U.S. patent application number 10/137953 was filed with the patent office on 2003-07-10 for cytokine-, stress-, and oncoprotein-activated human protein kinase kinases.
This patent application is currently assigned to University of Massachusetts, a Massachusetts corporation. Invention is credited to Davis, Roger J., Tournier, Cathy, Whitmarsh, Alan.
Application Number | 20030129606 10/137953 |
Document ID | / |
Family ID | 25393160 |
Filed Date | 2003-07-10 |
United States Patent
Application |
20030129606 |
Kind Code |
A1 |
Davis, Roger J. ; et
al. |
July 10, 2003 |
Cytokine-, stress-, and oncoprotein-activated human protein kinase
kinases
Abstract
Disclosed are human mitogen-activated (MAP) kinase kinase
isoforms (MKKs). MKKs mediate unique signal transduction pathways
that activate human MAP kinases p38 and JNK, which result in
activation of other factors, including activating transcription
factor-2 (ATF2) and c-Jun. The pathways are activated by a number
of factors, including cytokines and environmental stress. Methods
are provided for identifying reagents that modulate MKK function or
activity and for the use of such reagents in the treatment of
MKK-mediated disorders.
Inventors: |
Davis, Roger J.; (Princeton,
MA) ; Whitmarsh, Alan; (Shrewsbury, MA) ;
Tournier, Cathy; (Worcester, MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Assignee: |
University of Massachusetts, a
Massachusetts corporation
|
Family ID: |
25393160 |
Appl. No.: |
10/137953 |
Filed: |
May 3, 2002 |
Related U.S. Patent Documents
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10137953 |
May 3, 2002 |
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09593653 |
Jun 13, 2000 |
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09593653 |
Jun 13, 2000 |
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08888429 |
Jul 7, 1997 |
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6136596 |
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08888429 |
Jul 7, 1997 |
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08530950 |
Sep 19, 1995 |
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5736381 |
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08530950 |
Sep 19, 1995 |
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08446083 |
May 19, 1995 |
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5804427 |
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Current U.S.
Class: |
435/6.14 ;
435/15; 435/194; 435/320.1; 435/325; 435/69.1; 435/7.1;
536/23.2 |
Current CPC
Class: |
A61P 37/00 20180101;
A61P 29/00 20180101; C07K 14/4736 20130101; A61P 19/02 20180101;
A61P 9/02 20180101; A61P 11/00 20180101; C07K 14/005 20130101; C12Q
1/6883 20130101; C12N 9/1205 20130101; G01N 2500/02 20130101; C12N
9/12 20130101; A61P 13/12 20180101; A61P 17/02 20180101; A61P 37/06
20180101; C07K 14/82 20130101; A61P 9/10 20180101; C12Q 1/485
20130101; A61P 43/00 20180101; C07K 14/4705 20130101; A61K 38/00
20130101; A61P 31/04 20180101; C12N 2710/10322 20130101; C12N
2710/10332 20130101; C12Q 2600/158 20130101; A61P 1/16
20180101 |
Class at
Publication: |
435/6 ; 435/7.1;
435/69.1; 435/15; 435/320.1; 435/325; 435/194; 536/23.2 |
International
Class: |
C12Q 001/68; G01N
033/53; C12Q 001/48; C07H 021/04; C12P 021/02; C12N 005/06; C12N
009/12 |
Goverment Interests
[0002] This invention was made in part with National Cancer
Institute research grant CA 58396 and CA 65861. The Federal
government has certain rights in the invention.
Claims
What is claimed is:
1. A substantially pure mammalian mitogen-activated protein kinase
kinase (MKK) polypeptide having serine, threonine, and tyrosine
kinase activity, and phosphorylating mitogen-activated protein
(MAP) kinase JNK, but not p38.
2. A polypeptide of claim 1 comprising the amino acid sequence of
SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID
NO: 30, or SEQ ID NO: 32.
3. An isolated polynucleotide sequence encoding a polypeptide of
claim 1.
4. An isolated polynucleotide sequence of claim 3 comprising the
sequence of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO:
27, SEQ ID NO: 29, or SEQ ID NO: 31, or degenerate variants
thereof, or a polynucleotide sequence fully complementary to the
sequence of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO:
27, SEQ ID NO: 29, or SEQ ID NO: 31, or degenerate variants
thereof.
5. An isolated polynucleotide sequence of claim 3 comprising a
polynucleotide sequence that hybridizes under stringent
hybridization conditions to the sequence of SEQ ID NO: 17, SEQ ID
NO: 19, or SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:
31, or a complement thereof.
6. A recombinant expression vector containing a polynucleotide
sequence of claim 3.
7. A recombinant host cell comprising a polynucleotide sequence of
claim 3.
8. A purified antibody that binds specifically to a polypeptide of
claim 1.
9. A purified antibody that binds specifically to a polypeptide of
claim 2.
10. A method of measuring the activity of a mitogen-activated
protein kinase kinase (MKK7) in a biological test sample, said
method comprising: a) incubating said test sample with an MKK
substrate for the MKK polypeptide of claim 1 and labeled phosphate
under conditions sufficient to allow phosphorylation of said
substrate; and b) determining the rate of incorporation of labeled
phosphate into said substrate, wherein said rate of incorporation
is a measure of MKK7 activity.
11. A method of claim 10, wherein said MKK substrate is selected
from the group consisting of JNK MAP kinases, activating
transcription factor-2 (ATF2), ATFa, cAMP response element binding
protein (CRE-BPa), and c-Jun.
12. A method of claim 10, wherein said biological test sample is
fluid, cells, or tissue obtained from a mammal.
13. A method for measuring the synthesis of MKK7 in a biological
test sample, the method comprising the steps of: a) obtaining a
biological sample; b) contacting said biological sample with an
antibody that specifically binds an MKK7 polypeptide of claim 1;
and c) detecting said antibody bound to MKK7 polypeptide, wherein
the level of MKK7 synthesis is determined by the amount of bound
antibody.
14. A method for measuring the level of expression of MKK7 in a
test sample, the method comprising the steps of: a) isolating total
or polyadenylated RNA from the test sample; b) incubating the RNA
with a polynucleotide probe specific for an MKK7 polynucleotide of
claim 3; and c) determining the amount of said probe hybridized to
the RNA, wherein the level of expression of MKK7 is directly
related to the amount of MKK7 probe hybridized to the RNA.
15. A method for identifying a reagent that modulates MKK7
activity, said method comprising: a) obtaining a test sample
containing MKK7; b) incubating said test sample with an MKK
substrate for the MKK polypeptide of claim 1, a range of reagent
concentrations, and labeled phosphate under conditions sufficient
to allow phosphorylation of said subtrate when said reagent is not
present; c) detecting phosphorylation of said substrate; and d)
comparing the effect of said reagent on MKK7 activity relative to a
control, wherein any variation compared to control indicates a
reagent able to modulate MKK7 substrate phosphorylation.
16. A method of claim 15, wherein said MKK7 substrate is one or
more of JNK, ATF2, ATFa, CRE-BPa, and c-Jun.
17. A method of claim 15 wherein said modulation is inhibition of
MKK7 activity.
18. A method for identifying a reagent that modulates MKK7
synthesis, said method comprising: a) providing a sample capable of
MKK7 synthesis; b) incubating said sample with a range of reagent
concentrations under conditions that allow synthesis of MKK7 when
said reagent is not present; c) detecting an MKK7 polypeptide of
claim 1; and d) comparing the effect of said reagent on MKK7
synthesis relative to a control, wherein any variation compared to
control indicates a reagent able to modulate MKK7.
19. A method of claim 18 wherein said modulation is inhibition of
MKK7 synthesis.
20. A method for identifying a reagent that modulates MKK7
expression, said method comprising: a) providing a sample capable
of expressing MKK7; b) incubating said sample with a range of
concentrations of said reagent under conditions where MKK7 is
expressed in the absence of said reagent; c) isolating total or
polyadenylated RNA from the sample; d) incubating the RNA with a
polynucleotide probe specific for a MKK7 nucleic acid of claim 3;
and e) comparing the effect of said reagent on MKK7 RNA expression
relative to a control, wherein any variation compared to control
indicates a reagent able to modulate MKK7 expression.
21. A method of treating an MKK7-mediated disorder in a patient,
the method comprising administering to the patient a
therapeutically effective amount of a reagent that modulates MKK7
activity.
22. The method of claim 21, wherein said MKK7-mediated disorder is
selected from the group consisting of ischemic heart disease,
kidney failure, oxidative liver damage, respiratory distress
syndrome, heat and radiation burns, septic shock, rheumatoid
arthritis, autoimmune disorders, and inflammatory diseases.
23. A method of treating an MKK7-associated disorder in a patient,
comprising administering to the patient a therapeutically effective
amount of an MKK7 polypeptide.
24. The method of claim 23, wherein said MKK7-associated disorder
is ischemic heart disease, kidney failure, oxidative liver damage,
respiratory distress syndrome, heat and radiation burns, septic
shock, rheumatoid arthritis, autoimmune disorders, or inflammatory
diseases.
25. A substantially pure human mitogen-activated protein kinase
kinase (MKK) polypeptide having serine, threonine, and tyrosine
kinase activity, and phosphorylating human mitogen-activated
protein (MAP) kinase p38.
26. A polypeptide of claim 25 comprising the amino acid sequence of
SEQ ID NO: 2 or SEQ ID NO: 4.
27. A polypeptide of claim 25, further characterized in that said
polypeptide phosphorylates human mitogen-activated protein (MAP)
kinase JNK.
28. A polypeptide of claim 27, comprising the amino acid sequence
of SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.
29. A purified antibody which binds specifically to a polypeptide
of claim 25.
30. A purified antibody which binds specifically to a polypeptide
of claim 26.
31. A purified antibody which binds specifically to a polypeptide
of claim 27.
32. A purified antibody which binds specifically to a polypeptide
of claim 28.
33. A method of measuring the activity of a mitogen-activated
protein kinase kinase (MKK) in a biological test sample, said
method comprising: a) incubating said test sample with an MKK
substrate for the MKK polypeptide of claim 25 and labeled phosphate
under conditions sufficient to allow phosphorylation of said
substrate, and b) determining the rate of incorporation of labeled
phosphate into said substrate, wherein said rate of incorporation
is a measure of MKK activity.
34. A method of claim 33, wherein said MKK substrate is selected
from the group consisting of p38 and JNK MAP kinases, activating
transcription factor-2 (ATF2), kinases, activating transcription
factor-2 (ATF2), ATFa, cAMP response element binding protein
(CRE-BPa), and c-Jun.
35. A method of claim 33, wherein said biological test sample is
fluid, cells, or tissue obtained from a mammal.
36. A method for measuring the synthesis of MKK in a biological
test sample, comprising the steps of: a) fractionating proteins
present in said sample by gel electrophoresis; b) transferring the
proteins onto a membrane; and c) probing the proteins with a
labeled antibody specific to a MKK polypeptide of claim 25, wherein
the level of MKK synthesis is determined by the amount of bound
labeled antibody.
37. A method for measuring the level of expression of MKK in a test
sample, comprising the steps of: a) isolating polyadenylated RNA
from the test sample; b) incubating polyadenylated RNA with a
polynucleotide probe specific for a MKK polypeptide of claim 25; c)
determining the amount of said probe hybridized said polyadenylated
RNA, wherein the level of expression of MKK is directly related to
the amount of MKK probe hybridized to said RNA.
38. A method for identifying a reagent which modulates MKK
activity, said method comprising: a) using the method of claim 33;
b) comparing the effect of said reagent on MKK activity relative to
a control, wherein a reagent able to modulate MKK substrate
phosphorylation is identified.
39. A method of claim 38, wherein said MKK substrate is one or more
of p38, JNK, ATF2, ATFa, CRE-BPa, and c-Jun.
40. A method of claim 38, wherein said modulation is inhibition of
MKK activity.
41. A method for identifying a reagent which modulates MKK
synthesis, said method comprising: a) using the method of claim 36;
b) comparing the effect of said reagent on MKK synthesis relative
to a control, wherein a reagent able to modulate MKK synthesis is
identified.
42. A method of claim 41, wherein said MKK substrate is one or more
of p38, JNK, ATF2, ATFa, CRE-BPa, and c-Jun.
43. A method of claim 41, wherein said modulation is inhibition of
MKK synthesis.
44. A method for identifying a reagent which modulates MKK
expression, said method comprising: a) using the method of claim
37; b) comparing the effect of said reagent on MKK expression
relative to a control, wherein a reagent able to modulate MKK
expression is identified.
45. A method of treating an MKK-mediated disorder in a patient,
comprising administering to the patient a therapeutically effective
amount of a reagent that modulates MKK activity.
46. The method of claim 45, wherein said MKK-mediated disorder is
selected from the group consisting of ischemic heart disease,
kidney failure, oxidative liver damage, respiratory distress
syndrome, heat and radiation burns, septic shock, rheumatoid
arthritis, autoimmune disorders, and inflammatory diseases.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S. patent
Ser. No. 08/530,950, filed Sep. 19, 1995, which is a
continuation-in-part of pending application Ser. No. 08/446,083,
filed May 19, 1995, which applications are incorporated herein by
reference in their entirety.
BACKGROUND OF THE INVENTION
[0003] This invention relates to protein kinases.
[0004] Mitogen-activated protein (MAP) kinases are important
mediators of signal transduction from the cell surface to the
nucleus. Multiple MAP kinases have been described in yeast
including SMK1, HOG1, MPK1, FUS3, and KSS1. In mammals, the MAP
kinases identified are extracellular signal-regulated MAP kinase
(ERK), c-Jun amino-terminal kinase (JNK), and p38 kinase (Davis
(1994) Trends Biochem. Sci. 19:470). These MAP kinase isoforms are
activated by dual phosphorylation on threonine and tyrosine.
[0005] Activating Transcription Factor-2 (ATF2), ATFa, and cAMP
Response Element Binding Protein (CRE-BPa) are related
transcription factors that bind to similar sequences located in the
promoters of many genes (Ziff (1990) Trends in Genet. 6:69). The
binding of these transcription factors leads to increased
transcriptional activity. ATF2 binds to several viral proteins,
including the oncoprotein E1a (Liu and Green (1994) Nature
388:520), the hepatitis B virus X protein (Maguire et al. (1991)
Science 252:842), and the human T cell leukemia virus 1 tax protein
(Wagner and Green (1993) Science 262:395). ATF2 also interacts with
the tumor suppressor gene product Rb (Kim et al. (1992) Nature
358:331), the high mobility group protein HMG(I)Y (Du et al. (1993)
Cell 74:887), and the transcription factors nuclear NF-.kappa.B (Du
et al. (1993) Cell 74:887) and c-Jun (Benbrook and Jones (1990)
Oncogene 5:295).
SUMMARY OF THE INVENTION
[0006] The invention is based on the identification and isolation
of a new group of human mitogen-activated protein kinase kinases
(MKKs). The MKK isoforms described herein, MKK3, MKK6, MKK4
(including MKK4-.alpha., -.beta., and -.gamma.), MKK7 (including
murine MKK7, human MKK7, MKK7b, MKK7c, MKK7d, and MKK7e) have
serine, threonine, and tyrosine kinase activity. MKK3, MKK4, and
MKK6 specifically phosphorylate the human MAP kinase p38 at
Thr.sup.180 and Tyr.sup.182. The MKK4 isoforms also phosphorylate
the human MAP kinases JNK (including JNK1, JNK2, and JNK5) at
Thr.sup.183 and Tyr.sup.185. The MKK7 isoforms phosphorylate JNK at
Thr.sup.183 and Tyr.sup.185.
[0007] Accordingly, the invention features a substantially pure
human MKK polypeptide having serine, threonine, and tyrosine kinase
activity that specifically phosphorylates human p38 MAP kinase.
MKK3 has the amino acid sequence of SEQ ID NO: 2. The invention
further includes MKK6 having the amino acid sequence of SEQ ID NO:
4 and having serine, threonine, and tyrosine kinase activity that
specifically phosphorylates human p38 MAP kinase.
[0008] The invention further features a substantially pure human
MKK polypeptide having serine, threonine, and tyrosine kinase
activity that specifically phosphorylates human p38 MAP kinase and
JNK. MKK4 isoform MKK4-.alpha. has the amino acid sequence of SEQ
ID NO: 6. MKK4 isoform MKK4-.beta. has the amino acid sequence of
SEQ ID NO: 8. MKK4 isoform MKK4-.gamma. has the amino acid sequence
of SEQ ID NO: 10.
[0009] The invention also features a substantially pure MKK
polypeptide (MKK7) having serine, threonine, and tyrosine kinase
activity that specifically phosphorylates mitogen-activated protein
kinase JNK. MKK isoforms MKK7 (murine) and MKK7 (human) have the
amino acid sequences of SEQ ID NOS: 18 and 26, respectively. The
MKK7 isoforms MKK7b, MKK7c, MKK7d, and MKK7e have the amino acid
sequences of SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 30, and SEQ
ID NO: 32, respectively.
[0010] As used herein, the term "mitogen-activating protein kinase
kinase" or "MKK" means a protein kinase which possesses the
characteristic activity of phosphorylating and activating a human
mitogen-activating protein kinase. Examples of MKKs include MKK3
and MKK6, which specifically phosphorylate and activate p38 MAP
kinase at Thr.sup.180 and Tyr.sup.182, MKK4 isoforms which
specifically phosphorylate and activate p38 MAP kinase at
Thr.sup.180 and Tyr.sup.182, and JNK at Thr.sup.183 and
Tyr.sup.185, and MKK7 isoforms which specifically phosphorylate JNK
at Thr.sup.183 and Tyr.sup.185.
[0011] An "MKK7" is a mammalian isoform of mitogen-activated
protein kinase kinase (MKK) polypeptide having serine, threonine,
and tyrosine kinase activity, and phosphorylating mitogen-activated
protein (MAP) kinase JNK but not p38.
[0012] The invention includes the specific p38 and JNK MKKs
disclosed, as well as closely related MKKs which are identified and
isolated by the use of probes or antibodies prepared from the
polynucleotide and amino acid sequences disclosed for the MKKs of
the invention. This can be done using standard techniques, e.g., by
screening a genomic, cDNA, or combinatorial chemical library with a
probe having all or a part of the nucleic acid sequences of the
disclosed MKKs. The invention further includes synthetic
polynucleotides having all or part of the amino acid sequence of
the MKKs herein described.
[0013] The term "polypeptide" means any chain of amino acids,
regardless of length or post-translational modification (e.g.,
glycosylation or phosphorylation), and includes natural proteins as
well as synthetic or recombinant polypeptides and peptides.
[0014] The term "substantially pure," when referring to a
polypeptide, means a polypeptide that is at least 60%, by weight,
free from the proteins and naturally-occurring organic molecules
with which it is naturally associated. A substantially pure MKK
polypeptide (e.g., human) is at least 75%, more preferably at least
90%, and most preferably at least 99%, by weight, MKK polypeptide.
A substantially pure MKK can be obtained, for example, by
extraction from a natural source; by expression of a recombinant
nucleic acid encoding a MKK polypeptide, or by chemically
synthesizing the protein. Purity can be measured by any appropriate
method, e.g., column chromatography, polyacrylamide gel
electrophoresis, or HPLC analysis.
[0015] In one aspect, the invention features isolated
polynucleotides which encode the MKKs of the invention. In one
embodiment, the polynucleotide is the nucleotide sequence of SEQ ID
NO: 1. In other embodiments, the polynucleotide is the nucleotide
sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,
SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID
NO: 29, or SEQ ID NO: 31, respectively.
[0016] As used herein, "polynucleotide" refers to a nucleic acid
sequence or deoxyribonucleotides or ribonucleotides in the form of
a separate fragment or a component of a larger construct. DNA
encoding portions or all of the polypeptides of the invention can
be assembled from cDNA fragments or from oligonucleotides that
provide a synthetic gene which can be expressed in a recombinant
transcriptional unit. Polynucleotide sequences of the invention
include DNA, RNA, and cDNA sequences, and can be derived from
natural sources or synthetic sequences synthesized by methods known
to the art.
[0017] An "isolated" polynucleotide is a nucleic acid molecule that
is separated in some way from sequences in the naturally occurring
genome of an organism. Thus, the term "isolated polynucleotide"
includes any nucleic acid molecules that are not naturally
occuring. The term therefore includes, for example, a recombinant
polynucleotide which is incorporated into a vector, into an
autonomously replicating plasmid or virus, or into the genomic DNA
of a prokaryote or eukaryote, or which exists as a separate
molecule independent of other sequences. It also includes a
recombinant DNA which is part of a hybrid gene encoding additional
polypeptide sequences.
[0018] The isolated polynucleotide sequences of the invention also
include polynucleotide sequences that hybridize under stringent
conditions to the polynucleotide sequences specified herein. The
term "stringent conditions" means hybridization conditions that
guarantee specificity between hybridizing polynucleotide sequences,
such as those described herein, or more stringent conditions. One
skilled in the art can select posthybridization washing conditions,
including temperature and salt concentrations, which reduce the
number of nonspecific hybridizations such that only highly
complementary sequences are identified (Sambrook et al. (1989) in
Molecular Cloning, 2d ed.; Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.
[0019] The isolated polynucleotide sequences of the invention also
include sequences complementary to the polynucleotides encoding MKK
(antisense sequences). Antisense nucleic acids are DNA or RNA
molecules that are complementary to at least a portion of a
specific mRNA molecule (Weintraub (1990) Scientific American
262:40). The invention includes all antisense polynucleotides that
inhibit production of MKK polypeptides. In the cell, the antisense
nucleic acids hybridize to the corresponding mRNA, forming a
double-stranded molecule. Antisense oligomers of about 15
nucleotides are preferred, since they are easily synthesized and
introduced into a target MKK-producing cell. The use of antisense
methods to inhibit the translation of genes is known in the art,
and is described, e.g., in Marcus-Sakura Anal. Biochem., 172:289
(1988).
[0020] In addition, ribozyme nucleotide sequences for MKK are
included in the invention. Ribozymes are RNA molecules possessing
the ability to specifically cleave other single-stranded RNA in a
manner analogous to DNA restriction endonucleases. Through the
modification of nucleotide sequences encoding these RNAs, molecules
can be engineered to recognize specific nucleotide sequences in an
RNA molecule and cleave it (Cech (1988) J. Amer. Med. Assn.
260:3030). A major advantage of this approach is that, because they
are sequence-specific, only mRNAs with particular sequences are
inactivated.
[0021] There are two basic types of ribozymes, tetrahymena-type
(Hasselhoff (1988) Nature 334:585) and "hammerhead"-type.
Tetrahymena-type ribozymes recognize sequences which are four bases
in length, while "hammerhead"-type ribozymes recognize base
sequences 11-18 bases in length. The longer the sequence, the
greater the likelihood that the sequence will occur exclusively in
the target mRNA species. Consequently, hammerhead-type ribozymes
are preferable to tetrahymena-type ribozymes for inactivating a
specific mRNA species, and 18-base recognition sequences are
preferable to shorter recognition sequences.
[0022] The MKK polypeptides can also be used to produce antibodies
that are immunoreactive or bind epitopes of the MKK polypeptides.
Accordingly, one aspect of the invention features antibodies to the
MKK polypeptides of the invention. The antibodies of the invention
include polyclonal antibodies which include pooled monoclonal
antibodies with different epitopic specificities, as well as
distinct monoclonal antibody preparations. Monoclonal antibodies
are made from antigen-containing fragments of the MKK polypeptide
by methods known in the art (see, for example, Kohler et al. (1975)
Nature 256:495).
[0023] The term "antibody" as used herein includes intact molecules
as well as fragments thereof, such as Fa, F(ab').sub.2, and Fv,
which are capable of binding an epitopic determinant. Antibodies
that specifically bind MKK polypeptides can be prepared using
intact polypeptides or fragments containing small peptides of
interest as the immunizing antigen. The polypeptide or peptide used
to immunize an animal can be derived from translated cDNA or
chemically synthesized, and can be conjugated to a carrier protein,
if desired. Commonly used carriers that are chemically coupled to
peptides include bovine serum albumin and thyroglobulin. The
coupled peptide is then used to immunize the animal (e.g., a mouse,
a rat, or a rabbit).
[0024] A molecule (e.g., antibody) that "specifically binds" is one
that binds to a particular polypeptide, e.g., MKK7, but that does
not substantially recoginze or bind to other molecules in a sample,
e.g., a biological sample which includes MKK7. References to
constructs made of an antibody (or fragment thereof) coupled to a
compound comprising a detectable marker include constructs made by
any technique, including chemical means and recombinant
techniques.
[0025] The invention also features methods of identifying subjects
at risk for MKK-mediated disorders by measuring activation of the
MKK signal transduction pathway. Activation of the MKK signal
transduction pathway can be determined by measuring MKK synthesis;
activation of MKK isoforms; activation of MKK substrates p38 or JNK
isoforms; or activation of p38 and JNK substrates such as ATF2,
ATFa, CRE-BPa, and c-Jun. The term "JNK" or "JNK isoforms" includes
JNK1, JNK2, and JNK3. The term "MKK substrate" as used herein
includes MKK substrates, as well as MKK substrate substrates, e.g.,
p38, JNK, ATF2, and c-Jun.
[0026] In one embodiment, activation of the MKK signal transduction
pathway is determined by measuring activation of the appropriate
MKK signal transduction pathway substrates (for example, selected
from p38, JNK isoforms, ATF2, ATFa, CRE-BPa, or c-Jun). MKK
activity is measured by the rate of substrate phosphorylation as
determined by quantitation of the rate of labelled phosphorus
(e.g., [.sup.32]P or [.sup.33]P) incorporation. This can also be
measured using phosphorylation-specific reagents, such as
antibodies. The specificity of MKK substrate phosphorylation can be
tested by measuring p38 activation, JNK activation, or both, or by
employing mutated p38 or JNK molecules that lack the sites for MKK
phosphorylations. Altered phosphorylation of the substrate relative
to control values indicates alteration of the MKK signal
transduction pathway, and increased risk in a subject of an
MKK-mediated disorder. MKK activation of p38 and JNK can be
detected in a coupled assay with the MKK signal transduction
substrate ATF2, or related compounds such as ATFa and CRE-BPa.
Activation can also be detected with the substrate c-Jun. When ATF2
is included in the assay, it is present as an intact protein or as
a fragment of the intact protein, e.g., the activation domain
(residues 1-109, or a portion thereof). ATF2 is incubated with a
test sample in which MKK activity is to be measured and
[.gamma.-.sup.32P]ATP, under conditions sufficient to allow the
phosphorylation of ATF2. ATF2 is then isolated and the amount of
phosphorylation quantitated. In a specific embodiment, ATF2 is
isolated by immunoprecipitation, resolved by SDS-PAGE, and detected
by autoradiography.
[0027] In another embodiment, activation of the MKK signal
transduction pathway is determined by measuring the level of MKK
expression in a test sample. In a specific embodiment, the level of
MKK expression is measured by Western blot analysis. The proteins
present in a sample are fractionated by gel electrophoresis,
transferred to a membrane, and probed with labeled antibodies to
MKK. In another specific embodiment, the level of MKK expression is
measured by Northern blot analysis. Total cellular or
polyadenylated [poly(A).sup.+] mRNA is isolated from a test sample.
The RNA is fractionated by electrophoresis and transferred to a
membrane. The membrane is probed with labeled MKK cDNA. In another
embodiment, MKK expression is measured by quantitative PCR applied
to expressed mRNA.
[0028] The MKKs of the invention are useful for screening reagents
that modulate MKK activity. MKKs are activated by phosphorylation.
Accordingly, in one aspect, the invention features methods for
identifying a reagent which modulates MKK activity, by incubating
MKK with the test reagent and measuring the effect of the test
reagent on MKK synthesis, phosphorylation, function, or activity.
In one embodiment, the test reagent is incubated with MKK and
[.sup.32]P-ATP, and the rate of MKK phosphorylation determined, as
described above. In another embodiment, the test reagent is
incubated with a cell transfected with an MKK polynucleotide
expression vector, and the effect of the test reagent on MKK
transcription is measured by Northern blot analysis, as described
above. In a further embodiment, the effect of the test reagent on
MKK synthesis is measured by Western blot analysis using an
antibody to MKK. In still another embodiment, the effect of a
reagent on MKK activity is measured by incubating MKK with the test
reagent, [.sup.32]P-ATP, and a substrate in the MKK signal
transduction pathway, including one or more of p38, JNK, and ATF2.
The rate of substrate phosphorylation is determined as described
above.
[0029] The term "modulation of MKK activity" includes inhibitory or
stimulatory effects.
[0030] The invention is particularly useful for screening reagents
that inhibit MKK activity. Such reagents are useful for the
treatment or prevention of MKK-mediated disorders, for example,
inflammation and oxidative damage.
[0031] The invention further features a method of treating a
MKK-mediated disorder by administering to a subject in need
thereof, an effective dose of a therapeutic reagent that inhibits
the activity of MKK.
[0032] An "MKK-mediated disorder" is a pathological condition
resulting, at least in part, from excessive activation of an MKK
signal transduction pathway. The MKK signal transduction pathways
are activated by several factors, including inflammation and
stress. MKK-mediated disorders include, for example, ischemic heart
disease, burns due to heat or radiation (UV, X-ray, .gamma.,
.beta., etc.), kidney failure, liver damage due to oxidative stress
or alcohol, respiratory distress syndrome, septic shock, rheumatoid
arthritis, autoimmune disorders, and other types of inflammatory
diseases.
[0033] A "therapeutic reagent" any compound or molecule that
achieves the desired effect on an MKK-mediated disorder when
administered to a subject in need thereof.
[0034] MKK-mediated disorders further include proliferative
disorders, particularly disorders that are stress-related. Examples
of stress-related MKK-mediated proliferative disorders are
psoriasis, acquired immune deficiency syndrome, malignancies of
various tissues of the body, including malignancies of the skin,
bone marrow, lung, liver, breast, gastrointestinal system, and
genito-urinary tract. Preferably, therapeutic reagents inhibit the
activity or expression of MKK inhibit cell growth or cause
apoptosis.
[0035] A therapeutic reagent that "inhibits MKK activity"
interferes with a MKK-mediated signal transduction pathway. For
example, a therapeutic reagent can alter the protein kinase
activity of MKK, decrease the level of MKK transcription or
translation, e.g., an antisense polynucleotide able to bind MKK
mRNA, or suppress MKK phosphorylation of p38, JNK, or ATF2, thus
disrupting the MKK-mediated signal transduction pathway. Examples
of such reagents include antibodies that bind specifically to MKK
polypeptides, and fragments of MKK polypeptides that competitively
inhibit MKK polypeptide activity.
[0036] A therapeutic reagent that "enhances MKK activity"
supplements a MKK-mediated signal transduction pathway. Examples of
such reagents include the MKK polypeptides themselves, which can be
administered in instances where the MKK-mediated disorder is caused
by under expression of the MKK polypeptide, or expression of a
mutant MKK polypeptide. In addition, portions of DNA encoding an
MKK polypeptide can be introduced into cells that under express an
MKK polypeptide.
[0037] A "therapeutically effective amount" is an amount of a
reagent sufficient to decrease or prevent the symptoms associated
with the MKK-mediated disorder.
[0038] Therapeutic reagents for treatment of MKK-mediated disorders
identified by the methods of the invention are administered to a
subject in a number of ways known to the art, including
parenterally by injection, infusion, sustained-release injection or
implant, intravenously, intraperitoneally, intramuscularly,
subcutaneously, or transdermally. Epidermal disorders and disorders
of the epithelial tissues are treated by topical application of the
reagent. The reagent is mixed with other compounds to improve
stability and efficiency of delivery (e.g., liposomes,
preservatives, or dimethyl sulfoxide (DMSO)). Polynucleotide
sequences, including antisense sequences, can be therapeutically
administered by techniques known to the art resulting in
introduction into the cells of a subject suffering from the
MKK-mediated disorder. These methods include the use of viral
vectors (e.g., retrovirus, adenovirus, vaccinia virus, or herpes
virus), colloid dispersions, and liposomes.
[0039] The materials of the invention are ideally suited for the
preparation of a kit for the detection of the level or activity of
MKK. Accordingly, the invention features a kit comprising an
antibody that binds MKK, or a nucleic acid probe that hybridizes to
a MKK polynucleotide, and suitable buffers. The probe or monoclonal
antibody can be labeled to detect binding to a MKK polynucleotide
or protein. In a preferred embodiment, the kit features a labeled
antibody to MKK.
[0040] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0041] Other features and advantages of the invention will be
apparent from the detailed description, and from the claims.
DETAILED DESCRIPTION
[0042] The drawings will first be described.
[0043] Drawings
[0044] FIG. 1 is a comparison of the amino acid sequences of MKK3
(SEQ ID NO: 2), MKK4-.alpha. (SEQ ID NO: 6), the human MAP kinase
kinases MEK1 (SEQ ID NO: 11) and MEK2 (SEQ ID NO: 12), and the
yeast HOG1 MAP kinase kinase PBS2 (SEQ ID NO: 13). Sequences were
compared using the PILE-UP program (version 7.2; Wisconsin Genetics
Computer Group). The protein sequences are presented in single
letter code (A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H,
His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R,
Arg; S, Ser; T, Thr; V, Val; W, Trp, and Y, Tyr). The PBS2 sequence
is truncated at both the NH.sub.2-- (<) and COOH-- (>)
termini. Gaps introduced into the sequences to optimize the
alignment are illustrated by a dash. Identical residues are
indicated by a period. The sites of activating phosphorylation in
MEK are indicated by asterisks.
[0045] FIG. 2A is a dendrogram showing the relationship between
members of the human and yeast MAP kinase kinases. The dendrogram
was created by the unweighted pair-group method with the use of
arithmetic averages (PILE-UP program). The human (hu) MAP kinase
kinases MEK1, MEK2, MKK3, and MKK4; the Saccharomyces cerevisiae
(sc) MAP kinase kinases PBS2, MKK1, and STE7; and the Saccharomyces
pombe (sp) MAP kinase kinases WIS1 and BYR1 are presented.
[0046] FIG. 2B is a dendrogram showing the relationship between
MKKs. The dendrogram was created as described for FIG. 2A.
[0047] FIG. 3 is a schematic representation of the ERK, p38, and
JNK signal transduction pathways. MEK1 and MEK2 are activators of
the ERK subgroup of MAP kinase. MKK3 and MKK4 are activators of the
p38 MAP kinase. MKK4 is identified as an activator of both the p38
and JNK subgroups of MAP kinase.
[0048] FIGS. 4A-4D are a representation of the nucleic acid (SEQ ID
NO: 1) and amino acid sequences (SEQ ID NO: 2) for MKK3.
[0049] FIGS. 5A-5C are a representation of the nucleic acid (SEQ ID
NO: 3) and amino acid sequences (SEQ ID NO: 4) for MKK6.
[0050] FIGS. 6A-6F are a representation of the nucleic acid (SEQ ID
NO: 5) and amino acid sequences (SEQ ID NO: 6) for MKK4.alpha..
[0051] FIGS. 7A-7F are a representation of the nucleic acid (SEQ ID
NO: 7) and amino acid sequences (SEQ ID NO: 8) for MKK4.beta..
[0052] FIGS. 8A-8F are a representation of the nucleic acid (SEQ ID
NO: 9) and amino acid sequences (SEQ ID NO: 10) for
MKK4.gamma..
[0053] FIG. 9 is a representation of the deduced primary structure
of MKK7 (SEQ ID NO: 18) compared with hep (SEQ ID NO: 21), the MAP
kinase kinases MEK1 (MKK 1; SEQ ID NO: 11), MEK2 (MKK2; SEQ ID NO:
12), MKK3 (SEQ ID NO: 2), MKK4.gamma. (SEQ ID NO: 10), MKK5 (SEQ ID
NO: 22), and MKK6 (SEQ ID NO: 4) using the PILE-UP program (version
7,2; Wisconsin Genetics Computer Group). Gaps introduced into the
sequences to optimize the alignment are illustrated with a dash
(-). Identity is indicated with a dot (.). The sites of activating
phosphorylation of MAP kinase kinases (2, 27, 37, and 38) are
indicated with asterisks (*)
[0054] FIGS. 10A-10D are a representation of the nucleic acid (SEQ
ID NO: 17) and amino acid (SEQ ID NO: 18) sequences for MKK7.
[0055] FIGS. 11A-11D are a representation of the nucleic acid (SEQ
ID NO: 19) and amino acid (SEQ ID NO: 20) sequences of MKK7b.
[0056] FIGS. 12A-12B are a representation of the nucleic acid (SEQ
ID NO: 25) and amino acid (SEQ ID NO: 26) sequences of human
MKK7.
[0057] FIGS. 13A-13D are a representation of the nucleic acid (SEQ
ID NO: 27) and amino acid (SEQ ID NO: 28) sequences of murine
MKK7c.
[0058] FIGS. 14A-14D are a representation of the nucleic acid (SEQ
ID NO: 29) and amino acid (SEQ ID NO: 30) sequences of murine
MKK7d.
[0059] FIGS. 15A-15D are a representation of the nucleic acid (SEQ
ID NO: 31) and amino acid (SEQ ID NO: 32) sequences of murine
MKK7e.
[0060] FIG. 16A is a graph of data from a transfection assay in
which cells were co-transfected with AP-1 reporter plasmid
pTRE-Luciferase with expression vectors for MKK4, MKK7, JNK1,
JNK1(APF), or control vector.
[0061] FIG. 16B is a graph of a transfection assay in which cells
were co-transfected with a GAL4-ATF2 fusion vector and an
expression vector for MKK4, MKK7, JNK1, JNK1(APF), or control
vector.
HUMAN MITOGEN-ACTIVATED PROTEIN KINASE KINASES
[0062] The human MAP kinase kinases MKK3 and MKK4 (MKK3/4), and
MKK7, described herein mediate the transduction of specific signals
from the cell surface to the nucleus along specific pathways. These
signal transduction pathways are initiated by factors such as
cytokines, UV radiation, osmotic shock, and oxidative stress.
Activation of MKK3/4, MKK6, and MKK7 results in activation of the
MAP kinases. p38 is activated by MKK3 and MKK4. JNK is activated by
MKK4 and MKK7. p38 and JNK in turn activate a group of related
transcription factors such as ATF2, ATFa, and CRE-BPa. These
transcription factors in turn activate expression of specific
genes. For example, ATF2 in known to activate expression of human T
cell leukemia virus 1 (Wagner and Green (1993) Science 262:395),
transforming growth factor-b2 (Kim et al. (1992) supra),
interferon-.beta. (Du et al. (1993) Cell 74:887), and E-selectin
(DeLuca et al. (1994) J. Biol. Chem. 269:19193). In addition, ATF2
is implicated in the function of a T cell-specific enhancer
(Georgopoulos et al. (1992) Mol. Cell. Biol. 12:747).
[0063] The JNK group of MAP kinases is activated by exposure of
cells to environmental stress or by treatment of cells with
pro-inflammatory cytokines (Gupta et al. (1994) EMBO J.
15:2760-2770; Drijard et al. (1991) Cell 76:1025-1037; Kyriakis et
al. (1994) Nature 369:156-160; Sluss et al. (1994) Mol. Cell. Biol.
14:8376-8384; Kallunki et al. (1994) Genes & Dev. 8:2996-3007).
Targets of the JNK signal transduction pathway include the
transcription factors ATF2 and c-jun (Whitmarsh & Davis (1996)
J. Mol. Med. 74:589-607). These transcription factors are members
of the bZIP group that bind as homo- and hetero-dimeric complexes
to AP-1 and AP-1-like sites in the promoters of many genes (Curran
& Franza (1988) Cell 55:395-397). JNK binds to an
NH.sub.2-terminal region of ATF2 and c-Jun and phosphorylates two
sites within the activation domain of each transcription factor
(Drijard et al. (1994) Cell 76:1025-1037; van Dam et al. (1995)
EMBO J. 14:1798-1811; Livingstone et al. (1995) EMBO J.
14:1785-1797). This phosphorylation leads to increased
transcriptional activity (Whitmarsh, supra). Together, these
biochemical studies indicate that the JNK signal transduction
pathway contributes to the regulation of AP-1 transcriptional
activity in response to cytokines and environmental stress
(Whitmarsh, supra). Strong support for this hypothesis is provided
by genetic evidence indicating that the JNK signaling pathway is
required for the normal regulation of AP-1 transcriptional activity
(Yang et al. (1997) Proc. Natl. Acad. Sci. USA, 94:3004-3009).
[0064] JNK is activated by dual phosphorylation on Thr-183 and
Tyr-185 (Drijard, supra). MKK4 (also known as SEKI) was the first
MAP kinase kinase identified as a component of the JNK signal
transduction pathway (Drijard et al. (1995) Science 267:682-685;
Lin et al. (1995) Science 268:286-290; Sanchez et al. (1994) Nature
372:794-798). Biochemical studies demonstrate that MKK4
phosphorylates and activates JNK (Drijard et al. (1995) Science
267:682-685; Lin et al. (1995) Science 268:286-290; Sanchez et al.
(1994) Nature 372:794-798). However, the function of MKK4 may not
be restricted to the JNK signal transduction pathway because MKK4
also phosphorylates and activates p38 MAP kinase (Drijard et al.
(1995) Science 267:682-685; Lin et al. (1995) Science 268:286-290).
This specificity of MKK4 to activate both JNK and p38 MAP kinase
provides a mechanism that may account for the co-ordinate
activation of these MAP kinases in cells treated with cytokines or
environmental stress (Davis (1994) Trends Biochem. Sci.
19:470-473). However, this co-ordinate activation is not always
observed. For example, JNK activation in the liver correlates with
decreased p38 MAP kinase activity (Mendelson et al. (1996) Proc.
Natl. Acad. Sci. USA 93:12908-12913). These data suggest that the
properties of MKK4 are insufficient to account for the regulation
of JNK in vivo.
[0065] The isolation of human MKKs is described in Example 1,
Example 22, Drijard et al. ((1995) Science 267:682-685, hereby
specifically incorporated by reference), and Raingeaud et al.
((1995) Mol. Cell. Biol. 16:1247-1255). Distinctive regions of the
yeast PBS2 sequence were used to design polymerase chain reaction
(PCR) primers. Amplification of human brain mRNA with these primers
resulted in the formation of specific products which were cloned
into a plasmid vector and sequenced. Two different complementary
DNAs (cDNAs) that encoded human protein kinases were identified:
one encoding a 36 kD protein (MKK3), and one encoding a 44 kD
protein (MKK4). MKK4 includes 3 isoforms that vary slightly at the
NH.sub.2-terminal, identified as .alpha., .beta., and .gamma.. The
amino acid sequences of MKK3 (SEQ ID NO: 2), MKK4-.alpha. (SEQ ID
NO: 6), MKK4-.beta. (SEQ ID NO: 8), and MKK4-.gamma. (SEQ ID NO:
10) are shown in FIG. 1. The nucleic acid and amino acid sequences
of MKK3 (FIG. 4), MKK6 (FIG. 5), MKK4-.alpha. (FIG. 6), MKK4-.beta.
(FIG. 7), and MKK4-.gamma. (FIG. 8) are also provided. MKK6 was
isolated from a human skeletal muscle library by
cross-hybridization with MKK3. Except for differences at the
N-terminus, MKK6 is highly homologous to MKK3. Other human MKK3 and
MKK4 isoforms that exist can be identified by the method described
in Example 1.
[0066] The expression of these human MKK isoforms was examined by
Northern (RNA) blot analysis of mRNA isolated from eight adult
human tissues (Example 2). Both protein kinases were found to be
widely expressed in human tissues, with the highest expression seen
in skeletal muscle tissue.
[0067] The substrate specificity of MKK3 was investigated in an in
vitro phosphorylation assay with recombinant epitope-tagged MAP
kinases (JNK1, p38, and ERK2) as substrates (Example 3). MKK3
phosphorylated p38, but did not phosphorylate JNK1 or ERK2.
Phosphoaminoacid analysis of p38 demonstrated the presence of a
phosphothreonine and phosphotyrosine. Mutational analysis of p38
demonstrated that replacement of phosphorylation sites Thr.sup.180
and Tyr.sup.182 with Ala and Phe, respectively, blocked p38
phosphorylation. These results establish that MKK3 functions in
vitro as a p38 MAP kinase kinase.
[0068] Studies of the in vitro substrate specificity of MKK4 are
described in Example 4. MKK4 incubated with [.gamma.-.sup.32P] ATP,
and JNK1, p38, or ERK2 was found to phosphorylate both p38 and
JNK1. MKK4 activation of JNK and p38 was also studied by incubating
MKK4 with wild-type or mutated JNK1 or p38. The p38 substrate ATF2
was included in each assay. MKK4 was found to exhibit less
autophosphorylation than MKK3. MKK4 was also found to be a
substrate for activated MAP kinase. Unlike MKK3, MKK4 was also
found to activate JNK1. MKK4 incubated with wild-type JNK1, but not
mutated JNK1, resulted in increased phosphorylation of ATF2. These
results establish that MKK4 is a p38 MAP kinase kinase that also
phosphorylates the JNK subgroup of MAP kinases.
[0069] In vivo activation of p38 by UV-stimulated MKK3 is described
in Example 5. Cells expressing MKK3 were exposed in the presence or
absence of UV radiation. MKK3 was isolated by immunoprecipitation
and used for protein kinase assays with the substrates p38 or JNK.
ATF2 was included in some assays as a substrate for p38 and JNK.
MKK3 from non-activated cultured COS cells caused a small amount of
phosphorylation of p38 MAP kinase, resulting from basal activity of
MKK3. MKK3 from UV-irradiated cells caused increased
phosphorylation of p38 MAP kinase, but not of JNK1. An increase in
p38 activity was also detected in assays in which ATF2 was included
as a substrate. These results establish that MKK3 is activated by
UV radiation.
[0070] The effect of expression of MKK3 and MKK4 on p38 activity
was examined in COS-1 cells (Example 6). Cells were transfected
with a vector encoding p38 and a MEK1, MKK3, or MKK4. Some of the
cells were also exposed to EGF or UV radiation. p38 was isolated by
immunoprecipitation and assayed for activity with
[.gamma.-.sup.32P]ATP and ATF2. The expression of the ERK activator
MEK1 did not alter p38 phosphorylation of ATF2. In contrast,
expression of MKK3 or MKK4 caused increased activity of p38 MAP
kinase. The activation of p38 caused by MKK3 and MKK4 was similar
to that observed in UV-irradiated cells, and was much greater than
that detected in EGF-treated cells. These in vitro results provide
evidence that MKK3 and MKK4 activate p38 in vivo.
[0071] A series of experiments was conducted to examine the
potential regulation of ATF2 by JNK1. These experiments are
described in Gupta et al. (1995) Science 267:389-393, hereby
specifically incorporated by reference. The effect of UV radiation
on ATF2 phosphorylation was investigated in COS-1 cells transfected
with and without epitope-tagged JNK1 (Example 7). Cells were
exposed to UV radiation, and JNK1 and JNK2 visualized by in-gel
protein kinase assay with the substrate ATF2. JNK1 and JNK2 were
detected in transfected and non-transfected cells exposed to UV
radiation; however, JNK1 levels were higher in the transfected
cells. These results demonstrate that ATF2 is a substrate for the
JNK1 and JNK2 protein kinases, and that these protein kinases are
activated in cells exposed to UV light.
[0072] The site of JNK1 phosphorylation of ATF2 was examined by
deletion analysis (Example 8). Progressive NH.sub.2-terminal domain
deletion GST-ATF2 fusion proteins were generated, and
phosphorylation by JNK1 isolated from UV-irradiated cells was
examined. The results showed that JNK1 requires the presence of
ATF2 residues 1-60 for phosphorylation of the NH.sub.2-terminal
domain of ATF2.
[0073] The ATF2 residues required for binding of JNK1 were
similarly examined. JNK1 was incubated with immobilized ATF2,
unbound JNK1 was removed by extensive washing, and bound JNK1 was
detected by incubation with [.gamma.-.sup.32P]ATP. Results indicate
that residues 20 to 60 of ATF2 are required for binding and
phosphorylation by JNK1. A similar binding interaction between ATF2
and the 55 kD JNK2 protein kinase has also been observed.
[0074] Phosphorylation by JNK1 was shown to reduce the
electrophoretic mobility of ATF2 (Example 9). Phosphoamino acid
analysis of the full-length ATF2 molecule (residues 1-505)
demonstrated that JNK phosphorylated both Thr and Ser residues. The
major sites of Thr and Ser phosphorylation were located in the
NH.sub.2 and COOH terminal domains, respectively. The
NH.sub.2-terminal sites of phosphorylation were identified as
Thr.sup.69 and Thr.sup.71 by phosphopeptide mapping and mutational
analysis. These sites of Thr phosphorylation are located in a
region of ATF2 that is distinct from the sub-domain required for
JNK binding (residues 20 to 60).
[0075] The reduced electrophoretic mobility seen with
phosphorylation of ATF2 was investigated further (Example 10). JNK1
was activated in CHO cells expressing JNK1 by treatment with UV
radiation, pro-inflammatory cytokine interleukin-1 (IL-1), or
serum. A decreased electrophoretic mobility of JNK1-activated ATF2
was observed in cells treated with UV radiation and IL-1. Smaller
effects were seen after treatment of cells with serum. These
results indicate that ATF2 is an in viva substrate for JNK1.
[0076] The effect of UV radiation on the properties of wild-type
(Thr.sup.69, 71) and phosphorylation-defective (Ala.sup.69, 71)
ATF2 molecules was investigated (Example 11). Exposure to UV caused
a decrease in the electrophoretic mobility of both endogenous and
over-expressed wild-type ATF2. This change in electrophoretic
mobility was associated with increased ATF2 phosphorylation. Both
the electrophoretic mobility shift and increased phosphorylation
were blocked by the replacement of Thr.sup.69 and Thr.sup.71 with
Ala in ATF2. This mutation also blocked the phosphorylation of ATF2
on Thr residues in vivo.
[0077] Transcriptional activities of fusion proteins consisting of
the GAL4 DNA binding domain and wild-type or mutant ATF2 were
examined (Example 12). Point mutations at Thr.sup.69 and/or
Thr.sup.71 of ATF2 significantly decreased the transcriptional
activity of ATF2 relative to the wild-type molecule, indicating the
physiological relevance of phosphorylation at these sites for
activity.
[0078] The binding of JNK1 to the NH.sub.2-terminal activation
domain of ATF2 (described in Example 8) suggested that a
catalytically inactive JNK1 molecule could function as a dominant
inhibitor of the wild-type JNK1 molecule. This hypothesis was
investigated by examining the effect of a catalytically inactive
JNK1 molecule on ATF2 function (Example 13). A
catalytically-inactive JNK1 mutant was constructed by replacing the
sites of activating Thr.sup.183 and Tyr.sup.185 phosphorylation
with Ala and Phe, respectively (Ala.sup.183,Phe.sup.185, termed
"dominant-negative"). Expression of wild-type JNK1 caused a small
increase in serum-stimulated ATF2 transcriptional activity. In
contrast, dominant-negative JNK1 inhibited both control and
serum-stimulated ATF2 activity. This inhibitory effect results from
the non-productive binding of the JNK1 mutant to the ATF2
activation domain, effectively blocking ATF2 phosphorylation.
[0079] The tumor suppressor gene product Rb binds to ATF2 and
increases ATF2-stimulated gene expression (Kim et al. (1992) Nature
358:331). Similarly, the adenovirus oncoprotein E1A associates with
the DNA binding domain of ATF2 and increases ATF2-stimulated gene
expression by a mechanism that requires the NH.sub.2-terminal
activation domain of ATF2 (Liu and Green (1994) Nature 368:520).
ATF2 transcriptional activity was investigated with the luciferase
reporter gene system in control, Rb-treated, and E1A-treated cells
expressing wild-type or mutant ATF2 molecules (Example 14). Rb and
E1A were found to increase ATF2-stimulated gene expression of both
wild-type and mutant ATF2. However, mutant ATF2 caused a lower
level of reporter gene expression than did wild-type ATF2.
Together, these results indicate a requirement for ATF2
phosphorylation (on Thr.sup.69 and Thr.sup.71) plus either Rb or
E1A for maximal transcriptional activity. Thus, Rb and E1A act in
concert with ATF2 phosphorylation to control transcriptional
activity.
[0080] A series of experiments were conducted to examine the action
of p38 activation and to establish the relationship of the p38 MAP
kinase pathway to the ERK and JNK signal transduction pathways
(Raingeaud et al. (1995) J. Biol. Chem. 270:7420, hereby
specifically incorporated by reference). Initially, the substrate
specificity of p38 was investigated by incubating p38 with proteins
that have been demonstrated to be substrates for the ERK and/or JNK
groups of MAP kinases (Example 15). We examined the phosphorylation
of MBP (Erickson et al. (1990) J. Biol. Chem. 265:19728), EGF-R
(Northwood et al. (1991) J. Biol. Chem. 266:15266), cytoplasmic
phospholipase A.sub.2 (cPLA.sub.2) (Lin et al. (1993) Cell 72:269),
c-Myc (Alvarez et al. (1991) J. Biol. Chem. 266:15277), I.kappa.B,
c-Jun, and wild-type (Thr.sup.69, 71) or mutated (Ala.sup.69, 71)
ATF2. p38 phosphorylated MBP and EGF-R, and to a lesser extent
I.kappa.B, but not the other ERK substrates, demonstrating that the
substrate specificity of p38 differs from both the ERK and JNK
groups of MAP kinases. Wild-type ATF2, but not mutated ATF2
(Ala.sup.69, 71), was found to be an excellent p38 substrate.
[0081] The phosphorylation of ATF2 by p38 was associated with an
electrophoretic mobility shift of ATF2 during polyacrylamide gel
electrophoresis. We tested the hypothesis that p38 phosphorylates
ATF2 at the same sites as JNK1 by replacing Thr.sup.69 and
Thr.sup.71 with Ala (Ala.sup.69, 71). It was found that p38 did not
phosphorylate mutated ATF2, which demonstrates that p38
phosphorylates ATF2 within the NH2-terminal activation domain on
Thr.sup.69 and Thr.sup.71.
[0082] A comparison of the binding of JNK and p38 to ATF2 was
conducted by incubating extracts of cells expressing JNK1 or p38
with epitope alone (GST) or GST-ATF2 (residues 1-109 containing the
activation domain) (Example 16). Bound protein kinases were
detected by Western blot analysis. The results demonstrate that
both p38 and JNK bind to the ATF2 activation domain.
[0083] EGF and phorbol ester are potent activators of the ERK
signal transduction pathway (Egan and Weinberg (1993) Nature
365:781), causing maximal activation of the ERK sub-group of MAP
kinases. These treatments, however, cause only a small increase in
JNK protein kinase activity (Drijard et al. (1994) supra; Hibi et
al. (1993) supra). The effects of EGF or phorbol esters, as well UV
radiation, osmotic shock, interleukin-1, tumor necrosis factor, and
LPS, on p38 activity were all tested (Example 17). Significantly,
EGF and phorbol ester caused only a modest increase in p38 protein
kinase activity, whereas environmental stress (UV radiation and
osmotic shock) caused a marked increase in the activity of both p38
and JNK. Both p38 and JNK were activated in cells treated with
pro-inflammatory cytokines (TNF and IL-1) or endotoxic LPS.
Together, these results indicate that p38, like JNK, is activated
by a stress-induced signal transduction pathway.
[0084] ERKs and JNKs are activated by dual phosphorylation within
the motifs Thr-Glu-Tyr and Thr-Pro-Tyr, respectively. In contrast,
p38 contains the related sequence Thr-Gly-Tyr. To test whether this
motif is relevant to the activation of p38, the effect of the
replacement of Thr-Gly-Tyr with Ala-Gly-Phe was examined (Example
18). The effect of UV radiation on cells expressing wild-type
(Thr.sup.180,Tyr.sup.182) or mutant p38 (Ala.sup.180, Phe.sup.182)
was studied. Western blot analysis using an anti-phosphotyrosine
antibody demonstrated that exposure to UV radiation caused an
increase in the Tyr phosphorylation of p38. The increased Tyr
phosphorylation was confirmed by phosphoamino acid analysis of p38
isolated from [.gamma.-.sup.32P] phosphate-labeled cells. This
analysis also demonstrated that UV radiation caused increased Thr
phosphorylation of p38. Significantly, the increased
phosphorylation on Thr.sup.180 and Tyr.sup.182 was blocked by the
Ala.sup.180/Phe.sup.182 mutation. This result demonstrates that UV
radiation causes increased activation of p38 by dual
phosphorylation.
[0085] It has recently been demonstrated that ERK activity is
regulated by the mitogen-induced dual specificity phosphatases MKP1
and PAC1 (Ward et al. (1994) Nature 367:651). The activation of p38
by dual phosphorylation (Example 18) raises the possibility that
p38 may also be regulated by dual specificity phosphatases. We
examined the effect of MKP1 and PAC1 on p38 MAP kinase activation
(Example 19). Cells expressing human MKP1 and PAC1 were treated
with and without UV radiation, and p38 activity measured. The
expression of PAC1 or MKP1 was found to inhibit p38 activity. The
inhibitory effect of MKP1 was greater than PAC1. In contrast, cells
transfected with a catalytically inactive mutant phosphatase
(mutant PAC1 Cys.sup.257/Ser) did not inhibit p38 MAP kinase. These
results demonstrate that p38 can be regulated by dual specificity
phosphatases PAC1 and MKP1.
[0086] The sub-cellular distribution of p38 MAP kinase was examined
by indirect immunofluorescence microscopy (Example 20).
Epitope-tagged p38 MAP kinase was detected using the M2 monoclonal
antibody. Specific staining of cells transfected with
epitope-tagged p38 MAP kinase was observed at the cell surface, in
the cytoplasm, and in the nucleus. Marked changes in cell surface
and nuclear p38 MAP kinase were not observed following UV
irradiation, but an increase in the localization of cytoplasmic p38
MAP kinase to the perinuclear region was detected.
[0087] A series of experiments were conducted to study the
activation of JNK by hyper-osmotic media (Example 21). These
experiments were reported by Galcheva-Gargova et al. (1994) Science
265:806, hereby specifically incorporated by reference. CHO cells
expressing epitope-tagged JNK1 were incubated with 0-1000 mM
sorbitol, and JNK1 activity measured in an immune complex kinase
assay with the substrate c-Jun. Increased JNK1 activity was
observed in cells incubated for 1 hour with 100 mM sorbitol.
Increased JNK1 activity was observed within 5 minutes of exposure
to 300 mM sorbitol. Maximal activity was observed 15 to 30 minutes
after osmotic shock with a progressive decline in JNK1 activity at
later times. The activation of JNK by osmotic shock was studied in
cells expressing wild-type (Thr.sup.183, Tyr.sup.185) or mutated
(Ala.sup.183, Phe.sup.185) JNK1. JNK1 activity was measured after
incubation for 15 minutes with or without 300 mM sorbitol. Cells
expressing wild-type JNK1 showed increased JNK1 activity, while
cells expressing mutated JNK1 did not. These results demonstrate
that the JNK signal transduction pathway is activated in cultured
mammalian cells exposed to hyper-osmotic media.
[0088] The results of the above-described experiments are
illustrated in FIG. 3, which diagrams the ERK, p38, and JNK MAP
kinase signal transduction pathways. ERKs are potently activated by
treatment of cells with EGF or phorbol esters. In contrast, p38 is
only slightly activated under these conditions (Example 15).
However, UV radiation, osmotic stress, and inflammatory cytokines
cause a marked increase in p38 activity. This difference in the
pattern of activation of ERK and p38 suggests that these MAP
kinases are regulated by different signal transduction pathways.
The molecular basis for the separate identity of these signal
transduction pathways is established by the demonstration that the
MAP kinase kinases that activate ERK (MEK1 and MEK2) and p38 (MKK3,
MKK4, and MKK6) are distinct.
[0089] The isolation of murine and human MKK7 is described in
Example 22. Distinctive regions of the Drosophila MAP kinase kinase
hep sequence were used to design polymerase chain reaction (PCR)
primers. Amplification of murine testis mRNA with these primers
resulted in the formation of specific products which were cloned
into a plasmid vector and sequenced. One sequence related to hep
was identified and used to screen a murine testis library. Five
DNAs (cDNAs) that encoded protein kinases were identified: one
encoding a MAP protein kinase kinase (MKK7). The others encoded
various splice variants: MKK7b (a partial sequence appears in FIG.
11), MKK7c (FIG. 13), MKK7d (FIG. 14), MKK7e (FIG. 15). The deduced
amino acid sequences of MKK7 (SEQ ID NO: 18) and hep (SEQ ID NO:
21) are shown in FIG. 9, and compared to the MAP kinase kinases
MEK1 (SEQ ID NO: 11), MEK2 (SEQ ID NO: 12), MKK3 (SEQ ID NO: 2),
MKK4 (SEQ ID NO: 10), MKK5 (SEQ ID NO: 22), and MKK6 (SEQ ID NO:
4). A human MKK7 was identified by screening a human cDNA library
with a full-length (mouse) MKK7 cDNA probe. The identified partial
sequence (lacking the 3' end) is homologous to mouse MKK7c.
[0090] The expression of MKK7 and MKK4 isoforms was examined by
Northern (RNA) blot analysis of poly A+ mRNA isolated from eight
murine tissues (Example 23). Both protein kinases were found to be
widely expressed.
[0091] The substrate specificity of MKK7 was investigated in an in
vitro phosphorylation assay with recombinant, epitope-tagged MAP
kinases (JNK1, p38, and ERK2) as substrates (Example 24). MKK7
phosphorylated JNK, but did not phosphorylate p38 or ERK2. MKK7 was
phosphorylated by p38 and JNK1.
[0092] MKK7 was found to specifically activate JNK protein kinase
in vivo (Example 25). CHO cells were co-transfected with an
epitope-tagged MAP kinase (JNK1, p38, or ERK2) together with an
empty expression vector or an expression vector encoding MKK1,
MKK4, MKK6, or MKK7 and the product of the phosphorylation reaction
analyzed. MKK7 activated only JNK1, and did so to a greater extent
than did MKK4.
[0093] To test whether MKK7 could cause increased AP-1
transcriptional activity, a co-transfection assay was employed
(Example 26). Co-expression of MKK7 with JNK caused an increase in
AP-1 reporter gene expression that was greater than the increase
seen with MKK4 and JNK. A similar result was seen when ATF2 was
used as the reporter gene. In addition, MKK7 alone was able to
increase expression of ATF2 (FIG. 16).
[0094] MKK isoforms are useful for screening reagents which
modulate MKK activity. Described in the Use section following the
Examples are methods for identifying reagents capable of inhibiting
or activating MKK activity.
[0095] The discovery of human MKK isoforms and MKK-mediated signal
transduction pathways is clinically significant for the treatment
of MKK-mediated disorders. One use of the MKK isoforms is in a
method for screening reagents able to inhibit or prevent the
activation of the MKK-MAP kinase-ATF2 pathways.
EXAMPLES
[0096] The following examples are meant to illustrate, not limit,
the invention.
Example 1
[0097] MKK Protein Kinases
[0098] The primary sequences of MKK3 and MKK4 were deduced from the
sequence of cDNA clones isolated from a human fetal brain
library.
[0099] The primers TTYTAYGGNGCNTTYTTYATHGA (SEQ ID NO: 14) and
ATBCTYTCNGGNGCCATKTA (SEQ ID NO: 15) were designed based on the
sequence of PBS2 (Brewster et al. (1993) Science 259:1760; Maeda et
al. (1994) Nature 369:242). The primers were used in a PCR reaction
with human brain mRNA as template. Two sequences that encoded
fragments of PBS2-related protein kinases were identified.
Full-length human cDNA clones were isolated by screening of a human
fetal brain library (Drijard et al. (1995) Science 267:682-685).
The cDNA clones were examined by sequencing with an Applied
Biosystems model 373A machine. The largest clones obtained for MKK3
(2030 base pairs (bp)) and MKK4 (3576 bp) contained the entire
coding region of these protein kinases.
[0100] The primary structures of MKK3 (SEQ ID NO: 2) and
MKK4-.alpha. (SEQ ID NO: 6) are shown in FIG. 1. An in-frame
termination codon is located in the 5.dbd. untranslated region of
the MKK3 cDNA, but not in the 5' region of the MKK4 cDNA. The MKK4
protein sequence presented starts at the second in-frame initiation
codon.
[0101] These sequences were compared to those of the human MAP
kinase kinases MEK1 (SEQ ID NO : 11) and MEK2 (SEQ ID NO: 12)
(Zheng and Guan (1993) J. Biol. Chem 268:11435) and of the yeast
MAP kinase kinase PBS2 (SEQ ID NO: 13) (Boguslawaski and Polazzi
(1987) Proc. Natl. Acad. Sci. USA 84:5848) (FIG. 1). The identity
and similarity of the kinases with human MKK3 (between subdomains I
and XI) were calculated with the BESTFIT program (version 7.2;
Wisconsin Genetics Computer Group) (percent of identity to percent
of similarity): MEK1, 41%/63%; MEK2, 41%/62%; MKK4.alpha., 52%/73%;
and PBS2, 40%/59%). The identity and similarity of the kinases with
human MKK4.alpha. were calculated to be as follows (percent of
identity to percent of similarity): MEK1, 44%/63%; MEK2, 45%/61%;
MKK3, 52%/73%; and PBS2, 44%/58%.
[0102] The cDNA sequences of MKK3 and MKK4.gamma. have been
deposited in GenBank with accession numbers L36719 and L36870,
respectively. The MKK4.gamma. cDNA sequence contains both the cDNA
sequences of MKK4.alpha. and MKK4.beta., which are generated in
vivo from alternate splicing sites. One of ordinary skill in the
art can readily determine the amino acid sequences of MKK3 and MKK4
isoforms from the deposited cDNA sequences.
Example 2
[0103] Expression of MKK3 and MKK4 mRNA in Adult Human Tissue
[0104] Northern blot analysis was performed with polyadenylated
[poly(A)*] mRNA (2 .mu.g) isolated from human heart, brain,
placenta, lung, liver, muscle, kidney, and pancreas tissues. The
mRNA was fractionated by denaturing agarose gel electrophoresis and
was transferred to a nylon membrane. The blot was probed with the
MKK3 and MKK4 cDNA labeled by random priming with
[.alpha.-.sup.32P]ATP (deoxyadenosine triphosphate) (Amersham
International PLC). MKK3 and MKK4 were expressed in all tissues
examined; the highest expression of MKK3 and MKK4 was found in
skeletal muscle tissue.
[0105] The relation between members of the human and yeast MAP
kinase kinase group is presented as a dendrogram (FIG. 2). MKK3/4
form a unique subgroup of human MAP kinase kinases.
Example 3
[0106] In Vitro Phosphorylation of p38 MAP kinase by MKK3
[0107] GST-JNK1, and GST-ERK2 have been described (Drijard et al.
(1994) supra; Gupta et al. (1995) Science 267:389; Wartmann and
Davis (1994) J. Biol. Chem. 269:6695, each herein specifically
incorporated by reference). GST-p38 MAP kinase was prepared from
the expression vector pGSTag (Dressier et al. (1992) Biotechniques
13:866) and a PCR fragment containing the coding region of the p38
MAP kinase cDNA. GST-MKK3 and MKK4 were prepared with pGEX3X
(Pharmacia-LKB Biotechnology) and PCR fragments containing the
coding region of the MKK3 and MKK4 cDNAs. The GST fusion proteins
were purified by affinity chromatography with the use of
GSH-agarose (Smith and Johnson (1988) Gene 67:31). The expression
vectors pCMV-Flag-JNK1 and pCMV-MEK1 have been described (Drijard
et al. (1994) supra; Wartmann and Davis (1994) supra). The plasmid
pCMV-Flag-p38 MAP kinase was prepared with the expression vector
pCMV5 (Andersson et al. (1989) J. Biol. Chem. 264:8222) and the p38
MAP kinase cDNA. The expression vectors for MKK3 and MKK4 were
prepared by subcloning of the cDNAs into the polylinker of pCDNA3
(Invitrogen). The Flag epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
(SEQ ID NO: 16); Immunex, Seattle, Wash.) was inserted between
codons 1 and 2 of the kinases by insertional overlapping PCR (Ho et
al. (1989) Gene 77:51).
[0108] Protein kinase assays were performed in kinase buffer (25 mM
4-(2-hydroxyethyl)-1-piperazineethansulfonic acid, pH 7.4, 25 mM
.beta.-glycerophosphate, 25 mM MgCl.sub.2, 2 mM dithiothreitol, and
0.1 mM orthovanadate). Recombinant GST-MKK3 was incubated with
[.gamma.-.sup.32P]ATP and buffer, GST-JNK1, GST-p38 MAP kinase, or
GST-ERK2. The assays were initiated by the addition of 1 .mu.g of
substrate proteins and 50 .mu.M [.gamma.-.sup.32P]ATP (10 Ci/mmol)
in a final volume of 25 .mu.l. The reactions were terminated after
30 minutes at 25.degree. C. by addition of Laemmli sample buffer.
The phosphorylation of the substrate proteins was examined after
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by
autoradiography. Phosphoaminoacid analysis was performed by partial
acid hydrolysis and thin-layer chromatography (Drijard et al.
(1994) supra; Alvarez et al. (1991) J. Biol. Chem. 266:15277).
Autophosphorylation of MKK3 was observed in all groups. MKK3
phosphorylated p38 MAP kinase, but not JNK1 or ERK2.
[0109] A similar insertional overlapping PCR procedure was used to
replace Thr.sup.180 and Tyr.sup.182 of p38, with Ala and Phe,
respectively. The sequence of all plasmids was confirmed by
automated sequencing on an Applied Biosystems model 373A machine.
GST-MKK3 was incubated with [.gamma.-.sup.32P]ATP and buffer,
wild-type GST-p38 MAP kinase (TGY), or mutated GST-p38 MAP kinase
(AGF). The phosphorylated proteins were resolved by SDS-PAGE and
detected by autoradiography. Only phosphorylation of wild-type p38
was observed.
Example 4
[0110] In Vitro Phosphorylation and Activation of JNK and p38 MAP
Kinase by MKK4
[0111] Protein kinase assays were conducted as described in Example
3. Recombinant GST-MKK4 was incubated with [.gamma.-.sup.32P] ATP
and buffer, GST-JNK1, GST-p38 MAP kinase, or GST-ERK2. JNK1 and p38
were phosphorylated, as was MKK4 incubated with JNK1 and p38.
[0112] GST-MKK4 was incubated with [.gamma.-.sup.32P]ATP and
buffer, wild-type JNK1 (Thr.sup.183, Tyr.sup.185), or mutated
GST-JNK1 (Ala.sup.183, Phe.sup.185). The JNK1 substrate ATF2 (Gupta
et al. (1995) supra) was included in each incubation. ATF2 was
phosphorylated in the presence of MKK4 and wild-type JNK1. The
results establish that MKK4 phosphorylates and activates both p38
and JNK1.
Example 5
[0113] Phosphorylation and Activation of p38 MAP Kinase by
UV-stimulated MKK3
[0114] Epitope-tagged MKK3 was expressed in COS-1 cells maintained
in Dulbecco's modified Eagle's medium supplemented with fetal
bovine serum (5%)(Gibco-BRL). The cells were transfected with the
lipofectamine reagent according to the manufacturer's
recommendations (Gibco-BRL) and treated with UV radiation or EGF as
described (Drijard et al. (1994) supra).
[0115] The cells were exposed in the absence and presence of UV-C
(40 J/m.sup.2). The cells were solubilized with lysis buffer (20 mM
tris, pH 7.4, 1% TRITON.RTM. X-100, 10% glycerol, 137 mM NaCl, 2 mM
EDTA, 25 mM .beta.-glycerophosphate, 1 mM Na orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, and leupeptin (10 .mu.g/ml)) and
centrifuged at 100,000.times.g for 15 minutes at 4.degree. C. MKK3
was isolated by immunoprecipitation. The epitope-tagged protein
kinases were incubated for 1 hour at 4.degree. C. with the M2
antibody to the Flag epitope (IBI-Kodak) bound to protein
G-Sepharose (Pharmacia-LKB Biotechnology). The immunoprecipitates
were washed twice with lysis buffer and twice with kinase
buffer.
[0116] Protein kinase assays were conducted with the substrate
GST-p38 MAP kinase or JNK1. ATF2 was included in some assays. Basal
levels of MKK3 phosphorylation of p38 MAP kinase were observed.
UV-irradiation resulted in increased phosphorylation of p38 MAP
kinase, but not of JNK1. The increased p38 MAP kinase activity
resulted in increased phosphorylation of ATF2.
Example 6
[0117] Activation of p38 MAP Kinase in Cells Expressing MKK3 and
MKK4
[0118] COS-1 cells were transfected with epitope-tagged p38 MAP
kinase, together with an empty expression vector or an expression
vector encoding MEK1, MKK3, or MKK4.alpha.. Some of the cultures
were exposed to UV radiation (40 J/m.sup.2) or treated with 10 nM
EGF. p38 MAP kinase was isolated by immunoprecipitation with M2
monoclonal antibody, and the protein kinase activity was measured
in the immunecomplex with [.gamma.-.sup.32P] ATP and ATF2 as
substrates. The product of the phosphorylation reaction was
visualized after SDS-PAGE by autoradiography. ATF2 was not
phosphorylated in the control MEK1, or EGF-treated groups, but was
phosphorylated in the MKK3, MKK4, and UV-irradiated groups. MKK3
and MKK4 phosphorylation of ATF2 was similar to that seen with p38
MAP kinase isolated from UV-irradiated cells.
Example 7
[0119] Phosphorylation of ATF2 by JNK1 and JNK2
[0120] COS-1 cells were maintained in Dulbecco's modified Eagle's
medium supplemented with bovine serum albumin (5%) (Gibco-BRL).
Metabolic labeling with [.sup.32]P was performed by incubation of
cells for 3 hours in phosphate-free modified Eagle's medium (Flow
Laboratories Inc.) supplemented with [.sup.32p] orthophosphate (2
mCi/ml) (Dupont-NEN). COS-1 cells were transfected without (Mock)
and with epitope-tagged JNK1 (JNK1). Plasmid expression vectors
encoding the JNK1 cDNA have previously been described (Drijard et
al. (1994) Cell 76:1025, herein specifically incorporated by
reference). Plasmid DNA was transfected into COS-1 cells by the
lipofectamine method (Gibco-BRL). After 48 hours of incubation,
some cultures were exposed to 40 J/m.sup.2 UV radiation and
incubated for 1 hour at 37.degree. C.
[0121] Cells were lysed in 20 mM Tris, pH 7.5, 25 mM
.beta.-glycerophosphate, 10% glycerol, 1% Triton.RTM. X-100, 0.5%
(w/v) deoxycholate, 0.1% (w/v) SDS, 0.137 M NaCl, 2 mM
pyrophosphate, 1 mM orthovanadate, 2 mM EDTA, 10 .mu.g/ml
leupeptin, 1 mM PMSF. Soluble extracts were prepared by
centrifugation in a microfuge for 20 minutes at 4.degree. C. JNK1
immunoprecipitates were also prepared by reaction with a rabbit
antiserum prepared with recombinant JNK1 as an antigen.
[0122] In-gel protein kinase assays were performed with cell
lysates and JNK1 immunoprecipitates after SDS-PAGE by renaturation
of protein kinases, polymerization of the substrate (GST-ATF2,
residues 1-505) in the gel, and incubation with
[.gamma.-.sup.32P]ATP (Drijard et al. (1994) supra) The
incorporation of [.sup.32P] phosphate was visualized by
autoradiography and quantitated with a Phosphorimager and
ImageQuant software (Molecular Dynamics Inc., Sunnyvale, Calif.).
The cell lysates demonstrate the presence of 46 kD and 55 kD
protein kinases that phosphorylate ATF2 in extracts prepared from
UV-irradiated cells. The 46 kD and 55 kD protein kinases were
identified as JNK1 and JNK2, respectively.
Example 8
[0123] Binding of JNK1 to ATF2 and Phosphorylation of the
NH.sub.2-Terminal Activation Domain
[0124] The site of JNK1 phosphorylation of ATF2 was investigated by
generation of progressive NH.sub.2-terminal domain deletions of
ATF2. Plasmid expression vectors encoding ATF2 (pECE-ATF2) (Liu and
Green (1994) and (1990)), have been described. Bacterial expression
vectors for GST-ATF2 fusion proteins were constructed by
sub-cloning ATF2 cDNA fragments from a polymerase chain reaction
(PCR) into pGEX-3X (Pharmacia-LKB Biotechnology Inc.). The sequence
of all constructed plasmids was confirmed by automated sequencing
with an Applied Biosystems model 373A machine. The GST-ATF2
proteins were purified as described (Smith and Johnson (1988) Gene
67:31), resolved by SDS-PAGE and stained with Coomassie blue.
GST-ATF2 fusion proteins contained residues 1-505, 1-349, 350-505,
1-109, 20-109, 40-109, and 60-109.
[0125] The phosphorylation of GST-ATF2 fusion proteins by JNK1
isolated from UV-irradiated cells was examined in an immunocomplex
kinase assay. Immunecomplex kinase assays were performed with Flag
epitope-tagged JNK1 and the monoclonal antibody M2 (IBI-Kodak) as
described by Drijard et al. (1994) supra). Immunecomplex protein
kinase assays were also performed with a rabbit antiserum prepared
with recombinant JNK1 as an antigen. The cells were solubilized
with 20 mM Tris, pH 7.5, 10% glycerol, 1% Triton.RTM. X-100, 0.137
M NaCl, 25 mM .beta.-glycerophosphate, 2 mM EDTA, 1 mM
orthovanadate, 2 mM pyrophosphate, 10 .mu.g/ml leupeptin, and 1 mM
PMSF. JNK1 was immunoprecipitated with protein G-Sepharose bound to
a rabbit polyclonal antibody to JNK or the M2 monoclonal antibody
to the Flag epitope. The beads were washed three times with lysis
buffer and once with kinase buffer (20 mM Hepes, pH 7.6, 20 mM
MgCl.sub.2, 25 mM .beta.-glycerophosphate, 100 .mu.M Na
orthovanadate, 2 mM dithiothreitol). The kinase assays were
performed at 25.degree. C. for 10 minutes with 1 .mu.g of
substrate, 20 .mu.M adenosine triphosphate and 10 .mu.Ci of
[.gamma.-.sup.32P]ATP in 30 .mu.l of kinase buffer. The reactions
were terminated with Laemmli sample buffer and the products were
resolved by SDS-PAGE (10% gel). JNK1 phosphorylates GST-ATF2 fusion
proteins containing residues 1-505, 1-349, 1-109, 20-109, and
40-109, but not 60-109. These results indicate that the presence of
ATF2 residues 1-60 are required for phosphorylation by JNK.
[0126] The binding of immobilized GST-ATF2 fusion proteins was
examined in a solid-phase kinase assay as described by Hibi et al.
((1993) Genes Dev. 7:2135, herein specifically incorporated by
reference). JNK1 from UV-irradiated cells was incubated with
GST-ATF2 fusion proteins bound to GSH-agarose. The agarose beads
were washed extensively to remove the unbound JNK1. Phosphorylation
of the GST-ATF2 fusion proteins by the bound JNK1 protein kinase
was examined by addition of [.gamma.-.sup.32P]ATP. JNK1 bound
GST-ATF2 fusion proteins containing residues 1-505, 1-349, 1-109,
20-109, and 40-109, indicating that the presence of residues 20-60
were required for binding of JNK1 to ATF2.
Example 9
[0127] Phosphorylation of the NH.sub.2-Terminal Activation Domain
of ATF2 on Thr.sup.69 and Thr.sup.71 by JNK1
[0128] The effect of UV radiation on the properties of wild-type
(Thr.sup.69, 71) and phosphorylation-defective (Ala.sup.69, 71)
ATF2 molecules was examined. Mock-transfected and JNK1-transfected
COS cells were treated without and with 40 J/m.sup.2 UV radiation.
The epitope-tagged JNK1 was isolated by immunoprecipitation with
the M2 monoclonal antibody. The phosphorylation of GST-ATF2
(residues 1 to 109) was examined in an immunocomplex kinase assay
as described above. The GST-ATF2 was resolved from other proteins
by SDS-PAGE and stained with Coomassie blue. The phosphorylation of
GST-ATF2 was detected by autoradiography. JNK1-transfected cells,
but not mock-transfected cells, phosphorylated ATF2. JNK1
phosphorylation of ATF2 was greater in cells exposed to UV
radiation. Phosphorylation of ATF2 by JNK1 was associated with a
decreased electrophoretic mobility.
[0129] In a separate experiment, GST fusion proteins containing
full-length ATF2 (residues 1 to 505), an NH.sub.2-terminal fragment
(residues 1 to 109), and a COOH-terminal fragment (residues 95 to
505) were phosphorylated with JNK1 and the sites of phosphorylation
analyzed by phosphoamino acid analysis. The methods used for
phosphopeptide mapping and phosphoamino acid analysis have been
described (Alvarez et al. (1991) J. Biol. Chem. 266:15277). The
horizontal dimension of the peptide maps was electrophoresis and
the vertical dimension was chromatography. The NH.sub.2-terminal
sites of phosphorylation were identified as Thr.sup.69 and
Thr.sup.71 by phosphopeptide mapping and mutational analysis.
Site-directed mutagenesis was performed as described above,
replacing Thr.sup.69 and Thr.sup.71 with Ala. Phosphorylation of
mutated ATF2 was not observed.
Example 10
[0130] Reduced Electrophoretic Mobility of JNK-Activated ATF2
[0131] CHO cells were maintained in Ham's F12 medium supplemented
with 5% bovine serum albumin (Gibco-BRL). Cells were labeled and
transfected with JNK1 as described above. CHO cells were treated
with UV-C (40 J/m.sup.2), IL-1.alpha. (10 ng/ml) (Genzyme), or
fetal bovine serum (20%) (Gibco-BRL). The cells were incubated for
30 minutes at 37.degree. C. prior to harvesting. The
electrophoretic mobility of ATF2 after SDS-PAGE was examined by
protein immuno-blot analysis. A shift in ATF2 electrophoretic
mobility was observed in cells treated with UV, IL-1, and serum.
These results indicate that JNK1 activation is associated with an
electrophoretic mobility shift of ATF2, further suggesting that
ATF2 is an in vivo substrate for JNK1.
Example 11
[0132] Increased ATF2 Phosphorylation After Activation of JNK
[0133] COS-1 cells were transfected without (control) and with an
ATF2 expression vector (ATF2), as described above (Hai et al.
(1989) supra). The effect of exposure of the cells to 40 J/m.sup.2
UV-C was examined. After irradiation, the cells were incubated for
0 or 30 minutes (control) or 0, 15, 30, and 45 minutes (ATF2) at
37.degree. C. and then collected. The electrophoretic mobility of
ATF2 during SDS-PAGE was examined by protein immuno-blot analysis
as described above. The two electrophoretic mobility forms of ATF2
were observed in ATF2-transfected cells, but not in control
cells.
[0134] The phosphorylation state of wild-type (Thr.sup.69, 71) ATF2
and mutated (Ala.sup.69, 71) ATF2 was examined in cells labeled
with [.sup.32]P, treated without and with 40 J/m.sup.2 UV-C, and
then incubated at 37.degree. C. for 30 minutes (Hai et al. (1989)
supra). The ATF2 proteins were isolated by immunoprecipitation and
analyzed by SDS-PAGE and autoradiography. The phosphorylated ATF2
proteins were examined by phosphoamino acid analysis as described
above. Both forms of ATF2 contained phosphoserine, but only
wild-type ATF2 contained phosphothreonine.
[0135] Tryptic phosphopeptide mapping was used to compare ATF2
phosphorylated in vitro by JNK1 with ATF2 phosphorylated in COS-1
cells. A map was also prepared with a sample composed of equal
amounts of in vivo and in vitro phosphorylated ATF2 (Mix). Mutation
of ATF2 at Thr.sup.69 and Thr.sup.71 resulted in the loss of two
tryptic phosphopeptides in maps of ATF2 isolated from UV-irradiated
cells. These phosphopeptides correspond to mono- and
bis-phosphorylated peptides containing Thr.sup.69 and Thr.sup.71.
Both of these phosphopeptides were found in maps of ATF2
phosphorylated by JNK1 in vitro.
Example 12
[0136] Inhibition of ATF2-Stimulated Gene Expression by Mutation of
the Phosphorylation Sites Thr.sup.69 and Thr.sup.71
[0137] A fusion protein consisting of ATF2 and the GAL4 DNA binding
domain was expressed in CHO cells as described above. The activity
of the GAL4-ATF2 fusion protein was measured in co-transfection
assays with the reporter plasmid pG5E1bLuc (Seth et al. (1992) J.
Biol. Chem. 267:24796, hereby specifically incorporated by
reference). The reporter plasmid contains five GAL4 sites cloned
upstream of a minimal promoter element and the firefly luciferase
gene. Transfection efficiency was monitored with a control plasmid
that expresses .beta.-galactosidase (pCH110; Pharmacia-LKB
Biotechnology). The luciferase and .beta.-galactosidase activity
detected in cell extracts was measured as the mean activity ratio
of three experiments (Gupta et al. (1993) Proc. Natl. Acad. Sci.
USA 90:3216, hereby specifically incorporated by reference). The
results, shown in Table 1, demonstrate the importance of
phosphorylation at Thr.sup.69 and Thr.sup.71 for transcriptional
activity.
1TABLE 1 INHIBITION OF ATF-2 STIMULATED GENE EXPRESSION BY MUTATION
OF THE PHOSPHORYLATION SITES THR.sup.69,71 LUCIFERASE ACTIVITY
PROTEIN (Light Units/OD) GAL4 45 GAL4-ATF2 (wild type) 320,000
GAL4-ATF2 (Ala.sup.69) 24,000 GAL4-ATF2 (Ala.sup.71) 22,000
GAL4-ATF2 (Ala.sup.69,71) 29,000 GAL4-ATF2 (Glu.sup.69) 27,000
Example 13
[0138] Effect of Dominant-Negative JNK1 Mutant on ATF2 Function
[0139] The luciferase reporter plasmid system was used to determine
the effect of point mutations at the ATF2 phosphorylation sites
Thr.sup.69 and Thr.sup.71 in serum-treated CHO cells transfected
with wild-type (Thr.sup.183, Tyr.sup.185) or mutant (Ala.sup.183,
Phe.sup.185) JNK1. Control experiments were done with
mock-transfected cells. The CHO cells were serum-starved for 18
hours and then incubated without or with serum for 4 hours.
Expression of wild-type ATF2 caused a small increase in
serum-stimulated ATF2 transcriptional activity. In contrast, mutant
JNK1 inhibited both control and serum-stimulated ATF2 activity.
Example 14
[0140] Effect of Tumor Suppressor Gene Product Rb and Adenovirus
Oncoprotein E1A on ATF2-Stimulated Gene Expression
[0141] The effect of expression of the Rb tumor suppressor gene
product and adenovirus oncoprotein E1A on ATF2 transcriptional
activity were investigated with a luciferase reporter plasmid and
GAL4-ATF2 (residues 1-505), as described above. Cells were
transfected with wild-type (Thr.sup.69, 71) or mutated (Ala.sup.69,
71) ATF2. No effect of Rb or E1A on luciferase activity was
detected in the absence of GAL4-ATF2. Rb and E1A were found to
increase ATF2-stimulated gene expression of both wild-type and
mutated ATF2. However, mutated ATF2 caused a lower level of
reporter gene expression than did wild-type ATF2. These results
indicate a requirement for ATF2 phosphorylation (on Thr.sup.69 and
Thr.sup.71) plus either Rb or E1A for maximal transcriptional
activity.
Example 15
[0142] Substrate Specificity of p38 MAP Kinase
[0143] Substrate phosphorylation by p38 MAP kinase was examined by
incubation of bacterially-expressed p38 MAP kinase with I.kappa.B,
cMyc, EGF-R, cytoplasmic phospholipase A.sub.2 (cPLA.sub.2), c-jun,
and mutated ATF2 (Thr.sup.69, 71) and ATP(.gamma.-.sup.32P]
(Raingeaud et al. (1995) J. Biol. Chem 270:7420, herein
specifically incorporated by reference). GST-I.kappa.B was provided
by Dr D. Baltimore (Massachusetts Institute of Technology).
GST-cMyc (Alvarez et al. (1991) J. Biol. Chem. 266:15277),
GST-EGF-R (residues 647-688) (Koland et al. (1990) Biochem.
Biophys. Res. Commun. 166:90), and GST-c-Jun (Drijard et al. (1994)
supra) have been described. The phosphorylation reaction was
terminated after 30 minutes by addition of Laemmli sample buffer.
The phosphorylated proteins were resolved by SDS-PAGE and detected
by autoradiography. The rate phosphorylation of the substrate
proteins was quantitated by PhosphorImager (Molecular Dynamics
Inc.) analysis. The relative phosphorylation of ATF2, MBP, EGF-R,
and I.kappa.B was 1.0, 0.23, 0.04, and 0.001, respectively.
Example 16
[0144] Binding of p38 MAP Kinase to ATF2
[0145] Cell extracts expressing epitope-tagged JNK1 and p38 MAP
kinase were incubated with a GST fusion protein containing the
activation domain of ATF2 (residues 1-109) immobilized on GSH
agarose. The supernatant was removed and the agarose was washed
extensively. Western blot analysis of the supernatant and
agarose-bound fractions was conducted as follows: proteins were
fractionated by SDS-PAGE, electrophoretically transferred to an
Immobilon-P membrane, and probed with monoclonal antibodies to
phosphotyrosine (PY20) and the Flag epitope (M2). Immunocomplexes
were detected using enhanced chemiluminescence (Amersham
International PLC). Control experiments were performed using
immobilized GST.
Example 17
[0146] p38 MAP Kinase and JNK1 Activation by Pro-Inflammatory
Cytokines and Environmental Stress
[0147] The effect of phorbol ester, EGF, UV radiation, osmotic
stress, IL-1, tumor necrosis factor (TNF), and LPS on p38 MAP
kinase and JNK1 activity were measured in immunecomplex protein
kinase assays using ATP [.gamma.-.sup.32P] and ATF2 as substrates.
TNF.alpha. and IL-1.alpha. were from Genzyme Corp. Lipolysaccharide
(LPS) was isolated from lyophilized Salmonella minesota Re595
bacteria as described (Mathison et a. (1988) J. Clin. Invest.
81:1925). Phorbol myristate acetate was from Sigma. EGF was
purified from mouse salivary glands (Davis (1988) J. Biol. Chem.
263:9462). Kinase assays were performed using immunoprecipitates of
p38 and JNK. The immunocomplexes were washed twice with kinase
buffer (described above), and the assays initiated by the addition
of 1 .mu.g of ATF2 and 50 .mu.M [.gamma.-.sup.32P]ATP (10 Ci/mmol)
in a final volume of 25 .mu.l. The reactions were terminated after
30 minutes at 30.degree. C. by addition of Laemmli sample buffer.
The phosphorylation of ATF2 was examined after SDS-PAGE by
autoradiography, and the rate of ATF2 phosphorylation quantitated
by PhosphorImager analysis.
[0148] The results are shown in Table 2. Exposure of HeLa cells to
10 nM phorbol myristate acetate very weakly activated p38 and JNK1.
Similarly, treatment with 10 nM EGF only weakly activated p38 and
JNK1. By contrast, treatment with 40 J/m.sup.2 UV-C, 300 mM
sorbitol, 10 ng/ml interleukin-1, and 10 ng/ml TNF.alpha. strongly
activated p38 and JNK1 activity. The effect of LPS on the activity
of p38 was examined using CHO cells that express human CD14.
Exposure of CHO cells to 10 ng/ml LPS only slightly activated p38
and JNK1 activity.
2TABLE 2 p38 AND JNK1 ACTIVATION BY PRO-INFLAMMATORY CYTOKINES AND
ENVIRONMENTAL STRESS. Relative Protein Kinase Activity JNK p38
Control 1.0 1.0 Epidermal Growth Factor (10 nM) 1.9 2.1 Phorbol
Ester (10 nM) 2.3 2.9 Lipopolysaccharide (10 ng/ml) 3.6 3.7 Osmotic
Shock (300 mM sorbitol) 18.1 4.2 Tumor Necrosis Factor (10 ng/ml)
19.3 10.3 Interleukin-1 (10 ng/ml) 8.9 6.2 UV (40 J/M.sup.2) 7.4
17.1
Example 18
[0149] p38 MAP Kinase Activation by Dual Phosphorylation on Tyr and
Thr
[0150] COS-1 cells expressing wild-type (Thr.sup.180, Tyr.sup.182)
or mutated (Ala.sup.180, Phe.sup.182) p38 MAP kinase were treated
without and with UV-C (40 J/m.sup.2). The cells were harvested 30
minutes following exposure with or without UV radiation. Control
experiments were performed using mock-transfected cells. The level
of expression of epitope-tagged p38 MAP kinase and the state of Tyr
phosphorylation of p38 MAP kinase was examined by Western blot
analysis using the M2 monoclonal antibody and the phosphotyrosine
monoclonal antibody PY20. Immune complexes were detected by
enhanced chemiluminescence.
[0151] Wild-type and mutant p38 were expressed at similar levels.
Western blot analysis showed that UV radiation caused an increase
in the Tyr phosphorylation of p38. The increased Tyr
phosphorylation was confirmed by phosphoamino acid analysis of p38
isolated from (.sup.32P]phosphate-labeled cells. The results also
showed that UV radiation increased Thr phosphorylation of p38. The
increased phosphorylation on Tyr and Thr was blocked by mutated
p38. Wild-type and mutated p38 were isolated from the COS-1 cells
by immunoprecipitation. Protein kinase activity was measured in the
immune complex using [.gamma.-.sup.32P]ATP and GST-ATF2 as
substrates. The phosphorylated GST-ATF2 was detected after SDS-PAGE
by autoradiography. UV radiation resulted in a marked increase in
the activity of wild-type p38, while the mutant p38 was found to be
catalytically inactive. These results show that p38 is activated by
dual phosphorylation within the Thr-Gly-Tyr motif.
Example 19
[0152] MAP Kinase Phosphatase Inhibits p38 MAP Kinase
Activation
[0153] The cells were treated without and with 40 J/m.sup.2 UV-C.
Control experiments were performed using mock-transfected cells
(control) and cells transfected with the catalytically inactive
mutated phosphatase mPAC1 (Cys.sup.257/Ser) and human MKP1. The
activity of p38 MAP kinase was measured with an immunecomplex
protein kinase assay employing [.gamma.-.sup.32P]ATP and GST-ATF2
as substrates. The expression of PAC1 or MKP1 was found to inhibit
p38 phosphorylation, demonstrating that p38 can be regulated by the
dual specificity phosphatases PAC1 and MKP1.
Example 20
[0154] Subcellular Distribution of p38 MAP Kinase
[0155] Epitope-tagged p38 MAP kinase was expressed in COS cells.
The cells were treated without or with 40 J/m.sup.2 UV radiation
and then incubated for 60 minutes at 37.degree. C. The p38 MAP
kinase was detected by indirect immunofluorescence using the M2
monoclonal antibody. The images were acquired by digital imaging
microscopy and processed for image restoration.
[0156] Immunocytochemistry
[0157] Coverslips (22mm.times.22mm No. 1; Gold Seal Cover Glass;
Becton-Dickinson) were pre-treated by boiling in 0.1 N HCl for 10
minutes, rinsed in distilled water, autoclaved and coated with
0.01% poly-L-lysine (Sigma; St. Louis Mo.). The coverslips were
placed at the bottom of 35 mm multiwell tissue culture plates
(Becton Dickinson, UK). Transfected COS-1 cells were plated
directly on the coverslips and allowed to adhere overnight in
Dulbecco's modified Eagle's medium supplemented with 5% fetal calf
serum (Gibco-BRL). Twenty-four hours post-transfection, the cells
were rinsed once and incubated at 37.degree. C. for 30 minutes in
25 mM Hepes, pH 7.4, 137 mM NaCl, 6 mM KCl, 1 MM MgCl.sub.2, 1 mM
CaCl.sub.2, 10 mM glucose. The cells were rinsed once with
phosphate-buffered saline and the coverslips removed from the
tissue culture wells. Cells were fixed in fresh 4% paraformaldehyde
in phosphate-buffered saline for 15 minutes at 22.degree. C. The
cells were permeabilized with 0.25% Triton.RTM. X-100 in
phosphate-buffered saline for 5 minutes and washed three times in
DWB solution (150 mM NaCl, 15 mM Na citrate, pH 7.0, 2% horse
serum, 1% (w/v) bovine serum albumin, 0.05% Triton.RTM. X-100) for
5 minutes. The primary antibody (M2 anti-FLAG monoclonal antibody,
Eastman-Kodak Co., New Haven, Conn.) was diluted 1:250 in DWB and
applied to the cells in a humidified environment at 22.degree. C.
for 1 hour. The cells were again washed three times as above and
fluorescein isothiocyanate-conjugated goat anti-mouse Ig secondary
antibody (Kirkegaard & Perry Laboratories Inc. Gaithersburg,
Md.) was applied at a 1:250 dilution for 1 hour at 22.degree. C. in
a humidified environment. The cells were then washed three times in
DWB and then mounted onto slides with Gel-Mount (Biomeda Corp.
Foster City, Calif.) for immunofluorescence analysis. Control
experiments were performed to assess the specificity of the
observed immunofluorescence. No fluorescence was detected when the
transfected cells were stained in the absence of the primary M2
monoclonal antibody, or mock-transfected cells.
[0158] Digital Imaging Microscopy and Image Restoration
[0159] Digital images of the fluorescence distribution in single
cells were obtained using a Nikon 60.times. Planapo objective
(numerical aperture=1.4) on a Zeiss IM-35 microscope equipped for
epifluorescence as previously described (Carrington et al. (1990)
in: Non-invasive Techniques in Cell Biology, Fosbett &
Grinstein, eds., Wiley-Liss, NY; pp. 53-72; Fay et al. (1989) J.
Microsci. 153:133-149). Images of various focal planes were
obtained with a computer controlled focus mechanism and a
thermoelectrically cooled charged-coupled device camera (model 220;
Photometrics Ltd., Tucson, Ariz.). The exposure of the sample to
the excitation source was determined by a computer-controlled
shutter and wavelength selector system (MVI, Avon, Mass.). The
charge-coupled device camera and microscope functions were
controlled by a microcomputer, and the data acquired from the
camera were transferred to a Silicon Graphics model 4D/GTX
workstation (Mountainview, Calif.) for image processing. Images
were corrected for non-uniformities in sensitivity and for the dark
current of the charge coupled device detector. The calibration of
the microscopy blurring was determined by measuring the
instrument's point spread function as a series of optical sections
at 0.125 .mu.m intervals of a 0.3 .mu.m diameter fluorescently
labeled latex bead (Molecular Probes Inc.). The image restoration
algorithm used is based upon the theory of ill-posed problems and
obtains quantitative dye density values within the cell that are
substantially more accurate than those in an unprocessed image
(Carrington et al. (1990) supra; Fay et al. (1989) supra). After
image processing, individual optical sections of cells were
inspected and analyzed using computer graphics software on a
Silicon Graphics workstation. p38 MAP kinase was observed at the
cell surface, in the cytoplasm, and in the nucleus. After
irradiation, an increased localization of cytoplasmic p38 to the
perinuclear region was detected.
Example 21
[0160] Activation of the MKK Signal Transduction Pathway by Osmotic
Shock
[0161] CHO cells were co-transfected with the plasmid
pCMV-Flag-Jnk1 and pRSV-Neo (Drijard et al. (1994) supra). A stable
cell line expressing epitope-tagged Jnk1 (Flag; Immunex Corp.) was
isolated by selection with Geneticin (Gibco-BRL). The cells were
incubated with 0, 100, 150, 300, 600, or 1000 mM sorbitol for 1
hour at 37.degree. C. The cells were collected in lysis buffer (20
mM Tris, pH 7.4, 1% TRITON.RTM. X-100, 2 mM EDTA, 137 mM NaCl, 25
mM .beta.-glycerophosphate, 1 mM orthovanadate, 2 mM pyrophosphate,
10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 .mu.g/ml
leupeptin) and a soluble extract was obtained by centrifugation at
100,000 g for 30 minutes at 4.degree. C. The0 epitope-tagged JNK1
was isolated by immunoprecipitation with the monoclonal antibody M2
(Immunex Corp.). The immunoprecipitates were washed extensively
with lysis buffer. Immunecomplex kinase assays were done in 25
.mu.l of 25 mM Hepes, pH 7.4, 25 mM MgCl.sub.2, 25 mM
.beta.-glycerophosphate, 2 mM dithiothreitol, 100 .mu.M
orthovanadate, and 50 .mu.M ATP [.gamma.-.sup.32P] (10 Ci/mmole)
with 2.5 .mu.g of bacterially expressed c-Jun (residues 1-79) fused
to glutathione-S-transferase (GST) as a substrate. The
phosphorylation of c-Jun was examined after SDS-PAGE by
autoradiography and PhosphorImager (Molecular Dynamics Inc.)
analysis. JNK1 activation was observed at all concentrations of
sorbitol exposure.
[0162] The time course of JNK1 protein kinase activation was
measured in cells incubated in medium supplemented with 300 mM
sorbitol as described above. Increased JNK1 activity was observed
within 5 minutes of exposure to sorbitol, with maximum activity
occurring after 15-30 minutes.
[0163] Mutation of JNK1 at the phosphorylation sites Thr.sup.183
and Tyr.sup.185 blocked the activation of JNK1 protein kinase
activity by osmotic shock. CHO cells were transfected with vector,
wild-type JNK1 (Thr.sup.183, Tyr.sup.185), and mutated JNK1
(Ala.sup.183, Phe.sup.185). The cells were incubated in medium
supplemented without or with 300 mM sorbitol for 15 minutes before
measurement of JNK1 protein kinase activity as described above.
JNK1 activation was seen in the wild-type but not mutated JNK1.
Example 22
[0164] Molecular Cloning of MKK7
[0165] RT-PCR was employed to identify a fragment of a novel
mammalian MAP kinase kinase. The primers designed for the protocol,
ATNGCNGTNAARCARATG (SEQ ID NO; 23) and ATNCKYTCNGGNGCCATRTA (SEQ ID
NO: 24), were based on the sequence of the Drosophila MAP kinase
kinase hep (Glise et al. (1995) Cell 83:451-461). Murine testis
mRNA was used as the template. A single product (461 bp) was
detected following RT-PCR amplification of murine testis mRNA.
Sequence analysis identified this PCR product as a fragment of a
novel mammalian MAP kinase kinase. Full-length murine cDNA clones
were isolated by screening a murine testis library (Stratagene
Inc.). The cDNA clones were examined by sequencing with an Applied
Biosystems model 373A machine. A group of seven clones was
identified by sequence analysis to contain a single long open
reading frame that encoded a putative protein kinase (FIG. 9 and
FIG. 10; SEQ ID NO: 17 and SEQ ID NO: 18). In-frame termination
codons were detected in the 5' and 3' regions of these clones. This
sequence includes protein kinase sub-domains I-XI and is related to
the MAP kinase kinase group. The novel protein kinase was
designated MKK7. The sites of activating phosphorylation of MAP
kinase kinases located in sub-domain VIII are conserved in MKK7.
Comparison of MKK7 with other members of the mammalian MAP kinase
kinase group demonstrates that MKK7 is related to the JNK activator
MKK4.
[0166] One additional cDNA clone isolated from the .lambda. phage
library differed from the other seven clones. This clone contained
the same 3' untranslated region and coding region of MKK7, but had
a different 5' region that lacked an in-frame termination codon.
This clone represents an alternatively spliced form of MKK7 (MKK7b;
FIG. 11; SEQ ID NO: 19). The MKK7b cDNA clone does not have an
initiation codon in the alternative 5' region; this cDNA therefore
encodes the same MKK7 protein kinase as the other clones that were
isolated. However, if the MKK7b cDNA clone is not full-length it is
possible that additional 5' sequence may include an in-frame
initiation codon. If true, MKK7b is predicted to fuse the sequence
M-[?]-SPAPAPSQRAALQLPLANDGGSRSPSSESSPQHPTPPTRPRH-(SEQ ID NO: 33) to
the initiating methionine of MKK7 (FIG. 9). Although the Drosophila
MAP kinase kinase hep shares substantial sequence similarity with
MKK7, the sequence of the NH2-terminal extension of MKK7b is not
conserved in the hep protein kinase. Three additional clones
encoded MKK7 splice variants that differ in the 5' and 3' regions.
These clones (MKK7c (FIG. 13), MKK7d (FIG. 14), and MKK7e (FIG.
15)) are full-length because of the presence of in-frame
termination codons in the 5' and 3' regions.
[0167] A human cDNA library was screened with a full-length mouse
MKK7 cDNA probe. A single clone was identified and squenced. A
partial MKK7 sequence was identified (FIG. 12; SEQ ID NO: 25 and
SEQ ID NO: 26) that is missing the 3' end. The sequence is most
homologous to mouse MKK7c.
[0168] The sequences of MKK7, MKK7b, hep, and human MKK7 cDNAs have
been deposited in Genbank with accession numbers U93030, U93031,
U93032, and AF00319 respectively.
Example 23
[0169] Expression of MKK7
[0170] MKK7 expression was examined by Northern blot analysis of
mRNA isolated from different tissues. The analysis was done with
poly A+ mRNA (2 .mu.g) isolated from different tissues and
fractionated by denaturing agarose gel electrophoresis and
transferred to a nylon membrane (Clontech). The blot was probed
with MKK4 and MKK7 cDNAs labeled by random priming with
[.alpha.-.sup.32P]dATP (Amersham International PLC).
[0171] MKK7 was found to be widely expressed in murine tissues. A
single MKK7 transcript (approximately 4.0-kb) was detected in all
of the tissues examined, except for testis where two MKK7
transcripts (4.0 kb and 1.6 kb) were detected. The highest levels
of MKK7 expression were in testis. Significant expression of MKK7
was also observed in heart, brain, lung, liver, and kidney. This
contrasts with MKK4 expression which was highest in brain although
significant amounts of expression were observed in brain, liver,
muscle, heart, and kidney. Although MKK4 and MKK7 are co-expressed,
the relative abundance of each MAP kinase kinase is different in
each of the tissues examined.
Example 24
[0172] Specific Activation of JNK by MKK7 in vitro
[0173] To examine the specificity of MKK7, in vitro protein kinase
assays were performed. A bacterial MKK7 expression vector was
prepared by sub-cloning an MKK7 cDNA (Eco RI and Pvu II fragment)
into the Eco RI and Sma I sites of pGEX-5X1 (Pharmacia-LKB). The
glutathione-S-transferase (GST) fusion protein was purified by
affinity chromatography (Smith and Johnson (1988) Gene 67:31-40).
The recombinant proteins GST-ATF2 (Gupta et al. (1995) Science
267:389-393), GST-cJun (Drijard (1994) supra), GST-cMyc (Alvarez et
al. (1991) J. Biol. Chem. 266:15277-15285), GST-ERK2 (Seth et al.
(1992) J. Biol. Chem. 267:24796-24804), GST-p38, (Raingeaud et al.
(1995) J. Biol. Chem. 270:7420-7426), and GST-JNK1 (Drijard (1994)
supra) have been described.
[0174] Protein kinase assays were performed in kinase buffer (25 mM
4-(2-hydroxyethyl)-1-piperazineethansulfonic acid (pH 7.4), 25 mM
.beta.-glycerophosphate, 25 mM MgCl.sub.2, 2 mM dithiothreitol, 0.1
mM orthovanadate). The assays were initiated by the addition of 1
.mu.g of substrate proteins and 50 .mu.M [.gamma.-32P]ATP (10
Ci/mmol) in a final volume of 25 .mu.l. The reactions were
terminated after 30 minutes at 25.degree. C. by addition of Laemmli
sample buffer. The phosphorylation of the substrate proteins was
examined after SDS-polyacrylamide gel electrophoresis (PAGE) by
autoradiography.
[0175] Recombinant MAP kinases were incubated with GST (control) or
GST-MKK7 using the substrate ATP[.gamma.-.sup.32P]. Recombinant
MKK7 purified from bacteria was not observed to autophosphorylate.
Incubation of the recombinant MKK7 with MAP kinases demonstrated
that MKK7 phosphorylated JNK1, but not p38 or ERK2. MKK7 was
phosphorylated by p38 and JNK1. The significance of the
retrophosphorylation of the MAP kinase kinase by the MAP kinase is
unclear, but similar retrophosphorylation has been detected in
kinase assays using MKK4 (Drijard (1995) supra) and the Drosophila
JNK activator hep (Sluss (1996) supra).
[0176] To test whether the phosphorylation of JNK1 by MKK7 caused
increased protein kinase activity, experiments using ATF2 as the
JNK substrate were performed. GST-MKK7 was incubated in a protein
kinase assay with recombinant JNK1. JNK activity was measured by
including the JNK substrate ATF2 in each assay. ATF2 was not
phosphorylated by MKK7, but was weakly phosphorylated by JNK1.
Incubation of MKK7 with JNK1 caused phosphorylation of JNK1 and a
large increase in ATF2 phosphorylation. These data indicate that
MKK7 phosphorylates and activates JNK1. To confirm this conclusion,
the effect of replacement of the JNK dual phosphorylation motif
Thr-Pro-Tyr with Ala-Pro-Phe was examined. MKK7 did not
phosphorylate the mutated JNK1 (APF) protein. Furthermore, MKK7 did
not increase ATF2 phosphorylation by the mutated JNK1 protein
kinase. Thus, MKK7 is a JNK activator in vitro.
Example 25
[0177] Specific Activation of JNK by MKK7 In Vivo
[0178] To examine the specificity of MKK7 in vivo, cotransfection
assays were performed. CHO cells were maintained in Dulbecco's
modified Eagle's medium supplemented with fetal calf serum (5%;
Gibco-BRL). The cells were transfected with the lipofectamine
reagent according to the manufacturer's recommendations (Gibco-BRL)
(Drijard (1994) supra). Cells were co-transfected with vectors
encoding epitope-tagged JNK1 together with an empty expression
vector (control) or an expression vector encoding MKK4 or MKK7. The
epitope tag was derived from the hemagglutinin protein (HA) of the
influenza virus. JNK1 was isolated by immunoprecipitation of cell
lysates. The cells were solubilized with lysis buffer (20 mM Tris
(pH 7.4), 1% TRITON X-100.RTM., 10% glycerol, 137 mM NaCl, 2 mM
EDTA, 25 mM .beta.-glycerophosphate, 1 mM Na orthovanadate, 2 mM
pyrophosphate, 1 mM PMSF, 10 .mu.g/ml leupeptin) and centrifuged at
100,000.times.g for 15 minutes at 4.degree. C. The epitope-tagged
protein kinases were immunoprecipitated by incubation for 3 hours
at 4.degree. C. with an anti-HA monoclonal antibody bound to
protein-G Sepharose (Pharmacia-LKB Biotechnology Inc.). The
immunoprecipitates were washed three times with lysis buffer (Gupta
et al. (1995) Science 267:389-393). Protein kinase activity was
measured in the immunecomplex with [.gamma.-.sup.32P]ATP and c-Jun
as substrates. The product of the phosphorylation reaction was
visualized after SDS-PAGE by autoradiography. The ERK2 and p38 MAP
kinases were not activated by co-expressed MKK7. Control
experiments demonstrated that the ERK2 and p38 MAP kinases were
activated by their respective cognate MAP kinase kinases, MKK1 and
MKK6. In contrast, MKK7 did activate JNK1. Interestingly, the
activation of JNK1 by co-expressed MKK7 was greater than that
caused by the previously described JNK activator MKK4. Together,
these data establish that MKK7 can function as a specific activator
of JNK in cultured cells.
Example 26
[0179] Activation of the JNK Signal Transduction Pathway by
MKK7
[0180] The JNK signaling pathway is known to regulate AP-1
transcriptional activity (Whitmarsh (1996) supra). To test the
hypothesis that the expression of MKK7 would cause increased AP-1
transcriptional activity, a co-transfection assay was employed
using a luciferase reporter gene that contains three AP-1 sites
cloned upstream of a minimal promoter element (Rincon and Flavell
(1994) EMBO J. 13:4370-4381). Luciferase reporter gene expression
was measured in co-transfection assays using the 0.5 .mu.g of the
reporter plasmid pTRE-luciferase (Rincon (1994) supra) and 0.25
.mu.g of the .beta.-galactosidase expression vector pCH110
(Pharmacia-LKB). Experiments using GAL4 fusion proteins were
performed using 0.25 .mu.g of pGAL4-ATF2 (residues 1-109), 0.5
.mu.g of the reporter plasmid pG5E1bLuc, and 0.25 .mu.g of pCH110
(Gupta et al. (1995) supra). The effect of protein kinases was
examined by co-transfection with 0.3 .mu.g of an empty expression
vector or a protein kinase expression vector. The ERK2, p38, JNK1,
MKK1, MKK3, MKK4, and MKK6 expression vectors have been described.
The cells were harvested 36 hours post-transfection. The
.beta.-galactosidase and luciferase activity in the cell lysates
was measured as described (Gupta (1995) supra). Expression of MKK4,
MKK7, or JNK1 did not cause marked changes in AP-1 reporter gene
expression (FIG. 16A). In contrast, co-expression of MKK7 with JNK1
caused increased AP-1-dependent reporter gene expression.
Consistent with the observation that MKK4 causes weaker activation
of JNK than MKK7, co-expression of MKK4 with JNK caused a smaller
increase in AP-1 reporter gene expression (FIG. 16A). Together,
these data demonstrate that MKK7 can function as an activator of
the JNK signal transduction pathway.
[0181] To further examine the effect of MKK7 on transcriptional
activity, the effect of MKK7 on the transcription factor ATF2 was
investigated. Previous studies have demonstrated that ATF2 is a
target of the JNK signal transduction pathway (van Dam et al.
(1995) supra; Gupta et al. (1995) supra; Livingstone et al (1995)
supra). JNK phosphorylates two sites (Thr-69 and Thr-71) in the
NH.sub.2-terminal activation domain of ATF2 and increases
transcriptional activity. A GAL4 fusion protein strategy was
employed to monitor the transcriptional activity of the activation
domain of ATF2 (Gupta (1995) supra). Measurement of reporter gene
expression demonstrated that the co-expression of MKK4 with JNK1
caused increased transcriptional activity (FIG. 16B). A similar
level of reporter gene expression was caused by expression of MKK7
and a larger increase was detected when MKK7 was co-expressed with
JNK1. The more potent effect of MKK7, compared with MKK4, on
transcriptional activity is consistent with the relative effects of
MKK7 and MKK4 on JNK activation. To confirm that the increased
reporter gene expression was mediated by ATF2 phosphorylation, the
effect of replacement of the sites of ATF2 phosphorylation (Thr-69
and Thr-71) with Ala was examined. The mutated ATF2 protein was not
regulated by MKK4, MKK7, or JNK1 (FIG. 16B). Together, these data
demonstrate that MKK7 can regulate a physiological target of the
JNK signaling pathway.
[0182] Use
[0183] The MKK polypeptides and polynucleotides of the invention
are useful for identifying reagents that modulate the MKK signal
transduction pathways. Reagents that modulate an MKK signal
transduction pathway can be identified by their effect on MKK
synthesis, MKK phosphorylation, or MKK activity. For example, the
effect of a reagent on MKK activity can be measured by the in vitro
kinase assays described above. MKK is incubated without (control)
and with a test reagent under conditions sufficient to allow the
components to react, then the effect of the test reagent on kinase
activity is subsequently measured. Reagents that inhibit an MKK
signal transduction pathway can be used in the treatment of
MKK-mediated disorders. Reagents that stimulate an MKK signal
transduction pathway can be used in a number of ways, including
induction of programmed cell death (apoptosis) in tissues. For
example, the elimination of UV damaged cells can be used to prevent
cancer.
[0184] Generally, for identification of a reagent that inhibits the
MKK signal transduction pathway, a kinase assay (see, for example,
Example 3) is used. A range of reagent concentrations (e.g., 1.0 nM
to 100 mM) are added to a test system that includes an MKK
substrate and a radioactive marker such as [.gamma.-.sup.32P]ATP.
Appropriate substrate molecules include p38, JNK1, JNK2, or ATF2.
The incorporation of labelled phosphorus (e.g., [.sup.32]p or
[.sup.33]P) into the substrate is determined, and the results
obtained with the test reagent compared to control values. Of
particular interest are reagents that result in inhibition of
[.sup.32]P incorporation of about 80% or more. Phosphorylation may
also be examined using a reagent that is phosphorylation-dependent,
for example, an antibody. Phosphorylation-dependent antibodies may
be made using MKK7 phosphorylated on the activating sites,
Ser.sup.198 and Thr.sup.202. This may be accomplished by immunizing
animals with a synthetic peptide (for example, approximately 15
amino acids in length) corresponding to the MKK7 sequence with
phosphorylated Ser.sup.198 and Thr.sup.202. Methods of producing
such antibodies are known in the art. Such antibodies are useful
for the detection of activated MKK7 in tissues and cell extracts
(e.g. on Western blots) and may be used in a kit.
[0185] Assays that test the effect of a reagent on MKK synthesis
can also be used to identify compounds that inhibit MKK signal
transduction pathways. The effect of the test reagent on MKK
expression is measured by, for example, Western blot analysis with
an antibody specific for an MKK. Antibody binding is visualized by
autoradiography or chemiluminescence, and is quantitated. The
effect of the test reagent on MKK mRNA expression can be examined,
for example, by Northern blot analysis using a polynucleotide probe
or by polymerase chain reaction.
[0186] Reagents found to inhibit MKK signal transduction pathways
can be used as therapeutic agents for the treatment of MKK-mediated
disorders. Such reagents are also useful in drug design for
elucidation of the specific molecular features needed to inhibit
MKK signal transduction pathways.
[0187] In addition, the invention provides a method for the
treatment of MKK-mediated stress-related and inflammatory
disorders. The method includes administration of an effective
amount of a therapeutic reagent that inhibits MKK function.
Suitable reagents inhibit either MKK activity or expression. The
concentration of the reagent to be administered is determined based
on a number of factors, including the appropriate dosage, the route
of administration, and the specific condition being treated. The
appropriate dose of a reagent is determined by methods known to
those skilled in the art including routine experimentation to
optimize the dosage as necessary for the individual patient and
specific MKK-mediated disorder being treated. Specific
therapeutically effective amounts appropriate for administration
are readily determined by one of ordinary skill in the art (see,
for example, Remington's Pharmaceutical Sciences. 18th ed.,
Gennaro, ed., Mack Publishing Company, Easton, Pa., 1990). Dosages
may range from about 0.1-10 mg/kilo/day.
[0188] The invention provides methods for both acute and
prophylactic treatment of stress-related and inflammatory
disorders. For example, it is envisioned that ischemic heart
disease will be treated during episodes of ischemia and oxidative
stress following reperfusion. In addition, a patient at risk for
ischemia can be treated prior to ischemic episodes.
[0189] In another example, a therapeutic agent that inhibits MKK
function or activity is administered to control inflammatory
responses by inhibiting the secretion of inflammatory cytokines,
including TNF and IL-1.
[0190] Stress-related proliferative disorders can also be treated
by the method of the invention by administering a therapeutic
reagent that inhibits MKK function or activity. Such therapeutic
reagents can be used alone or in combination with other therapeutic
reagents, for example, with chemotherapeutic agents in the
treatment of malignancies. Indeed, the control of stress-activated
MKK by the therapeutic reagents provided by this invention can
modulate symptoms caused by other therapeutic strategies that
induce stress.
[0191] The therapeutic reagents employed are compounds which
inhibit MKK function or activity, including polynucleotides,
polypeptides, and other molecules such as antisense
oligonucleotides and ribozymes, which can be made according to the
invention and techniques known to the art. Polyclonal or monoclonal
antibodies (including fragments or derivatives thereof) that bind
epitopes of MKK also can be employed as therapeutic reagents.
Dominant-negative forms of MKK which effectively displace or
compete with MKK for substrate binding and/or phosphorylation can
be used to decrease protein kinase activity. Dominant-negative
forms can be created by mutations within the catalytic domain of
the protein kinases, using methods known in the art, and as
described above (Example 13). The catalytic residues are conserved
in all the MKK isoforms. For example, mutation of Lys.sup.76
inhibits MKK7 activity. Similarly, mutation of the conserved sites
of activating phosphorylation (Ser.sup.198, Thr.sup.202) inhibits
MKK7 activity. These kinase-inactive forms of MKK7 act as
dominant-negative inhibitors.
[0192] In some cases, augmentation of MKK activity is desirable,
e.g., induction of apoptosis. The methods of the invention can be
used to identify reagents capable of increasing MKK function or
activity. Alternatively, increased activity is achieved by
over-expression of MKK. When an MKK-mediated disorder is associated
with under-expression of MKK, or expression of a mutant MKK
polypeptide, a sense polynucleotide sequence (the DNA coding
strand) or MKK polypeptide can be introduced into the cell to
enhance normal MKK activity. If necessary, these treatments are
targeted to specific cells by their mode of administration. (e.g.,
by use of cell-type specific viral vectors), or by placing MKK7
nucleic acids in recombinant constructs with cell-type specific or
inducible promoters by methods known in the art. For example, MKK7
nucleic acid-containing vectors can be constructed by recombinant
DNA technology methods standard in the art. Vectors can be plasmid,
viral, or others known in the art, used for replication and
expression in mammalian cells. Expression of the sequence encoding
the MKK7 nucleic acid can be by any promoter known in the art to
act in mammalian, preferably human cells. Such promoters can be
inducible or constitutive. Such promoters include, but are not
limited to: the SV40 early promoter region (Bernoist et al., Nature
290:304, 1981); the promoter contained in the 3' long terminal
repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797,
1988); the herpes thymidine kinase promoter (Wagner et al., Proc.
Natl. Acad. Sci. USA 78:1441, 1981); or the regulatory sequences of
the metallothionein gene (Brinster et al., Nature 296:39,
1988).
[0193] The antibodies of the invention can be administered
parenterally by injection or by gradual infusion over time. The
monoclonal antibodies of the invention can be administered
intravenously, intraperitoneally, intramuscularly, subcutaneously,
intracavity, or transdermally.
[0194] Preparations for parenteral administration of a polypeptide
or an antibody of the invention include sterile aqueous or
non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous solvents are propylene glycol, polyethylene glycol,
vegetable oils such as olive oil, and injectable organic esters
such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose) and the like. Preservatives and
other additives can also be present, such as, for example,
antimicrobials, antioxidants, chelating agents, and inert gases,
and the like.
[0195] Polynucleotide sequences, including antisense sequences, can
be therapeutically administered by various techniques known to
those skilled in the art. Such therapy would achieve its
therapeutic effect by introduction of the MKK polynucleotide into
cells of mammals having a MKK-mediated disorder. Delivery of MKK
polynucleotides can be achieved using free polynucleotide or a
recombinant expression vector such as a chimeric virus or a
colloidal dispersion system. Especially preferred for therapeutic
delivery of nucleotide sequences is the use of targeted
liposomes.
[0196] Targeting of the therapeutic reagent to specific tissues is
desirable to increase the efficiency of delivery. The targeting can
be achieved by passive mechanisms via the route of administration.
Active targeting to specific tissues can also be employed. The use
of liposomes, colloidal suspensions, and viral vectors allows
targeting to specific tissues by changing the composition of the
formulation containing the therapeutic reagent, for example, by
including molecules that act as receptors for components of the
target tissues. Examples include sugars, glycoplipids,
polynucleotides, or proteins. These molecules can be included with
the therapeutic reagent. Alternatively, these molecules can be
included by indirect methods, for example, by inclusion of a
polynucleotide that encodes the molecule, or by use of packaging
systems that provide targeting molecules. Those skilled in the art
will know, or will ascertain with the use of the teaching provided
herein, which molecules and procedures will be useful for delivery
of the therapeutic reagent to specific tissues.
[0197] Transgenic Animals
[0198] MKK polypeptides can also be expressed in transgenic
animals. These animals represent a model system for the study of
disorders that are caused by or exacerbated by overexpression or
underexpression of MKK, and for the development of therapeutic
agents that modulate the expression or activity of MKK. For
example, dominant-negative and constitutively activated alleles
could be expressed in mice to establish physiological function.
[0199] Transgenic animals can be farm animals (pigs, goats, sheep,
cows, horses, rabbits, and the like) rodents (such as rats, guinea
pigs, and mice), non-human primates (for example, baboons, monkeys,
and chimpanzees), and domestic animals (for example, dogs and
cats). Transgenic mice are especially preferred.
[0200] Any technique known in the art can be used to introduce a
MKK transgene into animals to produce the founder lines of
transgenic animals. Such techniques include, but are not limited
to, pronuclear microinjection (U.S. Pat. No. 4,873,191); retrovirus
mediated gene transfer into germ lines (Van der Putten et al.,
Proc. Natl. Acad. Sci., USA 82:6148, 1985); gene targeting into
embryonic stem cells (Thompson et al., Cell 56:313, 1989); and
electroporation of embryos (Lo, Mol. Cell. Biol. 3:1803, 1983).
Especially useful are the methods described in Yang et al. (Proc.
Natl Acac. Sci. USA 94:3004-3009, 1997)
[0201] The present invention provides for transgenic animals that
carry the MKK transgene in all their cells, as well as animals that
carry the transgene in some, but not all of their cells. That is,
the invention provides for mosaic animals. The transgene can be
integrated as a single transgene or in concatamers, e.g.,
head-to-head tandems or head-to-tail tandems. The transgene can
also be selectively introduced into and activated in a particular
cell type (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232, 1992).
The regulatory sequences required for such a cell-type specific
activation will depend upon the particular cell type of interest,
and will be apparent to those of skill in the art.
[0202] When it is desired that the MKK transgene be integrated into
the chromosomal site of the endogenous MKK gene, gene targeting is
preferred. Briefly, when such a technique is to be used, vectors
containing some nucleotide sequences homologous to an endogenous
MKK gene are designed for the purpose of integrating, via
homologous recombination with chromosomal sequences, into and
disrupting the function of the nucleotide sequence of the
endogenous gene. The transgene also can be selectively introduced
into a particular cell type, thus inactivating the endogenous MKK
gene in only that cell type (Gu et al., Science 265:103, 1984). The
regulatory sequences required for such a cell-type specific
inactivation will depend upon the particular cell type of interest,
and will be apparent to those of skill in the art. These techniques
are useful for preparing "knock outs" having no functional MKK
gene.
[0203] Once transgenic animals have been generated, the expression
of the recombinant MKK gene can be assayed utilizing standard
techniques. Initial screening may be accomplished by Southern blot
analysis or PCR techniques to determine whether integration of the
transgene has taken place. The level of mRNA expression of the
transgene in the tissues of the transgenic animals may also be
assessed using techniques which include, but are not limited to,
Northern blot analysis of tissue samples obtained from the animal,
in situ hybridization analysis, and RT-PCR. Samples of MKK
gene-expressing tissue can also be evaluated immunocytochemically
using antibodies specific for the MKK transgene product.
[0204] For a review of techniques that can be used to generate and
assess transgenic animals, skilled artisans can consult Gordon
(Intl. Rev. Cytol. 115:171-229, 1989), and may obtain additional
guidance from, for example: Hogan et al. Manipulating the Mouse
Embryo, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1986);,
Krimpenfort et al. (Bio/Technology 9:86, 1991), Palmiter et al.
(Cell 41:343, 1985), Kraemer et al. (Genetic Manipulation of the
Early Mammalian Embryo, Cold Spring Harbor Press, Cold Spring
Harbor, N.Y., 1985), Hammer et al. (Nature 315:680, 1985), Purcel
et al. (Science, 244:1281, 1986), Wagner et al. (U.S. Pat. No.
5,175,385), and Krimpenfort et al. (U.S. Pat. No. 5,175,384) (the
latter two publications are hereby incorporated by reference).
[0205] Other Embodiments
[0206] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof,
that the foregoing description is intended to illustrate and not
limit the scope of the invention, which is defined by the scope of
the appended claims. Other aspects, advantages, and modifications
are within the scope of the following claims.
Sequence CWU 1
1
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