the first aspect, the invention
further provides nucleic acid constructs which include a MEKK1
nucleic acid molecule described herein. In certain embodiments, the
nucleic acid molecules of the invention are operatively linked to
native or heterologous regulatory sequences. Also included are
vectors and host cells containing the MEKK1 nucleic acid molecules
of the invention, e.g., vectors and host cells suitable for
producing MEKK1 nucleic acid molecules and polypeptides.
[0015] In another related embodiment of the first aspect, the
invention provides nucleic acid fragments suitable as primers or
hybridization probes for the detection of MEKK1-encoding nucleic
acids.
[0016] In still another related embodiment of the first aspect,
isolated nucleic acid molecules that are antisense to a MEKK1
encoding nucleic acid molecule are provided.
[0017] In a second aspect, the invention features MEKK1
polypeptides, and biologically active or antigenic fragments
thereof, that are useful, e.g., as reagents or targets, in assays
applicable to treatment and diagnosis of MEKK1 mediated or related
disorders. In another embodiment, the invention provides MEKK1
polypeptides having a MEKK1 activity. Preferred polypeptides are
MEKK1 proteins including at least one processed/cleaved domain,
e.g., amino acid residues 876-1512 of SEQ ID NO:2, and/or one
kinase or catalytic domain, e.g., amino acid residues 1191-1512 of
SEQ ID NO:2, and, preferably, having a MEKK1 activity, e.g., a
MEKK1 activity as described herein.
[0018] In another embodiment of the second aspect, the invention
provides MEKK1 polypeptides, e.g., a MEKK1 polypeptide having the
amino acid sequence shown in SEQ ID NO:2; the amino acid sequence
encoded by the cDNA insert of the plasmid deposited with ATCC
Accession Number PTA-1836; an amino acid sequence that is
substantially identical to the amino acid sequence shown in SEQ ID
NO:2; or an amino acid sequence encoded by a nucleic acid molecule
having a polynucleotide sequence which hybridizes under stringent
hybridization conditions to a nucleic acid molecule comprising the
polynucleotide sequence of SEQ ID NO:1 or 3, or the sequence of the
DNA insert of the plasmid deposited with ATCC Accession Number
PTA-1836, wherein the nucleic acid encodes a full length MEKK1
protein.
[0019] In a related embodiment of the second aspect, the invention
further provides nucleic acid constructs that include a MEKK1
nucleic acid molecule described herein.
[0020] In a related embodiment of the second aspect, the invention
provides MEKK1 polypeptides or fragments operatively linked to
non-MEKK1 polypeptides to form fusion proteins.
[0021] In a third aspect, the invention features antibodies and
antigen-binding fragments thereof, that react with, or more
preferably specifically bind MEKK1 polypeptides.
[0022] In a fourth aspect, the invention provides methods of
screening for compounds that modulate the expression or activity of
the MEKK1 polypeptides or nucleic acids.
[0023] In a fifth aspect, the invention provides a process for
modulating MEKK1 polypeptide or nucleic acid expression or
activity, e.g., using the aforementioned screened compounds. In
certain embodiments, the methods involve treatment of conditions
related to aberrant activity or expression of the MEKK1
polypeptides or nucleic acids, such as conditions involving
aberrant or deficient cellular adhesion, proliferation or
differentiation.
[0024] The invention also provides assays for determining the
activity of, or the presence or absence of, MEKK1 polypeptides or
nucleic acid molecules in a biological sample, including for
disease diagnosis.
[0025] In a sixth aspect, the invention provides assays for
determining the presence or absence of a genetic alteration in a
MEKK1 polypeptide or nucleic acid molecule, including for disease
diagnosis.
[0026] It is to be understood from the foregoing general
description that the following detailed description is exemplary
and explanatory, and intended to provide further explanation of the
invention claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The accompanying drawings are included to provide a further
understanding of the invention, and are incorporated in, and
constitute a part of this specification that illustrate several
embodiments of the invention, and together with the description
serve to explain the principles of the invention.
[0028] FIG. 1 depicts the nucleic acid sequence (SEQ ID NO:1) of
human MEKK1 and the predicted amino acid sequence (SEQ ID NO:2) of
human MEKK1. The methionine-initiated open reading frame of human
MEKK1 is shown in SEQ ID NO:3, and starts at nucleotide 7 and goes
to nucleotide 4545 of SEQ ID NO:1.
[0029] FIG. 2 depicts a summary of the alignment of the human MEKK1
protein (SEQ ID NO:2) with a partial human MEKK1 protein (GenBank
Accession Number AF042838), rat MEKK1 (GenBank Accession Number
U48596), and mouse MEKK1 (GenBank Accession Number 117340).
[0030] FIG. 3 shows the results of a Western blot for MEKK1 and NIK
protein expression levels using an antiflag antibody.
[0031] FIG. 4 shows a graph of the results of a CAT ELISA to
measure NF.kappa..circle-solid.B activation by MEKK1.
[0032] FIG. 5 shows the results of a Western blot of
immunoprecipitates of cell lysates of transfected cells expressing
different tags using either anti-myc or anti-flag antibodies.
DETAILED DESCRIPTION
[0033] The patent applications, patents and literature references
cited herein indicate the knowledge of those of ordinary skill in
this field and are hereby incorporated by reference in their
entirety. In the case of inconsistencies between any reference
cited herein and the specific teachings of the present disclosure,
this disclosure will prevail. Similarly, any inconsistencies
between an art-understood meaning of a term and a meaning of a term
as specifically taught in the present disclosure will be resolved
in favor of this disclosure.
[0034] The human full-length MEKK1 nucleic acid sequence (FIG. 1;
SEQ ID NO:1), which is approximately 5245 nucleotides long
including untranslated regions, contains a predicted
methionine-initiated coding sequence of about 4536 nucleotides
(nucleotides 7-4545 of SEQ ID NO:1, which correspond to nucleotides
14539 of SEQ ID NO:3). As used herein, "MEKK1 nucleic acid" and
"MEKK1 gene" comprise nucleic acid sequences 1-5245 of SEQ ID NO:1.
The coding sequence encodes a 1512 amino acid protein (SEQ ID
NO:2). The open reading frame (ORF) analysis of the human MEKK1
protein of SEQ ID NO:2 indicates that the protein does not include
a signal sequence nor does it include any transmembrane
domains.
[0035] Human MEKK1 contains an activation loop between the kinase
subdomain VII and VIII, specifically the two threonine amino acid
residues 1400 and 1412. (Deak et al., Biochem. J. 322:185-192
(1997); and Siow et al., J. Biol. Chem. 272:7586-7594 (1997).) The
ORF analysis of human MEKK1 also showed the following sites:
[0036] one predicted glycosaminoglycan attachment site (PS00002) at
about amino acids 101 to 104 of SEQ ID NO:2;
[0037] four predicted cAMP- and cGMP-dependent protein kinase
phosphorylation sites (PS00004) at about amino acids 272 to 275,
395 to 398, 585 to 588, 1023 to 1026 of SEQ ID NO:2;
[0038] twenty-two predicted protein kinase C phosphorylation sites
(PS00005) at about amino acids 169 to 171, 258 to 260, 261 to 263,
281 to 283, 304 to 306, 379 to 381, 394 to 396, 397 to 399, 434 to
436, 514 to 516, 531 to 533, 657 to 659, 816 to 818, 823 to 825,
911 to 913, 928 to 930, 1021 to 1023, 1039 to 1041, 1070 to 1072,
1088 to 1090, 1148 to 1150, and 1166 to 1168 of SEQ ID NO:2;
[0039] nineteen predicted casein kinase II phosphorylation sites
(PS00006) located at about amino acids 20 to 23, 232 to 235, 300 to
303, 429 to 432, 434 to 437, 457 to 460, 507 to 510, 660 to 672,
705 to 708, 782 to 785, 900 to 903, 923 to 926, 947 to 950, 997 to
1000, 1089 to 1092, 1114 to 1117, 1130 to 1133, 1166 to 1169, and
1280 to 1283 of SEQ ID NO:2;
[0040] one predicted tyrosine kinase phosphorylation site(PS00007)
at about amino acids 1160 to 1167 of SEQ ID NO:2;
[0041] twenty-three predicted N-myristoylation sites (PS00008) from
about amino acid 6 to 11, 16 to 21, 25 to 30, 100 to 105, 121 to
126, 222 to 227, 254 to 259, 530 to 535, 570 to 575, 604 to 609,
611 to 616, 617 to 622, 872 to 877, 879 to 884, 914 to 919, 1096 to
1101, 1246 to 1251, 1252 to 1257, 1266 to 1271, 1328 to 1333, 1399
to 1404, 1407 to 1412, 1423 to 1428 of SEQ ID NO:2; and
[0042] one amidation site (PS00009) from about amino acid residue
246 to 249 of SEQ ID NO: 2.
[0043] For general information regarding PFAM identifiers, PS
prefix and PF prefix domain identification numbers, refer to
Sonnhammer et al., Protein 28:405420 (1997) and
http://www.psc.edu/general/software/packages- /pfam/pfam.html.
[0044] A plasmid containing the nucleotide sequence encoding human
MEKK1 was deposited with American Type Culture Collection (ATCC),
10801 University Boulevard, Manassas, Va. 20110-2209, on May 18,
2000 and assigned Accession Number PTA-1836. This deposit will be
maintained under the terms of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Procedure. This deposit was made merely as a
convenience for those of skill in the art and is not an admission
that a deposit is required under 35 U.S.C. .sctn.112.
[0045] The cloning method used herein was successful, compared to
other methods tried previously in the art, for several reasons.
Among these reasons were the screening of a lambda gt11 phage
library. Second, the cDNA library used herein had a large insert,
5'-stretched, which enabled more full-length clones to be
identified. Finally, the cDNA was made from normal human cDNA,
while others in the art had unsuccessfully used three tumor cell
lines to try and piece together the sequence.
[0046] The MEKK1 protein contains a significant number of
structural characteristics in common within the kinase catalytic
domain with other members of the MEKK family. The term "family",
when referring to the protein and nucleic acid molecules of the
invention, means two or more proteins or nucleic acid molecules
having a common structural domain or motif and having sufficient
amino acid or nucleotide sequence homology as defined herein. Such
family members can be naturally or non-naturally occurring and can
be from either the same or different species. For example, a family
can contain a first protein of human origin as well as other
distinct proteins of human origin, or alternatively, can contain
homologues of non-human origin, e.g., rat or mouse proteins.
Members of a family can also have common functional
characteristics.
[0047] In some embodiments, a MEKK1 protein includes at least one
phosphorylation site, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more protein
kinase C phosphorylation sites; and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19 or more casein kinase II
phosphorylation sites; 1, 2, 3,4 or more cAMP- and cGMP-dependent
protein kinase phosphorylation sites; and at least one tyrosine
kinase phosphorylation site. The MEKK1 protein can additionally
include at least one, glycosaminoglycan attachment site; at least
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and preferably eighteen to
twenty-three N-myristoylation sites; and at least one amidation
site.
[0048] As the MEKK1 polypeptides of the invention may modulate
MEKK1-mediated activities, they may be useful for developing novel
diagnostic and therapeutic agents for MEKK1-mediated or related
disorders, as described below.
[0049] As used herein, a "MEKK1 activity", "biological activity of
MEKK1", or "functional activity of MEKK1", refers to an activity
exerted by a MEKK1 protein, polypeptide, or nucleic acid molecule
on, e.g., a MEKK1-responsive cell or on a MEKK1 substrate, e.g., a
protein substrate, as determined in vivo or in vitro. In one
embodiment, a MEKK1 activity is a direct activity, such as an
association with a MEKK1 target molecule. A "target molecule" or
"binding partner" is a molecule with which a MEKK1 protein binds or
interacts in nature. A MEKK1 activity can also be an indirect
activity, e.g., a cellular signaling activity mediated by
interaction of the MEKK1 protein with a MEKK1 receptor.
[0050] A MEKK1 protein of the invention may display activities
including mediating (1) activation of I.kappa.B kinase which leads
to NF.kappa.B activation; (2) activation of ERK1 and ERK2 and
activation of the c-Myc transcription factor independent of JNK
activity; (3) apoptosis by both JNK-dependent and JNK-independent
mechanisms; and (4) AP-1 activation via JNK-dependent pathway. For
example, MEKK1 proteins may regulate processes involved, for
example, in embryonic development or tissue differentiation.
Examples of such embryonic development and tissue differentiation
include neural development (such as axonal growth and/or guidance
or growth), as well as tissue maintenance and function. In
addition, MEKK1 may be involved in pathological conditions, such as
neuronal degeneration, neoplastic transformation, and tumor
progression.
[0051] Based on the above-described sequence similarities, the
MEKK1 molecules of the present invention are predicted in some
instances to have similar biological activities as other MEKK
family members. Thus, the MEKK1 molecules and modulators of MEKK1
(particularly inhibitors of MEKK1) can serve as novel diagnostic
targets and therapeutic agents for controlling protein-protein
interaction disorders and signal transduction disorders, such as
cellular proliferative and/or differentiative disorders selected
from the group consisting of hyperplasia, neoplasia, and cancer, as
well as degenerative diseases, such a neurodegenerative diseases,
autoimmune diseases, inflammatory conditions, and allergic
responses.
[0052] Examples of cellular proliferative and/or differentiative
disorders include cancer, e.g., carcinoma, sarcoma, metastatic
disorders or hematopoietic neoplastic disorders, e.g., leukemias. A
metastatic tumor can arise from a multitude of primary tumor types,
including, but not limited to, those of prostate, colon, lung,
breast and liver origin.
[0053] As used herein, the terms "cancer", "hyperproliferative" and
"neoplastic" refer to cells having the capacity for autonomous
growth, i.e., an abnormal state or condition characterized by
rapidly proliferating cell growth. Hyperproliferative and
neoplastic disease states may be categorized as pathologic, i.e.,
characterizing or constituting a disease state, or may be
categorized as non-pathologic, i.e., a deviation from normal but
not associated with a disease state. The term is meant to include
all types of cancerous growths or oncogenic processes, and
metastatic tissues or malignantly transformed cells, tissues, or
organs, irrespective of histopathologic type or stage of
invasiveness. "Pathologic hyperproliferative" cells occur in
disease states characterized by malignant tumor growth. Examples of
non-pathologic hyperproliferative cells include proliferation of
cells associated with wound repair.
[0054] The terms "cancer" or "neoplasms" include malignancies of
the various organ systems, including, for example, lung, breast,
thyroid, lymphoid system, gastrointestinal system, and
genito-urinary tract, as well as adenocarcinomas, which include
malignancies such as most colon cancers, renal-cell carcinoma,
prostate cancer and/or testicular tumors, non-small cell carcinoma
of the lung, cancer of the small intestine and cancer of the
esophagus.
[0055] The term "carcinoma" is art recognized and refers to
malignancies of epithelial or endocrine tissues including
respiratory system carcinomas, gastrointestinal system carcinomas,
genitourinary system carcinomas, testicular carcinomas, breast
carcinomas, prostatic carcinomas, endocrine system carcinomas, and
melanomas. Exemplary carcinomas include those forming from tissue
of the cervix, lung, prostate, breast, head and neck, colon and
ovary. The term also includes carcinosarcomas, e.g., which include
malignant tumors composed of carcinomatous and sarcomatous tissues.
An "adenocarcinoma" refers to a carcinoma derived from glandular
tissue or in which the tumor cells form recognizable glandular
structures.
[0056] The term "sarcoma" is art recognized and refers to malignant
tumors of mesenchymal derivation.
[0057] Additional examples of proliferative disorders include
hematopoietic neoplastic disorders. As used herein, the term
"hematopoietic neoplastic disorders" includes diseases involving
hyperplastic/neoplastic cells of hematopoietic origin, e.g.,
arising from myeloid, lymphoid or erythroid lineages, or precursor
cells thereof. Preferably, the diseases arise from poorly
differentiated acute leukemias, e.g., erythroblastic leukemia and
acute megakaryoblastic leukemia. Additional exemplary myeloid
disorders include, but are not limited to, acute promyeloid
leukemia (APML), acute myelogenous leukemia (AML) and chronic
myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit
Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include,
but are not limited to acute lymphoblastic leukemia (ALL) which
includes B-lineage ALL and T-lineage ALL, chronic lymphocytic
leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia
(HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of
malignant lymphomas include, but are not limited to, non-Hodgkin
lymphoma and variants thereof, peripheral T cell lymphomas, adult T
cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL),
large granular lymphocytic leukemia (LGF), Hodgkin's disease, and
Reed-Sternberg disease.
[0058] Examples of inflammatory conditions within the scope of the
invention include, but are not limited to, asthma and chronic
obstructive pulmonary disease, rheumatoid arthritis, multiple
sclerosis, inflammatory bowel disease, e.g., Crohn's disease and
ulcerative colitis, and complications arising from organ
transplantation.
[0059] The MEKK1 protein, and derivatives and other variants of the
sequence in SEQ ID NO:2 thereof, are collectively referred to as
"polypeptides or proteins of the invention" or "MEKK1 polypeptides
or proteins". Nucleic acid molecules encoding such polypeptides or
proteins are collectively referred to as "nucleic acids of the
invention" or "MEKK1 nucleic acids." MEKK1 molecules refer to MEKK1
nucleic acids, polypeptides, and antibodies.
[0060] As used herein, the term "nucleic acid molecule" includes
DNA molecules (e.g., a cDNA or genomic DNA) and RNA molecules
(e.g., an mRNA) and analogs of the DNA or RNA generated, e.g., by
the use of nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA.
[0061] The term "isolated or purified nucleic acid molecule"
includes nucleic acid molecules which are separated from other
nucleic acid molecules which are present in the natural source of
the nucleic acid. For example, with regard to genomic DNA, the term
"isolated" includes nucleic acid molecules which are separated from
the chromosome with which the genomic DNA is naturally associated.
Preferably, an "isolated" nucleic acid is free of sequences which
naturally flank the nucleic acid (i.e., sequences located at the 5'
and/or 3' ends of the nucleic acid) in the genomic DNA of the
organism from which the nucleic acid is derived. For example, in
various embodiments, the isolated nucleic acid molecule can contain
less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of
5' and/or 3' nucleotide sequences 5 which naturally flank the
nucleic acid molecule in genomic DNA of the cell from which the
nucleic acid is derived. Moreover, an "isolated" nucleic acid
molecule, such as a cDNA molecule, can be substantially free of
other cellular material, or culture mediums when produced by
recombinant techniques, or substantially free of chemical
precursors or other chemicals when chemically synthesized.
[0062] As used herein, the term "hybridizes under stringent
conditions" describes conditions for hybridization and washing.
Stringent conditions are known to those skilled in the art and can
be found, e.g., in Current Protocols in Molecular Biology, John
Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous
methods are described in that reference and either can be used. A
preferred example of stringent hybridization conditions are
hybridization in 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 50.degree. C. Another example of
stringent hybridization conditions are hybridization in 6.times.
sodium chloride/sodium citrate (SSC) at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
55.degree. C. A further example of stringent hybridization
conditions are hybridization in 6.times. sodium chloride/sodium
citrate (SSC) at about 45.degree. C., followed by one or more
washes in 0.2.times.SSC, 0.1% SDS at 60.degree. C. Preferably,
stringent hybridization conditions are hybridization in 6.times.
sodium chloride/sodium citrate (SSC) at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
65.degree. C. Particularly preferred stringency conditions (and the
conditions that should be used if the practitioner is uncertain
about what conditions should be applied to determine if a molecule
is within a hybridization limitation of the invention) are 0.5M
Sodium Phosphate, 7% SDS at 65.degree. C., followed by one or more
washes at 0.2.times.SSC, 1% SDS at 65.degree. C. Preferably, an
isolated nucleic acid molecule of the invention that hybridizes
under stringent conditions to the sequence of SEQ ID NO:1 or 3,
corresponds to a naturally-occurring nucleic acid molecule.
[0063] As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural
protein).
[0064] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules which include an open reading frame
encoding a MEKK1 protein, preferably a mammalian MEKK1 protein, and
can further include non-coding regulatory sequences, and
introns.
[0065] An "isolated" or "purified" polypeptide or protein is
substantially free of cellular material or other contaminating
proteins from the cell or tissue source from which the protein is
derived, or substantially free from chemical precursors or other
chemicals when chemically synthesized. In one embodiment, the
language "substantially free" means preparation of MEKK1 protein
having less than about 30%, 20%, 10% and more preferably 5% (by dry
weight), of non-MEKK1 protein (also referred to herein as a
"contaminating protein"), or of chemical precursors or non-MEKK1
chemicals. When the MEKK1 protein, or a biologically active portion
thereof, is recombinantly produced, it is also preferably
substantially free of culture medium, i.e., culture medium
represents less than about 20%, more preferably less than about
10%, and most preferably less than about 5% of the volume of the
protein preparation. The invention includes isolated or purified
preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry
weight.
[0066] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of MEKK1 (e.g., the sequence
of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert
of the plasmid deposited with ATCC as Accession Number PTA-1836)
without abolishing or, more preferably, without substantially
altering a biological activity of MEKK1, whereas an "essential"
amino acid residue results in such a change. For example, amino
acid residues that are conserved among the polypeptides of the
present invention are predicted to be particularly unamenable to
alteration.
[0067] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in a MEKK1 protein is
preferably replaced with another amino acid residue from the same
side chain family. Alternatively, in another embodiment, mutations
can be introduced randomly along all or part of a MEKK1 coding
sequence, such as by saturation mutagenesis, and the resultant
mutant polypeptides can be screened for MEKK1 biological activity
to identify mutant polypeptides that retain activity. Following
mutagenesis of SEQ ID NO:1 or 3, or the nucleotide sequence of the
DNA insert of the plasmid deposited with ATCC as Accession Number
PTA-1836, the encoded protein can be expressed recombinantly and
the activity of the protein can be determined.
[0068] As used herein, a "biologically active portion" of a MEKK1
protein includes a fragment of a MEKK1 protein which participates
in an interaction between a MEKK1 molecule and a non-MEKK1
molecule. Biologically active portions of a MEKK1 protein include
peptides comprising amino acid sequences sufficiently homologous
to, or derived from, the amino acid sequence of the MEKK1 protein,
e.g., the amino acid sequence shown in SEQ ID NO:2, which include
less amino acids than the full-length MEKK1 proteins, and exhibit
at least one activity of a MEKK1 protein. Typically, biologically
active portions comprise a domain or motif with at least one
activity of the MEKK1 protein, e.g., signal transduction, cell-cell
adhesion, cell migration, patterning, cellular growth and/or
differentiation. A biologically active portion of a MEKK1 protein
can be a polypeptide which is, for example, 10, 25, 50, 100, 200 or
more amino acids in length. Biologically active portions of a MEKK1
protein can be used as targets for developing agents which modulate
a MEKK1 mediated activity, e.g., protein-protein interaction,
cell-cell adhesion, or signal transduction.
[0069] Calculations of homology or sequence identity between
sequences (the terms are used interchangeably herein) are performed
as follows.
[0070] To determine the percent identity of two amino acid
sequences, or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, 90%, 95%, 96%, 97%,
98%, 99%, or 100% of the length of the reference sequence (e.g.,
when aligning a second sequence to the MEKK1 amino acid sequence of
SEQ ID NO:2 having 1512 amino acid residues, at least 1450,
preferably at least 1475, more preferably at least 1490, even more
preferably at least 1500, and even more preferably at least 1506,
or 1512 amino acid residues are aligned). The amino acid residues
or nucleotides at corresponding amino acid positions or nucleotide
positions are then compared. When a position in the first sequence
is occupied by the same amino acid residue or nucleotide as the
corresponding position in the second sequence, then the molecules
are identical at that position (as used herein amino acid or
nucleic acid "identity" is equivalent to amino acid or nucleic acid
"homology"). The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences, taking into account the number of gaps, and the length
of each gap, which need to be introduced for optimal alignment of
the two sequences.
[0071] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm
which has been incorporated into the GAP program in the GCG
software package (available at http://www.gcg.com), using either a
Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14,
12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In
yet another preferred embodiment, the percent identity between two
nucleotide sequences is determined using the GAP program in the GCG
software package (available at http://www.gcg.com), using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred
set of parameters (and the one that should be used if the
practitioner is uncertain about what parameters should be applied
to determine if a molecule is within a sequence identity or
homology limitation of the invention) are a Blossum 62 scoring
matrix with a gap penalty of 12, a gap extend penalty of 4, and a
frameshift gap penalty of 5.
[0072] The percent identity between two amino acid or nucleotide
sequences can be determined using the algorithm of E. Meyers and W.
Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into
the ALIGN program (version 2.0), using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4.
[0073] The nucleic acid and protein sequences described herein can
be used as a "query sequence" to perform a search against public
databases to, for example, identify other family members or related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol.
215:403-10 (1990). BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to MEKK1 nucleic acid molecules of the
invention. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to MEKK1 protein molecules of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al., Nucleic Acids Res.
25(17):3389-3402 (1997). When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. See
http://www.ncbi.nlm.nih.gov.
[0074] "Misexpression" or "aberrant expression", as used herein,
refers to a non-wild type pattern of gene expression, at the RNA or
protein level. It includes expression at non-wild type levels,
i.e., over- or under-expression; a pattern of expression that
differs from wild type in terms of the time or stage at which the
gene is expressed, e.g., increased or decreased expression (as
compared with wild type) at a predetermined developmental period or
stage; a pattern of expression that differs from wild type in terms
of decreased expression (as compared with wild type) in a
predetermined cell type or tissue type; a pattern of expression
that differs from wild type in terms of the splicing size, amino
acid sequence, post-transitional modification, or biological
activity of the expressed polypeptide; a pattern of expression that
differs from wild type in terms of the effect of an environmental
stimulus or extracellular stimulus on expression of the gene, e.g.,
a pattern of increased or decreased expression (as compared with
wild type) in the presence of an increase or decrease in the
strength of the stimulus.
[0075] "Subject", as used herein, can refer to a mammal, e.g., a
human, or to an experimental or animal or disease model. The
subject can also be a non-human animal, e.g., a horse, cow, goat,
or other domestic animal.
[0076] A "purified preparation of cells", as used herein, refers
to, in the case of plant or animal cells, an in vitro preparation
of cells and not an entire intact plant or animal. In the case of
cultured cells or microbial cells, it consists of a preparation of
at least 10% and more preferably 50% of the subject cells.
[0077] Various aspects and embodiments of the invention are
described in further detail below.
[0078] Isolated Nucleic Acid Molecules
[0079] In one aspect, the invention provides, an isolated or
purified, nucleic to acid molecule that encodes a MEKK1 polypeptide
described herein, e.g., a full-length MEKK1 protein or a fragment
thereof, e.g., a biologically active portion of MEKK1 protein. Also
included is a nucleic acid fragment suitable for use as a
hybridization probe, which can be used, e.g., to a identify nucleic
acid molecule encoding a polypeptide of the invention, MEKK1 mRNA,
and fragments suitable for use as primers, e.g., PCR primers for
the amplification or mutation of nucleic acid molecules.
[0080] In one embodiment, an isolated nucleic acid molecule of the
invention includes the nucleotide sequence shown in SEQ ID NO:1, or
the nucleotide sequence of the DNA insert of the plasmid deposited
with ATCC as Accession Number PTA-1836, or a portion of any of
these nucleotide sequences. In one embodiment, the nucleic acid
molecule includes sequences encoding the human MEKK1 protein (i.e.,
"the coding region", from nucleotides 74539 of SEQ ID NO:1), as
well as 5' untranslated sequence corresponding to nucleotides 1-6
of SEQ ID NO:1 and 3' untranslated sequence corresponding to
nucleotides 4546 to 5245 of SEQ ID NO:1. Alternatively, the nucleic
acid molecule can include only the coding region of SEQ ID NO:1
(e.g., nucleotides 7-4545, corresponding to nucleotides 1-4539 of
SEQ ID NO:3) and, e.g., no flanking sequences which normally
accompany the subject sequence. In another embodiment, the nucleic
acid molecule encodes a sequence corresponding to the mature
protein from about amino acid 1 to amino acid 1512 of SEQ ID
NO:2.
[0081] In another embodiment, an isolated nucleic acid molecule of
the invention includes a nucleic acid molecule which is a
complement of the nucleotide sequence shown in SEQ ID NO:1 or 3, or
the nucleotide sequence of the cDNA insert of the plasmid deposited
with ATCC as Accession Number PTA-1836. In other embodiments, the
nucleic acid molecule of the invention is sufficiently
complementary to the nucleotide sequence shown in SEQ ID NO:1 or 3,
or the nucleotide sequence of the cDNA insert of the plasmid
deposited with ATCC as Accession Number PTA-1836 such that it can
hybridize to the nucleotide sequence shown in SEQ ID NO:1 or 3, or
the nucleotide sequence of the cDNA insert of the plasmid deposited
with ATCC as Accession Number PTA-1836, thereby forming a stable
duplex.
[0082] In one embodiment, an isolated nucleic acid molecule of the
present invention includes a nucleotide sequence which is at least
about: 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%,93%, 94%,95%,
96%, 97%, 98%, 99%, or more homologous to the entire length of the
nucleotide sequence shown in SEQ ID NO:1 or 3, or the entire length
of the nucleotide sequence of the cDNA insert of the plasmid
deposited with ATCC as Accession Number PTA-1836, or a portion,
preferably of the same length, of any of these nucleotide
sequences.
[0083] MEKK1 Nucleic Acid Fragments
[0084] A nucleic acid molecule of the invention can include only a
portion of the nucleic acid sequence of SEQ ID NO:1 or 3, or the
nucleotide sequence of the cDNA insert of the plasmid deposited
with ATCC as Accession Number PTA-1836. For example, such a nucleic
acid molecule can include a fragment that can be used as a probe or
primer or a fragment encoding a portion of a MEKK1 protein, e.g.,
an immunogenic or biologically active portion of a MEKK1 protein. A
fragment can comprise nucleotides 3576-4542 of SEQ ID NO:1, which
encodes a kinase or catalytic domain of human MEKK1. A fragment can
comprise nucleotides 2632-4542 of SEQ ID NO:1, which encodes a
cleaved/processed domain of human MEKK1. The nucleotide sequence
determined from the cloning of the MEKK1 gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning other MEKK1 family members, or fragments thereof, as
well as MEKK1 homologues, or fragments thereof, from other
species.
[0085] In another embodiment, a nucleic acid molecule includes a
nucleotide sequence that includes part, or all, of the coding
region and extends into either (or both) the 5' or 3' noncoding
region. Thus, the following nucleic acid molecules are
provided:
[0086] a nucleic acid molecule comprising from about 10 to about 64
contiguous nucleotides from the nucleic acid sequence
ATGGCGGCGGCGGCGGGGAATCGCGCCTCGTCGGGATTCCCGGGCGC CAGGGCTA and having
at least 80% homology to the nucleic acid sequence shown in SEQ ID
NO:1;
[0087] a nucleic acid molecule comprising from about 10 to about 64
contiguous nucleotides from the nucleic acid sequence
GAGAAAATGGCGGCGGCGGCGGGGAATCGCGCCTCGTCGGGATTCCC GGGCGCCAGGGCTA and
having at least 80% homology to the nucleic acid sequence shown in
SEQ ID NO:1;
[0088] a nucleic acid molecule comprising the nucleic acid sequence
GCGCGCCCGCG and having at least 80% homology to the nucleic acid
sequence shown in SEQ ID NO:1;
[0089] a nucleic acid molecule comprising the nucleic acid sequence
CCGCGAGCCGCGGCGGC and having at least 80% homology to the nucleic
acid sequence shown in SEQ ID NO:1;
[0090] a nucleic acid molecule comprising the nucleic acid sequence
GCACGTGGA and having at least 80% homology to the nucleic acid
sequence shown in SEQ ID NO:1;
[0091] a nucleic acid molecule comprising the nucleic acid sequence
CTACGTCTA and having at least 80% homology to the nucleic acid
sequence shown in SEQ ID NO:1;
[0092] a nucleic acid molecule comprising the nucleic acid sequence
CCAGTTCCA and having at least 80% homology to the nucleic acid
sequence shown in SEQ ID NO:1;
[0093] a nucleic acid molecule comprising the nucleic acid sequence
GCTATTGC and having at least 80% homology to the nucleic acid
sequence shown in SEQ ID NO:1;
[0094] a nucleic acid molecule comprising the nucleic acid sequence
TTTGGATGGTCA and having at least 80% homology to the nucleic acid
sequence shown in SEQ ID NO:1;
[0095] a nucleic acid molecule comprising the nucleic acid sequence
GGACAGCTTC and having at least 80% homology to the nucleic acid
sequence shown in SEQ ID NO:1;
[0096] a nucleic acid molecule comprising the nucleic acid sequence
CCCCTGAGTGC and having at least 80% homology to the nucleic acid
sequence shown in SEQ ID NO:1;
[0097] a nucleic acid molecule comprising the nucleic acid sequence
GCCAGCATTT and having at least 80% homology to the nucleic acid
sequence shown in SEQ ID NO:1;
[0098] a nucleic acid molecule comprising the nucleic acid sequence
CATCTAGACCT and having at least 80% homology to the nucleic acid
sequence shown in SEQ ID NO:1;
[0099] a nucleic acid molecule comprising the nucleic acid sequence
GGCTGTAGCA and having at least 80% homology to the nucleic acid
sequence shown in SEQ ID NO:1;
[0100] a nucleic acid molecule comprising the nucleic acid sequence
GTAATGCTGT and having at least 80% homology to the nucleic acid
sequence shown in SEQ ID NO:1;
[0101] a nucleic acid molecule comprising the nucleic acid sequence
CCCAGTGAC and having at least 80% homology to the nucleic acid
sequence shown in SEQ ID NO:1;
[0102] a nucleic acid molecule comprising the nucleic acid sequence
GGATGCCCTCCCCAT and having at least 80% homology to the nucleic
acid sequence shown in SEQ ID NO:1; and
[0103] a nucleic acid molecule comprising the nucleic acid sequence
GGCCTTTCG and having at least 80% homology to the nucleic acid
sequence shown in SEQ ID NO:1.
[0104] A nucleic acid fragment can include a sequence encoding a
domain, region, or functional site described herein. A nucleic acid
fragment can also include one or more domain, region, or functional
site described herein. Thus, for example, a nucleic acid fragment
can contain a sequence encoding a protein kinase domain.
[0105] MEKK1 probes and primers are provided. Typically a
probe/primer is an isolated or purified oligonucleotide. The
oligonucleotide typically includes a region of nucleotide sequence
that hybridizes under stringent conditions to at least about 7, 12
or 15, preferably about 20 or 25, more preferably about 30, 35, 40,
45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or
antisense sequence of SEQ ID NO:1 or 3, or the nucleotide sequence
of the DNA insert of the plasmid deposited with ATCC as Accession
Number PTA-1836, or of a naturally occurring allelic variant or
mutant of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number
PTA-1836.
[0106] In a preferred embodiment the nucleic acid is a probe which
is at least 5 or 10, and less than 200, more preferably less than
100, or less than 50, base pairs in length. It should be identical,
or differ by 1, or less than in 5 or 10 bases, from a sequence
disclosed herein. If alignment is needed for this comparison the
sequences should be aligned for maximum homology.
[0107] "Looped" out sequences from deletions or insertions, or
mismatches, are considered differences.
[0108] In another embodiment, a set of primers is provided, e.g.,
primers suitable for use in a PCR, which can be used to amplify a
nucleic acid sequence which encodes a selected region of a MEKK1
sequence, e.g., a domain, region, site or other sequence described
herein. The primers should be at least 5, 10, or 50 base pairs in
length and less than 100, or less than 200, base pairs in length.
The primers should be identical, or differ by one base from a
sequence disclosed herein or from a naturally occurring variant,
e.g., primers suitable for amplifying all or a portion of any of
the following regions are provided:
[0109] a kinase or catalytic domain comprising amino acids 1191 to
1512 of SEQ ID NO:2; and
[0110] a processed or cleaved domain comprising amino acids 876 to
1512 of SEQ ID NO:2.
[0111] A nucleic acid fragment can encode an epitope bearing region
of a polypeptide described herein.
[0112] A nucleic acid fragment encoding a "biologically active
portion of a MEKK1 polypeptide" can be prepared by isolating a
portion of the nucleotide sequence of SEQ ID NO:1 or 3, or the
nucleotide sequence of the cDNA insert of the plasmid deposited
with ATCC as Accession Number PTA-1836, which encodes a polypeptide
having a MEKK1 biological activity (e.g., the biological activities
of the MEKK1 proteins are described herein), expressing the encoded
portion of the MEKK1 protein (e.g., by recombinant expression in
vitro) and assessing the activity of the encoded portion of the
MEKK1 protein. For example, a nucleic acid fragment encoding a
biologically active portion of MEKK1 includes a protein kinase
domain, e.g., amino acid residues 1191 to 1512 of SEQ ID NO:2. A
nucleic acid fragment encoding a biologically active portion of a
MEKK1 polypeptide, may comprise a nucleotide sequence which is
greater than 900 or more nucleotides in length.
[0113] MEKK1 Nucleic Acid Variants
[0114] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequence shown in SEQ ID NO:1 or 3,
or the nucleotide sequence of the cDNA insert of the plasmid
deposited with ATCC as Accession Number PTA-1836. Such differences
can be due to degeneracy of the genetic code and result in a
nucleic acid which encodes the same MEKK1 proteins as those encoded
by the nucleotide sequence disclosed herein. In another embodiment,
an isolated nucleic acid molecule of the invention has a nucleotide
sequence encoding a protein having an amino acid sequence which
differs, by at least 1, but less than 5, 10, 20, 50, 75, or 100
amino acid residues from that shown in SEQ ID NO:2. If alignment is
needed for this comparison the sequences should be aligned for
maximum homology. "Looped" out sequences from deletions or
insertions, or mismatches, are considered differences.
[0115] Nucleic acids of the inventor can be chosen for having
codons, which are preferred, or non-preferred, for a particular
expression system. For example, the nucleic acid can be one in
which at least one codon, preferably, at least 10% or 20% of the
codons, has been altered such that the sequence is optimized for
expression in bacteria, e.g., E. coli, yeast, human, insect, or
mammalian cells, e.g., Chinese hamster ovary (CHO) or SV40
transformed simian (COS) cells.
[0116] Nucleic acid variants can be naturally occurring, such as
allelic variants (same locus), homologs (different locus), and
orthologs (different organism), or can be non-naturally occurring.
Non-naturally occurring variants can be made by mutagenesis
techniques, including those applied to polynucleotides, cells, or
organisms. The variants can contain nucleotide substitutions,
deletions, inversions and insertions. Variation can occur in either
or both the coding and non-coding regions. The variations can
produce both conservative and non-conservative amino acid
substitutions, as compared to the encoded product.
[0117] In a preferred embodiment, the nucleic acid differs from
that of SEQ ID NO:1 or 3, or the sequence of the cDNA insert of the
plasmid deposited with ATCC as Accession Number PTA-1836, e.g., as
follows: by at least one but less than 10, 20, 30, or 40
nucleotides; at least one but less than 1%, 5%, 10% or 20% of the
in the subject nucleic acid. If it is necessary for this analysis,
the sequences should be aligned for maximum homology. "Looped" out
sequences from deletions or insertions, or mismatches, are
considered differences.
[0118] Orthologs, homologs, and allelic variants can be identified
using methods known in the art. These variants comprise a
nucleotide sequence encoding a polypeptide that is 50%, at least
about 55%, typically at least about 70-75%, more typically at least
about 80-85%, and most typically at least about 90-95% or more,
identical to the amino acid sequence shown in SEQ ID NO:2 or a
fragment of this sequence. Such nucleic acid molecules can readily
be identified as being able to hybridize under stringent
conditions, to the nucleotide sequence shown in SEQ ID NO:1 or SEQ
ID NO:3, or a fragment of the sequence. Nucleic acid molecules
corresponding to orthologs, homologs, and allelic variants of the
MEKK1 cDNAs of the invention can further be isolated by mapping to
the same chromosome or locus as the MEKK1 gene.
[0119] Preferred variants include those that are correlated with
protein binding activities.
[0120] The invention also provides allelic variants of human MEKK1.
An "allelic variant," as used herein, is a protein having at least
about 75% amino acid sequence, preferably at least about 85%, more
preferably at least about 95%, and most preferably at least about
99% identity to the amino acid sequence of MEKK1, or to a fragment
thereof, or to a protein conjugate thereof which retains the
biological activity of MEKK1. Allelic variants of MEKK1 include
both functional and non-functional proteins. Functional allelic
variants are naturally occurring amino acid sequence variants of
the MEKK1 protein within a population that maintain the ability to
bind MEKK1 binding partners. Functional allelic variants will
typically contain only conservative substitution of one or more
amino acids of SEQ ID NO:2, or substitution, deletion or insertion
of non-critical residues in non-critical regions of the protein.
Non-functional allelic variants are naturally-occurring amino acid
sequence variants of the MEKK1, e.g., human MEKK1, protein within a
population that do not have the ability to bind MEKK1 binding
partners. Non-functional allelic variants will typically contain a
non-conservative substitution, a deletion, or insertion, or
premature truncation of the amino acid sequence of SEQ ID NO:2, or
a substitution, insertion, or deletion in critical residues or
critical regions of the protein.
[0121] This aspect of the inventions further includes MEKK1 allelic
variant expression elements. Such elements include, without
limitation, isolated or recombinant nucleic acid sequences encoding
MEKK1, or nucleic acid sequences specifically homologous or
complementary thereof, vectors comprising and such any such nucleic
acid sequences, and recombinant expression vectors which express
MEKK1 or antisense transcripts or dominant mutants thereof.
[0122] Moreover, nucleic acid molecules encoding other MEKK1 family
members and, thus, which have a nucleotide sequence which differs
from the MEKK1 sequences of SEQ ID NO:1 or 3, or the nucleotide
sequence of the cDNA insert of the plasmid deposited with ATCC as
Accession Number PTA-1836 are intended to be within the scope of
the invention.
[0123] "Dominant negative mutant" as used herein refers to a
nucleic acid coding region sequence which has been changed with
regard to at least one position in the sequence, relative to the
corresponding wild type native version, preferably at a position
which encodes a changed amino acid residue position at an active
site required for biological and/or pharmacological activity in the
native peptide. Dominant negative mutant embodiments of the
invention, for example, include peptides comprising a sequence as
depicted in SEQ ID No:2, wherein one or more positions
corresponding to SEQ ID NO:2, e.g., lysine at position 1273, are
substituted or deleted. Dominant negative mutants are, moreover,
defined to be included within the scope of the disclosure of the
variants sections above. Such dominant negative mutants can be
prepared by art recognized procedures (see, e.g., Townsley et al.,
Proc. Natl. Acad. Sci., USA. 94:2362-2367 (1997)).
[0124] Antisense Nucleic Acid Molecules, Ribozymes and Modified
MEKK1 Nucleic Acid Molecules
[0125] In another aspect, the invention features, an isolated
nucleic acid molecule which is antisense to MEKK1. An "antisense"
nucleic acid can include a nucleotide sequence which is
complementary to a "sense" nucleic acid encoding a protein, e.g.,
complementary to the coding strand of a double-stranded cDNA
molecule or complementary to an mRNA sequence. The antisense
nucleic acid can be complementary to an entire MEKK1 coding strand,
or to only a portion thereof (e.g., the coding region of human
MEKK1 corresponding to SEQ ID NO:3). In another embodiment, the
antisense nucleic acid molecule is antisense to a "noncoding
region" of the coding strand of a nucleotide sequence encoding
MEKK1 (e.g., the 5' and 3' untranslated regions).
[0126] An antisense nucleic acid can be designed such that it is
complementary to the entire coding region of MEKK1 mRNA, but more
preferably is an oligonucleotide which is antisense to only a
portion of the coding or noncoding region of MEKK1 mRNA. For
example, the antisense oligonucleotide can be complementary to the
region surrounding the translation start site of MEKK1 mRNA, e.g.,
between the -10 and +10 regions of the target gene nucleotide
sequence of interest. An antisense oligonucleotide can be, for
example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, or more nucleotides in length.
[0127] An antisense nucleic acid of the invention can be
constructed using chemical synthesis and enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed between the antisense and sense nucleic acids,
e.g., phosphorothioate derivatives and acridine substituted
nucleotides can be used. The antisense nucleic acid also can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0128] The antisense nucleic acid molecules of the invention are
typically administered to a subject (e.g., by direct injection at a
tissue site), or generated in situ such that they hybridize with or
bind to cellular mRNA and/or genomic DNA encoding a MEKK1 protein
to thereby inhibit expression of the protein, e.g., by inhibiting
transcription and/or translation. Alternatively, antisense nucleic
acid molecules can be modified to target selected cells and then
administered systemically. For systemic administration, antisense
molecules can be modified such that they specifically bind to
receptors or antigens expressed on a selected cell surface, e.g.,
by linking the antisense nucleic acid molecules to peptides or
antibodies which bind to cell surface receptors or antigens. The
antisense nucleic acid molecules can also be delivered to cells
using the vectors described herein. To achieve sufficient
intracellular concentrations of the antisense molecules, vector
constructs in which the antisense nucleic acid molecule is placed
under the control of a strong pol II or pol III promoter are
preferred.
[0129] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other
(Gaultier et al., Nucleic Acids. Res. 15:6625-6641(1987)). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al., Nucleic Acids Res.
15:6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et al.,
FEBS Lett. 215:327-330(1987)).
[0130] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. A ribozyme having specificity for a
MEKK1-encoding nucleic acid can include one or more sequences
complementary to the nucleotide sequence of a MEKK1 cDNA disclosed
herein (i.e., SEQ ID NO:1 or 3), and a sequence having known
catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No.
5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591). For
example, a derivative of a Tetrahymena L-19 IVS RNA can be
constructed in which the nucleotide sequence of the active site is
complementary to the nucleotide sequence to be cleaved in a
MEKK1-encoding mRNA. (See, e.g., Cech et al., U.S. Pat. No.
4,987,071; and Cech et al., U.S. Pat. No. 5,116,742.)
Alternatively, MEKK1 mRNA can be used to select a catalytic RNA
having a specific ribonuclease activity from a pool of RNA
molecules. (See, e.g., Bartel et al., Science 261:1411-1418.
(1993))
[0131] MEKK1 gene expression can be inhibited by targeting
nucleotide sequences complementary to the regulatory region of the
MEKK1 (e.g., the MEKK1 promoter and/or enhancers) to form triple
helical structures that prevent transcription of the MEKK1 gene in
target cells. See, generally, Helene, Anticancer Drug Des.
6(6):569-84 (1991); Helene et al., Ann. N.Y. Acad. Sci. 660:27-36
(1992); and Maher, Bioassays 14(12):807-15 (1992). The potential
sequences that can be targeted for triple helix formation can be
increased by creating a so called "switchback" nucleic acid
molecule. Switchback molecules are synthesized in an alternating
5'-3', 3'-5' manner, such that they base pair with first one strand
of a duplex and then the other, eliminating the necessity for a
sizeable stretch of either purines or pyrimidines to be present on
one strand of a duplex.
[0132] The invention also provides detectably labeled
oligonucleotide primer and probe molecules. Typically, such labels
are chemiluminescent, fluorescent, radioactive, or
colorimetric.
[0133] A MEKK1 nucleic acid molecule can be modified at the base
moiety, sugar moiety or phosphate backbone to improve, e.g., the
stability, hybridization, or solubility of the molecule. For
example, the deoxyribose phosphate backbone of the nucleic acid
molecules can be modified to generate peptide nucleic acids (see
Hyrup et al., Bioorganic & Medicinal Chemistry 4 (1): 5-23
(1996)). As used herein, the terms "peptide nucleic acid" or "PNA"
refers to a nucleic acid mimic, e.g., a DNA mimic, in which the
deoxyribose phosphate backbone is replaced by a pseudopeptide
backbone and only the four natural nucleobases are retained. The
neutral backbone of a PNA can allow for specific hybridization to
DNA and RNA under conditions of low ionic strength. The synthesis
of PNA oligomers can be performed using standard solid phase
peptide synthesis protocols as described in Hyrup et al., (1996)
supra; Perry-O'Keefe et al., Proc. Natl. Acad. Sci. 93: 14670-675
(1996).
[0134] PNAs of MEKK1 nucleic acid molecules can be used in
therapeutic and diagnostic applications. For example, PNAs can be
used as antisense or antigene agents for sequence-specific
modulation of gene expression by, for example, inducing
transcription or translation arrest or inhibiting replication. PNAs
of MEKK1 nucleic acid molecules can also be used in the analysis of
single base pair mutations in a gene (e.g., by PNA-directed PCR
clamping); as `artificial restriction enzymes` when used in
combination with other enzymes, (e.g., S1 nucleases (Hyrup B.
(1996) supra)); or as probes or primers for DNA sequencing or
hybridization (Hyrup et al., (1996) supra; Perry-O'Keefe
supra).
[0135] In other embodiments, the oligonucleotide may include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci.
USA 86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci.
USA 84:648-652 (1987); PCT Publication No. W088/09810) or the
blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In
addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (See, e.g., Krol et al.,
Bio-Techniques 6:958-976 (1988)) or intercalating agents. (See,
e.g., Zon Pharm. Res. 5:539-549 (1988)). To this end, the
oligonucleotide may be conjugated to another molecule, (e.g., a
peptide, hybridization triggered cross-linking agent, transport
agent, or hybridization-triggered cleavage agent).
[0136] The invention also includes molecular beacon oligonucleotide
primer and probe molecules having at least one region which is
complementary to a MEKK1 nucleic acid of the invention, two
complementary regions one having a fluorophore and one a quencher
such that the molecular beacon is useful for quantitating the
presence of the MEKK1 nucleic acid of the invention in a sample.
Molecular beacon nucleic acids are described, for example, in
Lizardi et al., U.S. Pat. No. 5,854,033; Nazarenko et al., U.S.
Pat. No. 5,866,336, and Livak et al., U.S. Pat. No. 5,876,930.
[0137] Isolated MEKK1 Polypeptides
[0138] In another aspect, the invention features an isolated MEKK1
protein, or fragment, e.g., a biologically active portion, for use
as immunogens or antigens to raise or test (or more generally, to
bind) anti-MEKK1 antibodies. MEKK1 protein can be isolated from
cels or tissue sources using standard protein purification
techniques. MEKK1 protein or fragments thereof can be produced by
recombinant DNA techniques or synthesized chemically.
[0139] Polypeptides of the invention include those which arise as a
result of the existence of multiple genes, alternative
transcription events, alternative RNA splicing events, and
alternative translational and postranslational events. The
polypeptide can be expressed in systems, e.g., cultured cells,
which result in substantially the same postranslational
modifications present when expressed the polypeptide is expressed
in a native cell, or in systems which result in the alteration or
omission of postranslational modifications, e.g., gycolsylation or
cleavage, present when expressed in a native cell. As used herein,
"MEKK1 protein" and "MEKK1 polypeptide" comprises the amino acid
sequences of SEQ ID NO:2.
[0140] In a preferred embodiment, a MEKK1 polypeptide has one or
more of the following characteristics:
[0141] (i) it has the ability to modulate a signal transduction
pathway;
[0142] (ii) it modulates a protein binding interaction;
[0143] (iii) it has a molecular weight, e.g., a deduced molecular
weight, of SEQ ID NO:2;
[0144] (iv) it has an amino acid composition or other physical
characteristics of SEQ ID NO:2;
[0145] (v) it has an overall sequence similarity of at least 60%,
preferably at least 70%, more preferably at least 80, 90, 95%, 96%,
97%, 98%, or 99% with a polypeptide of SEQ ID NO:2;
[0146] (vi) it has a kinase domain which is preferably about 70%,
80%, 90% or 95% homologous with amino acid residues 1191 to 1512 of
SEQ ID NO:2; or
[0147] (vii) it has a processed/cleaved domain which is preferably
about 70%, 80%, 90% or 95% homologous with amino acid residues 876
to 1512 of SEQ ID NO:2.
[0148] In other embodiments, a MEKK1 polypeptide is a polypeptide
with one or more of the following characteristics:
[0149] a polypeptide comprising from about 5 to about 19 contiguous
amino acids from the amino acid sequence MAAAAGNRASSSGFPGARAT and
having at least 80% homology to the amino acid sequence shown in
SEQ ID NO:2;
[0150] a polypeptide comprising from about 5 to about 19 contiguous
amino acids from the amino acid sequence EKMAAAAGNRASSSGFPGARAT and
having at least 80% homology to the amino acid sequence shown in
SEQ ID NO:2;
[0151] a polypeptide comprising the amino acid sequence SAPAA and
having at least 80% homology to the. amino acid sequence shown in
SEQ ID NO:2;
[0152] a polypeptide comprising the amino acid sequence ASRGG and
having at least 80% homology to the amino acid sequence shown in
SEQ ID NO:2;
[0153] a polypeptide comprising the amino acid sequence CARGT and
having at least 80% homology to the amino acid sequence shown in
SEQ ID NO:2;
[0154] a polypeptide comprising the amino acid sequence VSSSTH and
having at least 80% homology to the amino acid sequence shown in
SEQ ID NO:2;
[0155] a polypeptide comprising the amino acid sequence LMAIADE and
having at least 80% homology to the amino acid sequence shown in
SEQ ID NO:2;
[0156] a polypeptide comprising the amino acid sequence
TLDGQQDSFLQASVPNNYLETTENSSPECT and having at least 80% homology to
the amino acid sequence shown in SEQ ID NO:2;
[0157] a polypeptide comprising the amino acid sequence LASISV and
having at least 80% homology to the amino. acid sequence shown in
SEQ ID NO:2;
[0158] a polypeptide comprising the amino acid sequence
SFGCSSNSSNAVIPSDE and having at least 80% homology to the amino
acid sequence shown in SEQ ID NO:2; or
[0159] a polypeptide comprising the amino acid sequence
SQDALPIVPQLQVENGEDIIIIQQDTPETLPGHTKAKQPYREDT and having at least
80% homology to the amino acid sequence shown in SEQ ID NO:2.
[0160] Some embodiments of the MEKK1 protein, or fragment thereof,
differ from the corresponding sequence in SEQ ID NO:2. In one
embodiment it differs by at least one but by less than 15, 10 or 5
amino acid residues. In another it differs from the corresponding
sequence in SEQ ID NO:2 by at least one residue but less than 20%,
15%, 10% or 5% of the residues in it differ from the corresponding
sequence in SEQ ID NO:2. (If this comparison requires alignment the
sequences should be aligned for maximum homology. "Looped" out
sequences from deletions or insertions, or mismatches, are
considered differences.) The differences are, preferably,
differences or changes at a non-essential residue or a conservative
substitution. In a preferred embodiment the differences are not in
a protein kinase domain.
[0161] Other embodiments include a protein that contain one or more
changes in amino acid sequence, e.g., a change in an amino acid
residue which is not essential for activity. Such MEKK1 proteins
differ in amino acid sequence from SEQ ID NO:2, yet retain
biological activity.
[0162] In one embodiment, a biologically active portion of a MEKK1
protein includes a protein kinase domain. Moreover, other
biologically active portions, in which other regions of the protein
are deleted, can be prepared by recombinant techniques and
evaluated for one or more of the functional activities of a native
MEKK1 protein.
[0163] In a preferred embodiment, the MEKK1 protein has the amino
acid sequence shown in SEQ ID NO:2. In other embodiments, the MEKK1
protein is substantially identical to SEQ ID NO:2. In yet another
embodiment, the MEKK1 protein is substantially identical to SEQ ID
NO:2 and retains the functional activity of the protein of SEQ ID
NO:2, as described in detail in subsection I above. Thus, the
following nucleic acid molecules are provided:
[0164] MEKK1 Chimeric or Fusion Proteins
[0165] In another aspect, the invention provides MEKK1 chimeric or
fusion proteins. As used herein, a MEKK1 "chimeric protein" or
"fusion protein" includes a MEKK1 polypeptide linked to a non-MEKK1
polypeptide. A "non-MEKK1 polypeptide" refers to a polypeptide
having an amino acid sequence corresponding to a protein which is
not substantially homologous to the MEKK1 protein, e.g., a protein
which is different from the MEKK1 protein and which is derived from
the same or a different organism. The MEKK1 polypeptide of the
fusion protein can correspond to all or a portion e.g., a fragment
described herein of a MEKK1 amino acid sequence. In a preferred
embodiment, a MEKK1 fusion protein includes at least one (or two)
biologically active portion of a MEKK1 protein. The non-MEKK1
polypeptide can be fused to the N-terminus or C-terminus of the
MEKK1 polypeptide.
[0166] The fusion protein can include a moiety which has a high
affinity for a ligand. For example, the fusion protein can be a
GST-MEKK1 fusion protein in which the MEKK1 sequences are fused to
the C-terminus of the GST sequences. Such fusion proteins can
facilitate the purification of recombinant MEKK1. Alternatively,
the fusion protein can be a MEKK1 protein containing a heterologous
signal sequence at its N-terminus. In certain host cells (e.g.,
mammalian host cells), expression and/or secretion of MEKK1 can be
increased through use of a heterologous signal sequence.
[0167] Fusion proteins can include all or a part of a serum
protein, e.g., an IgG constant region, or human serum albumin.
[0168] The MEKK1 fusion proteins of the invention can be
incorporated into pharmaceutical compositions and administered to a
subject in vivo. The MEKK1 fusion proteins can be used to affect
the bioavailability of a MEKK1 substrate. MEKK1 fusion proteins may
be useful therapeutically for the treatment of disorders caused by,
for example, (i) aberrant modification or mutation of a gene
encoding a MEKK1 protein; (ii) mis-regulation of the MEKK1 gene;
and (iii) aberrant post-translational modification of a MEKK1
protein.
[0169] Moreover, the MEKK1-fusion proteins of the invention can be
used as immunogens to produce anti-MEKK1 antibodies in a subject,
to purify MEKK1 ligands and in screening assays to identify
molecules which inhibit the interaction of MEKK1 with a MEKK1
substrate.
[0170] Expression vectors are commercially available that already
encode a fusion moiety (e.g., a GST polypeptide). A MEKK1-encoding
nucleic acid can be cloned into such an expression vector such that
the fusion moiety is linked in-frame to the MEKK1 protein.
[0171] Variants of MEKK1 Proteins
[0172] In another aspect, the invention also features a variant of
a MEKK1 polypeptide, e.g., which functions as an agonist (mimetics)
or as an antagonist. Variants of the MEKK1 proteins can be
generated by mutagenesis, e.g., discrete point mutation, the
insertion or deletion of sequences or the truncation of a MEKK1
protein. An agonist of the MEKK1 proteins can retain substantially
the same, or a subset, of the biological activities of the
naturally occurring form of a MEKK1 protein. An antagonist of a
MEKK1 protein can inhibit one or more of the activities of the
naturally occurring form of the MEKK1 protein by, for example,
competitively modulating a MEKK1-mediated activity of a MEKK1
protein. Thus, specific biological effects can be elicited by
treatment with a variant of limited function. Preferably, treatment
of a subject with a variant having a subset of the biological
activities of the naturally occurring form of the protein has fewer
side effects in a subject relative to treatment with the naturally
occurring form of the MEKK1 protein.
[0173] Variants of a MEKK1 protein can be identified by screening
combinatorial libraries of mutants, e.g., truncation mutants, of a
MEKK1 protein for agonist or antagonist activity.
[0174] Libraries of fragments e.g., N-terminal, C-terminal, or
internal fragments, of a MEKK1 protein coding sequence can be used
to generate a variegated population of fragments for screening and
subsequent selection of variants of a MEKK1 protein.
[0175] Variants in which a cysteine residues is added or deleted or
in which a residue which is glycosylated is added or deleted are
particularly preferred.
[0176] Methods for screening gene products of combinatorial
libraries made by point mutations or truncation, and for screening
cDNA libraries for gene products having a selected property.
Recursive ensemble mutagenesis (REM), a new technique which
enhances the frequency of functional mutants in the libraries, can
be used in combination with the screening assays to identify MEKK1
variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA
89:7811-7815; Delgrave et al., Protein Engineering 6(3):327-331
(1993)).
[0177] Cell based assays can be exploited to analyze a variegated
MEKK1 library. For example, a library of expression vectors can be
transfected into a cell line, e.g., a cell line which ordinarily
responds to MEKK1 in a substrate-dependent manner. The transfected
cells are then contacted with MEKK1 and the effect of the
expression of the mutant on signaling by the MEKK1 substrate can be
detected, e.g., by measuring binding to a growth factor or cell
surface receptor. Plasmid DNA can then be recovered from the cells
which score for inhibition, or alternatively, potentiation of
signaling by the MEKK1 substrate, and the individual clones further
characterized.
[0178] In another aspect, the invention features a method of making
a MEKK1 polypeptide, e.g., a peptide having a non-wild type
activity, e.g., an antagonist, agonist, or super agonist of a
naturally occurring MEKK1 polypeptide, e.g., a naturally occurring
MEKK1 polypeptide. The method includes: altering the sequence of a
MEKK1 polypeptide, e.g., altering the sequence, e.g., by
substitution or deletion of one or more residues of a non-conserved
region, a domain or residue disclosed herein, and testing the
altered polypeptide for the desired activity.
[0179] In another aspect, the invention features a method of making
a fragment or analog of a MEKK1 polypeptide a biological activity
of a naturally occurring MEKK1 polypeptide. The method includes:
altering the sequence, e.g., by substitution or deletion of one or
more residues, of a MEKK1 polypeptide, e.g., altering the sequence
of a non-conserved region, or a domain or residue described herein,
and testing the altered polypeptide for the desired activity.
[0180] Anti-MEKK1 Antibodies
[0181] In another aspect, the invention provides an anti-MEKK1
antibody. The term "antibody" as used herein refers to an
immunoglobulin molecule or immunologically active portion thereof,
i.e., an antigen-binding portion. Examples of immunologically
active portions of immunoglobulin molecules include F(ab) and
F(ab').sub.2 fragments which can be generated by treating the
antibody with an enzyme such as pepsin.
[0182] The antibody can be a polyclonal, monoclonal, recombinant,
e.g., a chimeric or humanized, fully human, non-human, e.g.,
murine, or single chain antibody. In a preferred embodiment it has
effector function and can fix complement. The antibody can be
coupled to a toxin or imaging agent.
[0183] A full-length MEKK1 protein or, antigenic peptide fragment
of MEKK1 can be used as an immunogen or can be used to identify
anti-MEKK1 antibodies made with other immunogens, e.g., cells,
membrane preparations, and the like. The antigenic peptide of MEKK1
should include at least 8 amino acid residues of the amino acid
sequence shown in SEQ ID NO:2 and encompasses an epitope of MEKK1.
Preferably, the antigenic peptide includes at least 10 amino acid
residues, more preferably at least 15 amino acid residues, even
more preferably at least 20 amino acid residues, and most
preferably at least 30 amino acid residues.
[0184] Antibodies reactive with, or specific for, any of the
regions or domains described herein are provided.
[0185] Preferred epitopes encompassed by the antigenic peptide are
regions of MEKK1 are located on the surface of the protein, e.g.,
hydrophilic regions, as well as regions with high antigenicity. For
example, an Emini surface probability analysis of the human MEKK1
protein sequence can be used to indicate the regions that have a
particularly high probability of being localized to the surface of
the MEKK1 protein and are thus likely to constitute surface
residues useful for targeting antibody production.
[0186] In a preferred embodiment the antibody binds an epitope on
any domain or region on MEKK1 proteins described herein.
[0187] Chimeric, humanized, but most preferably, completely human
antibodies are desirable for applications which include repeated
administration, e.g., therapeutic treatment (and some diagnostic
applications) of human patients.
[0188] The anti-MEKK1 antibody can be a single chain antibody. A
single-chain antibody (scFV) may be engineered (see, for example,
Colcher et al., Ann N Y Acad Sci Jun 30;880:263-80 (1999); and
Reiter, Clin Cancer Res February;2(2):245-52 (1996)). The single
chain antibody can be dimerized or multimerized to generate
multivalent antibodies having specificities for different epitopes
of the same target MEKK1 protein.
[0189] In a preferred embodiment, the antibody has reduced ability
or no ability to bind an Fc receptor, for example, where it is an
isotype or subtype, fragment or other mutant, which does not
support binding to an Fc receptor, or where it has a mutagenized or
deleted Fc receptor binding region.
[0190] An anti-MEKK1 antibody (e.g., monoclonal antibody) can be
used to isolate MEKK1 by standard techniques, such as affinity
chromatography or immunoprecipitation. Moreover, an anti-MEKK1
antibody can be used to detect MEKK1 protein (e.g., in a cellular
lysate or cell supernatant) in order to evaluate the abundance and
pattern of expression of the protein. Anti-MEKK1 antibodies can be
used diagnostically to monitor protein levels in tissue as part of
a clinical testing procedure, e.g., to, for example, determine the
efficacy of a given treatment regimen. Detection can be facilitated
by coupling (i.e., physically linking) the antibody to a detectable
substance (i.e., antibody labeling). Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0191] Recombinant Expression Vectors, Host Cells and Genetically
Engineered Cells
[0192] In another aspect, the invention includes, vectors,
preferably expression vectors, containing a nucleic acid encoding a
polypeptide described herein. As used herein, the term "vector"
refers to a nucleic acid molecule capable of transporting another
nucleic acid to which it has been linked and can include a plasmid,
cosmid or viral vector. The vector can be capable of autonomous
replication or it can integrate into a host DNA. Viral vectors
include, e.g., replication defective retroviruses, adenoviruses and
adeno-associated viruses.
[0193] A vector can include a MEKK1 nucleic acid in a form suitable
for expression of the nucleic acid in a host cell. Preferably the
recombinant expression vector includes one or more regulatory
sequences operatively linked to the nucleic acid sequence to be
expressed. The term "regulatory sequence" includes promoters,
enhancers and other expression control elements (e.g.,
polyadenylation signals). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence, as well as
tissue-specific regulatory and/or inducible sequences. The design
of the expression vector can depend on such factors as the choice
of the host cell to be transformed, the level of expression of
protein desired, and the like. The expression vectors of the
invention can be introduced into host cells to thereby produce
proteins or polypeptides, including fusion proteins or
polypeptides, encoded by nucleic acids as described herein (e.g.,
MEKK1 proteins, mutant forms of MEKK1 proteins, fusion proteins,
and the like).
[0194] The recombinant expression vectors of the invention can be
designed for expression of MEKK1 proteins in prokaryotic or
eukaryotic cells. For example, polypeptides of the invention can be
expressed in E. coli, insect cells (e.g., using baculovirus
expression vectors), yeast cells or mammalian cells. Suitable host
cells are discussed further in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
[0195] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, a proteolytic cleavage site is
introduced at the junction of the fusion moiety and the recombinant
protein to enable separation of the recombinant protein from the
fusion moiety subsequent to purification of the fusion protein.
Such enzymes, and their cognate recognition sequences, include
Factor Xa, thrombin and enterokinase. Typical fusion expression
vectors include pGEX (Pharmacia Biotech Inc; Smith et al., Gene
67:3140 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and
pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione
S-transferase (GST), maltose E binding protein, or protein A,
respectively, to the target recombinant protein.
[0196] Purified fusion proteins can be used in MEKK1 activity
assays, (e.g., direct assays or competitive assays described in
detail below), or to generate antibodies specific for MEKK1
proteins. In a preferred embodiment, a fusion protein expressed in
a retroviral expression vector of the present invention can be used
to infect bone marrow cells which are subsequently transplanted
into irradiated recipients. The pathology of the subject recipient
is then examined after sufficient time has passed (e.g., six (6)
weeks).
[0197] To maximize recombinant protein expression in, e.g., E.
coli, is to express the protein in a host bacteria with an impaired
capacity to proteolytically cleave the recombinant protein
(Gottesman, S., Gene Expression Technology: Methods in Enzymology
185, Academic Press, San Diego, Calif. (1990) 119-128). Another
strategy is to alter the sequence of the nucleic acid to be
inserted into an expression vector so that the individual codons
for each amino acid are those preferentially utilized in the host
bacteria, e.g., E. coli (Wada et al., Nucleic Acids Res.
20:2111-2118 (1992)). Such alteration of nucleic acid sequences of
the invention can be carried out by standard DNA synthesis
techniques.
[0198] The MEKK1 expression vector can be a yeast expression
vector, a vector for expression in insect cells, e.g., a
baculovirus expression vector or a vector suitable for expression
in mammalian cells.
[0199] When used in mammalian cells, the expression vector's
control functions are often provided by viral regulatory elements.
For example, commonly used promoters are derived from polyoma,
Adenovirus 2, cytomegalovirus and Simian Virus 40.
[0200] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al., Genes Dev.
1:268-277 (1987)), lymphoid-specific promoters (Calame et al., Adv.
Immunol. 43:235-275 (1988)), in particular promoters of T cell
receptors (Winoto et al., EMBO J, 8:729-733 (1989)) and
immunoglobulins (Banerji et al., Cell 33:729-740 (1983)); (Queen et
al., Cell 33:741-748 (1983)), neuron-specific promoters (e.g., the
neurofilament promoter; Byrne et al., Proc. Natl. Acad. Sci. USA
86:5473-5477 (1989)), pancreas-specific promoters (Edlund et al.,
Science 230:912-916 (1985)), and mammary gland-specific promoters
(e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European
Application Publication No. 264,166). Developmentally-regulated
promoters are also encompassed, for example, the murine hox
promoters (Kessel et al., Science 249:374-379 (1990)) and the
fetoprotein promoter (Campes et al., Genes Dev. 3:537-546
(1989)).
[0201] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. Regulatory sequences
(e.g., viral promoters and/or enhancers) operatively linked to a
nucleic acid cloned in the antisense orientation can be chosen
which direct the constitutive, tissue specific or cell type
specific expression of antisense RNA in a variety of cell types.
The antisense expression vector can be in the form of a recombinant
plasmid, phagemid or attenuated virus. For a discussion of the
regulation of gene expression using antisense genes see Weintraub
et al., Antisense RNA as a molecular tool for genetic analysis,
Reviews--Trends in Genetics, Vol. 1(1) (1986).
[0202] Another aspect the invention provides a host cell which
includes a nucleic acid molecule described herein, e.g., a MEKK1
nucleic acid molecule within a recombinant expression vector or a
MEKK1 nucleic acid molecule containing sequences which allow it to
homologously recombine into a specific site of the host cell's
genome. The terms "host cell" and "recombinant host cell" are used
interchangeably herein. Such terms refer not only to the particular
subject cell but to the progeny or potential progeny of such a
cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not, in fact, be identical to the parent cell, but
are still included within the scope of the term as used herein.
[0203] A host cell can be any suitable prokaryotic or eukaryotic
cell. For example, a MEKK1 protein can be expressed in bacterial
cells such as E. coli, insect cells, yeast or mammalian cells, such
as CHO or COS cells. Other suitable host cells are known to those
skilled in the art.
[0204] Vector DNA can be introduced into host cells via
conventional transformation or transfection techniques. As used
herein, the terms "transformation" and "transfection" are intended
to refer to a variety of art-recognized techniques for introducing
foreign nucleic acid (e.g., DNA) into a host cell, including
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation
[0205] A host cell of the invention can be used to produce (i.e.,
express) a MEKK1 protein. Accordingly, the invention further
provides methods for producing a MEKK1 protein using the host cells
of the invention. In one embodiment, the method includes culturing
the host cell of the invention (into which a recombinant expression
vector encoding a MEKK1 protein has been introduced) in a suitable
medium such that a MEKK1 protein is produced. In another
embodiment, the method further includes isolating a MEKK1 protein
from the medium or the host cell.
[0206] In another aspect, the invention features, a cell or
purified preparation of cells which include a MEKK1 transgene, or
which otherwise misexpress MEKK1. The cell preparation can consist
of human or non human cells, e.g., rodent cells, e.g., mouse or rat
cells, rabbit cells, or pig cells. In preferred embodiments, the
cell or cells include a MEKK1 transgene, e.g., a heterologous form
of a MEKK1, e.g., a gene derived from humans (in the case of a
non-human cell). The MEKK1 transgene can be misexpressed, e.g.,
overexpressed or underexpressed. In other preferred embodiments,
the cell or cells include a gene which misexpress an endogenous
MEKK1, e.g., a gene the expression of which is disrupted, e.g., a
knockout. Such cells can serve as a model for studying disorders
which are related to mutated or mis-expressed MEKK1 alleles or for
use in drug screening.
[0207] In another aspect, the invention features, a human cell,
e.g., a hematopoietic stem cell, transformed with nucleic acid
which encodes a subject MEKK1 polypeptide.
[0208] Also provided are cells, preferably human cells, e.g., human
hematopoietic or fibroblast cells, in which an endogenous MEKK1 is
under the control of a regulatory sequence that does not normally
control the expression of the endogenous MEKK1 gene. The expression
characteristics of an endogenous gene within a cell, e.g., a cell
line or microorganism, can be modified by inserting a heterologous
DNA regulatory element into the genome of the cell such that the
inserted regulatory element is operably linked to the endogenous
MEKK1 gene. For example, an endogenous MEKK1 gene which is
"transcriptionally silent," e.g., not normally expressed, or
expressed only at very low levels, may be activated by inserting a
regulatory element which is capable of promoting the expression of
a normally expressed gene product in that cell. Techniques such as
targeted homologous recombination, can be used to insert the
heterologous DNA as described in, e.g., Chappel, U.S. Pat. No.
5,272,071; WO91/06667, published in May 16, 1991.
[0209] Transgenic Animals
[0210] The invention provides non-human transgenic animals. Such
animals are useful for studying the function and/or activity of a
MEKK1 protein and for identifying and/or evaluating modulators of
MEKK1 activity. As used herein, a "transgenic animal" is a
non-human animal, preferably a mammal, more preferably a rodent
such as a rat or mouse, in which one or more of the cells of the
animal includes a transgene. Other examples of transgenic animals
include non-human primates, sheep, dogs, cows, goats, chickens,
amphibians, and the like. A transgene is exogenous DNA or a
rearrangement, e.g., a deletion of endogenous chromosomal DNA,
which preferably is integrated into or occurs in the genome of the
cells of a transgenic animal. A transgene can direct the expression
of an encoded gene product in one or more cell types or tissues of
the transgenic animal, other transgenes, e.g., a knockout, reduce
expression. Thus, a transgenic animal can be one in which an
endogenous MEKK1 gene has been altered by, e.g., by homologous
recombination between the endogenous gene and an exogenous DNA
molecule introduced into a cell of the animal, e.g., an embryonic
cell of the animal, prior to development of the animal.
[0211] Intronic sequences and polyadenylation signals can also be
included in the transgene to increase the efficiency of expression
of the transgene. A tissue-specific regulatory sequence(s) can be
operably linked to a transgene of the invention to direct
expression of a MEKK1 protein to particular cells. A transgenic
founder animal can be identified based upon the presence of a MEKK1
transgene in its genome and/or expression of MEKK1 mRNA in tissues
or cells of the animals. A transgenic founder animal can then be
used to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene encoding a MEKK1 protein
can further be bred to other transgenic animals carrying other
transgenes.
[0212] MEKK1 proteins or polypeptides can be expressed in
transgenic animals or plants, e.g., a nucleic acid encoding the
protein or polypeptide can be introduced into the genome of an
animal. In preferred embodiments the nucleic acid is placed under
the control of a tissue specific promoter, e.g., a milk or egg
specific promoter, and recovered from the milk or eggs produced by
the animal. Suitable animals are mice, pigs, cows, goats, and
sheep.
[0213] The invention also includes a population of cells from a
transgenic animal, e.g., as discussed below.
[0214] Uses
[0215] The nucleic acid molecules, proteins, protein homologues,
and antibodies described herein can be used in one or more of the
following methods: a) screening assays; b) predictive medicine
(e.g., diagnostic assays, prognostic assays, monitoring clinical
trials, and pharmacogenetics); and c) methods of treatment (e.g.,
therapeutic and prophylactic); and d) methods of identifying other
important small molecules and potential drug targets within MEKK1
signaling pathway. The isolated nucleic acid molecules of the
invention can be used, for example, to express a MEKK1 protein
(e.g., via a recombinant expression vector in a host cell in gene
therapy applications), to detect a MEKK1 mRNA (e.g., in a
biological sample) or a genetic alteration in a MEKK1 gene, and to
modulate MEKK1 activity, as described further below. The MEKK1
proteins can be used to treat disorders characterized by
insufficient or excessive production of a MEKK1 substrate or
production of MEKK1 inhibitors. In addition, the MEKK1 proteins can
be used to screen for naturally occurring MEKK1 substrates, to
screen for small molecules, drugs or compounds which regulate or
modulate MEKK1 activity, as well as to treat disorders
characterized by insufficient or excessive production of MEKK1
protein or production of MEKK1 protein forms which have decreased,
aberrant or unwanted activity compared to MEKK1 wild type protein
(e.g., a proliferative disorder). Moreover, the anti-MEKK1
antibodies of the invention can be used to detect and isolate MEKK1
proteins, regulate the bioavailability of MEKK1 proteins, and
modulate MEKK1 activity.
[0216] A method of evaluating a compound for the ability to
interact with, e.g., bind, a subject MEKK1 polypeptide is provided.
The method includes: contacting the compound with the subject MEKK1
polypeptide; and evaluating ability of the compound to interact
with, e.g., to bind or form a complex with the subject MEKK1
polypeptide. This method can be performed in vitro, e.g., in a cell
free system, or in vivo, e.g., in a two-hybrid interaction trap
assay. This method can be used to identify naturally occurring
molecules which interact with subject MEKK1 polypeptide. It can
also be used to find natural or synthetic inhibitors of subject
MEKK1 polypeptide. Screening methods are discussed in more detail
below.
[0217] Screening Assays:
[0218] The invention provides methods (also referred to herein as
"screening assays") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., proteins, peptides,
peptidomimetics, peptoids, small molecules or other drugs) which
bind to MEKK1 proteins, have a stimulatory or inhibitory effect on,
for example, MEKK1 expression or MEKK1 activity, or have a
stimulatory or inhibitory effect on, for example, the expression or
activity of a MEKK1 substrate. Compounds thus identified can be
used to modulate the activity of target gene products (e.g., MEKK1
genes) in a therapeutic protocol, to elaborate the biological
function of the target gene product, or to identify compounds that
disrupt normal target gene interactions.
[0219] In one embodiment, the invention provides assays for
screening candidate or test compounds which are substrates of a
MEKK1 protein or polypeptide or a biologically active portion
thereof. In another embodiment, the invention provides assays for
screening candidate or test compounds which bind to or modulate the
activity of a MEKK1 protein or polypeptide or a biologically active
portion thereof.
[0220] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries; peptoid
libraries (libraries of molecules having the functionalities of
peptides, but with a novel, non-peptide backbone which are
resistant to enzymatic degradation but which nevertheless remain
bioactive)(see, e.g., Zuckermann, J. Med. Chem. 1994, 37: 2678-85
(1994)); spatially addressable parallel solid phase or solution
phase libraries; synthetic library methods requiring deconvolution;
the `one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library and peptoid library approaches are limited to peptide
libraries, while the other four approaches are applicable to
peptide, non-peptide oligomer or small molecule libraries of
compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).
[0221] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al., Proc. Natl.
Acad. Sci. U.S.A. 90:6909 (1993); Erb et al., Proc. Natl. Acad.
Sci. USA 91:11422 (1994); Zuckermann et al., J. Med. Chem. 37:2678
(1994); Cho et al., Science 261:1303; Carrell et al., Angew. Chem.
Int. Ed. Engl. 33:2059 (1994); Carell et al., Angew. Chem. Int. Ed.
Engl. 33:2061 (1994); and in Gallop et al., J. Med. Chem. 37:1233
(1994).
[0222] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al., Proc Natl Acad Sci USA
89:1865-1869 (1992)) or on phage (Scott et al., Science 249:386-390
(1990)); (Devlin, Science 249:404-406 (1990)); (Cwirla et al.,
Proc. Natl. Acad. Sci. 87:6378-6382 (1990)); (Felici, J. Mol. Biol.
222:301-310 (1991)); (Ladner supra.).
[0223] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a MEKK1 protein or biologically active portion
thereof is contacted with a test compound, and the ability of the
test compound to modulate MEKK1 activity is determined. Determining
the ability of the test compound to modulate MEKK1 activity can be
accomplished by monitoring, for example, binding of MEKK1 to ATP,
IKK, JNK-1, JNK-2, and JNK-3. The cell, for example, can be of
mammalian origin, e.g., a human HELA and Jurkat cell. The ability
of the test compound to modulate MEKK1 binding to a compound, e.g.,
a MEKK1 substrate, or to bind to MEKK1 can also be evaluated. This
can be accomplished, for example, by coupling the compound, e.g.,
the substrate, with a radioisotope or enzymatic label such that
binding of the compound, e.g., the substrate, to MEKK1 can be
determined by detecting the labeled compound, e.g., substrate, in a
complex. Alternatively, MEKK1 could be coupled with a radioisotope
or enzymatic label to monitor the ability of a test compound to
modulate MEKK1 binding to a MEKK1 substrate in a complex. For
example, compounds (e.g., MEKK1 substrates) can be labeled with
.sup.125I, .sup.35S, .sup.14C, or .sup.3H, either directly or
indirectly, and the radioisotope detected by direct counting of
radioemmission or by scintillation counting. Alternatively,
compounds can be enzymatically labeled with, for example,
horseradish peroxidase, alkaline phosphatase, or luciferase, and
the enzymatic label detected by determination of conversion of an
appropriate substrate to product.
[0224] The ability of a compound (e.g., a MEKK1 substrate) to
interact with MEKK1 with or without the labeling of any of the
interactants can be evaluated. For example, a microphysiometer can
be used to detect the interaction of a compound with MEKK1 without
the labeling of either the compound or the MEKK1. McConnell et al.,
Science 257:1906-1912 (1992). As used herein, a 37
microphysiometer" (e.g., Cytosensor) is an analytical instrument
that measures the rate at which a cell acidifies its environment
using a light-addressable potentiometric sensor (LAPS). Changes in
this acidification rate can be used as an indicator of the
interaction between a compound and MEKK1.
[0225] In yet another embodiment, a cell-free assay is provided in
which a MEKK1 protein or biologically active portion thereof is
contacted with a test compound and the ability of the test compound
to bind to the MEKK1 protein or biologically active portion thereof
is evaluated. Preferred biologically active portions of the MEKK1
proteins to be used in assays of the present invention indude
fragments which participate in interactions with non-MEKK1
molecules, e.g., fragments with high surface probability
scores.
[0226] Soluble and/or membrane-bound forms of isolated proteins
(e.g., MEKK1 proteins or biologically active portions thereof) can
be used in the cell-free assays of the invention. When
membrane-bound forms of the protein are used, it may be desirable
to utilize a solubilizing agent. Examples of such solubilizing
agents include non-ionic detergents such as n-octylglucoside,
n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamide, Triton.RTM. X-100, Triton.RTM. X-114,
Thesit.RTM., Isotridecypoly(ethylene glycol ether)n,
3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),
3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane
sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane
sulfonate.
[0227] Cell-free assays involve preparing a reaction mixture of the
target gene protein and the test compound under conditions and for
a time sufficient to allow the two components to interact and bind,
thus forming a complex that can be removed and/or detected.
[0228] The biological interaction between two molecules can also be
detected, e.g., using fluorescence energy transfer (FET) (see, for
example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos
et al., U.S. Pat. No. 4,868,103) scintillation proximity assay
(SPA) and homogeneous time resolved fluorescence (HTRF). A
fluorophore label on the first, `donor` molecule is selected such
that its emitted fluorescent energy will be absorbed by a
fluorescent label on a second, `acceptor` molecule, which in turn
is able to fluoresce due to the absorbed energy. Alternately, the
`donor` protein molecule may simply utilize the natural fluorescent
energy of tryptophan residues. Labels are chosen that emit
different wavelengths of light, such that the `acceptor` molecule
label may be differentiated from that of the `donor`. Since the
efficiency of energy transfer between the labels is related to the
distance separating the molecules, the spatial relationship between
the molecules can be assessed. In a situation in which binding
occurs between the molecules, the fluorescent emission of the
`acceptor` molecule label in the assay should be maximal. An FET
binding event can be conveniently measured through standard
fluorometric detection means well known in the art (e.g., using a
fluorimeter).
[0229] In another embodiment, determining the ability of the MEKK1
protein to bind to a target molecule can be accomplished using
real-time Biomolecular Interaction Analysis (BIA) (see, e.g.,
Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345
and Szabo et al., Curr. Opin. Struct. Biol. 5:699-705 (1995)).
"Surface plasmon resonance" or "BIA" detects biospecific
interactions in real time, without labeling any of the interactants
(e.g., BIAcore). Changes in the mass at the binding surface
(indicative of a binding event) result in alterations of the
refractive index of light near the surface (the optical phenomenon
of surface plasmon resonance (SPR)), resulting in a detectable
signal which can be used as an indication of real-time reactions
between biological molecules.
[0230] In one embodiment, the target gene product or the test
substance is anchored onto a solid phase. The target gene
product/test compound complexes anchored on the solid phase can be
detected at the end of the reaction. Preferably, the target gene
product can be anchored onto a solid surface, and the test
compound, (which is not anchored), can be labeled, either directly
or indirectly, with detectable labels discussed herein.
[0231] It may be desirable to immobilize either MEKK1 or an
anti-MEKK1 antibody or its target molecule to facilitate separation
of complexed from uncomplexed forms of one or both of the proteins,
as well as to accommodate automation of the assay. Binding of a
test compound to a MEKK1 protein, or interaction of a MEKK1 protein
with a target molecule in the presence and absence of a candidate
compound, can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtiter plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows one or both
of the proteins to be bound to a matrix. For example,
glutathione-S-transferase/MEKK1 fusion proteins or
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione derivatized microtiter plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or MEKK1 protein, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtiter plate wells are washed to remove any
unbound components, the matrix immobilized in the case of beads,
complex determined either directly or indirectly, for example, as
described above. Alternatively, the complexes can be dissociated
from the matrix, and the level of MEKK1 binding or activity
determined using standard techniques.
[0232] Other techniques for immobilizing either a MEKK1 protein or
a target molecule on matrices include using conjugation of biotin
and streptavidin. Biotinylated MEKK1 protein or target molecules
can be prepared from biotin-NHS (N-hydroxy-succinimide) using
techniques known in the art (e.g., biotinylation kit, Pierce
Chemicals, Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
[0233] In order to conduct the assay, the non-immobilized component
is added to the coated surface containing the anchored component.
After the reaction is complete, unreacted components are removed
(e.g., by washing) under conditions such that any complexes formed
will remain immobilized on the solid surface. The detection of
complexes anchored on the solid surface can be accomplished in a
number of ways. Where the previously non-immobilized component is
pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the previously
non-immobilized component is not pre-labeled, an indirect label can
be used to detect complexes anchored on the surface; e.g., using a
labeled antibody specific for the immobilized component (the
antibody, in turn, can be directly labeled or indirectly labeled
with, e.g., a labeled anti-Ig antibody).
[0234] In one embodiment, this assay is performed utilizing
antibodies reactive with MEKK1 protein or target molecules but
which do not interfere with binding of the MEKK1 protein to its
target molecule. Such antibodies can be derivatized to the wells of
the plate, and unbound target or MEKK1 protein trapped in the wells
by antibody conjugation. Methods for detecting such complexes, in
addition to those described above for the GST-immobilized
complexes, include immunodetection of complexes using antibodies
reactive with the MEKK1 protein or target molecule, as well as
enzyme-linked assays which rely on detecting an enzymatic activity
associated with the MEKK1 protein or target molecule.
[0235] Alternatively, cell free assays can be conducted in a liquid
phase. In such an assay, the reaction products are separated from
unreacted components, by any of a number of standard techniques,
including but not limited to: differential centrifugation (see, for
example, Rivas, G., and Minton, A. P., Trends Biochem Sci 1993
August;18(8):284-7); chromatography (gel filtration chromatography,
ion-exchange chromatography); electrophoresis (see, e.g., Ausubel
et al., eds. Current Protocols in Molecular Biology, J. Wiley: New
York (1999).); and immunoprecipitation (see, for example, Ausubel
et al., eds. Current Protocols in Molecular Biology, J. Wiley: New
York (1999)). Such resins and chromatographic techniques are known
to one skilled in the art (see, e.g., Heegaard, N. H., J Mol
Recognit 1998 Winter;11(1-6):141-8; Hage, D. S., and Tweed, S. A. J
Chromatogr B Biomed Sci Appl 1997 October 10;699(1-2):499-525).
Further, fluorescence energy transfer may also be conveniently
utilized, as described herein, to detect binding without further
purification of the complex from solution.
[0236] In a preferred embodiment, the assay includes contacting the
MEKK1 protein or biologically active portion thereof with a known
compound which binds MEKK1 to form an assay mixture, contacting the
assay mixture with a test compound, and determining the ability of
the test compound to interact with a MEKK1 protein, wherein
determining the ability of the test compound to interact with a
MEKK1 protein includes determining the ability of the test compound
to preferentially bind to MEKK1 or biologically active portion
thereof, or to modulate the activity of a target molecule, as
compared to the known compound.
[0237] The target gene products of the invention can interact in
vivo with one or more cellular or extracellular macromolecules,
such as proteins. For the purposes of this discussion, such
cellular and extracellular macromolecules are referred to herein as
"binding partners." Compounds that disrupt such interactions can be
useful in regulating the activity of the target gene product. Such
compounds can include, but are not limited to, molecules such as
antibodies, peptides, and small molecules. The preferred target
genes/products for use in this embodiment are the MEKK1 genes
herein identified. In an alternative embodiment, the invention
provides methods for determining the ability of the test compound
to modulate the activity of a MEKK1 protein through modulation of
the activity of a downstream effector of a MEKK1 target molecule.
For example, the activity of the effector molecule on an
appropriate target can be determined, or the binding of the
effector to an appropriate target can be determined, as previously
described.
[0238] To identify compounds that interfere with the interaction
between the target gene product and its cellular or extracellular
binding partner(s), a reaction mixture containing the target gene
product and the binding partner is prepared, under conditions and
for a time sufficient, to allow the two products to form complex.
In order to test an inhibitory agent, the reaction mixture is
provided in the presence and absence of the test compound. The test
compound can be initially included in the reaction mixture, or can
be added at a time subsequent to the addition of the target gene
and its cellular or extracellular binding partner. Control reaction
mixtures are incubated without the test compound or with a placebo.
The formation of any complexes between the target gene product and
the cellular or extracellular binding partner is then detected. The
formation of a complex in the control reaction, but not in the
reaction mixture containing the test compound, indicates that the
compound interferes with the interaction of the target gene product
and the interactive binding partner. Additionally, complex
formation within reaction mixtures containing the test compound and
normal target gene product can also be compared to complex
formation within reaction mixtures containing the test compound and
mutant target gene product. This comparison can be important in
those cases wherein it is desirable to identify compounds that
disrupt interactions of mutant but not normal target gene
products.
[0239] These assays can be conducted in a heterogeneous or
homogeneous format. Heterogeneous assays involve anchoring either
the target gene product or the binding partner onto a solid phase,
and detecting complexes anchored on the solid phase at the end of
the reaction. In homogeneous assays, the entire reaction is carried
out in a liquid phase. In either approach, the order of addition of
reactants can be varied to obtain different information about the
compounds being tested. For example, test compounds that interfere
with the interaction between the target gene products and the
binding partners, e.g., by competition, can be identified by
conducting the reaction in the presence of the test substance.
Alternatively, test compounds that disrupt preformed complexes,
e.g., compounds with higher binding constants that displace one of
the components from the complex, can be tested by adding the test
compound to the reaction mixture after complexes have been formed.
The various formats are briefly described below.
[0240] In a heterogeneous assay system, either the target gene
product or the interactive cellular or extracellular binding
partner, is anchored onto a solid surface (e.g., a microtiter
plate), while the non-anchored species is labeled, either directly
or indirectly. The anchored species can be immobilized by
non-covalent or covalent attachments. Alternatively, an immobilized
antibody specific for the species to be anchored can be used to
anchor the species to the solid surface.
[0241] In order to conduct the assay, the partner of the
immobilized species is exposed to the coated surface with or
without the test compound. After the reaction is complete,
unreacted components are removed (e.g., by washing) and any
complexes formed will remain immobilized on the solid surface.
Where the non-immobilized species is pre-labeled, the detection of
label immobilized on the surface indicates that complexes were
formed. Where the non-immobilized species is not pre-labeled, an
indirect label can be used to detect complexes anchored on the
surface; e.g., using a labeled antibody specific for the initially
non-immobilized species (the antibody, in turn, can be directly
labeled or indirectly labeled with, e.g., a labeled anti-Ig
antibody). Depending upon the order of addition of reaction
components, test compounds that inhibit complex formation or that
disrupt preformed complexes can be detected.
[0242] Alternatively, the reaction can be conducted in a liquid
phase in the presence or absence of the test compound, the reaction
products separated from unreacted components, and complexes
detected; e.g., using an immobilized antibody specific for one of
the binding components to anchor any complexes formed in solution,
and a labeled antibody specific for the other partner to detect
anchored complexes. Again, depending upon the order of addition of
reactants to the liquid phase, test compounds that inhibit complex
or that disrupt preformed complexes can be identified.
[0243] In an alternate embodiment of the invention, a homogeneous
assay can be used. For example, a preformed complex of the target
gene product and the interactive cellular or extracellular binding
partner product is prepared in that either the target gene products
or their binding partners are labeled, but the signal generated by
the label is quenched due to complex formation (see, e.g., U.S.
Pat. No. 4,109,496 that utilizes this approach for immunoassays).
The addition of a test substance that competes with and displaces
one of the species from the preformed complex will result in the
generation of a signal above background. In this way, test
substances that disrupt target gene product-binding partner
interaction can be identified.
[0244] In yet another aspect, the MEKK1 proteins can be used as
"bait proteins" in a two-hybrid assay or three-hybrid assay (see,
e;g., U.S. Pat. No. 5,283,317; Zervos et al., Cell 72:223-232
(1993); Madura et al., J. Biol. Chem. 268:12046-12054 (1993);
Bartel et al., Biotechniques 14:920-924 (1993); Iwabuchi et al.,
Oncogene 8:1693-1696 (1993); and Brent WO94/10300), to identify
other proteins, which bind to or interact with MEKK1
("MEKK1-binding proteins" or "MEKK1-bp") and are involved in MEKK1
activity. Such MEKK1-bps can be activators or inhibitors of signals
by the MEKK1 proteins or MEKK1 targets as, for example, downstream
elements of a MEKK1-mediated signaling pathway.
[0245] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a MEKK1
protein is fused to a gene encoding the DNA binding domain of a
known transcription factor (e.g., GAL-4). In the other construct, a
DNA sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
(Alternatively, the MEKK1 protein can be the fused to the activator
domain.) If the "bait" and the "prey" proteins are able to
interact, in vivo, forming a MEKK1-dependent complex, the
DNA-binding and activation domains of the transcription factor are
brought into close proximity. This proximity allows transcription
of a reporter gene (e.g., LacZ) which is operably linked to a
transcriptional regulatory site responsive to the transcription
factor. Expression of the reporter gene can be detected and cell
colonies containing the functional transcription factor can be
isolated and used to obtain the cloned gene which encodes the
protein which interacts with the MEKK1 protein.
[0246] In another embodiment, modulators of MEKK1 expression are
identified. For example, a cell or cell free mixture is contacted
with a candidate compound and the expression of MEKK1 mRNA or
protein evaluated relative to the level of expression of MEKK1 mRNA
or protein in the absence of the candidate compound. When
expression of MEKK1 mRNA or protein is greater in the presence of
the candidate compound than in its absence, the candidate compound
is identified as a stimulator of MEKK1 mRNA or protein expression.
Alternatively, when expression of MEKK1 mRNA or protein is less
(statistically significantly less) in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of MEKK1 mRNA or protein expression. The level of
MEKK1 mRNA or protein expression can be determined by methods
described herein for detecting MEKK1 mRNA or protein.
[0247] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulating agent can be identified using a cell-based or a cell
free assay, and the ability of the agent to modulate the activity
of a MEKK1 protein can be confirmed in vivo.
[0248] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein (e.g., a MEKK1 modulating agent, an antisense
MEKK1 nucleic acid molecule, a MEKK1-specific antibody, or a
MEKK1-binding partner) in an appropriate animal model to determine
the efficacy, toxicity, side effects, or mechanism of action, of
treatment with such an agent. Furthermore, novel agents identified
by the above-described screening assays can be used for treatments
as described herein.
[0249] Detection Assays
[0250] Portions or fragments of the nucleic acid sequences
identified herein can be used as polynucleotide reagents. For
example, these sequences can be used to: (i) map their respective
genes on a chromosome e.g., to locate gene regions associated with
genetic disease or to associate MEKK1 with a disease; (ii) identify
an individual from a minute biological sample (tissue typing); and
(iii) aid in forensic identification of a biological sample. These
applications are described in the subsections below.
[0251] Chromosome Mapping
[0252] The MEKK1 nucleotide sequences or portions thereof can be
used to map the location of the MEKK1 genes on a chromosome. This
process is called chromosome mapping. Chromosome mapping is useful
in correlating the MEKK1 sequences with genes associated with
disease.
[0253] Briefly, MEKK1 genes can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp in length) from the
MEKK1 nucleotide sequences. These primers can then be used for PCR
screening of somatic cell hybrids containing individual human
chromosomes. Only those hybrids containing the human gene
corresponding to the MEKK1 sequences will yield an amplified
fragment.
[0254] A panel of somatic cell hybrids in which each cell line
contains either a single human chromosome or a small number of
human chromosomes, and a full set of mouse chromosomes, can allow
easy mapping of individual genes to specific human chromosomes.
(D'Eustachio et al., Science 220:919-924 (1983)).
[0255] Other mapping strategies e.g., in situ hybridization
(described in Fan, Proc. Natl. Acad. Sci. USA, 87:6223-27 (1990)),
pre-screening with labeled flow-sorted chromosomes, and
pre-selection by hybridization to chromosome specific cDNA
libraries can be used to map MEKK1 to a chromosomal location.
[0256] Fluorescence in situ hybridization (FISH) of a DNA sequence
to a metaphase chromosomal spread can further be used to provide a
precise chromosomal location in one step. The FISH technique can be
used with a DNA sequence as short as 500 or 600 bases. However,
clones larger than 1,000 bases have a higher likelihood of binding
to a unique chromosomal location with sufficient signal intensity
for simple detection. Preferably 1,000 bases, and more preferably
2,000 bases will suffice to get good results at a reasonable amount
of time. For a review of this technique, see Verma et al., Human
Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York
(1988)).
[0257] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0258] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. (Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between a gene and a disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, for
example, Egeland et al., Nature, 325:783-787 (1987).
[0259] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the MEKK1 gene, can be determined. If a mutation is observed in
some or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes, such as deletions or translocations
that are visible from chromosome spreads or detectable using PCR
based on that DNA sequence. Ultimately, complete sequencing of
genes from several individuals can be performed to confirm the
presence of a mutation and to distinguish mutations from
polymorphisms.
[0260] Tissue Typing
[0261] MEKK1 sequences can be used to identify individuals from
biological samples using, e.g., restriction fragment length
polymorphism (RFLP). In this technique, an individual's genomic DNA
is digested with one or more restriction enzymes, the fragments
separated, e.g., in a Southern blot, and probed to yield bands for
identification. The sequences of the present invention are useful
as additional DNA markers for RFLP (described in U.S. Pat. No.
5,272,057).
[0262] Furthermore, the sequences of the present invention can also
be used to determine the actual base-by-base DNA sequence of
selected portions of an individual's genome. Thus, the MEKK1
nucleotide sequences described herein can be used to prepare two
PCR primers from the 5' and 3' ends of the sequences. These primers
can then be used to amplify an individual's DNA and subsequently
sequence it. Panels of corresponding DNA sequences from
individuals, prepared in this manner, can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences.
[0263] Allelic variation occurs to some degree in the coding
regions of these sequences, and to a greater degree in the
noncoding regions. Each of the sequences described herein can, to
some degree, be used as a standard against which DNA from an
individual can be compared for identification purposes. Because
greater numbers of polymorphisms occur in the noncoding regions,
fewer sequences are necessary to differentiate individuals. The
noncoding sequences of SEQ ID NO:1 can provide positive individual
identification with a panel of perhaps 10 to 1,000 primers which
each yield a noncoding amplified sequence of 100 bases. If
predicted coding sequences, such as those in SEQ ID NO:3 are used,
a more appropriate number of primers for positive individual
identification would be 500-2,000.
[0264] If a panel of reagents from MEKK1 nucleotide sequences
described herein is used to generate a unique identification
database for an individual, those same reagents can later be used
to identify tissue from that individual. Using the unique
identification database, positive identification of the individual,
living or dead, can be made from extremely small tissue
samples.
[0265] Use of Partial MEKK1 Sequences in Forensic Biology
[0266] DNA-based identification techniques can also be used in
forensic biology. To make such an identification, PCR technology
can be used to amplify DNA sequences taken from very small
biological samples such as tissues, e.g., hair or skin, or body
fluids, e.g., blood, saliva, or semen found at a crime scene. The
amplified sequence can then be compared to a standard, thereby
allowing identification of the origin of the biological sample.
[0267] The sequences of the present invention can be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, which can enhance the
reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" (i.e., another DNA
sequence that is unique to a particular individual). As mentioned
above, actual base sequence information can be used for
identification as an accurate alternative to patterns formed by
restriction enzyme generated fragments. Sequences targeted to
noncoding regions of SEQ ID NO:1 (e.g., fragments derived from the
noncoding regions of SEQ ID NO:1 having a length of at least 20
bases, preferably at least 30 bases) are particularly appropriate
for this use.
[0268] The MEKK1 nucleotide sequences described herein can further
be used to provide polynucleotide reagents, e.g., labeled or
labelable probes which can be used in, for example, an in situ
hybridization technique, to identify a specific tissue. This can be
very useful in cases where a forensic pathologist is presented with
a tissue of unknown origin. Panels of such MEKK1 probes can be used
to identify tissue by species and/or by organ type.
[0269] In a similar fashion, these reagents, e.g., MEKK1 primers or
probes can be used to screen tissue culture for contamination
(i.e., screen for the presence of a mixture of different types of
cells in a culture).
[0270] Predictive Medicine
[0271] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
and monitoring clinical trials are used for prognostic (predictive)
purposes to thereby treat an individual.
[0272] Generally, the invention provides, a method of determining
if a subject is at risk for a disorder related to a lesion in or
the misexpression of a gene which encodes MEKK1 or a signal
transduction protein or a cell adhesion protein.
[0273] The method includes one or more of the following:
[0274] detecting, in a tissue of the subject, the presence or
absence of a mutation which affects the expression of the MEKK1
gene, or detecting the presence or absence of a mutation in a
region which controls the expression of the gene, e.g., a mutation
in the 5' control region;
[0275] detecting, in a tissue of the subject, the presence or
absence of a mutation which alters the structure of the MEKK1
gene;
[0276] detecting, in a tissue of the subject, the misexpression of
the MEKK1 gene, at the mRNA level, e.g., detecting a non-wild type
level of a mRNA;
[0277] detecting, in a tissue of the subject, the misexpression of
the gene, at the protein level, e.g., detecting a non-wild type
level of a MEKK1 polypeptide.
[0278] In preferred embodiments the method includes: ascertaining
the existence of at least one of: a deletion of one or more
nucleotides from the MEKK1 gene; an insertion of one or more
nucleotides into the gene, a point mutation, e.g., a substitution
of one or more nucleotides of the gene, a gross chromosomal
rearrangement of the gene, e.g., a translocation, inversion, or
deletion.
[0279] For example, detecting the genetic lesion can include: (i)
providing a probe/primer including an oligonucleotide containing a
region of nucleotide sequence which hybridizes to a sense or
antisense sequence from SEQ ID NO:1 or SEQ ID NO:3, or naturally
occurring mutants thereof, or 5' or 3' flanking sequences naturally
associated with the MEKK1 gene; (ii) exposing the probe/primer to
nucleic acid of the tissue; and detecting, by hybridization, e.g.,
in situ hybridization, of the probe/primer to the nucleic acid, the
presence or absence of the genetic lesion.
[0280] In preferred embodiments detecting the misexpression
includes ascertaining the existence of at least one of: an
alteration in the level of a messenger RNA transcript of the MEKK1
gene; the presence of a non-wild type splicing pattern of a
messenger RNA transcript of the gene; or a non-wild type level of
MEKK1.
[0281] Methods of the invention can be used prenatally or to
determine if a subject's offspring will be at risk for a
disorder.
[0282] In preferred embodiments the method includes determining the
structure of a MEKK1 gene, an abnormal structure being indicative
of risk for the disorder.
[0283] In preferred embodiments the method includes contacting a
sample form the subject with an antibody to the MEKK1 protein or a
nucleic acid, which hybridizes specifically with the gene. There
and other embodiments are discussed below.
[0284] Diagnostic and Prognostic Assays
[0285] The presence, level, or absence of MEKK1 protein or nucleic
acid in a biological sample can be evaluated by obtaining a
biological sample from a test subject and contacting the biological
sample with a compound or an agent capable of detecting MEKK1
protein or nucleic acid (e.g., mRNA or genomic DNA) that encodes
MEKK1 protein such that the presence of MEKK1 protein or nucleic
acid is detected in the biological sample. The term "biological
sample" includes tissues, cells and biological fluids isolated from
a subject, as well as tissues, cells and fluids present within a
subject. A preferred biological sample is serum. The level of
expression of the MEKK1 gene can be measured in a number of ways,
including, but not limited to: measuring the mRNA encoded by the
MEKK1 genes; measuring the amount of protein encoded by the MEKK1
genes; or measuring the activity of the protein encoded by the
MEKK1 genes.
[0286] The level of mRNA corresponding to the MEKK1 gene in a cell
can be determined both by in situ and by in vitro formats.
[0287] The isolated mRNA can be used in hybridization or
amplification assays that include, but are not limited to, Southern
or Northern analyses, polymerase chain reaction analyses and probe
arrays. One preferred diagnostic method for the detection of mRNA
levels involves contacting the isolated mRNA with a nucleic acid
molecule (probe) that can hybridize to the mRNA encoded by the gene
being detected. The nucleic acid probe can be, for example, a
full-length MEKK1 nucleic acid, such as the nucleic acid of SEQ ID
NO:1, or the DNA insert of the plasmid deposited with ATCC as
Accession Number PTA-1836, or a portion thereof, such as an
oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500
nucleotides in length and sufficient to specifically hybridize
under stringent conditions to MEKK1 mRNA or genomic DNA. Other
suitable probes for use in the diagnostic assays are described
herein.
[0288] In one format, mRNA (or cDNA) is immobilized on a surface
and contacted with the probes, for example by running the isolated
mRNA on an agarose gel and transferring the mRNA from the gel to a
membrane, such as nitrocellulose. In an alternative format, the
probes are immobilized on a surface and the mRNA (or cDNA) is
contacted with the probes, for example, in a two-dimensional gene
chip array. A skilled artisan can adapt known mRNA detection
methods for use in detecting the level of mRNA encoded by the MEKK1
genes.
[0289] The level of mRNA in a sample that is encoded by one of
MEKK1 can be evaluated with nucleic acid amplification, e.g., by
rtPCR (Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain
reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA 88:189-193),
self sustained sequence replication (Guatelli et al., Proc. Natl.
Acad. Sci. USA 87:1874-1878 (1990)), transcriptional amplification
system (Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173-1177
(1989)), Q-Beta Replicase (Lizardi et al., Bio/Technology 6:1197
(1988)), rolling circle replication (Lizardi et al., U.S. Pat. No.
5,854,033) or any other nucleic acid amplification method, followed
by the detection of the amplified molecules using techniques known
in the art. As used herein, amplification primers are defined as
being a pair of nucleic acid molecules that can anneal to 5' or 3'
regions of a gene (plus and minus strands, respectively, or
vice-versa) and contain a short region in between. In general,
amplification primers are from about 10 to 30 nucleotides in length
and flank a region from about 50 to 200 nucleotides in length.
Under appropriate conditions and with appropriate reagents, such
primers permit the amplification of a nucleic acid molecule
comprising the nucleotide sequence flanked by the primers.
[0290] For in situ methods, a cell or tissue sample can be
prepared/processed and immobilized on a support, typically a glass
slide, and then contacted with a probe that can hybridize to mRNA
that encodes the MEKK1 gene being analyzed.
[0291] In another embodiment, the methods further contacting a
control sample with a compound or agent capable of detecting MEKK1
mRNA, or genomic DNA, and comparing the presence of MEKK1 mRNA or
genomic DNA in the control sample with the presence of MEKK1 mRNA
or genomic DNA in the test sample.
[0292] A variety of methods can be used to determine the level of
protein encoded by MEKK1. In general, these methods include
contacting an agent that selectively binds to the protein, such as
an antibody with a sample, to evaluate the level of protein in the
sample. In a preferred embodiment, the antibody bears a detectable
label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or
F(ab').sub.2) can be used. The term "labeled", with regard to the
probe or antibody, is intended to encompass direct labeling of the
probe or antibody by coupling (i.e., physically linking) a
detectable substance to the probe or antibody, as well as indirect
labeling of the probe or antibody by reactivity with a detectable
substance. Examples of detectable substances are provided
herein.
[0293] The detection methods can be used to detect MEKK1 protein in
a biological sample in vitro as well as in vivo. In vitro
techniques for detection of MEKK1 protein include enzyme linked
immunosorbent assays (ELISAs), immunoprecipitations,
immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay
(RIA), and Western blot analysis. In vivo techniques for detection
of MEKK1 protein include introducing into a subject a labeled
anti-MEKK1 antibody. For example, the antibody can be labeled with
a radioactive marker whose presence and location in a subject can
be detected by standard imaging techniques.
[0294] In another embodiment, the methods further include
contacting the control sample with a compound or agent capable of
detecting MEKK1 protein, and comparing the presence of MEKK1
protein in the control sample with the presence of MEKK1 protein in
the test sample.
[0295] The invention also includes kits for detecting the presence
of MEKK1 in a biological sample. For example, the kit can include a
compound or agent capable of detecting MEKK1 protein or mRNA in a
biological sample; and a standard. The compound or agent can be
packaged in a suitable container. The kit can further comprise
instructions for using the kit to detect MEKK1 protein or nucleic
acid.
[0296] For antibody-based kits, the kit can include: (1) a first
antibody (e.g., attached to a solid support) which binds to a
polypeptide corresponding to a marker of the invention; and,
optionally, (2) a second, different antibody which binds to either
the polypeptide or the first antibody and is conjugated to a
detectable agent.
[0297] For oligonucleotide-based kits, the kit can include: (1) an
oligonucleotide, e.g., a detectably labeled oligonucleotide, which
hybridizes to a nucleic acid sequence encoding a polypeptide
corresponding to a marker of the invention or (2) a pair of primers
useful for amplifying a nucleic acid molecule corresponding to a
marker of the invention. The kit can also includes a buffering
agent, a preservative, or a protein stabilizing agent. The kit can
also includes components necessary for detecting the detectable
agent (e.g., an enzyme or a substrate). The kit can also contain a
control sample or a series of control samples which can be assayed
and compared to the test sample contained. Each component of the
kit can be enclosed within an individual container and all of the
various containers can be within a single package, along with
instructions for interpreting the results of the assays performed
using the kit.
[0298] The diagnostic methods described herein can identify
subjects having, or at risk of developing, a disease or disorder
associated with misexpressed or aberrant or unwanted MEKK1
expression or activity. As used herein, the term "unwanted"
includes an unwanted phenomenon involved in a biological response
such as pain or deregulated cell proliferation.
[0299] In one embodiment, a disease or disorder associated with
aberrant or unwanted MEKK1 expression or activity is identified. A
test sample is obtained from a subject and MEKK1 protein or nucleic
acid (e.g., mRNA or genomic DNA) is evaluated, wherein the level,
e.g., the presence or absence, of MEKK1 protein or nucleic acid is
diagnostic for a subject having or at risk of developing a disease
or disorder associated with aberrant or unwanted MEKK1 expression
or activity. As used herein, a "test sample" refers to a biological
sample obtained from a subject of interest, including a biological
fluid (e.g., serum), cell sample, or tissue.
[0300] The prognostic assays described herein can be used to
determine whether a subject can be administered an agent (e.g., an
agonist, antagonist, peptidomimetic, protein, peptide, nucleic
acid, small molecule, or other drug candidate) to treat a disease
or disorder associated with aberrant or unwanted MEKK1 expression
or activity. For example, such methods can be used to determine
whether a subject can be effectively treated with an agent for a
cell signaling or cell adhesion disorder.
[0301] The methods of the invention can also be used to detect
genetic alterations in a MEKK1 gene, thereby determining if a
subject with the altered gene is at risk for a disorder
characterized by misregulation in MEKK1 protein activity or nucleic
acid expression, such as a cell signaling or cell adhesion
disorder. In preferred embodiments, the methods include detecting,
in a sample from the subject, the presence or absence of a genetic
alteration characterized by at least one of an alteration affecting
the integrity of a gene encoding a MEKK1-protein, or the
misexpression of the MEKK1 gene. For example, such genetic
alterations can be detected by ascertaining the existence of at
least one of (1) a deletion of one or more nucleotides from a MEKK1
gene; (2) an addition of one or more nucleotides to a MEKK1 gene;
(3) a substitution of one or more nucleotides of a MEKK1 gene; (4)
a chromosomal rearrangement of a MEKK1 gene; (5) an alteration in
the level of a messenger RNA transcript of a MEKK1 gene; (6)
aberrant modification of a MEKK1 gene, such as of the methylation
pattern of the genomic DNA; (7) the presence of a non-wild type
splicing pattern of a messenger RNA transcript of a MEKK1 gene; (8)
a non-wild type level of a MEKK1-protein; (9) allelic loss of a
MEKK1 gene; and (10) inappropriate post-translational modification
of a MEKK1-protein.
[0302] An alteration can be detected without a probe/primer in a
polymerase chain reaction, such as anchor PCR or RACE PCR, or,
alternatively, in a ligation chain reaction (LCR), the latter of
which can be particularly useful for detecting point mutations in
the MEKK1 gene. This method can include the steps of collecting a
sample of cells from a subject, isolating nucleic acid (e.g.,
genomic DNA, mRNA, or both) from the sample, contacting the nucleic
acid sample with one or more primers which specifically hybridize
to a MEKK1 gene under conditions such that hybridization and
amplification of the MEKK1 gene (if present) occurs, and detecting
the presence or absence of an amplification product, or detecting
the size of the amplification product and comparing the length to a
control sample. It is anticipated that PCR and/or LCR may be
desirable to use as a preliminary amplification step in conjunction
with any of the techniques used for detecting mutations described
herein.
[0303] Alternative amplification methods include: self sustained
sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA
87:1874-1878 (1990)), transcriptional amplification system (Kwoh et
al., Proc. Natl. Acad. Sci. USA 86:1173-1177 (1989)), Q-Beta
Replicase (Lizardi et al., Bio-Technology 6:1197 (1988)), or other
nucleic acid amplification methods, followed by the detection of
the amplified molecules using techniques known to those of skill in
the art.
[0304] In another embodiment, mutations in a MEKK1 gene from a
sample cell can be identified by detecting alterations in
restriction enzyme cleavage patterns. For example, sample and
control DNA are isolated, optionally amplified, digested with one
or more restriction endonucleases, and fragment length sizes are
determined, e.g., by gel electrophoresis and compared. Differences
in fragment length sizes between sample and control DNA indicates
mutations in the sample DNA. Moreover, the use of sequence specific
ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used
to score for the presence of specific mutations by development or
loss of a ribozyme cleavage site.
[0305] In other embodiments, genetic mutations in MEKK1 can be
identified by hybridizing a sample and control nucleic acids, e.g.,
DNA or RNA, two dimensional arrays, e.g., chip based arrays. Such
arrays include a plurality of addresses, each of which is
positionally distinguishable from the other. A different probe is
located at each address of the plurality. The arrays can have a
high density of addresses, e.g., can contain hundreds or thousands
of oligonucleotides probes (Cronin et al. Human Mutation 7: 244-255
(1996); Kozal et al. Nature Medicine 2: 753-759 (1996)). For
example, genetic mutations in MEKK1 can be identified in two
dimensional arrays containing light-generated DNA probes as
described in Cronin, M. T. et al. supra. Briefly, a first
hybridization array of probes can be used to scan through long
stretches of DNA in a sample and control to identify base changes
between the sequences by making linear arrays of sequential
overlapping probes. This step allows the identification of point
mutations. This step is followed by a second hybridization array
that allows the characterization of specific mutations by using
smaller, specialized probe arrays complementary to all variants or
mutations detected. Each mutation array is composed of parallel
probe sets, one complementary to the wild-type gene and the other
complementary to the mutant gene.
[0306] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
MEKK1 gene and detect mutations by comparing the sequence of the
sample MEKK1 with the corresponding wild-type (control) sequence.
Automated sequencing procedures can be utilized when performing the
diagnostic assays (Biotechniques 19:448 (1995)), including
sequencing by mass spectrometry.
[0307] Other methods for detecting mutations in the MEKK1 gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers
et al., Science 230:1242 (1985); Cotton et al., Proc. Natl Acad Sci
USA 85:4397 (1988); Saleeba et al., Methods Enzymol. 217:286-295
(1992)).
[0308] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in MEKK1
cDNAs obtained from samples of cells. For example, the mutY enzyme
of E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et
al., Carcinogenesis 15:1657-1662 (1994); U.S. Pat. No.
5,459,039).
[0309] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in MEKK1 genes. For
example, single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al., Proc Natl. Acad. Sci
USA: 86:2766 (1989), see also Cotton, Mutat. Res. 285:125-144
(1993); and Hayashi, Genet. Anal. Tech. Appl. 9:73-79 (1992)).
Single-stranded DNA fragments of sample and control MEKK1 nucleic
acids will be denatured and allowed to renature. The secondary
structure of single-stranded nucleic acids varies according to
sequence, the resulting alteration in electrophoretic mobility
enables the detection of even a single base change. The DNA
fragments may be labeled or detected with labeled probes. The
sensitivity of the assay may be enhanced by using RNA (rather than
DNA), in which the secondary structure is more sensitive to a
change in sequence. In a preferred embodiment, the subject method
utilizes heteroduplex analysis to separate double stranded
heteroduplex molecules on the basis of changes in electrophoretic
mobility (Keen et al., Trends Genet 7:5 (1991)).
[0310] In yet another embodiment, the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al., Nature 313:495 (1985)). When DGGE is used as
the method of analysis, DNA will be modified to insure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner, Biophys Chem
265:12753 (1987)).
[0311] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension (Saiki et al., Nature 324:163 (1986)); Saiki et al.,
Proc. Natl Acad. Sci USA 86:6230 (1989)).
[0312] Alternatively, allele specific amplification technology
which depends on selective PCR amplification may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may carry the mutation of
interest in the center of the molecule (so that amplification
depends on differential hybridization) (Gibbs et al., Nucleic Acids
Res. 17:2437-2448 (1989)) or at the extreme 3' end of one primer
where, under appropriate conditions, mismatch can prevent, or
reduce polymerase extension (Prossner, Tibtech 11:238 (1993)). In
addition it may be desirable to introduce a novel restriction site
in the region of the mutation to create cleavage-based detection
(Gasparini et al., Mol. Cell Probes 6:1 (1992)). It is anticipated
that in certain embodiments amplification may also be performed
using Taq ligase for amplification (Barany, Proc. Natl. Acad. Sci
USA 88:189 (1991)). In such cases, ligation will occur only if
there is a perfect match at the 3' end of the 5' sequence making it
possible to detect the presence of a known mutation at a specific
site by looking for the presence or absence of amplification.
[0313] The methods described herein may be performed, for example,
by utilizing prepackaged diagnostic kits comprising at least one
probe nucleic acid or antibody reagent described herein, which may
be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving a MEKK1 gene.
[0314] Pharmaceutical Compositions
[0315] The nucleic acid and polypeptides, as well as anti-MEKK1
antibodies (also referred to herein as "active compounds") of the
invention can be incorporated into pharmaceutical compositions.
Such compositions typically include the nucleic acid molecule,
protein, or antibody and a pharmaceutically acceptable carrier. As
used herein the language "pharmaceutically acceptable carrier"
includes solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. Supplementary
active compounds can also be incorporated into the
compositions.
[0316] A pharmaceutical composition is formulated to be compatible
with its intended route of administration. Examples of routes of
administration include parenteral, e.g., intravenous, intradermal,
subcutaneous, oral (e.g., inhalation), transdermal (topical),
transmucosal, and rectal administration. Solutions or suspensions
used for parenteral, intradermal, or subcutaneous application can
include the following components: a sterile diluent such as water
for injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0317] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It should be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0318] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0319] Oral compositions generally include an inert diluent or an
edible carrier. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients and used in
the form of tablets, troches, or capsules, e.g., gelatin capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash. Pharmaceutically compatible binding agents,
and/or adjuvant materials can be included as part of the
composition. The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0320] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0321] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0322] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0323] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0324] It is advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
[0325] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. Compounds which exhibit
high therapeutic indeces are preferred. While compounds that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets such compounds to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects.
[0326] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC50 (i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
[0327] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25
mg/kg body weight, more preferably about 0.1 to 20 mg/kg body
weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The
protein or polypeptide can be administered one time per week for
between about 1 to 10 weeks, preferably between 2 to 8 weeks, more
preferably between about 3 to 7 weeks, and even more preferably for
about 4, 5, or 6 weeks. The skilled artisan will appreciate that
certain factors may influence the dosage and timing required to
effectively treat a subject, including but not limited to the
severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases
present. Moreover, treatment of a subject with a therapeutically
effective amount of a protein, polypeptide, or antibody can include
a single treatment or, preferably, can include a series of
treatments.
[0328] For antibodies, the preferred dosage is 0.1 mg/kg of body
weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act
in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually
appropriate. Generally, partially human antibodies and fully human
antibodies have a longer half-life within the human body than other
antibodies. Accordingly, lower dosages and less frequent
administration is often possible. Modifications such as lipidation
can be used to stabilize antibodies and to enhance uptake and
tissue penetration (e.g., into the brain). A method for lipidation
of antibodies is described by Cruikshank et al., (J. Acquired
Immune Deficiency Syndromes and Human Retrovirology 14:193
(1997)).
[0329] The present invention encompasses agents which modulate
expression or activity. An agent may, for example, be a small
molecule. For example, such small molecules include, but are not
limited to, peptides, peptidomimetics (e.g., peptoids), amino
acids, amino acid analogs, is polynucleotides, polynucleotide
analogs, nucleotides, nucleotide analogs, organic or inorganic
compounds (i.e,. including heteroorganic and organometallic
compounds) having a molecular weight less than about 10,000 grams
per mole, organic or inorganic compounds having a molecular weight
less than about 5,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 1,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 500 grams per mole, and salts, esters, and other
pharmaceutically acceptable forms of such compounds.
[0330] Exemplary doses include milligram or microgram amounts of
the small molecule per kilogram of subject or sample weight (e.g.,
about 1 microgram per kilogram to about 500 milligrams per
kilogram, about 100 micrograms per kilogram to about 5 milligrams
per kilogram, or about 1 microgram per kilogram to about 50
micrograms per kilogram. It is furthermore understood that
appropriate doses of a small molecule depend upon the potency of
the small molecule with respect to the expression or activity to be
modulated. When one or more of these small molecules is to be
administered to an animal (e.g., a human) in order to modulate
expression or activity of a polypeptide or nucleic acid of the
invention, a physician, veterinarian, or researcher may, for
example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. In
addition, it is understood that the specific dose level for any
particular animal subject will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[0331] An antibody (or fragment thereof) may be conjugated to a
therapeutic moiety such as a cytotoxin, a therapeutic agent or a
radioactive metal ion. A cytotoxin or cytotoxic agent includes any
agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin); antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
[0332] The conjugates of the invention can be used for modifying a
given biological response, the drug moiety is not to be construed
as limited to classical chemical therapeutic agents. For example,
the drug moiety may be a protein or polypeptide possessing a
desired biological activity. Such proteins may include, for
example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor,
alpha.-interferon, .beta.-interferon, nerve growth factor, platelet
derived growth factor, tissue plasminogen activator; or, biological
response modifiers such as, for example, lymphokines, interleukin-1
("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"),
granulocyte macrophase colony stimulating factor ("GM-CSF"),
granulocyte colony stimulating factor ("G-CSF"), or other growth
factors.
[0333] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980.
[0334] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al., Proc. Natl. Acad.
Sci. USA 91:3054-3057 (1994)). The pharmaceutical preparation of
the gene therapy vector can include the gene therapy vector in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery vector can be produced intact from
recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can include one or more cells which produce the gene
delivery system.
[0335] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0336] Methods of Treatment:
[0337] The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant or unwanted MEKK1 expression or activity. With regards to
both prophylactic and therapeutic methods of treatment, such
treatments may be specifically tailored or modified, based on
knowledge obtained from the field of pharmacogenomics.
[0338] "Pharmacogenomics", as used herein, refers to the
application of genomics technologies such as gene sequencing,
statistical genetics, and gene expression analysis to drugs in
clinical development and on the market. More specifically, the term
refers the study of how a patient's genes determine his or her
response to a drug (e.g., a patient's "drug response phenotype", or
"drug response genotype".) Thus, another aspect of the invention
provides methods for tailoring an individual's prophylactic or
therapeutic treatment with either the MEKK1 molecules of the
present invention or MEKK1 modulators according to that
individual's drug response genotype. Pharmacogenomics allows a
clinician or physician to target prophylactic or therapeutic
treatments to patients who will most benefit from the treatment and
to avoid treatment of patients who will experience toxic
drug-related side effects.
[0339] In one aspect, the invention provides a-method for
preventing in a subject, a disease or condition associated with an
aberrant or unwanted MEKK1 expression or activity, by administering
to the subject a MEKK1 or an agent which modulates MEKK1 expression
or at least one MEKK1 activity. Subjects at risk for a disease
which is caused or contributed to by aberrant or unwanted MEKK1
expression or activity can be identified by, for example, any or a
combination of diagnostic or prognostic assays as described herein.
Administration of a prophylactic agent can occur prior to the
manifestation of symptoms characteristic of the MEKK1 aberrance,
such that a disease or disorder is prevented or, alternatively,
delayed in its progression. Depending on the type of MEKK1
aberrance, for example, a MEKK1, agonist or MEKK1 antagonist agent
can be used for treating the subject. The appropriate agent can be
determined based on screening assays described herein.
[0340] It is possible that some MEKK1 disorders can be caused, at
least in part, by an abnormal level of gene product, or by the
presence of a gene product exhibiting abnormal activity. As such,
the reduction in the level and/or activity of such gene products
would bring about the amelioration of disorder symptoms.
[0341] The MEKK1 molecules can act as novel diagnostic targets and
therapeutic agents for controlling one or more of cellular
proliferative and/or differentiative disorders as described above,
disorders associated with bone metabolism, immune disorders,
hematopoietic disorders, cardiovascular disorders, liver disorders,
viral diseases, pain or metabolic disorders.
[0342] The MEKK1 nucleic acid and protein of the invention can be
used to treat and/or diagnose a variety of medical disorders that
is subject to regulation or cure by manipulating a signal
transduction pathway in a cell involved in the disorder. Examples
of disorders or diseases include, but are not limited to, disorders
that result from aberrant cell growth or aberrant production of
secreted cellular products. As used herein, "aberrant cell growth"
means any disease or disorder in which an abnormal amount of cell
growth is observed. "Aberrant production of secreted cellular
products" means any disease or disorder in which an abnormal amount
of secreted cellular products is observed. Therefore, diseases
involving aberrant cell growth and aberrant production of secreted
cellular products are selected from the group consisting of
hyperplasia, neoplasia, and cancer, as well as degenerative
diseases, such a neurodegenerative diseases, autoimmune diseases,
inflammatory responses, and allergic responses. By "inhibits
aberrant cell growth" means any decrease in cell number or size,
including, without limitation, the decrease in cell number, a
decrease on the rate of cell division, an increase in the rate of
cell death, and a decrease in cell size. Standard methods for
measuring cell growth include standard apoptosis assays (e.g.,
TUNEL assays, DNA fragmentation, trypan blue exclusion) and cell
proliferation assays (e.g., .sup.3H-thymidine incorporation).
[0343] Preferred cancers subject to treatment using a method of the
present invention include, but are not limited to, small cell
carcinomas, non-small cell lung carcinomas with overexpressed EGF
receptors, breast cancers with overexpressed EGF or Neu receptors,
tumors having overexpressed growth factor receptors of established
autocrine loops and tumors having overexpressed growth factor
receptors of established paracrine loops. According to the present
invention, the term ""treatment" means the regulation of the
progression of a medical disorder or the complete removal of a
medical disorder (e.g., a cure). Treatment of a medical disorder
can comprise regulating the signal transduction activity of a cell
in such a manner that a cell involved in the medical disorder no
longer responds to extracellular stimuli (e.g., growth factors or
cytokines), or the killing of a cell involved in the medical
disorder through cellular apoptosis.
[0344] The present invention relates to a method of inducing and/or
maintaining a differentiated state, enhancing survival, and/or
promoting (or alternatively inhibiting) proliferation of a cell
responsive to a growth factor, morphogen or other environmental cue
which affects the cell through at least one signal transduction
pathway which includes a MEKK1 protein. In general, the method
comprises contacting cells with an amount of an agent which
significantly (statistically) modulates MEKK-dependent signaling by
the factor. For instance, it is contemplated by the invention that,
in light of the present finding of an apparently broad involvement
of members of the MEKK protein family in signal pathways implicated
in the formation of ordered spatial arrangements of differentiated
tissues in vertebrates, the subject method could be used to
generate and/or maintain an array of different vertebrate tissue
both in vitro and in vivo. A "MEKK1 therapeutic" whether inductive
or anti-inductive with respect to signaling by a MEKK1-dependent
pathway, can be, as appropriate, any of the preparations described
above, including isolated polypeptides, gene therapy constructs,
antisense molecules, peptidomimetics or agents identified in the
drug assays provided herein.
[0345] There are a wide variety of pathological cell proliferative
conditions for which MEKK1 therapeutics of the present invention
are useful. For instance, such agents provide therapeutic benefits
where the general strategy being the inhibition of an anomalous
cell proliferation. Diseases that might benefit from this
methodology include, but are not limited to, various cancers and
leukemias, psoriasis, bone diseases, fibroproliferative disorders
such as involving connective tissues, atherosclerosis and other
smooth muscle proliferative disorders, autoimmune disease,
inflammatory conditions such as rheumatoid arthritis, multiple
sclerosis and complications arising from organ transplantation,
allergic responses, as well as respiratory inflammation (e.g.,
asthma and chronic obstructive pulmonary disease), and inflammatory
bowel disease (e.g., Crohn's disease and ulcerative colitis).
[0346] In addition to proliferative disorders, the present
invention contemplates the use of MEKK1 therapeutics for the
treatment of differentiative disorders which result from, for
example, dedifferentiation of tissue which may optionally be
accompanied by apoptosis.
[0347] Additionally, MEKK1 molecules may play an important role in
the etiology of certain viral diseases, including, but not limited
to, Hepatitis B, Hepatitis C, and Herpes Simplex Virus (HSV).
Modulators of MEKK1 activity could be used to control viral
diseases. The modulators can be used in the treatment and/or
diagnosis of viral infected tissue or virus-associated tissue
fibrosis, especially liver and liver fibrosis. Also, MEKK1
modulators can be used in the treatment and/or diagnosis of
virus-associated carcinoma, especially hepatocellular cancer.
[0348] As discussed, successful treatment of MEKK1 disorders can be
brought about by techniques that serve to inhibit the expression or
activity of target gene products. For example, compounds, e.g., an
agent identified using an assays described above, that proves to
exhibit negative modulatory activity, can be used in accordance
with the invention to prevent and/or ameliorate symptoms of MEKK1
disorders. Such molecules can include, but are not limited to,
peptides, phosphopeptides, small organic or inorganic molecules, or
antibodies (including, for example, polyclonal, monoclonal,
humanized, anti-idiotypic, chimeric or single chain antibodies, and
Fab, F(ab').sub.2 and Fab expression library fragments, scFV
molecules, and epitope-binding fragments thereof).
[0349] Further, antisense and ribozyme molecules that inhibit
expression of the target gene can also be used in accordance with
the invention to reduce the level of target gene expression, thus
effectively reducing the level of target gene activity. Still
further, triple helix molecules can be utilized in reducing the
level of target gene activity. Antisense, ribozyme and triple helix
molecules are discussed above.
[0350] It is possible that the use of antisense, ribozyme, and/or
triple helix molecules to reduce or inhibit mutant gene expression
can also reduce or inhibit the transcription (triple helix) and/or
translation (antisense, ribozyme) of mRNA produced by normal target
gene alleles, such that the concentration of normal target gene
product present can be lower than is necessary for a normal
phenotype. In such cases, nucleic acid molecules that encode and
express target gene polypeptides exhibiting normal target gene
activity can be introduced into cells via gene therapy method.
Alternatively, in instances in that the target gene encodes an
extracellular protein, it can be preferable to co-administer normal
target gene protein into the cell or tissue in order to maintain
the requisite level of cellular or tissue target gene activity.
[0351] Another method by which nucleic acid molecules may be
utilized in treating or preventing a disease characterized by MEKK1
expression is through the use of aptamer molecules specific for
MEKK1 protein. Aptamers are nucleic acid molecules having a
tertiary structure which permits them to specifically bind to
protein ligands (see, e.g., Osborne et al., Curr. Opin. Chem Biol.,
1:5-9 (1997); and Patel, Curr. Opin. Chem. Biol. 1:32-46 (1997)).
Since nucleic acid molecules may in many cases be more conveniently
introduced into target cells than therapeutic protein molecules may
be, aptamers offer a method by which MEKK1 protein activity may be
specifically decreased without the introduction of drugs or other
molecules which may have pluripotent effects.
[0352] Antibodies can be generated that are both specific for
target gene product and that reduce target gene product activity.
Such antibodies may, therefore, by administered in instances
whereby negative modulatory techniques are appropriate for the
treatment of MEKK1 disorders. For a description of antibodies, see
the Antibody section above.
[0353] In circumstances wherein injection of an animal or a human
subject with a MEKK1 protein or epitope for stimulating antibody
production is harmful to the subject, it is possible to generate an
immune response against MEKK1 through the use of anti-idiotypic
antibodies (see, for example, Herlyn, D. Ann Med 1999;31(1):66-78;
and Bhattacharya-Chatterjee- , M., and Foon, K. A. Cancer Treat Res
1998;94:51-68). If an anti-idiotypic antibody is introduced into a
mammal or human subject, it should stimulate the production of
anti-anti-idiotypic antibodies, which should be specific to the
MEKK1 protein. Vaccines directed to a disease characterized by
MEKK1 expression may also be generated in this fashion.
[0354] Instances where the target antigen is intracellular and
whole antibodies are used, internalizing antibodies may be
preferred. Lipofectin or liposomes can be used to deliver the
antibody or a fragment of the Fab region that binds to the target
antigen into cells. Where fragments of the antibody are used, the
smallest inhibitory fragment that binds to the target antigen is
preferred. For example, peptides having an amino acid sequence
corresponding to the Fv region of the antibody can be used.
Alternatively, single chain neutralizing antibodies that bind to
intracellular target antigens can also be administered. Such single
chain antibodies can be administered, for example, by expressing
nucleotide sequences encoding single-chain antibodies within the
target cell population (see e.g., Marasco et al., Proc. Natl. Acad.
Sci. USA 90:7889-7893 (1993)).
[0355] The identified compounds that inhibit target gene
expression, synthesis and/or activity can be administered to a
patient at therapeutically effective doses to prevent, treat or
ameliorate MEKK1 disorders. A therapeutically effective dose refers
to that amount of the compound sufficient to result in amelioration
of symptoms of the disorders.
[0356] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
that exhibit large therapeutic indices are preferred. While
compounds that exhibit toxic side effects can be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0357] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage can vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose can be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound that achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma can
be measured, for example, by high performance liquid
chromatography.
[0358] Another example of determination of effective dose for an
individual is the ability to directly assay levels of "free" and
"bound" compound in the serum of the test subject. Such assays may
utilize antibody mimics and/or "biosensors" that have been created
through molecular imprinting techniques. The compound which is able
to modulate MEKK1 activity is used as a template, or "imprinting
molecule", to spatially organize polymerizable monomers prior to
their polymerization with catalytic reagents. The subsequent
removal of the imprinted molecule leaves a polymer matrix which
contains a repeated "negative image" of the compound and is able to
selectively rebind the molecule under biological assay conditions.
A detailed review of this technique can be seen in Ansell, R. J. et
al (1996) Current Opinion in Biotechnology 7:89-94 and in Shea, K.
J. (1994) Trends in Polymer Science 2:166-173. Such "imprinted"
affinity matrixes are amenable to ligand-binding assays, whereby
the immobilized monoclonal antibody component is replaced by an
appropriately imprinted matrix. An example of the use of such
matrixes in this way can be seen in Vlatakis, G. et al (1993)
Nature 361:645-647. Through the use of isotope-labeling, the "free"
concentration of compound which modulates the expression or
activity of MEKK1 can be readily monitored and used in calculations
of IC.sub.50.
[0359] Such "imprinted" affinity matrixes can also be designed to
include fluorescent groups whose photon-emitting properties
measurably change upon local and selective binding of target
compound. These changes can be readily assayed in real time using
appropriate fiberoptic devices, in turn allowing the dose in a test
subject to be quickly optimized based on its individual IC. A
rudimentary example of such a "biosensor" is discussed in Kriz, D.
et al (1995) Analytical Chemistry 67:2142-2144.
[0360] Another aspect of the invention pertains to methods of
modulating MEKK1 expression or activity for therapeutic purposes.
Accordingly, in an exemplary embodiment, the modulatory method of
the invention involves contacting a cell with a MEKK1 or agent that
modulates one or more of the activities of MEKK1 protein activity
associated with the cell. An agent that modulates MEKK1 protein
activity can be an agent as described herein, such as a nucleic
acid or a protein, a naturally-occurring target molecule of a MEKK1
protein (e.g., a MEKK1 substrate or receptor), a MEKK1 antibody, a
MEKK1 agonist or antagonist, a peptidomimetic of a MEKK1 agonist or
antagonist, or other small molecule.
[0361] In one embodiment, the agent stimulates one or MEKK1
activities. Examples of such stimulatory agents include active
MEKK1 protein and a nucleic acid molecule encoding MEKK1. In
another embodiment, the agent inhibits one or more MEKK1
activities. Examples of such inhibitory agents include antisense
MEKK1 nucleic acid molecules, anti-MEKK1 antibodies, and MEKK1
inhibitors. These modulatory methods can be performed in vitro
(e.g., by culturing the cell with the agent) or, alternatively, in
vivo (e.g., by administering the agent to a subject). As such, the
present invention provides methods of treating an individual
afflicted with a disease or disorder characterized by aberrant or
unwanted expression or activity of a MEKK1 protein or nucleic acid
molecule. In one embodiment, the method involves administering an
agent (e.g., an agent identified by a screening assay described
herein), or combination of agents that modulates (e.g., upregulates
or downregulates) MEKK1 expression or activity. In another
embodiment, the method involves administering a MEKK1 protein or
nucleic acid molecule as therapy to compensate for reduced,
aberrant, or unwanted MEKK1 expression or activity.
[0362] Stimulation of MEKK1 activity is desirable in situations in
which MEKK1 is abnormally downregulated and/or in which increased
MEKK1 activity is likely to have a beneficial effect. For example,
stimulation of MEKK1 activity is desirable in situations in which a
MEKK1 is downregulated and/or in which increased MEKK1 activity is
likely to have a beneficial effect. Likewise, inhibition of MEKK1
activity is desirable in situations in which MEKK1 is abnormally
upregulated and/or in which decreased MEKK1 activity is likely to
have a beneficial effect.
[0363] Pharmacogenomics
[0364] The MEKK1 molecules of the present invention, as well as
agents, or modulators which have a stimulatory or inhibitory effect
on MEKK1 activity (e.g., MEKK1 gene expression) as identified by a
screening assay described herein can be administered to individuals
to treat (prophylactically or therapeutically) MEKK1 associated
disorders (e.g., cell signaling or cell adhesion) associated with
aberrant or unwanted MEKK1 activity. In conjunction with such
treatment, pharmacogenomics (i.e., the study of the relationship
between an individual's genotype and that individual's response to
a foreign compound or drug) may be considered. Differences in
metabolism of therapeutics can lead to severe toxicity or
therapeutic failure by altering the relation between dose and blood
concentration of the pharmacologically active drug. Thus, a
physician or clinician may consider applying knowledge obtained in
relevant pharmacogenomics studies in determining whether to
administer a MEKK1 molecule or MEKK1 modulator as well as tailoring
the dosage and/or therapeutic regimen of treatment with a MEKK1
molecule or MEKK1 modulator.
[0365] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, for
example, Eichelbaum et al., Clin. Exp. Pharmacol. Physiol.
23(10-11) :983-985 (1996) and Linder et al., Clin. Chem.
43(2):254-266 (1997). In general, two types of pharmacogenetic
conditions can be differentiated. Genetic conditions transmitted as
a single factor altering the way drugs act on the body (altered
drug action) or genetic conditions transmitted as single factors
altering the way the body acts on drugs (altered drug metabolism).
These pharmacogenetic conditions can occur either as rare genetic
defects or as naturally-occurring polymorphisms.
[0366] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association", relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants.) Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high resolution map can be generated from a
combination of some ten-million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, a "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP may occur once per every 1000
bases of DNA. A SNP may be involved in a disease process, however,
the vast majority may not be disease-associated. Given a genetic
map based on the occurrence of such SNPs, individuals can be
grouped into genetic categories depending on a particular pattern
of SNPs in their individual genome. In such a manner, treatment
regimens can be tailored to groups of genetically similar
individuals, taking into account traits that may be common among
such genetically similar individuals.
[0367] Alternatively, a method termed the "candidate gene
approach", can be utilized to identify genes that predict drug
response. According to this method, if a gene that encodes a drug's
target is known (e.g., a MEKK1 protein of the present invention),
all common variants of that gene can be fairly easily identified in
the population and it can be determined if having one version of
the gene versus another is associated with a particular drug
response.
[0368] Alternatively, a method termed the "gene expression
profiling", can be utilized to identify genes that predict drug
response. For example, the gene expression of an animal dosed with
a drug (e.g., a MEKK1 molecule or MEKK1 modulator of the present
invention) can give an indication whether gene pathways related to
toxicity have been turned on.
[0369] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate
dosage and treatment regimens for prophylactic or therapeutic
treatment of an individual. This knowledge, when applied to dosing
or drug selection, can avoid adverse reactions or therapeutic
failure and thus enhance therapeutic or prophylactic efficiency
when treating a subject with a MEKK1 molecule or MEKK1 modulator,
such as a modulator identified by one of the exemplary screening
assays described herein.
[0370] The present invention further provides methods for
identifying new agents, or combinations, that are based on
identifying agents that modulate the activity of one or more of the
gene products encoded by one or more of the MEKK1 genes of the
present invention, wherein these products may be associated with
resistance of the cells to a therapeutic agent. Specifically, the
activity of the proteins encoded by the MEKK1 genes of the present
invention can be used as a basis for identifying agents for
overcoming agent resistance. By blocking the activity of one or
more of the resistance proteins, target cells, e.g., epithelial
cells, will become sensitive to treatment with an agent to which
the unmodified target cells were resistant.
[0371] Monitoring the influence of agents (e.g., drugs) on the
expression or activity of a MEKK1 protein can be applied in
clinical trials. For example, the effectiveness of an agent
determined by a screening assay as described herein to increase
MEKK1 gene expression, protein levels, or upregulate MEKK1
activity, can be monitored in clinical trials of subjects
exhibiting decreased MEKK1 gene expression, protein levels, or
downregulated MEKK1 activity. Alternatively, the effectiveness of
an agent determined by a screening assay to decrease MEKK1 gene
expression, protein levels, or downregulate MEKK1 activity, can be
monitored in clinical trials of subjects exhibiting increased MEKK1
gene expression, protein levels, or upregulated MEKK1 activity. In
such clinical trials, the expression or activity of a MEKK1 gene,
and preferably, other genes that have been implicated in, for
example, a MEKK1-associated disorder can be used as a "read out" or
markers of the phenotype of a particular cell.
OTHER EMBODIMENTS
[0372] In another aspect, the invention features, a method of
analyzing a plurality of capture probes. The method can be used,
e.g., to analyze gene expression. The method includes: providing a
two dimensional array having a plurality of addresses, each address
of the plurality being positionally distinguishable from each other
address of the plurality, and each address of the plurality having
a unique capture probe, e.g., a nucleic acid or peptide sequence;
contacting the array with a MEKK1, preferably purified, nucleic
acid, preferably purified, polypeptide, preferably purified, or
antibody, and thereby evaluating the plurality of capture probes.
Binding, e.g., in the case of a nucleic acid, hybridization with a
capture probe at an address of the plurality, is detected, e.g., by
signal generated from a label attached to the MEKK1 nucleic acid,
polypeptide, or antibody.
[0373] The capture probes can be a set of nucleic acids from a
selected sample, e.g., a sample of nucleic acids derived from a
control or non-stimulated tissue or cell.
[0374] The method can include contacting the MEKK1 nucleic acid,
polypeptide, or antibody with a first array having a plurality of
capture probes and a second array having a different plurality of
capture probes. The results of each hybridization can be compared,
e.g., to analyze differences in expression between a first and
second sample. The first plurality of capture probes can be from a
control sample, e.g., a wild type, normal, or non-diseased,
non-stimulated, sample, e.g., a biological fluid, tissue, or cell
sample. The second plurality of capture probes can be from an
experimental sample, e.g., a mutant type, at risk, disease-state or
disorder-state, or stimulated, sample, e.g., a biological fluid,
tissue, or cell sample.
[0375] The plurality of capture probes can be a plurality of
nucleic acid probes each of which specifically hybridizes, with an
allele of MEKK1. Such methods can be used to diagnose a subject,
e.g., to evaluate risk for a disease or disorder, to evaluate
suitability of a selected treatment for a subject, or to evaluate
whether a subject has a disease or disorder.
[0376] The method can be used to detect SNPs, as described
above.
[0377] In another aspect, the invention features, a method of
analyzing a plurality of probes. The method is useful, e.g., for
analyzing gene expression. The method includes: providing a two
dimensional array having a plurality of addresses, each address of
the plurality being positionally distinguishable from each other
address of the plurality having a unique capture probe, e.g.,
wherein the capture probes are from a cell or subject which express
MEKK1 or from a cell or subject in which a MEKK1 mediated response
has been elicited, eg., by contact of the cell with MEKK1 nucleic
acid or protein, or administration to the cell or subject MEKK1
nucleic acid or protein; contacting the array with one or more
inquiry probe, wherein an inquiry probe can be a nucleic acid,
polypeptide, or antibody (which is preferably other than MEKK1
nucleic acid, polypeptide, or antibody); providing a two
dimensional array having a plurality of addresses, each address of
the plurality being positionally distinguishable from each other
address of the plurality, and each address of the plurality having
a unique capture probe, e.g., wherein the capture probes are from a
cell or subject which does not express MEKK1 (or does not express
as highly as in the case of the MEKK1 positive plurality of capture
probes) or from a cell or subject which in which a MEKK1 mediated
response has not been elicited (or has been elicited to a lesser
extent than in the first sample); contacting the array with one or
more inquiry probes (which is preferably other than a MEKK1 nucleic
acid, polypeptide, or antibody), and thereby evaluating the
plurality of capture probes. Binding, e.g., in the case of a
nucleic acid, hybridization with a capture probe at an address of
the plurality, is detected, e.g., by signal generated from a label
attached to the nucleic acid, polypeptide, or antibody.
[0378] In another aspect, the invention features, a method of
analyzing MEKK1, e.g., analyzing structure, function, or
relatedness to other nucleic acid or amino acid sequences. The
method includes: providing a MEKK1 nucleic acid or amino acid;
comparing the MEKK1 sequence with one or more preferably a
plurality of sequences from a collection of sequences, e.g., a
nucleic acid or protein sequence database; to thereby analyze
MEKK1. The method can include evaluating the sequence identity
between a MEKK1 sequence and a database sequence. The method can be
performed by accessing the database at a second site, e.g., over
the internet.
[0379] In another aspect, the invention features, a set of
oligonucleotides, useful, e.g., for identifying SNP's, or
identifying specific alleles of MEKK1. The set includes a plurality
of oligonucleotides, each of which has a different nucleotide at an
interrogation position, e.g., an SNP or the site of a mutation. In
a preferred embodiment, the oligonucleotides of the plurality
identical in sequence with one another (except for differences in
length). The oligonucleotides can be provided with differential
labels, such that an oligonucleotides which hybridizes to one
allele provides a signal that is distinguishable from an
oligonucleotides which hybridizes to a second allele.
[0380] The sequence of a MEKK1 molecules is provided in a variety
of mediums to facilitate use thereof. A sequence can be provided as
a manufacture, other than an isolated nucleic acid or amino acid
molecule, which contains a MEKK1. Such a manufacture can provide a
nucleotide or amino acid sequence, e.g., an open reading frame, in
a form which allows examination of the manufacture using means not
directly applicable to examining the nucleotide or amino acid
sequences, or a subset thereof, as they exists in nature or in
purified form.
[0381] A MEKK1 nucleotide or amino acid sequence can be recorded on
computer readable media. As used herein, "computer readable media"
refers to any medium that can be read and accessed directly by a
computer. Such media include, but are not limited to: magnetic
storage media, such as floppy discs, hard disc storage medium, and
magnetic tape; optical storage media such as CD-ROM; electrical
storage media such as RAM and ROM; and hybrids of these categories
such as magnetic/optical storage media.
[0382] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide or amino acid sequence of the present
invention. The choice of the data storage structure will generally
be based on the means chosen to access the stored information. In
addition, a variety of data processor programs and formats can be
used to store the nucleotide sequence information of the present
invention on computer readable medium. The sequence information can
be represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. The
skilled artisan can readily adapt any number of data processor
structuring formats (eg., text file or database) in order to obtain
computer readable medium having recorded thereon the nucleotide
sequence information of the present invention.
[0383] By providing the nucleotide or amino acid sequences of the
invention in computer readable form, the skilled artisan can
routinely access the sequence information for a variety of
purposes. For example, one skilled in the art can use the
nucleotide or amino acid sequences of the invention in computer
readable form to compare a target sequence or target structural
motif with the sequence information stored within the data storage
means. A search is used to identify fragments or regions of the
sequences of the invention which match a particular target sequence
or target motif.
[0384] As used herein, a "target sequence" can be any DNA or amino
acid sequence of six or more nucleotides or two or more amino
acids. A skilled artisan can readily recognize that the longer a
target sequence is, the less likely a target sequence will be
present as a random occurrence in the database. Typical sequence
lengths of a target sequence are from about 10 to 100 amino acids
or from about 30 to 300 nucleotide residues. However, it is well
recognized that commercially important fragments, such as sequence
fragments involved in gene expression and protein processing, may
be of shorter length.
[0385] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium for analysis and comparison to other
sequences. A variety of known algorithms are disclosed publicly and
a variety of commercially available software for conducting search
means are and can be used in the computer-based systems of the
present invention. Examples of such software include, but are not
limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA).
[0386] Thus, the invention features a method of making a computer
readable record of a sequence of a MEKK1 sequence that includes
recording the sequence on a computer readable matrix. In a
preferred embodiment the record includes one or more of the
following: identification of an ORF; identification of a domain,
region, or site; identification of the start of transcription;
identification of the transcription terminator; the full length
amino acid sequence of the protein, or a mature form thereof; the
5' end of the translated region; or 3' regulatory elements.
[0387] In another aspect, the invention features, a method of
analyzing a sequence. The method includes: providing a MEKK1
sequence, or record, in computer readable form; comparing a second
sequence to the gene name sequence; thereby analyzing a sequence.
Comparison can include comparing to sequences for sequence identity
or determining if one sequence is included within the other, e.g.,
determining if the MEKK1 sequence includes a sequence being
compared. In a preferred embodiment the MEKK1 or second sequence is
stored on a first computer, e.g., at a first site and the
comparison is performed, read, or recorded on a second computer,
e.g., at a second site. For example, the MEKK1 or second sequence
can be stored in a public or proprietary database in one computer,
and the results of the comparison performed, read, or recorded on a
second computer. In a preferred embodiment the record includes one
or more of the following: identification of an ORF; identification
of a domain, region, or site; identification of the start of
transcription; identification of the transcription terminator; the
full length amino acid sequence of the protein, or a mature form
thereof; the 5' end of the translated region; or 3' regulatory
elements.
[0388] This invention is further illustrated by the following
examples that should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application are incorporated herein by
reference.
EXAMPLES
Example 1
Cloning the Full-Length Human MEKK1 Gene
[0389] The full-length human MEKK1 cDNA was isolated by screening a
human placenta cDNA library. This cDNA encodes a 4.5 kb open
reading frame which predicts a 164.5 kDa protein.
[0390] The human Placenta 5'-STRETCHPLUS cDNA library was purchased
from Clontech Laboratories, Inc. (Palo Alto, Calif.). The library
vector was phage lambda gt11. Approximately 500,000 plaques were
screened for human MEKK1 clones according to the Clontech lambda
library protocol. Phage were plated on 15-150 mm LB plates
supplemented with 10 mM Mg.sub.2SO.sub.4 and incubated at
42.degree. C. Duplicates of 80-S BA nitrocellulose filters
(Schleicher & Schuell, Keene, N H) were lifted from the 15
plates, then treated to denature and renature the DNA on the
filters, and then oven-baked at 80.degree. C. for 2 hours. The
filters were then incubated in a prehybridization solution of 50%
formamide, 5.times.SSPE, 5.times. Denhardt's solution, 0.1% SDS,
and 0.1 mg/ml denatured herring sperm DNA for 4 hours, then labeled
DNA probe was added. The DNA probe was about a 500 bps DNA fragment
of SacII and XmmI digested EST clone (W96411) (Research Genetics,
Huntsville, Ala.) of partial human MEKK1 DNA. The DNA probe was
labeled with .sup.32P-dCTP using the Prime-It.RTM. II kit from
Stratagene (La Jolla, Calif.). Ten positive plaques were picked for
secondary screening. Six positives were confirmed. Lambda phage DNA
was prepared using a Lambda phage DNA Wizard Prep kit (Promega,
Madison, Wis.). Restriction digestion with EcoRI revealed that the
longest insert was about a 5.5 kb sequence contained in one of the
six positive phage DNAs. The insert was cloned into bacterial
plasmid pBluescript SK+ at the EcoRI site and named pM6. The entire
cDNA insert was sequenced. The resulting nucleotide sequence (SEQ
ID NO:1) was aligned to the known full-length rat MEKK1 sequence
and determined to be the full-length human MEKK1 by homology.
[0391] The human MEKK1 sequence, (FIG. 1; SEQ ID NO:1), which is
approximately 5245 nucleotides long, including untranslated
regions, contains a predicted methionine-initiated coding sequence
of about 4539 nucleotides (nucleotides 7-4542 of SEQ ID NO:1, which
corresponds to numbered nucleotides 1-4539 of SEQ ID NO:3). The
coding sequence encodes a 1512 amino acid protein (SEQ ID
NO:2).
[0392] FIG. 2 shows the amino acid sequence alignment of the full
length human MEKK1 protein against other known MEKK1 proteins of
other species. This 1512 amino acid of human MEKK1 is 89% identical
to the rat MEKK1 amino acid sequence.
Example 2
The Full-length MEKK1 is an Activator of NF.kappa.B
[0393] The importance of MEKK1 function in the AP-1 pathway is well
established. (Gottschalf et al., J. Exp. Med. 178:1681 (1993); Want
et al., J. Mol. Cell. Biol. 14:11153 (1994); Rao, Immunol. Today
15:274 (1994); Angel et al., Biochem. Biophys. Acta. 1072:129
(1991).) However, the involvement of MEKK1 in
NF.kappa..circle-solid.B has been unclear. As described herein, a
NF.kappa.B reporter assay was performed to examine the effect of
MEKK1 in this pathway. The full-length human MEKK1 cDNA was
subcloned into the mammalian cell expression vector pcDNA3
(Invitrogen, Carlsbad, Calif.). This pcDNA3 vector was also
constructed using oligo directed-mutagenesis to have a 9-amino acid
FLAG epitope fused at the carboxy-terminus of the MEKK1 protein. By
using an antibody specific for the FLAG epitope, expression of the
human MEKK1 full-length protein can be determined.
[0394] HeLa cells were split at 1.5.times.10.sup.5 per 35 mm plates
24 hours before MEKK1 transfection using the CaPO.sub.4 method
(Invitrogen, Carlsbad, Calif.). HeLa cells were transfected with
increasing amounts of pcDAN3 expressing the C-terminal FLAG-tagged
human MEKK1 and co-transfected with 1 ug of an NF.kappa.B reporter
p(PRDII)4 DNA containing the chloramphenicol acetyltransferase gene
under the control of a promoter containing four
NF.kappa..circle-solid.B binding sites. Total DNA concentrations
were kept constant at 4 .mu.g by adding empty pcDNA3. As a positive
control, a pcDNA3 vector expressing a N-terminal FLAG-tagged NIK
protein was used. NIK, a NF.kappa.B inducing kinase, is known to be
a very potent activator of NF.kappa.B. After 24 hours, the cells
were washed three time with PBS and lysed in reporter lysis buffer
(Promega, Madison, Wis.). Lysates were harvested by scraping and
were transferred to Eppendorf tubes. The protein concentration of
each cell extract was determined by Coomassie Plus Protein Assay
Reagent (Pierce, Rockford, Ill.).
[0395] The expression levels of transfected MEKK1 and NIK proteins
were examined by Western blot analysis (FIG. 3). Ten micrograms of
each sample was applied onto a 4-12% SDS-PAGE gradient gel, then
transferred to a nitrocellulose membrane. Anti-FLAG antibody was
used as the primary antibody; the secondary antibody was
HRP-conjugated anti-mouse antibody (Amersham Pharmacia Biotech,
England). The Western blot was developed with ECL reagents
(Amersham Pharmacia Biotech, England).
[0396] The protein expression levels are shown in the Western blot
of FIG. 3. Increasing amounts of MEKK1 were synthesized in HeLa
cells which correlated with the amount of cDNA3-MEKK1flag DNA used
in the transfection. Expressed full-length MEKK1 protein migrated
on the SDS gel with an apparent molecular weight of 196 kDa.
[0397] To assay NF.kappa.B activity, the levels of chloramphenicol
acetyltransferase protein was measured by CAT ELISA (Roche
Molecular Biochemicals, Indianapolis, Ind.) (FIG. 4). CAT ELISA was
performed using 8 ..mu.g of each extract. The amount of CAT protein
in the 8 .mu.g extract was calculated relative to the CAT standard
and was graphed along the Y axis as the amount of CAT protein per 8
..mu.g extract. The X axis indicates increasing microgram amounts
of transfected pcDNA3-flagMEKK1 or pcDNA3-flagNIK DNAs.
[0398] The results show that transient expression of the
full-length human MEKK1 protein increased the NF..kappa.B activity
in cells. The level of NF.kappa..circle-solid.B activation changes
in a dose-dependent manner with the level of MEKK1 protein
expression, although the extent of NF..kappa.B activation is
apparently weaker compare than that of expressed NIK protein. This
example demonstrates that full-length human MEKK1 protein functions
as an activator of NF.kappa.B in cells.
Example 3
The Full-Length MEKK1 Interacts with IKK1 in Cells
[0399] One crucial step in NF.kappa.B activation pathway is the
signal-induced phosphorylation of I.kappa.B proteins by the
I.kappa.B kinase complex. To further characterize MEKK1 function in
NF.kappa..circle-solid.B activation, MEKK1 was assayed for direct
activation of the I.kappa.B kinase by measuring MEKK1 interaction
with components of the I.kappa.B kinase complex.
[0400] Transfections were performed exactly according to the
manufacturer's protocol using LipofectAMINE PLUS Regent (Life
Technologies, Rockville, Md.). Human 293 cells were split at
2.times.10.sup.6 per 100 mm plate 24 hours before transfection. The
expression vectors were 4 ug each of the following DNAs:
pcDNA3MEKK1flag, pcDNA3flagHANIK in the presence or absence of
pcDNAmycIKK1 or pcDNA3mycIKK2. After 28 hours, cells were washed
with PBS and resuspended in a lysis buffer (50 mM HEPES pH 7.9, 100
mM NaCl, 10% glycerol, 1 mM EDTA, 20 .mu..M.alpha.
glycerolphosphate, 1 mM NaPO.sub.4, 1 mM sodium metabisulphite, 20
mM PNPP, 0.1% NP-40, and protease inhibitor cocktail). Cell lysates
were incubated with 15 .l of anti-flag antibody M2 resin (Sigma,
St. Louis, Mo.) for 4 hours at 4.degree. C. The M2 resin was then
washed three times with the lysis buffer. Proteins that contained
the flag epitope remained bound to the anti-flag M2 beads along
with other proteins that specifically associated with the
flag-tagged protein. Immunoprecipitates were resolved on a 4-12%
SDS-PAGE gel and transferred to nitrocellulose membranes. Western
blot analysis was performed first with anti-myc antibody (Santa
Cruz Biotech, Santa Cruz, Calif.) for the presence of mycIKK1 and
mycIKK2 proteins in the immune complex. Then, anti-flag antibody
was used to detect flag-tagged MEKK1 and flag-tagged NIK proteins
(Sigma, St. Louis, Mo.).
[0401] The results are shown in FIG. 5. NIK protein was used as a
positive control because it is known to activate the I.kappa.B
kinase by interacting with IKK1 and IKK2. As in the flag-NIK
immunoprecipitates, the myc-tagged IKK1 protein was detected
specifically in the flag-tagged full-length MEKK1 immune complex.
Therefore, the full-length MEKK1 is capable of interacting with
IKK1. In contrast, the expression level of MEKK1 was extremely low
when co-expressed with IKK2 protein, therefore the association
between IKK2 and MEKK1 could not be observed. This example shows
that at least one component of the I.kappa.B kinase interacts with
MEKK1 protein.
[0402] Equivalents
[0403] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
28 1 5245 DNA Human CDS (7)..(4545) 1 gagaaa atg gcg gcg gcg gcg
ggg aat cgc gcc tcg tcg tcg gga ttc 48 Met Ala Ala Ala Ala Gly Asn
Arg Ala Ser Ser Ser Gly Phe 1 5 10 ccg ggc gcc agg gct acg agc cct
gag gca ggc ggc ggc gga gga gcc 96 Pro Gly Ala Arg Ala Thr Ser Pro
Glu Ala Gly Gly Gly Gly Gly Ala 15 20 25 30 ctc aag gcg agc agc gcg
ccc gcg gct gcc gcg gga ctg ctg cgg gag 144 Leu Lys Ala Ser Ser Ala
Pro Ala Ala Ala Ala Gly Leu Leu Arg Glu 35 40 45 gcg ggc agc ggg
ggc cgc gag cgg gcg gac tgg cgg cgg cgg cag ctg 192 Ala Gly Ser Gly
Gly Arg Glu Arg Ala Asp Trp Arg Arg Arg Gln Leu 50 55 60 cgc aaa
gtg cgg agt gtg gag ctg gac cag ctg cct gag cag ccg ctc 240 Arg Lys
Val Arg Ser Val Glu Leu Asp Gln Leu Pro Glu Gln Pro Leu 65 70 75
ttc ctt gcc gcc tca ccg ccg gcc tcc tcg act tcc ccg tcg ccg gag 288
Phe Leu Ala Ala Ser Pro Pro Ala Ser Ser Thr Ser Pro Ser Pro Glu 80
85 90 ccc gcg gac gca gcg ggg agt ggg acc ggc ttc cag cct gtg gcg
gtg 336 Pro Ala Asp Ala Ala Gly Ser Gly Thr Gly Phe Gln Pro Val Ala
Val 95 100 105 110 ccg ccg ccc cac gga gcc gcg agc cgc ggc ggc gcc
cac ctt acc gag 384 Pro Pro Pro His Gly Ala Ala Ser Arg Gly Gly Ala
His Leu Thr Glu 115 120 125 tcg gtg gcg gcg ccg gac agc ggc gcc tcg
agt ccc gca gcg gcc gag 432 Ser Val Ala Ala Pro Asp Ser Gly Ala Ser
Ser Pro Ala Ala Ala Glu 130 135 140 ccc ggg gag aag cgg gcg ccc gcc
gcc gag ccg tct cct gca gcg gcc 480 Pro Gly Glu Lys Arg Ala Pro Ala
Ala Glu Pro Ser Pro Ala Ala Ala 145 150 155 ccc gcc ggt cgt gag atg
gag aat aaa gaa act ctc aaa ggg ttg cac 528 Pro Ala Gly Arg Glu Met
Glu Asn Lys Glu Thr Leu Lys Gly Leu His 160 165 170 aag atg gat gat
cgt cca gag gaa cga atg atc agg gag aaa ctg aag 576 Lys Met Asp Asp
Arg Pro Glu Glu Arg Met Ile Arg Glu Lys Leu Lys 175 180 185 190 gca
acc tgt atg cca gcc tgg aag cac gaa tgg ttg gaa agg aga aat 624 Ala
Thr Cys Met Pro Ala Trp Lys His Glu Trp Leu Glu Arg Arg Asn 195 200
205 agg cga ggg cct gtg gtg gta aaa cca atc cca gtt aaa gga gat gga
672 Arg Arg Gly Pro Val Val Val Lys Pro Ile Pro Val Lys Gly Asp Gly
210 215 220 tct gaa atg aat cac tta gca gct gag tct cca gga gag gtc
cag gca 720 Ser Glu Met Asn His Leu Ala Ala Glu Ser Pro Gly Glu Val
Gln Ala 225 230 235 agt gcg gct tca cca gct tcc aaa ggc cga cgc agt
cct tct cct ggc 768 Ser Ala Ala Ser Pro Ala Ser Lys Gly Arg Arg Ser
Pro Ser Pro Gly 240 245 250 aac tcc cca tca ggt cgc aca gtg aaa tca
gaa tct cca gga gta agg 816 Asn Ser Pro Ser Gly Arg Thr Val Lys Ser
Glu Ser Pro Gly Val Arg 255 260 265 270 aga aaa aga gtt tcc cca gtg
cct ttt cag agt ggc aga atc aca cca 864 Arg Lys Arg Val Ser Pro Val
Pro Phe Gln Ser Gly Arg Ile Thr Pro 275 280 285 ccc cga aga gcc cct
tca cca gat ggc ttc tca cca tat agc cct gag 912 Pro Arg Arg Ala Pro
Ser Pro Asp Gly Phe Ser Pro Tyr Ser Pro Glu 290 295 300 gaa aca aac
cgc cgt gtt aac aaa gtg atg cgg gcc aga ctg tac tta 960 Glu Thr Asn
Arg Arg Val Asn Lys Val Met Arg Ala Arg Leu Tyr Leu 305 310 315 ctg
cag cag ata ggg cct aac tct ttc ctg att gga gga gac agc cca 1008
Leu Gln Gln Ile Gly Pro Asn Ser Phe Leu Ile Gly Gly Asp Ser Pro 320
325 330 gac aat aaa tac cgg gtg ttt att ggg cct cag aac tgc agc tgt
gca 1056 Asp Asn Lys Tyr Arg Val Phe Ile Gly Pro Gln Asn Cys Ser
Cys Ala 335 340 345 350 cgt gga aca ttc tgt att cat ctg cta ttt gtg
atg ctc cgg gtg ttt 1104 Arg Gly Thr Phe Cys Ile His Leu Leu Phe
Val Met Leu Arg Val Phe 355 360 365 caa cta gaa cct tca gac cca atg
tta tgg aga aaa act tta aag aat 1152 Gln Leu Glu Pro Ser Asp Pro
Met Leu Trp Arg Lys Thr Leu Lys Asn 370 375 380 ttt gag gtt gag agt
ttg ttc cag aaa tat cac agt agg cgt agc tca 1200 Phe Glu Val Glu
Ser Leu Phe Gln Lys Tyr His Ser Arg Arg Ser Ser 385 390 395 agg atc
aaa gct cca tct cgt aac acc atc cag aag ttt gtt tca cgc 1248 Arg
Ile Lys Ala Pro Ser Arg Asn Thr Ile Gln Lys Phe Val Ser Arg 400 405
410 atg tca aat tct cat aca ttg tca tca tct agt act tct acg tct agt
1296 Met Ser Asn Ser His Thr Leu Ser Ser Ser Ser Thr Ser Thr Ser
Ser 415 420 425 430 tca gaa aac agc ata aag gat gaa gag gaa cag atg
tgt cct att tgc 1344 Ser Glu Asn Ser Ile Lys Asp Glu Glu Glu Gln
Met Cys Pro Ile Cys 435 440 445 ttg ttg ggc atg ctt gat gaa gaa agt
ctt aca gtg tgt gaa gac ggc 1392 Leu Leu Gly Met Leu Asp Glu Glu
Ser Leu Thr Val Cys Glu Asp Gly 450 455 460 tgc agg aac aag ctg cac
cac cac tgc atg tca att tgg gca gaa gag 1440 Cys Arg Asn Lys Leu
His His His Cys Met Ser Ile Trp Ala Glu Glu 465 470 475 tgt aga aga
aat aga gaa cct tta ata tgt ccc ctt tgt aga tct aag 1488 Cys Arg
Arg Asn Arg Glu Pro Leu Ile Cys Pro Leu Cys Arg Ser Lys 480 485 490
tgg aga tct cat gat ttc tac agc cac gag ttg tca agt cct gtg gat
1536 Trp Arg Ser His Asp Phe Tyr Ser His Glu Leu Ser Ser Pro Val
Asp 495 500 505 510 tcc cct tct tcc ctc aga gct gca cag cag caa acc
gta cag cag cag 1584 Ser Pro Ser Ser Leu Arg Ala Ala Gln Gln Gln
Thr Val Gln Gln Gln 515 520 525 cct ttg gct gga tca cga agg aat caa
gag agc aat ttt aac ctt act 1632 Pro Leu Ala Gly Ser Arg Arg Asn
Gln Glu Ser Asn Phe Asn Leu Thr 530 535 540 cat tat gga act cag caa
atc cct cct gct tac aaa gat tta gct gag 1680 His Tyr Gly Thr Gln
Gln Ile Pro Pro Ala Tyr Lys Asp Leu Ala Glu 545 550 555 cca tgg att
cag gtg ttt gga atg gaa ctc gtt ggc tgc tta ttt tct 1728 Pro Trp
Ile Gln Val Phe Gly Met Glu Leu Val Gly Cys Leu Phe Ser 560 565 570
aga aac tgg aat gtg aga gag atg gcc ctc agg cgt ctt tcc cat gat
1776 Arg Asn Trp Asn Val Arg Glu Met Ala Leu Arg Arg Leu Ser His
Asp 575 580 585 590 gtc agt ggg gcc ctg ctg ttg gca aat ggg gag agc
act gga aat tct 1824 Val Ser Gly Ala Leu Leu Leu Ala Asn Gly Glu
Ser Thr Gly Asn Ser 595 600 605 ggg ggc agc agt gga agc agc ccg agt
ggg gga gcc acc agt ggg tct 1872 Gly Gly Ser Ser Gly Ser Ser Pro
Ser Gly Gly Ala Thr Ser Gly Ser 610 615 620 tcc cag acc agt atc tca
gga gat gtg gtg gag gca tgc tgc agc gtt 1920 Ser Gln Thr Ser Ile
Ser Gly Asp Val Val Glu Ala Cys Cys Ser Val 625 630 635 ctg tca atg
gtc tgt gct gac cct gtc tac aaa gtg tac gtt gct gct 1968 Leu Ser
Met Val Cys Ala Asp Pro Val Tyr Lys Val Tyr Val Ala Ala 640 645 650
tta aaa aca ttg aga gcc atg ctg gta tat act cct tgc cac agt tta
2016 Leu Lys Thr Leu Arg Ala Met Leu Val Tyr Thr Pro Cys His Ser
Leu 655 660 665 670 gcg gaa aga atc aaa ctt cag aga ctt ctc cag cca
gtt gta gac acc 2064 Ala Glu Arg Ile Lys Leu Gln Arg Leu Leu Gln
Pro Val Val Asp Thr 675 680 685 atc cta gtc aaa tgt gca gat gcc aat
agc cgc aca agt cag ctg tcc 2112 Ile Leu Val Lys Cys Ala Asp Ala
Asn Ser Arg Thr Ser Gln Leu Ser 690 695 700 ata tca aca ctg ttg gaa
ctg tgc aaa ggc caa gca gga gag ttg gca 2160 Ile Ser Thr Leu Leu
Glu Leu Cys Lys Gly Gln Ala Gly Glu Leu Ala 705 710 715 gtt ggc aga
gaa ata cta aaa gct gga tcc att ggt att ggt ggt gtt 2208 Val Gly
Arg Glu Ile Leu Lys Ala Gly Ser Ile Gly Ile Gly Gly Val 720 725 730
gat tat gtc tta aat tgt att ctt gga aac caa act gaa tca aac aat
2256 Asp Tyr Val Leu Asn Cys Ile Leu Gly Asn Gln Thr Glu Ser Asn
Asn 735 740 745 750 tgg caa gaa ctt ctt ggc cgc ctt tgt ctt ata gat
aga ctg ttg ttg 2304 Trp Gln Glu Leu Leu Gly Arg Leu Cys Leu Ile
Asp Arg Leu Leu Leu 755 760 765 gaa ttt cct gct gaa ttt tat cct cat
att gtc agt act gat gtt tca 2352 Glu Phe Pro Ala Glu Phe Tyr Pro
His Ile Val Ser Thr Asp Val Ser 770 775 780 caa gct gag cct gtt gaa
atc agg tat aag aag ctg ctg tcc ctc tta 2400 Gln Ala Glu Pro Val
Glu Ile Arg Tyr Lys Lys Leu Leu Ser Leu Leu 785 790 795 acc ttt gct
ttg cag tcc att gat aat tcc cac tca atg gtt ggc aaa 2448 Thr Phe
Ala Leu Gln Ser Ile Asp Asn Ser His Ser Met Val Gly Lys 800 805 810
ctt tcc aga agg atc tac ttg agt tct gca aga atg gtt act aca gta
2496 Leu Ser Arg Arg Ile Tyr Leu Ser Ser Ala Arg Met Val Thr Thr
Val 815 820 825 830 ccc cat gtg ttt tca aaa ctg tta gaa atg ctg agt
gtt tcc agt tcc 2544 Pro His Val Phe Ser Lys Leu Leu Glu Met Leu
Ser Val Ser Ser Ser 835 840 845 act cac ttc acc agg atg cgt cgc cgt
ttg atg gct att gca gat gag 2592 Thr His Phe Thr Arg Met Arg Arg
Arg Leu Met Ala Ile Ala Asp Glu 850 855 860 gtg gaa att gcc gaa gcc
atc cag ttg ggc gta gaa gac act ttg gat 2640 Val Glu Ile Ala Glu
Ala Ile Gln Leu Gly Val Glu Asp Thr Leu Asp 865 870 875 ggt caa cag
gac agc ttc ttg cag gca tct gtt ccc aac aac tat ctg 2688 Gly Gln
Gln Asp Ser Phe Leu Gln Ala Ser Val Pro Asn Asn Tyr Leu 880 885 890
gaa acc aca gag aac agt tcc cct gag tgc aca gtc cat tta gag aaa
2736 Glu Thr Thr Glu Asn Ser Ser Pro Glu Cys Thr Val His Leu Glu
Lys 895 900 905 910 act gga aaa gga tta tgt gct aca aaa ttg agt gcc
agt tca gag gac 2784 Thr Gly Lys Gly Leu Cys Ala Thr Lys Leu Ser
Ala Ser Ser Glu Asp 915 920 925 att tct gag aga ctg gcc agc att tca
gta gga cct tct agt tca aca 2832 Ile Ser Glu Arg Leu Ala Ser Ile
Ser Val Gly Pro Ser Ser Ser Thr 930 935 940 aca aca aca aca aca aca
aca gag caa cca aag cca atg gtt caa aca 2880 Thr Thr Thr Thr Thr
Thr Thr Glu Gln Pro Lys Pro Met Val Gln Thr 945 950 955 aaa ggc aga
ccc cac agt cag tgt ttg aac tcc tct cct tta tct cat 2928 Lys Gly
Arg Pro His Ser Gln Cys Leu Asn Ser Ser Pro Leu Ser His 960 965 970
cat tcc caa tta atg ttt cca gcc ttg tca acc cct tct tct tct acc
2976 His Ser Gln Leu Met Phe Pro Ala Leu Ser Thr Pro Ser Ser Ser
Thr 975 980 985 990 cca tct gta cca gct ggc act gca aca gat gtc tct
aag cat aga ctt 3024 Pro Ser Val Pro Ala Gly Thr Ala Thr Asp Val
Ser Lys His Arg Leu 995 1000 1005 cag gga ttc att ccc tgc aga ata
cct tct gca tct cct caa aca 3069 Gln Gly Phe Ile Pro Cys Arg Ile
Pro Ser Ala Ser Pro Gln Thr 1010 1015 1020 cag cgc aag ttt tct cta
caa ttc cac aga aac tgt cct gaa aac 3114 Gln Arg Lys Phe Ser Leu
Gln Phe His Arg Asn Cys Pro Glu Asn 1025 1030 1035 aaa gac tca gat
aaa ctt tcc cca gtc ttt act cag tca aga ccc 3159 Lys Asp Ser Asp
Lys Leu Ser Pro Val Phe Thr Gln Ser Arg Pro 1040 1045 1050 ttg ccc
tcc agt aac ata cac agg cca aag cca tct aga cct acc 3204 Leu Pro
Ser Ser Asn Ile His Arg Pro Lys Pro Ser Arg Pro Thr 1055 1060 1065
cca ggt aat aca agt aaa cag gga gat ccc tca aaa aat agc atg 3249
Pro Gly Asn Thr Ser Lys Gln Gly Asp Pro Ser Lys Asn Ser Met 1070
1075 1080 aca ctt gat ctg aac agt agt tcc aaa tgt gat gac agc ttt
ggc 3294 Thr Leu Asp Leu Asn Ser Ser Ser Lys Cys Asp Asp Ser Phe
Gly 1085 1090 1095 tgt agc agc aat agt agt aat gct gtt ata ccc agt
gac gag aca 3339 Cys Ser Ser Asn Ser Ser Asn Ala Val Ile Pro Ser
Asp Glu Thr 1100 1105 1110 gtg ttc acc cca gta gag gag aaa tgc aga
tta gat gtc aat aca 3384 Val Phe Thr Pro Val Glu Glu Lys Cys Arg
Leu Asp Val Asn Thr 1115 1120 1125 gag ctc aac tcc agt att gag gac
ctt ctt gaa gca tct atg cct 3429 Glu Leu Asn Ser Ser Ile Glu Asp
Leu Leu Glu Ala Ser Met Pro 1130 1135 1140 tca agt gat aca aca gta
act ttt aag tca gaa gtt gct gtc ctg 3474 Ser Ser Asp Thr Thr Val
Thr Phe Lys Ser Glu Val Ala Val Leu 1145 1150 1155 tct cct gaa aag
gct gaa aat gat gat acc tac aaa gat gat gtg 3519 Ser Pro Glu Lys
Ala Glu Asn Asp Asp Thr Tyr Lys Asp Asp Val 1160 1165 1170 aat cat
aat caa aag tgc aaa gag aag atg gaa gct gaa gaa gaa 3564 Asn His
Asn Gln Lys Cys Lys Glu Lys Met Glu Ala Glu Glu Glu 1175 1180 1185
gaa gct tta gca att gcc atg gca atg tca gcg tct cag gat gcc 3609
Glu Ala Leu Ala Ile Ala Met Ala Met Ser Ala Ser Gln Asp Ala 1190
1195 1200 ctc ccc ata gtt cct cag ctg cag gtt gaa aat gga gaa gat
atc 3654 Leu Pro Ile Val Pro Gln Leu Gln Val Glu Asn Gly Glu Asp
Ile 1205 1210 1215 atc att att caa cag gat aca cca gag act cta cca
gga cat acc 3699 Ile Ile Ile Gln Gln Asp Thr Pro Glu Thr Leu Pro
Gly His Thr 1220 1225 1230 aaa gca aaa caa ccg tat aga gaa gac act
gaa tgg ctg aaa ggt 3744 Lys Ala Lys Gln Pro Tyr Arg Glu Asp Thr
Glu Trp Leu Lys Gly 1235 1240 1245 caa cag ata ggc ctt gga gca ttt
tct tct tgt tat cag gct caa 3789 Gln Gln Ile Gly Leu Gly Ala Phe
Ser Ser Cys Tyr Gln Ala Gln 1250 1255 1260 gat gtg gga act gga act
tta atg gct gtt aaa cag gtg act tat 3834 Asp Val Gly Thr Gly Thr
Leu Met Ala Val Lys Gln Val Thr Tyr 1265 1270 1275 gtc aga aac aca
tct tct gag caa gaa gaa gta gta gaa gca cta 3879 Val Arg Asn Thr
Ser Ser Glu Gln Glu Glu Val Val Glu Ala Leu 1280 1285 1290 aga gaa
gag ata aga atg atg agc cat ctg aat cat cca aac atc 3924 Arg Glu
Glu Ile Arg Met Met Ser His Leu Asn His Pro Asn Ile 1295 1300 1305
att agg atg ttg gga gcc acg tgt gag aag agc aat tac aat ctc 3969
Ile Arg Met Leu Gly Ala Thr Cys Glu Lys Ser Asn Tyr Asn Leu 1310
1315 1320 ttc att gaa tgg atg gca ggg gga tcg gtg gct cat ttg ctg
agt 4014 Phe Ile Glu Trp Met Ala Gly Gly Ser Val Ala His Leu Leu
Ser 1325 1330 1335 aaa tat gga gcc ttc aaa gaa tca gta gtt att aac
tac act gaa 4059 Lys Tyr Gly Ala Phe Lys Glu Ser Val Val Ile Asn
Tyr Thr Glu 1340 1345 1350 cag tta ctc cgt ggc ctt tcg tat ctc cat
gaa aac caa atc att 4104 Gln Leu Leu Arg Gly Leu Ser Tyr Leu His
Glu Asn Gln Ile Ile 1355 1360 1365 cac aga gat gtc aaa ggt gcc aat
ttg cta att gac agc act ggt 4149 His Arg Asp Val Lys Gly Ala Asn
Leu Leu Ile Asp Ser Thr Gly 1370 1375 1380 cag aga cta aga att gca
gat ttt gga gct gca gcc agg ttg gca 4194 Gln Arg Leu Arg Ile Ala
Asp Phe Gly Ala Ala Ala Arg Leu Ala 1385 1390 1395 tca aaa gga act
ggt gca gga gag ttt cag gga caa tta ctg ggg 4239 Ser Lys Gly Thr
Gly Ala Gly Glu Phe Gln Gly Gln Leu Leu Gly 1400 1405 1410 aca att
gca ttt atg gca cct gag gta cta aga ggt caa cag tat 4284 Thr Ile
Ala Phe Met Ala Pro Glu Val Leu Arg Gly Gln Gln Tyr 1415 1420 1425
gga agg agc tgt gat gta tgg agt gtt ggc tgt gct att ata gaa 4329
Gly Arg Ser Cys Asp Val Trp Ser Val Gly Cys Ala Ile Ile Glu 1430
1435 1440 atg gct tgt gca aaa cca cca tgg aat gca gaa aaa cac tcc
aat 4374 Met Ala Cys Ala Lys Pro Pro Trp Asn Ala Glu Lys His Ser
Asn 1445 1450 1455 cat ctt gct ttg ata ttt aag att gct agt gca act
act gct cca 4419 His Leu Ala Leu Ile Phe Lys Ile Ala Ser Ala Thr
Thr Ala Pro 1460 1465 1470 tcg atc cct tca cat ttg tct cct ggt tta
cga gat gtg gct ctt 4464 Ser Ile Pro Ser His Leu Ser Pro Gly Leu
Arg
Asp Val Ala Leu 1475 1480 1485 cgt tgt tta gaa ctt caa cct cag gac
aga cct cca tca aga gag 4509 Arg Cys Leu Glu Leu Gln Pro Gln Asp
Arg Pro Pro Ser Arg Glu 1490 1495 1500 cta ctg aag cat cca gtc ttt
cgt act aca tgg tag ccaattatgc 4555 Leu Leu Lys His Pro Val Phe Arg
Thr Thr Trp 1505 1510 agatcaacta cagtagaaac aggatgctca acaagagaaa
aaaaacttgt ggggaaccac 4615 attgatattc tactggccat gatgccactg
aacagctatg aacgaggcca gtggggaacc 4675 cttacctaag tatgtgattg
acaaatcatg atctgtacct aagctcagta tgcaaaagcc 4735 caaactagtg
cagaaactgt aaactgtgcc tttcaaagaa ctggccctag gtgaacagga 4795
aaacaatgaa gtttgcatga ctaaattgca gaagcataat tttatttttt tggagcactt
4855 tttcagcaat attagcggct gaggggctca ggatctattt taatatttca
attattcttc 4915 catttcatat agtgatcaca agcagggggt tctgcaattc
cgttcaaatt ttttgtcact 4975 ggctataaaa tcagtatctg cctcttttag
gtcagagtat gctatgagta gcaatacata 5035 catatatttt taaaagttga
tacttcttta tgacccacag ttgaccttta ttttcttaaa 5095 taccagggca
gttgtggctc attgtgcatt ttactgttgg cccattcatt tcgtttttgg 5155
aaattatggt tttgtatttt catgtttatt tacattcatt tttgtttatt cagggaaagc
5215 tgatcttttt tttcaaacca aaaaaaaaaa 5245 2 1512 PRT Human 2 Met
Ala Ala Ala Ala Gly Asn Arg Ala Ser Ser Ser Gly Phe Pro Gly 1 5 10
15 Ala Arg Ala Thr Ser Pro Glu Ala Gly Gly Gly Gly Gly Ala Leu Lys
20 25 30 Ala Ser Ser Ala Pro Ala Ala Ala Ala Gly Leu Leu Arg Glu
Ala Gly 35 40 45 Ser Gly Gly Arg Glu Arg Ala Asp Trp Arg Arg Arg
Gln Leu Arg Lys 50 55 60 Val Arg Ser Val Glu Leu Asp Gln Leu Pro
Glu Gln Pro Leu Phe Leu 65 70 75 80 Ala Ala Ser Pro Pro Ala Ser Ser
Thr Ser Pro Ser Pro Glu Pro Ala 85 90 95 Asp Ala Ala Gly Ser Gly
Thr Gly Phe Gln Pro Val Ala Val Pro Pro 100 105 110 Pro His Gly Ala
Ala Ser Arg Gly Gly Ala His Leu Thr Glu Ser Val 115 120 125 Ala Ala
Pro Asp Ser Gly Ala Ser Ser Pro Ala Ala Ala Glu Pro Gly 130 135 140
Glu Lys Arg Ala Pro Ala Ala Glu Pro Ser Pro Ala Ala Ala Pro Ala 145
150 155 160 Gly Arg Glu Met Glu Asn Lys Glu Thr Leu Lys Gly Leu His
Lys Met 165 170 175 Asp Asp Arg Pro Glu Glu Arg Met Ile Arg Glu Lys
Leu Lys Ala Thr 180 185 190 Cys Met Pro Ala Trp Lys His Glu Trp Leu
Glu Arg Arg Asn Arg Arg 195 200 205 Gly Pro Val Val Val Lys Pro Ile
Pro Val Lys Gly Asp Gly Ser Glu 210 215 220 Met Asn His Leu Ala Ala
Glu Ser Pro Gly Glu Val Gln Ala Ser Ala 225 230 235 240 Ala Ser Pro
Ala Ser Lys Gly Arg Arg Ser Pro Ser Pro Gly Asn Ser 245 250 255 Pro
Ser Gly Arg Thr Val Lys Ser Glu Ser Pro Gly Val Arg Arg Lys 260 265
270 Arg Val Ser Pro Val Pro Phe Gln Ser Gly Arg Ile Thr Pro Pro Arg
275 280 285 Arg Ala Pro Ser Pro Asp Gly Phe Ser Pro Tyr Ser Pro Glu
Glu Thr 290 295 300 Asn Arg Arg Val Asn Lys Val Met Arg Ala Arg Leu
Tyr Leu Leu Gln 305 310 315 320 Gln Ile Gly Pro Asn Ser Phe Leu Ile
Gly Gly Asp Ser Pro Asp Asn 325 330 335 Lys Tyr Arg Val Phe Ile Gly
Pro Gln Asn Cys Ser Cys Ala Arg Gly 340 345 350 Thr Phe Cys Ile His
Leu Leu Phe Val Met Leu Arg Val Phe Gln Leu 355 360 365 Glu Pro Ser
Asp Pro Met Leu Trp Arg Lys Thr Leu Lys Asn Phe Glu 370 375 380 Val
Glu Ser Leu Phe Gln Lys Tyr His Ser Arg Arg Ser Ser Arg Ile 385 390
395 400 Lys Ala Pro Ser Arg Asn Thr Ile Gln Lys Phe Val Ser Arg Met
Ser 405 410 415 Asn Ser His Thr Leu Ser Ser Ser Ser Thr Ser Thr Ser
Ser Ser Glu 420 425 430 Asn Ser Ile Lys Asp Glu Glu Glu Gln Met Cys
Pro Ile Cys Leu Leu 435 440 445 Gly Met Leu Asp Glu Glu Ser Leu Thr
Val Cys Glu Asp Gly Cys Arg 450 455 460 Asn Lys Leu His His His Cys
Met Ser Ile Trp Ala Glu Glu Cys Arg 465 470 475 480 Arg Asn Arg Glu
Pro Leu Ile Cys Pro Leu Cys Arg Ser Lys Trp Arg 485 490 495 Ser His
Asp Phe Tyr Ser His Glu Leu Ser Ser Pro Val Asp Ser Pro 500 505 510
Ser Ser Leu Arg Ala Ala Gln Gln Gln Thr Val Gln Gln Gln Pro Leu 515
520 525 Ala Gly Ser Arg Arg Asn Gln Glu Ser Asn Phe Asn Leu Thr His
Tyr 530 535 540 Gly Thr Gln Gln Ile Pro Pro Ala Tyr Lys Asp Leu Ala
Glu Pro Trp 545 550 555 560 Ile Gln Val Phe Gly Met Glu Leu Val Gly
Cys Leu Phe Ser Arg Asn 565 570 575 Trp Asn Val Arg Glu Met Ala Leu
Arg Arg Leu Ser His Asp Val Ser 580 585 590 Gly Ala Leu Leu Leu Ala
Asn Gly Glu Ser Thr Gly Asn Ser Gly Gly 595 600 605 Ser Ser Gly Ser
Ser Pro Ser Gly Gly Ala Thr Ser Gly Ser Ser Gln 610 615 620 Thr Ser
Ile Ser Gly Asp Val Val Glu Ala Cys Cys Ser Val Leu Ser 625 630 635
640 Met Val Cys Ala Asp Pro Val Tyr Lys Val Tyr Val Ala Ala Leu Lys
645 650 655 Thr Leu Arg Ala Met Leu Val Tyr Thr Pro Cys His Ser Leu
Ala Glu 660 665 670 Arg Ile Lys Leu Gln Arg Leu Leu Gln Pro Val Val
Asp Thr Ile Leu 675 680 685 Val Lys Cys Ala Asp Ala Asn Ser Arg Thr
Ser Gln Leu Ser Ile Ser 690 695 700 Thr Leu Leu Glu Leu Cys Lys Gly
Gln Ala Gly Glu Leu Ala Val Gly 705 710 715 720 Arg Glu Ile Leu Lys
Ala Gly Ser Ile Gly Ile Gly Gly Val Asp Tyr 725 730 735 Val Leu Asn
Cys Ile Leu Gly Asn Gln Thr Glu Ser Asn Asn Trp Gln 740 745 750 Glu
Leu Leu Gly Arg Leu Cys Leu Ile Asp Arg Leu Leu Leu Glu Phe 755 760
765 Pro Ala Glu Phe Tyr Pro His Ile Val Ser Thr Asp Val Ser Gln Ala
770 775 780 Glu Pro Val Glu Ile Arg Tyr Lys Lys Leu Leu Ser Leu Leu
Thr Phe 785 790 795 800 Ala Leu Gln Ser Ile Asp Asn Ser His Ser Met
Val Gly Lys Leu Ser 805 810 815 Arg Arg Ile Tyr Leu Ser Ser Ala Arg
Met Val Thr Thr Val Pro His 820 825 830 Val Phe Ser Lys Leu Leu Glu
Met Leu Ser Val Ser Ser Ser Thr His 835 840 845 Phe Thr Arg Met Arg
Arg Arg Leu Met Ala Ile Ala Asp Glu Val Glu 850 855 860 Ile Ala Glu
Ala Ile Gln Leu Gly Val Glu Asp Thr Leu Asp Gly Gln 865 870 875 880
Gln Asp Ser Phe Leu Gln Ala Ser Val Pro Asn Asn Tyr Leu Glu Thr 885
890 895 Thr Glu Asn Ser Ser Pro Glu Cys Thr Val His Leu Glu Lys Thr
Gly 900 905 910 Lys Gly Leu Cys Ala Thr Lys Leu Ser Ala Ser Ser Glu
Asp Ile Ser 915 920 925 Glu Arg Leu Ala Ser Ile Ser Val Gly Pro Ser
Ser Ser Thr Thr Thr 930 935 940 Thr Thr Thr Thr Thr Glu Gln Pro Lys
Pro Met Val Gln Thr Lys Gly 945 950 955 960 Arg Pro His Ser Gln Cys
Leu Asn Ser Ser Pro Leu Ser His His Ser 965 970 975 Gln Leu Met Phe
Pro Ala Leu Ser Thr Pro Ser Ser Ser Thr Pro Ser 980 985 990 Val Pro
Ala Gly Thr Ala Thr Asp Val Ser Lys His Arg Leu Gln Gly 995 1000
1005 Phe Ile Pro Cys Arg Ile Pro Ser Ala Ser Pro Gln Thr Gln Arg
1010 1015 1020 Lys Phe Ser Leu Gln Phe His Arg Asn Cys Pro Glu Asn
Lys Asp 1025 1030 1035 Ser Asp Lys Leu Ser Pro Val Phe Thr Gln Ser
Arg Pro Leu Pro 1040 1045 1050 Ser Ser Asn Ile His Arg Pro Lys Pro
Ser Arg Pro Thr Pro Gly 1055 1060 1065 Asn Thr Ser Lys Gln Gly Asp
Pro Ser Lys Asn Ser Met Thr Leu 1070 1075 1080 Asp Leu Asn Ser Ser
Ser Lys Cys Asp Asp Ser Phe Gly Cys Ser 1085 1090 1095 Ser Asn Ser
Ser Asn Ala Val Ile Pro Ser Asp Glu Thr Val Phe 1100 1105 1110 Thr
Pro Val Glu Glu Lys Cys Arg Leu Asp Val Asn Thr Glu Leu 1115 1120
1125 Asn Ser Ser Ile Glu Asp Leu Leu Glu Ala Ser Met Pro Ser Ser
1130 1135 1140 Asp Thr Thr Val Thr Phe Lys Ser Glu Val Ala Val Leu
Ser Pro 1145 1150 1155 Glu Lys Ala Glu Asn Asp Asp Thr Tyr Lys Asp
Asp Val Asn His 1160 1165 1170 Asn Gln Lys Cys Lys Glu Lys Met Glu
Ala Glu Glu Glu Glu Ala 1175 1180 1185 Leu Ala Ile Ala Met Ala Met
Ser Ala Ser Gln Asp Ala Leu Pro 1190 1195 1200 Ile Val Pro Gln Leu
Gln Val Glu Asn Gly Glu Asp Ile Ile Ile 1205 1210 1215 Ile Gln Gln
Asp Thr Pro Glu Thr Leu Pro Gly His Thr Lys Ala 1220 1225 1230 Lys
Gln Pro Tyr Arg Glu Asp Thr Glu Trp Leu Lys Gly Gln Gln 1235 1240
1245 Ile Gly Leu Gly Ala Phe Ser Ser Cys Tyr Gln Ala Gln Asp Val
1250 1255 1260 Gly Thr Gly Thr Leu Met Ala Val Lys Gln Val Thr Tyr
Val Arg 1265 1270 1275 Asn Thr Ser Ser Glu Gln Glu Glu Val Val Glu
Ala Leu Arg Glu 1280 1285 1290 Glu Ile Arg Met Met Ser His Leu Asn
His Pro Asn Ile Ile Arg 1295 1300 1305 Met Leu Gly Ala Thr Cys Glu
Lys Ser Asn Tyr Asn Leu Phe Ile 1310 1315 1320 Glu Trp Met Ala Gly
Gly Ser Val Ala His Leu Leu Ser Lys Tyr 1325 1330 1335 Gly Ala Phe
Lys Glu Ser Val Val Ile Asn Tyr Thr Glu Gln Leu 1340 1345 1350 Leu
Arg Gly Leu Ser Tyr Leu His Glu Asn Gln Ile Ile His Arg 1355 1360
1365 Asp Val Lys Gly Ala Asn Leu Leu Ile Asp Ser Thr Gly Gln Arg
1370 1375 1380 Leu Arg Ile Ala Asp Phe Gly Ala Ala Ala Arg Leu Ala
Ser Lys 1385 1390 1395 Gly Thr Gly Ala Gly Glu Phe Gln Gly Gln Leu
Leu Gly Thr Ile 1400 1405 1410 Ala Phe Met Ala Pro Glu Val Leu Arg
Gly Gln Gln Tyr Gly Arg 1415 1420 1425 Ser Cys Asp Val Trp Ser Val
Gly Cys Ala Ile Ile Glu Met Ala 1430 1435 1440 Cys Ala Lys Pro Pro
Trp Asn Ala Glu Lys His Ser Asn His Leu 1445 1450 1455 Ala Leu Ile
Phe Lys Ile Ala Ser Ala Thr Thr Ala Pro Ser Ile 1460 1465 1470 Pro
Ser His Leu Ser Pro Gly Leu Arg Asp Val Ala Leu Arg Cys 1475 1480
1485 Leu Glu Leu Gln Pro Gln Asp Arg Pro Pro Ser Arg Glu Leu Leu
1490 1495 1500 Lys His Pro Val Phe Arg Thr Thr Trp 1505 1510 3 1495
PRT partial human MEKK1 protein 3 Pro Ser Pro Glu Ala Gly Gly Gly
Gly Gly Ala Leu Lys Ala Ser Ser 1 5 10 15 Ala Arg Ala Ala Ala Ala
Gly Leu Leu Arg Glu Ala Gly Ser Gly Gly 20 25 30 Arg Glu Arg Ala
Asp Trp Arg Arg Arg Gln Leu Arg Lys Val Arg Ser 35 40 45 Val Glu
Leu Asp Gln Leu Pro Glu Gln Pro Leu Phe Leu Ala Ala Ser 50 55 60
Pro Pro Ala Ser Ser Thr Ser Pro Ser Pro Glu Pro Ala Asp Ala Ala 65
70 75 80 Gly Ser Gly Thr Gly Phe Gln Pro Val Ala Val Pro Pro Pro
His Gly 85 90 95 Ala Ala Ser Arg Arg Gly Ala His Leu Thr Glu Ser
Val Ala Ala Pro 100 105 110 Asp Ser Gly Ala Ser Ser Pro Ala Ala Ala
Glu Pro Gly Glu Lys Arg 115 120 125 Ala Pro Ala Ala Glu Pro Ser Pro
Ala Ala Ala Pro Ala Gly Arg Glu 130 135 140 Met Glu Asn Lys Glu Thr
Leu Lys Gly Leu His Lys Met Asp Asp Arg 145 150 155 160 Pro Glu Glu
Arg Met Ile Arg Glu Lys Leu Lys Ala Thr Cys Met Pro 165 170 175 Ala
Trp Lys His Glu Trp Leu Glu Arg Arg Asn Arg Arg Gly Pro Val 180 185
190 Val Val Lys Pro Ile Pro Val Lys Gly Asp Gly Ser Glu Met Asn His
195 200 205 Leu Ala Ala Glu Ser Pro Gly Glu Val Gln Ala Ser Ala Ala
Ser Pro 210 215 220 Ala Ser Lys Gly Arg Arg Ser Pro Ser Pro Gly Asn
Ser Pro Ser Gly 225 230 235 240 Arg Thr Val Lys Ser Glu Ser Pro Gly
Val Arg Arg Lys Arg Val Ser 245 250 255 Pro Val Pro Phe Gln Ser Gly
Arg Ile Thr Pro Pro Arg Arg Ala Pro 260 265 270 Ser Pro Asp Gly Phe
Ser Pro Tyr Ser Pro Glu Glu Thr Asn Arg Arg 275 280 285 Val Asn Lys
Val Met Arg Ala Arg Leu Tyr Leu Leu Gln Gln Ile Gly 290 295 300 Pro
Asn Ser Phe Leu Ile Gly Gly Asp Ser Pro Asp Asn Lys Tyr Arg 305 310
315 320 Val Phe Ile Gly Pro Gln Asn Cys Ser Cys Ala His Gly Thr Phe
Cys 325 330 335 Ile His Leu Leu Phe Val Met Leu Arg Val Phe Gln Leu
Glu Pro Ser 340 345 350 Asp Pro Met Leu Trp Arg Lys Thr Leu Lys Asn
Phe Glu Val Glu Ser 355 360 365 Leu Phe Gln Lys Tyr His Ser Arg Arg
Ser Ser Arg Ile Lys Ala Pro 370 375 380 Ser Arg Asn Thr Ile Gln Lys
Phe Val Ser Arg Met Ser Asn Ser His 385 390 395 400 Thr Leu Ser Ser
Ser Ser Thr Ser Thr Ser Ser Ser Glu Asn Ser Ile 405 410 415 Lys Asp
Glu Glu Glu Gln Met Cys Pro Ile Cys Leu Leu Gly Met Leu 420 425 430
Asp Glu Glu Ser Leu Thr Val Cys Glu Asp Gly Cys Arg Asn Lys Leu 435
440 445 His His His Cys Met Ser Ile Trp Ala Glu Glu Cys Arg Arg Asn
Arg 450 455 460 Glu Pro Leu Ile Cys Pro Leu Cys Arg Ser Lys Trp Arg
Ser His Asp 465 470 475 480 Phe Tyr Ser His Glu Leu Ser Ser Pro Val
Asp Ser Pro Ser Ser Leu 485 490 495 Arg Ala Ala Gln Gln Gln Thr Val
Gln Gln Gln Pro Leu Ala Gly Ser 500 505 510 Arg Arg Asn Gln Glu Ser
Asn Phe Asn Leu Thr His Tyr Gly Thr Gln 515 520 525 Gln Ile Pro Pro
Ala Tyr Lys Asp Leu Ala Glu Pro Trp Ile Gln Val 530 535 540 Phe Gly
Met Glu Leu Val Gly Cys Leu Phe Ser Arg Asn Trp Asn Val 545 550 555
560 Arg Glu Met Ala Leu Arg Arg Leu Ser His Asp Val Ser Gly Ala Leu
565 570 575 Leu Leu Ala Asn Gly Glu Ser Thr Gly Asn Ser Gly Gly Ser
Ser Gly 580 585 590 Ser Ser Pro Ser Gly Gly Ala Thr Ser Gly Ser Ser
Gln Thr Ser Ile 595 600 605 Ser Gly Asp Val Val Glu Ala Cys Cys Ser
Val Leu Ser Met Val Cys 610 615 620 Ala Asp Pro Val Tyr Lys Val Tyr
Val Ala Ala Leu Lys Thr Leu Arg 625 630 635 640 Ala Met Leu Val Tyr
Thr Pro Cys His Ser Leu Ala Glu Arg Ile Lys 645 650 655 Leu Gln Arg
Leu Leu Gln Pro Val Val Asp Thr Ile Leu Val Lys Cys 660 665 670 Ala
Asp Ala Asn Ser Arg Thr Ser Gln Leu Ser Ile Ser Thr Leu Leu 675 680
685 Glu Leu Cys Lys Gly Gln Ala Gly Glu Leu Ala Val Gly Arg Glu Ile
690 695 700 Leu Lys Ala Gly Ser Ile Gly Ile Gly Gly Val Asp Tyr Val
Leu Asn 705 710 715 720 Cys Ile Leu Gly Asn Gln Thr Glu Ser Asn Asn
Trp Gln Glu Leu Leu 725 730 735 Gly Arg Leu Cys Leu Ile Asp Arg Leu
Leu Leu Glu Phe Pro Ala Glu
740 745 750 Phe Tyr Pro His Ile Val Ser Thr Asp Val Ser Gln Ala Glu
Pro Val 755 760 765 Glu Ile Arg Tyr Lys Lys Leu Leu Ser Leu Leu Thr
Phe Ala Leu Gln 770 775 780 Ser Ile Asp Asn Ser His Ser Met Val Gly
Lys Leu Ser Arg Arg Ile 785 790 795 800 Tyr Leu Ser Ser Ala Arg Met
Val Thr Thr Val Pro His Val Phe Ser 805 810 815 Lys Leu Leu Glu Met
Leu Ser Val Ser Ser Val Ser Thr His Phe Thr 820 825 830 Arg Met Arg
Arg Arg Leu Met Ala Tyr Ala Asp Glu Val Glu Ile Ala 835 840 845 Glu
Ala Ile Gln Leu Gly Val Glu Asp Thr Leu Gln Arg Gln Gln His 850 855
860 Asn Ser Phe Cys Arg His Leu Phe Pro Thr Thr Ile Trp Lys Pro Gln
865 870 875 880 Arg Thr Val Pro Leu Glu Cys Thr Val His Leu Glu Lys
Thr Gly Lys 885 890 895 Gly Leu Cys Ala Thr Lys Leu Ser Ala Ser Ser
Glu Asp Ile Ser Glu 900 905 910 Arg Leu Ala Arg Ile Ser Val Gly Pro
Ser Ser Ser Thr Thr Thr Thr 915 920 925 Thr Thr Thr Thr Glu Gln Pro
Lys Pro Met Val Gln Thr Lys Gly Arg 930 935 940 Pro His Ser Gln Cys
Leu Asn Ser Ser Pro Leu Ser His His Ser Gln 945 950 955 960 Leu Met
Phe Pro Ala Leu Ser Thr Pro Ser Ser Ser Thr Pro Ser Val 965 970 975
Pro Ala Gly Thr Ala Thr Asp Val Ser Lys His Arg Leu Gln Gly Phe 980
985 990 Ile Pro Cys Arg Ile Pro Ser Ala Ser Pro Gln Thr Gln Arg Lys
Phe 995 1000 1005 Ser Leu Gln Phe His Arg Asn Cys Pro Glu Asn Lys
Asp Ser Asp 1010 1015 1020 Lys Leu Ser Pro Val Phe Thr Gln Ser Arg
Pro Leu Pro Ser Ser 1025 1030 1035 Asn Ile His Arg Pro Lys Pro Ser
Arg Pro Thr Pro Gly Asn Thr 1040 1045 1050 Ser Lys Gln Gly Asp Pro
Ser Lys Asn Ser Met Thr Leu Asp Leu 1055 1060 1065 Asn Ser Ser Ser
Lys Cys Asp Asp Ser Phe Gly Leu Ser Ser Asn 1070 1075 1080 Ser Ser
Asn Cys Cys Tyr Thr Ser Asp Glu Thr Val Phe Thr Pro 1085 1090 1095
Val Glu Glu Lys Cys Arg Leu Asp Val Asn Thr Glu Leu Asn Ser 1100
1105 1110 Ser Ile Glu Asp Leu Leu Glu Ala Ser Met Pro Ser Ser Asp
Thr 1115 1120 1125 Thr Val Thr Phe Lys Ser Glu Val Ala Val Leu Ser
Pro Glu Lys 1130 1135 1140 Ala Glu Asn Asp Asp Thr Tyr Lys Asp Asp
Val Asn His Asn Gln 1145 1150 1155 Lys Cys Lys Glu Lys Met Glu Ala
Glu Glu Glu Glu Ala Leu Ala 1160 1165 1170 Ile Ala Met Ala Met Ser
Ala Ser Gln Val Ala Leu Pro Ile Val 1175 1180 1185 Pro Gln Leu Gln
Val Glu Asn Gly Glu Asp Ile Ile Ile Ile Gln 1190 1195 1200 Gln Asp
Thr Pro Glu Thr Leu Pro Gly His Thr Lys Ala Lys Gln 1205 1210 1215
Pro Tyr Arg Glu Asp Thr Glu Trp Leu Lys Gly Gln Gln Ile Gly 1220
1225 1230 Leu Gly Ala Phe Ser Ser Cys Tyr Gln Ala Gln Asp Val Gly
Thr 1235 1240 1245 Gly Thr Leu Met Ala Val Lys Gln Val Thr Tyr Val
Arg Asn Thr 1250 1255 1260 Ser Ser Glu Gln Glu Glu Val Val Glu Ala
Leu Arg Glu Glu Ile 1265 1270 1275 Arg Met Met Ser His Leu Asn His
Pro Asn Ile Ile Arg Met Leu 1280 1285 1290 Gly Ala Thr Cys Glu Lys
Ser Asn Tyr Asn Leu Phe Ile Glu Trp 1295 1300 1305 Met Ala Gly Gly
Ser Val Ala His Leu Leu Ser Lys Tyr Gly Ala 1310 1315 1320 Phe Lys
Glu Ser Val Val Ile Asn Tyr Thr Glu Gln Leu Leu Arg 1325 1330 1335
Gly Leu Ser Tyr Leu His Glu Asn Gln Ile Ile His Arg Asp Val 1340
1345 1350 Lys Gly Ala Asn Leu Leu Ile Asp Ser Thr Gly Gln Arg Leu
Arg 1355 1360 1365 Ile Ala Asp Phe Gly Ala Ala Ala Arg Leu Ala Ser
Lys Gly Thr 1370 1375 1380 Gly Ala Gly Glu Phe Gln Gly Gln Leu Leu
Gly Thr Ile Ala Phe 1385 1390 1395 Met Ala Pro Glu Val Leu Arg Gly
Gln Gln Tyr Gly Arg Ser Cys 1400 1405 1410 Asp Val Trp Ser Val Gly
Cys Ala Ile Ile Glu Met Ala Cys Ala 1415 1420 1425 Lys Pro Pro Trp
Asn Ala Glu Lys His Ser Asn His Leu Ala Leu 1430 1435 1440 Ile Phe
Lys Ile Ala Ser Ala Thr Thr Ala Pro Ser Ile Pro Ser 1445 1450 1455
His Leu Ser Pro Gly Leu Arg Asp Val Ala Leu Arg Cys Leu Glu 1460
1465 1470 Leu Gln Pro Gln Asp Arg Pro Pro Ser Arg Glu Leu Leu Lys
His 1475 1480 1485 Pro Val Phe Arg Thr Thr Trp 1490 1495 4 1492 PRT
rat MEKK1 4 Met Ala Ala Ala Ala Gly Asp Arg Ala Ser Ser Ser Gly Phe
Pro Gly 1 5 10 15 Ala Ala Ala Ala Ser Pro Glu Ala Gly Gly Gly Gly
Gly Ala Leu Gln 20 25 30 Gly Ser Gly Ala Pro Ala Ala Gly Ala Gly
Leu Leu Arg Glu Thr Gly 35 40 45 Ser Ala Gly Arg Glu Arg Ala Asp
Trp Arg Arg Gln Gln Leu Arg Lys 50 55 60 Val Arg Ser Val Glu Leu
Asp Gln Leu Pro Glu Gln Pro Leu Phe Leu 65 70 75 80 Thr Ala Ser Pro
Pro Cys Pro Ser Thr Ser Pro Ser Pro Glu Pro Ala 85 90 95 Asp Ala
Ala Ala Gly Ala Ser Gly Phe Gln Pro Ala Ala Gly Pro Pro 100 105 110
Pro Pro Gly Ala Ala Ser Arg Cys Gly Ser His Ser Ala Glu Leu Ala 115
120 125 Ala Ala Arg Asp Ser Gly Ala Arg Ser Pro Ala Gly Ala Glu Pro
Pro 130 135 140 Ser Ala Ala Ala Pro Ser Gly Arg Glu Met Glu Asn Lys
Glu Thr Leu 145 150 155 160 Lys Gly Leu His Lys Met Asp Asp Arg Pro
Glu Glu Arg Met Ile Arg 165 170 175 Glu Lys Leu Lys Ala Thr Cys Met
Pro Ala Trp Lys His Glu Trp Leu 180 185 190 Glu Arg Arg Asn Arg Arg
Gly Pro Val Val Val Lys Pro Ile Pro Ile 195 200 205 Lys Gly Asp Gly
Ser Glu Met Ser Asn Leu Ala Ala Glu Leu Gln Gly 210 215 220 Glu Gly
Gln Ala Gly Ser Ala Ala Pro Ala Pro Lys Gly Arg Arg Ser 225 230 235
240 Pro Ser Pro Gly Ser Ser Pro Ser Gly Arg Ser Gly Lys Pro Glu Ser
245 250 255 Pro Gly Val Arg Arg Lys Arg Val Ser Pro Val Pro Phe Gln
Ser Gly 260 265 270 Arg Ile Thr Pro Pro Arg Arg Ala Pro Ser Pro Asp
Gly Phe Ser Pro 275 280 285 Ser Pro Glu Glu Thr Ser Arg Arg Val Asn
Lys Val Met Arg Ala Arg 290 295 300 Leu Tyr Leu Leu Gln Gln Ile Gly
Pro Asn Ser Phe Leu Ile Gly Gly 305 310 315 320 Asp Ser Pro Asp Asn
Lys Tyr Arg Val Phe Ile Gly Pro Gln Asn Cys 325 330 335 Ser Cys Gly
Arg Gly Thr Phe Cys Ile His Leu Leu Phe Val Met Leu 340 345 350 Arg
Val Phe Gln Leu Glu Pro Ser Asp Pro Met Leu Trp Arg Lys Thr 355 360
365 Leu Lys Asn Phe Glu Val Glu Ser Leu Phe Gln Lys Tyr His Ser Arg
370 375 380 Arg Ser Ser Arg Ile Lys Ala Pro Ser Arg Asn Thr Ile Gln
Lys Phe 385 390 395 400 Val Ser Arg Met Ser Asn Cys His Thr Leu Ser
Ser Ser Ser Thr Ser 405 410 415 Thr Ser Ser Ser Glu Asn Ser Ile Lys
Asp Glu Glu Glu Gln Met Cys 420 425 430 Pro Ile Cys Leu Leu Gly Met
Leu Asp Glu Glu Ser Leu Thr Val Cys 435 440 445 Glu Asp Gly Cys Arg
Asn Lys Leu His His His Cys Met Ser Ile Trp 450 455 460 Ala Glu Glu
Cys Arg Arg Asn Arg Glu Pro Leu Ile Cys Pro Leu Cys 465 470 475 480
Arg Ser Lys Trp Arg Ser His Asp Phe Tyr Ser His Glu Leu Ser Ser 485
490 495 Pro Val Asp Ser Pro Thr Ser Leu Arg Gly Val Gln Gln Pro Ser
Ser 500 505 510 Pro Gln Gln Pro Val Ala Gly Ser Gln Arg Arg Asn Gln
Glu Ser Asn 515 520 525 Phe Asn Leu Thr His Tyr Gly Thr Gln Gln Ile
Pro Pro Ala Tyr Lys 530 535 540 Asp Leu Ala Glu Pro Trp Ile Gln Ala
Phe Gly Met Glu Leu Val Gly 545 550 555 560 Cys Leu Phe Ser Arg Asn
Trp Asn Val Arg Glu Met Ala Leu Arg Arg 565 570 575 Leu Ser His Asp
Val Ser Gly Ala Leu Leu Leu Ala Asn Gly Glu Ser 580 585 590 Thr Gly
Thr Ser Gly Gly Gly Ser Gly Gly Ser Leu Ser Ala Gly Ala 595 600 605
Ala Ser Gly Ser Ser Gln Pro Ser Ile Ser Gly Asp Val Val Glu Ala 610
615 620 Phe Cys Ser Val Leu Ser Ile Val Cys Ala Asp Pro Val Tyr Lys
Val 625 630 635 640 Tyr Val Ala Ala Leu Lys Thr Leu Arg Ala Met Leu
Val Tyr Thr Pro 645 650 655 Cys His Ser Leu Ala Glu Arg Ile Lys Leu
Gln Arg Leu Leu Arg Pro 660 665 670 Val Val Asp Thr Ile Leu Val Lys
Cys Ala Asp Ala Asn Ser Arg Thr 675 680 685 Ser Gln Leu Ser Ile Ser
Thr Leu Leu Glu Leu Cys Lys Gly Gln Ala 690 695 700 Gly Glu Leu Ala
Val Gly Arg Glu Ile Leu Lys Ala Gly Ser Ile Gly 705 710 715 720 Val
Gly Gly Val Asp Tyr Val Leu Ser Cys Ile Leu Gly Asn Gln Ala 725 730
735 Glu Ser Asn Asn Trp Gln Glu Leu Leu Gly Arg Leu Cys Leu Ile Asp
740 745 750 Arg Leu Leu Leu Glu Ile Ser Ala Glu Phe Tyr Pro His Ile
Val Ser 755 760 765 Thr Asp Val Ser Gln Ala Glu Pro Val Glu Ile Arg
Tyr Lys Lys Leu 770 775 780 Leu Ser Leu Leu Ala Phe Ala Leu Gln Ser
Ile Asp Asn Ser His Ser 785 790 795 800 Met Val Gly Lys Leu Ser Arg
Arg Ile Tyr Leu Ser Ser Ala Arg Met 805 810 815 Val Thr Thr Val Pro
Pro Leu Phe Ser Lys Leu Val Thr Met Leu Ser 820 825 830 Ala Ser Gly
Ser Ser His Phe Ala Arg Met Arg Arg Arg Leu Met Ala 835 840 845 Ile
Ala Asp Glu Val Glu Ile Ala Glu Val Ile Gln Leu Gly Ser Glu 850 855
860 Asp Thr Leu Asp Gly Gln Gln Asp Ser Ser Gln Ala Leu Ala Pro Pro
865 870 875 880 Arg Tyr Pro Glu Ser Ser Ser Leu Glu His Thr Ala His
Val Glu Lys 885 890 895 Thr Gly Lys Gly Leu Lys Ala Thr Arg Leu Ser
Ala Ser Ser Glu Asp 900 905 910 Ile Ser Asp Arg Leu Ala Gly Val Ser
Val Gly Leu Pro Ser Ser Ala 915 920 925 Thr Thr Glu Gln Pro Lys Pro
Thr Val Gln Thr Lys Gly Arg Pro His 930 935 940 Ser Gln Cys Leu Asn
Ser Ser Pro Leu Ser Pro Pro Gln Leu Met Phe 945 950 955 960 Pro Ala
Ile Ser Ala Pro Cys Ser Ser Ala Pro Ser Val Pro Ala Gly 965 970 975
Ser Val Thr Asp Ala Ser Lys His Arg Pro Arg Ala Phe Val Pro Cys 980
985 990 Lys Ile Pro Ser Ala Ser Pro Gln Thr Gln Arg Lys Phe Ser Leu
Gln 995 1000 1005 Phe Gln Arg Thr Cys Ser Glu Asn Arg Asp Ser Glu
Lys Leu Ser 1010 1015 1020 Pro Val Phe Thr Gln Ser Arg Pro Pro Pro
Ser Ser Asn Ile His 1025 1030 1035 Arg Ala Lys Ala Ser Arg Pro Val
Pro Gly Ser Thr Ser Lys Leu 1040 1045 1050 Gly Asp Ala Ser Lys Asn
Ser Met Thr Leu Asp Leu Asn Ser Ala 1055 1060 1065 Ser Gln Cys Asp
Asp Ser Phe Gly Ser Gly Ser Asn Ser Gly Ser 1070 1075 1080 Ala Val
Ile Pro Ser Glu Glu Thr Ala Phe Thr Pro Ala Glu Asp 1085 1090 1095
Lys Cys Arg Leu Asp Val Asn Pro Glu Leu Asn Ser Ser Ile Glu 1100
1105 1110 Asp Leu Leu Glu Ala Ser Met Pro Ser Ser Asp Thr Thr Val
Thr 1115 1120 1125 Phe Lys Ser Glu Val Ala Val Leu Ser Pro Glu Lys
Ala Glu Ser 1130 1135 1140 Asp Asp Thr Tyr Lys Asp Asp Val Asn His
Asn Gln Lys Cys Lys 1145 1150 1155 Glu Lys Met Glu Ala Glu Glu Glu
Glu Ala Leu Ala Ile Ala Met 1160 1165 1170 Ala Met Ser Ala Ser Gln
Asp Ala Leu Pro Ile Val Pro Gln Leu 1175 1180 1185 Gln Val Glu Asn
Gly Glu Asp Ile Ile Ile Ile Gln Gln Asp Thr 1190 1195 1200 Pro Glu
Thr Leu Pro Gly His Thr Lys Ala Asn Glu Pro Tyr Arg 1205 1210 1215
Glu Asp Thr Glu Trp Leu Lys Gly Gln Gln Ile Gly Leu Gly Ala 1220
1225 1230 Phe Ser Ser Cys Tyr Gln Ala Gln Asp Val Gly Thr Gly Thr
Leu 1235 1240 1245 Met Ala Val Lys Gln Val Thr Tyr Val Arg Asn Thr
Ser Ser Glu 1250 1255 1260 Gln Glu Glu Val Val Glu Ala Leu Arg Glu
Glu Ile Arg Met Met 1265 1270 1275 Ser His Leu Asn His Pro Asn Ile
Ile Arg Met Leu Gly Ala Thr 1280 1285 1290 Cys Glu Lys Ser Asn Tyr
Asn Leu Phe Ile Glu Trp Met Ala Gly 1295 1300 1305 Ala Ser Val Ala
His Leu Leu Ser Lys Tyr Gly Ala Phe Lys Glu 1310 1315 1320 Ser Val
Val Ile Asn Tyr Thr Glu Gln Leu Leu Arg Gly Leu Ser 1325 1330 1335
Tyr Leu His Glu Asn Gln Ile Ile His Arg Asp Val Lys Gly Ala 1340
1345 1350 Asn Leu Leu Ile Asp Ser Thr Gly Gln Arg Leu Arg Ile Ala
Asp 1355 1360 1365 Phe Gly Ala Ala Ala Arg Leu Ala Ser Lys Gly Thr
Gly Ala Gly 1370 1375 1380 Glu Phe Gln Gly Gln Leu Leu Gly Thr Ile
Ala Phe Met Ala Pro 1385 1390 1395 Glu Val Leu Arg Gly Gln Gln Tyr
Gly Arg Ser Cys Asp Val Trp 1400 1405 1410 Ser Val Gly Cys Ala Ile
Ile Glu Met Ala Cys Ala Lys Pro Pro 1415 1420 1425 Trp Asn Ala Glu
Lys His Ser Asn His Leu Ala Leu Ile Phe Lys 1430 1435 1440 Ile Ala
Ser Ala Thr Thr Ala Pro Ser Ile Pro Ser His Leu Ser 1445 1450 1455
Pro Gly Leu Arg Asp Val Ala Leu Arg Cys Leu Glu Leu Gln Pro 1460
1465 1470 Gln Asp Arg Pro Pro Ser Arg Glu Leu Leu Lys His Pro Val
Phe 1475 1480 1485 Arg Thr Thr Trp 1490 5 1492 PRT mouse MEKK1 5
Met Ala Ala Ala Ala Gly Asp Arg Ala Ser Ser Ser Gly Phe Pro Gly 1 5
10 15 Ala Ala Ala Ala Ser Pro Glu Ala Gly Gly Gly Gly Gly Gly Gly
Gly 20 25 30 Ala Leu Gln Gly Ser Gly Ala Pro Ala Ala Gly Ala Ala
Gly Leu Leu 35 40 45 Arg Glu Pro Gly Ser Ala Gly Arg Glu Arg Ala
Asp Trp Arg Arg Arg 50 55 60 Gln Leu Arg Lys Val Arg Ser Val Glu
Leu Asp Gln Leu Pro Glu Gln 65 70 75 80 Pro Leu Phe Leu Ala Ala Ala
Ser Pro Pro Cys Pro Ser Thr Ser Pro 85 90 95 Ser Pro Glu Pro Ala
Asp Ala Ala Ala Gly Ala Ser Arg Phe Gln Pro 100 105 110 Ala Ala Gly
Pro Pro Pro Pro Gly Ala Ala Ser Arg Cys Gly Ser His 115 120 125 Ser
Ala Glu Leu Ala Ala Ala Arg Asp Ser Gly Ala Arg Ser Pro Ala 130 135
140 Gly Ala Glu Pro Pro Ser Ala Ala Ala Pro Ser Gly Arg Glu Met Glu
145 150 155 160 Asn Lys Glu Thr Leu Lys Gly Leu His Lys Met Glu Asp
Arg Pro Glu 165 170 175 Glu Arg Met Ile
Arg Glu Lys Leu Lys Ala Thr Cys Met Pro Ala Trp 180 185 190 Lys His
Glu Trp Leu Glu Arg Arg Asn Arg Arg Gly Pro Val Val Val 195 200 205
Lys Pro Ile Pro Ile Lys Gly Asp Gly Ser Glu Val Asn Asn Leu Ala 210
215 220 Ala Glu Pro Gln Gly Glu Gly Gln Ala Gly Ser Ala Ala Pro Ala
Pro 225 230 235 240 Lys Gly Arg Arg Ser Pro Ser Pro Gly Ser Ser Pro
Ser Gly Arg Ser 245 250 255 Val Lys Pro Glu Ser Pro Gly Val Arg Arg
Lys Arg Val Ser Pro Val 260 265 270 Pro Phe Gln Ser Gly Arg Ile Thr
Pro Pro Arg Arg Ala Pro Ser Pro 275 280 285 Asp Gly Phe Ser Pro Tyr
Ser Pro Glu Glu Thr Ser Arg Arg Val Asn 290 295 300 Lys Val Met Arg
Ala Arg Leu Tyr Leu Leu Gln Gln Ile Gly Pro Asn 305 310 315 320 Ser
Phe Leu Ile Gly Gly Asp Ser Pro Asp Asn Lys Tyr Arg Val Phe 325 330
335 Ile Gly Pro Gln Asn Cys Ser Cys Gly Arg Gly Ala Phe Cys Ile His
340 345 350 Leu Leu Phe Val Met Leu Arg Val Phe Gln Leu Glu Pro Ser
Asp Pro 355 360 365 Met Leu Trp Arg Lys Thr Leu Lys Asn Phe Glu Val
Glu Ser Leu Phe 370 375 380 Gln Lys Tyr His Ser Arg Arg Ser Ser Arg
Ile Lys Ala Pro Ser Arg 385 390 395 400 Asn Thr Ile Gln Lys Phe Val
Ser Arg Met Ser Asn Ser His Thr Leu 405 410 415 Ser Ser Ser Ser Thr
Ser Thr Ser Ser Ser Glu Asn Ser Ile Lys Asp 420 425 430 Glu Glu Glu
Gln Met Cys Pro Ile Cys Leu Leu Gly Met Leu Asp Glu 435 440 445 Glu
Ser Leu Thr Val Cys Glu Asp Gly Cys Arg Asn Lys Leu His His 450 455
460 His Cys Met Ser Ile Trp Ala Glu Glu Cys Arg Arg Asn Arg Glu Pro
465 470 475 480 Leu Ile Cys Pro Leu Cys Arg Ser Trp Arg Ser His Asp
Phe Tyr Ser 485 490 495 His Glu Leu Ser Ser Pro Val Glu Ser Pro Ala
Ser Leu Arg Ala Val 500 505 510 Gln Gln Pro Ser Ser Pro Gln Gln Pro
Val Ala Gly Ser Gln Arg Arg 515 520 525 Asn Gln Glu Ser Ser Phe Asn
Leu Thr His Phe Gly Thr Gln Gln Ile 530 535 540 Pro Ser Ala Tyr Lys
Asp Leu Ala Glu Pro Trp Ile Gln Val Phe Gly 545 550 555 560 Met Glu
Leu Val Gly Cys Leu Phe Ser Arg Asn Trp Asn Val Arg Glu 565 570 575
Met Ala Leu Arg Arg Leu Ser His Asp Val Ser Gly Ala Leu Leu Leu 580
585 590 Ala Asn Gly Glu Ser Thr Gly Asn Ser Gly Gly Gly Ser Gly Gly
Ser 595 600 605 Leu Ser Ala Gly Ala Ala Ser Gly Ser Ser Gln Pro Ser
Ile Ser Gly 610 615 620 Asp Val Val Glu Ala Cys Cys Ser Val Leu Ser
Ile Val Cys Ala Asp 625 630 635 640 Pro Val Tyr Lys Val Tyr Val Ala
Ala Leu Lys Thr Leu Arg Ala Met 645 650 655 Leu Val Tyr Thr Pro Cys
His Ser Leu Ala Glu Arg Ile Lys Leu Gln 660 665 670 Arg Leu Leu Arg
Pro Val Val Asp Thr Ile Leu Val Lys Cys Ala Asp 675 680 685 Ala Asn
Ser Arg Thr Ser Gln Leu Ser Ile Ser Thr Val Leu Glu Leu 690 695 700
Cys Lys Gly Gln Ala Gly Glu Leu Ala Val Gly Arg Glu Ile Leu Lys 705
710 715 720 Ala Gly Ser Ile Gly Val Gly Gly Val Asp Tyr Val Leu Ser
Cys Ile 725 730 735 Leu Gly Asn Gln Ala Glu Ser Asn Asn Trp Gln Glu
Leu Leu Gly Arg 740 745 750 Leu Cys Leu Ile Asp Arg Leu Leu Leu Glu
Phe Pro Ala Glu Phe Tyr 755 760 765 Pro His Ile Val Ser Thr Asp Val
Ser Gln Ala Glu Pro Val Glu Ile 770 775 780 Arg Tyr Lys Lys Leu Leu
Ser Leu Leu Thr Phe Ala Leu Gln Ser Ile 785 790 795 800 Asp Asn Ser
His Ser Met Val Gly Lys Leu Ser Arg Arg Ile Tyr Leu 805 810 815 Ser
Ser Ala Arg Met Val Thr Ala Val Pro Ala Val Phe Ser Lys Leu 820 825
830 Val Thr Met Leu Asn Ala Ser Gly Ser Thr His Phe Thr Arg Met Arg
835 840 845 Arg Arg Leu Met Ala Ile Ala Asp Glu Val Glu Ile Ala Glu
Val Ile 850 855 860 Gln Leu Gly Val Glu Asp Thr Val Asp Gly His Gln
Asp Ser Leu Gln 865 870 875 880 Ala Val Ala Pro Thr Ser Cys Leu Glu
Asn Ser Ser Leu Glu His Thr 885 890 895 Val His Arg Glu Lys Thr Gly
Lys Gly Leu Ser Ala Thr Arg Leu Ser 900 905 910 Ala Ser Ser Glu Asp
Ile Ser Asp Arg Leu Ala Gly Val Ser Val Gly 915 920 925 Leu Pro Ser
Ser Thr Thr Thr Glu Gln Pro Lys Pro Ala Val Gln Thr 930 935 940 Lys
Gly Arg Pro His Ser Gln Cys Leu Asn Ser Ser Pro Leu Ser His 945 950
955 960 Ala Gln Leu Met Phe Pro Ala Pro Ser Ala Pro Cys Ser Ser Ala
Pro 965 970 975 Ser Val Pro Asp Ile Ser Lys His Arg Pro Gln Ala Phe
Val Pro Cys 980 985 990 Lys Ile Pro Ser Ala Ser Pro Gln Thr Gln Arg
Lys Phe Ser Leu Gln 995 1000 1005 Phe Gln Arg Asn Cys Ser Glu His
Arg Asp Ser Asp Gln Leu Ser 1010 1015 1020 Pro Val Phe Thr Gln Ser
Arg Pro Pro Pro Ser Ser Asn Ile His 1025 1030 1035 Arg Pro Lys Pro
Ser Arg Pro Val Pro Gly Ser Thr Ser Lys Leu 1040 1045 1050 Gly Asp
Ala Thr Lys Ser Ser Met Thr Leu Asp Leu Gly Ser Ala 1055 1060 1065
Ser Arg Cys Asp Asp Ser Phe Gly Gly Gly Gly Asn Ser Gly Asn 1070
1075 1080 Ala Val Ile Pro Ser Asp Glu Thr Val Phe Thr Pro Val Glu
Asp 1085 1090 1095 Lys Cys Arg Leu Asp Val Asn Thr Glu Leu Asn Ser
Ser Ile Glu 1100 1105 1110 Asp Leu Leu Glu Ala Ser Met Pro Ser Ser
Asp Thr Thr Val Thr 1115 1120 1125 Phe Lys Ser Glu Val Ala Val Leu
Ser Pro Glu Lys Ala Glu Asn 1130 1135 1140 Asp Asp Thr Tyr Lys Asp
Asp Val Asn His Asn Gln Lys Cys Lys 1145 1150 1155 Glu Lys Met Glu
Ala Glu Glu Glu Glu Ala Leu Ala Ile Ala Met 1160 1165 1170 Ala Met
Ser Ala Ser Gln Asp Ala Leu Pro Ile Val Pro Gln Leu 1175 1180 1185
Gln Val Glu Asn Gly Glu Asp Ile Ile Ile Ile Gln Gln Asp Thr 1190
1195 1200 Pro Glu Thr Leu Pro Gly His Thr Lys Ala Lys Gln Pro Tyr
Arg 1205 1210 1215 Glu Asp Ala Glu Trp Leu Lys Gly Gln Gln Ile Gly
Leu Gly Ala 1220 1225 1230 Phe Ser Ser Cys Tyr Gln Ala Gln Asp Val
Gly Thr Gly Thr Leu 1235 1240 1245 Met Ala Val Lys Gln Val Thr Tyr
Val Arg Asn Thr Ser Ser Glu 1250 1255 1260 Gln Glu Glu Val Val Glu
Ala Leu Arg Glu Glu Ile Arg Met Met 1265 1270 1275 Gly His Leu Asn
His Pro Asn Ile Ile Arg Met Leu Gly Ala Thr 1280 1285 1290 Cys Glu
Lys Ser Asn Tyr Asn Leu Phe Ile Glu Trp Met Ala Gly 1295 1300 1305
Gly Ser Val Ala His Leu Leu Ser Lys Tyr Gly Ala Phe Lys Glu 1310
1315 1320 Ser Val Val Ile Asn Tyr Thr Glu Gln Leu Leu Arg Gly Leu
Ser 1325 1330 1335 Tyr Leu His Glu Asn Gln Ile Ile His Arg Asp Val
Lys Gly Ala 1340 1345 1350 Asn Leu Leu Ile Asp Ser Thr Gly Gln Arg
Leu Arg Ile Ala Asp 1355 1360 1365 Phe Gly Ala Ala Ala Arg Leu Ala
Ser Lys Gly Thr Gly Ala Gly 1370 1375 1380 Glu Phe Gln Gly Gln Leu
Leu Gly Thr Ile Ala Phe Met Ala Pro 1385 1390 1395 Glu Val Leu Arg
Gly Gln Gln Tyr Gly Arg Ser Cys Asp Val Trp 1400 1405 1410 Ser Val
Gly Cys Ala Ile Ile Glu Met Ala Cys Ala Lys Pro Pro 1415 1420 1425
Trp Asn Ala Glu Lys His Ser Asn His Leu Ala Leu Ile Phe Lys 1430
1435 1440 Ile Ala Ser Ala Thr Thr Ala Pro Ser Ile Pro Ser His Leu
Ser 1445 1450 1455 Pro Gly Leu Arg Asp Val Ala Val Arg Cys Leu Glu
Leu Gln Pro 1460 1465 1470 Gln Asp Arg Pro Pro Ser Arg Glu Leu Leu
Lys His Pro Val Phe 1475 1480 1485 Arg Thr Thr Trp 1490 6 55 DNA
Human 6 atggcggcgg cggcggggaa tcgcgcctcg tcgggattcc cgggcgccag
ggcta 55 7 61 DNA Human 7 gagaaaatgg cggcggcggc ggggaatcgc
gcctcgtcgg gattcccggg cgccagggct 60 a 61 8 11 DNA Human 8
gcgcgcccgc g 11 9 17 DNA Human 9 ccgcgagccg cggcggc 17 10 12 DNA
Human 10 tttggatggt ca 12 11 10 DNA Human 11 ggacagcttc 10 12 11
DNA Human 12 cccctgagtg c 11 13 10 DNA Human 13 gccagcattt 10 14 11
DNA Human 14 catctagacc t 11 15 10 DNA Human 15 ggctgtagca 10 16 10
DNA Human 16 gtaatgctgt 10 17 15 DNA Human 17 ggatgccctc cccat 15
18 20 PRT Human 18 Met Ala Ala Ala Ala Gly Asn Arg Ala Ser Ser Ser
Gly Phe Pro Gly 1 5 10 15 Ala Arg Ala Thr 20 19 22 PRT Human 19 Glu
Lys Met Ala Ala Ala Ala Gly Asn Arg Ala Ser Ser Ser Gly Phe 1 5 10
15 Pro Gly Ala Arg Ala Thr 20 20 5 PRT Human 20 Ser Ala Pro Ala Ala
1 5 21 5 PRT Human 21 Ala Ser Arg Gly Gly 1 5 22 5 PRT Human 22 Cys
Ala Arg Gly Thr 1 5 23 6 PRT Human 23 Val Ser Ser Ser Thr His 1 5
24 7 PRT Human 24 Leu Met Ala Ile Ala Asp Glu 1 5 25 30 PRT Human
25 Thr Leu Asp Gly Gln Gln Asp Ser Phe Leu Gln Ala Ser Val Pro Asn
1 5 10 15 Asn Tyr Leu Glu Thr Thr Glu Asn Ser Ser Pro Glu Cys Thr
20 25 30 26 6 PRT Human 26 Leu Ala Ser Ile Ser Val 1 5 27 17 PRT
Human 27 Ser Phe Gly Cys Ser Ser Asn Ser Ser Asn Ala Val Ile Pro
Ser Asp 1 5 10 15 Glu 28 44 PRT Human 28 Ser Gln Asp Ala Leu Pro
Ile Val Pro Gln Leu Gln Val Glu Asn Gly 1 5 10 15 Glu Asp Ile Ile
Ile Ile Gln Gln Asp Thr Pro Glu Thr Leu Pro Gly 20 25 30 His Thr
Lys Ala Lys Gln Pro Tyr Arg Glu Asp Thr 35 40
* * * * *
References