U.S. patent application number 11/867504 was filed with the patent office on 2008-05-08 for novel germinal kinase proteins, compositions and methods of use.
This patent application is currently assigned to Rigel Pharmaceuticals, Incorporated. Invention is credited to Cindy Leo, Ying Luo, Xiang Xu, Simon Yu.
Application Number | 20080108098 11/867504 |
Document ID | / |
Family ID | 21847308 |
Filed Date | 2008-05-08 |
United States Patent
Application |
20080108098 |
Kind Code |
A1 |
Leo; Cindy ; et al. |
May 8, 2008 |
NOVEL GERMINAL KINASE PROTEINS, COMPOSITIONS AND METHODS OF USE
Abstract
The present invention provides compositions and methods for
modulating cell proliferation, survival, morphology, and migration.
Nucleic acids encoding proteins, and proteins so encoded which are
capable of modulating proliferation, survival, morphology, and
migration in mammalian cells are provided. Compositions and methods
for the treatment of disorders related to cell proliferation,
survival, morphology and migration are also provided. Prophylactics
and methods for the prevention of such disorders are also provided.
Also provided are compositions are methods for diagnostic and
prognostic determination of such disorders. Further provided are
assays for the identification of bioactive agents capable of
modulating proliferation, survival, morphology, and migration in
mammalian cells.
Inventors: |
Leo; Cindy; (San Francisco,
CA) ; Luo; Ying; (Pudong New Area, CN) ; Xu;
Xiang; (South San Francisco, CA) ; Yu; Simon;
(Newark, CA) |
Correspondence
Address: |
Klarquist Sparkman, LLP
121 SW Salmon St
Floor 16
Portland
OR
97204
US
|
Assignee: |
Rigel Pharmaceuticals,
Incorporated
South San Francisco
CA
|
Family ID: |
21847308 |
Appl. No.: |
11/867504 |
Filed: |
October 4, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10493164 |
Sep 20, 2004 |
7300780 |
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PCT/US02/33845 |
Oct 21, 2002 |
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11867504 |
Oct 4, 2007 |
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10029115 |
Oct 19, 2001 |
7265214 |
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10493164 |
Sep 20, 2004 |
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Current U.S.
Class: |
435/15 |
Current CPC
Class: |
G01N 33/5041 20130101;
C12Q 1/485 20130101; G01N 2500/02 20130101; C12N 9/1205
20130101 |
Class at
Publication: |
435/015 |
International
Class: |
C12Q 1/48 20060101
C12Q001/48 |
Claims
1-15. (canceled)
16. A method of identifying a modulator of phosphorylation by a
misshapen/NIKs-related kinase 3 (MINK3) protein, wherein the MINK3
protein has at least 98% identity to an amino acid sequence
selected from the group consisting of SEQ ID NOs:1, 3, and 5, the
method comprising the steps of contacting a MINK3 phosphorylation
substrate with the MINK3 protein and a candidate bioactive agent,
determining the effect of the candidate bioactive agent on
phosphorylation of the substrate by the MINK3 protein as compared
to a control without the candidate bioactive agent, thereby
identifying the modulator of phosphorylation by the MINK3
protein.
17. The method of claim 16, wherein the MINK3 phosphorylation
substrate is an 9extracellular signal response kinase (ERK)
protein.
18. The method of claim 16, wherein the MINK3 phosphorylation
substrate is a c-Jun N-terminal kinase (JNK) protein.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/493,164, filed Sep. 20, 2004, which is a
National Stage of International Application No. PCT/US2003/033845,
filed Oct. 21, 2002, which is a continuation of U.S. application
Ser. No. 10/029,115, filed Oct. 19, 2001, now issued as U.S. Pat.
No. 7,265,214, herein incorporated by reference in its
entirety.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] Not applicable.
FIELD OF THE INVENTION
[0003] The present invention provides compositions and methods for
modulating cell proliferation, survival, morphology, and migration.
Nucleic acids encoding proteins and proteins so encoded which are
capable of modulating proliferation, survival, morphology and
migration in mammalian cells are provided. Compositions and methods
for the treatment of disorders related to cell proliferation,
survival, morphology and migration are also provided. Prophylactics
and methods for the prevention of such disorders are also provided.
Also provided are compositions and methods for diagnostic and
prognostic determination of such disorders. Further provided are
assays for the identification of bioactive agents capable of
modulating proliferation, survival, morphology and migration in
mammalian cells.
BACKGROUND OF THE INVENTION
[0004] Cells cycle through various stages of growth, starting with
the M phase, where mitosis and cytoplasmic division (cytokinesis)
occurs. The M phase is followed by the G1 phase, in which the cells
resume a high rate of biosynthesis and growth. The S phase begins
with DNA synthesis, and ends when the DNA content of the nucleus
has doubled. The cell then enters G2 phase, which ends when mitosis
starts, signaled by the appearance of condensed chromosomes.
Terminally differentiated cells are arrested in the G1 phase, and
no longer undergo cell division.
[0005] The hallmark of a malignant cell is uncontrolled
proliferation. This phenotype is acquired through the accumulation
of gene mutations, the majority of which promote passage through
the cell cycle. Cancer cells ignore growth regulatory signals and
remain committed to cell division. Classic oncogenes, such as ras,
lead to inappropriate transition from G1 to S phase of the cell
cycle, mimicking proliferative extracellular signals. Cell cycle
checkpoint controls ensure faithful replication and segregation of
the genome. The loss of cell cycle checkpoint control results in
genomic instability, greatly accelerating the accumulation of
mutations which drive malignant transformation. Thus, modulating
cell cycle checkpoint pathways and other such pathways with
therapeutic agents could exploit the differences between normal and
tumor cells, both improving the selectivity of radio- and
chemotherapy, and leading to novel cancer treatments, including
treatment for metastatic cancers. As another example, it would be
useful to control entry into apoptosis.
[0006] On the other hand, it is also sometimes desirable to enhance
proliferation of cells in a controlled manner. For example,
proliferation of cells is useful in wound healing and where growth
of tissue is desirable. Thus, identifying modulators which promote,
enhance or deter the inhibition of proliferation is desirable.
[0007] Proteins of general interest that have been reported on
include kinases. The Step 20 family of kinases can be divided into
two structurally distinct subfamilies. The first subfamily contains
a C-terminal catalytic domain and an N-terminal binding site for
the small G proteins Rac1 and Cdc42 (Herskowitz, Cell, 80:187-197
(1995)). The yeast serine/threonine kinase Step 20 and its
mammalian homologue, p21 Activated Kinase 1 (PAK1), belong to this
subfamily. Step 20 initiates a mitogen-activated protein kinase
(MAPK) cascade that includes Ste11 (MAPKKK), Step 7 (MAPKK), and
FUS3/KSS1 (MAPK) in response to activation of the small G protein
Cdc42, as well as signals from the hetero-trimeric G proteins
coupled to pheromone receptors (Herskowitz, Cell, 80:187-197
(1995)). Similar to Step 20, PAK1 has been reported to be a Cdc42
and Rac1 effector molecule and specifically regulates the c-Jun
N-terminal kinase (JNK) pathway, one of the mammalian MAPK pathways
(Bagrodia, et. al., J. Biol. Chem., 270:27995-27998 (1995);
Kyriakis, et al., J. Biol. Chem., 271:24313-24316 (1996)). The JNK
pathway is activated by a variety of stress inducing agents,
including osmotic and heat shock, UV irradiation, protein
inhibitors and pro-inflammatory cytokines such as tumor necrosis
factor (TNF) (Ip, et al., Curr. Opin. Cell Biol., 10:205-219
(1998)). JNKs are activated through threonine and tyrosine
phosphorylation by MEK4 and MEK7 (MAPKK), which are in turn
phosphorylated and activated by MAPKKKs including MEK kinase 1
(MEKK1), and mixed lineage kinases MLK2 and MLK3 (Ip, et al., Curr.
Opin. Cell Biol., 10:205-219 (1998)). In addition to the activation
of the JNK pathway, PAK1 has also been reported to be a regulator
of the actin cytoskeleton (Sells, et al., Curr. Biol., 7:202-210
(1997)).
[0008] The second subgroup of Step 20 family of kinases is
represented by the family of germinal center kinases (GCK)
(Kyriakis, J. Biol. Chem., 274:5259-5262 (1999)). In contrast to
Step 20 and PAK1, GCK family members have an N-terminal kinase
domain and a C-terminal regulatory region. Many GCK family members,
including GCK, germinal center kinase related protein (GCKR),
meatopoietic protein kinase (HPK) 1, GCK-like kinase (GLK),
HPK/GCK-like kinase (HGK) and NCK interacting kinase (NIK), have
also been reported to activate the JNK pathway when overexpressed
in 293 cells (Pombo, et al., Nature, 377:750-754 (1995); Shi, et
al., J. Biol. Chem., 272:32102-32107 (1997); Kiefer, et al., EMBO
J., 15:7013-7025 (1996); Diener, et al., Proc. Natl. Acad. Sci.
USA, 94:9687-9692 (1997); Yao, et al., J. Biol. Chem.,
274:2118-2125 (1999); Su, et al., EMBO J., 16:1279-1290 (1997)).
Among those, GCK and GCKR have been implicated in mediating
TNF-induced JNK activation through TNF receptor associated factor 2
(Traf2) (Pombo, et al., Nature, 377:750-754 (1995); Diener, et al.,
Proc. Natl. Acad. Sci. USA, 94:9687-9692 (1997); Yuasa, et al., J.
Biol. Chem., 273:22681-22692 (1998)). NCK interacting kinase (NIK)
interacts with the SH2-SH3 domain containing adapter protein NCK
and has been proposed to link protein tyrosine kinase signals to
JNK activation (Su, et al., EMBO J., 16:1279-1290 (1997)).
[0009] A kinase related to TNIK has been reported on. MINK
(misshapen/NIKs-related kinase) protein and nucleic acid have been
previously described (Ippeita et. al., FEBS Letters, 469:19-23,
2000). MINK1 is a gck kinase family member which is upregulated
during brain development (Ippeita et. al., FEBS Letters, 469:19-23,
2000).
[0010] One study reports on a GCK family kinase from Dictyostelium
that can phosphorylate Severin in vitro. (Eichinger, et al., J.
Biol. Chem., 273:12952-12959 (1998)). Severin is an F-actin
fragmenting and capping enzyme that regulates Dictyostelium
motility. TNIK, a mammalian GCK, has been shown to regulate the
cytoskeleton, particularly to destabilize F-actin (Fu et al., JBC
274:30729-30737, 1999).
[0011] The Rho, rac and cdc42 small GTPases have been shown to
regulate actin polymerization and the formation of multimolecular
focal complexes (for example, see Nobes et al., Cell 81:53-62,
1995, and references therein; incorporated herein by reference).
Further, PAK1 has been shown to regulate actin cytoskeleton
organization, possibly through the phosphorylation and inhibition
of the myosin light chain kinase Sanders et al., Science
283:2083-2085, 1999).
[0012] In addition, intracellular signaling mechanisms affecting
cytoskeletal organization and underlying cell migration in response
to extracellular cues have been studied in some detail (for review,
see Maghazachi et al., Int. J. Biochem. Cell Biol. 32:931-943,
2000).
[0013] Several kinases and other intracellular signaling molecules
have also been implicated in the control of apoptosis and cell
survival in mammalian cells. For example, the JNK family of kinases
has been implicated in both apoptosis and cell survival, the
particular effect being dependent on the cellular context (for
review, see Ip et al., Curr. Opin. Cell Biol. 10:205-219,
1998).
[0014] The role of GCKs in the immune system is of particular
interest. Although GCKs are expressed widely, in B lymphocytic
follicular tissue, GCK expression is largely restricted to the
germinal center (Katz et al., JBC 269:16802-16809, 1994). In
germinal centers, B lymphocytes undergo differentiation and
selection, which is induced in part by ligands including members of
the TNF family. These ligands activate GCKs which in turn activate
other protein kinases that induce lymphocyte development (reviewed
in Kyriakis, JBC 274:5259-5262, 1999).
[0015] The integrity of intracellular signal transduction pathways
and their appropriate regulation is essential for B cell and T cell
development and function. An understanding of these signaling
pathways is therefore desirable to provide means for
therapeutically modulating lymphocyte function in a variety of
disorders characterized by hyper immune responses (e.g. auto-immune
disorders) or hypo immune responses (e.g. immunodeficiency
disorders). Such understanding is also desirable to provide for the
modulation of normal but undesirable immune responses, for example
following transplant immunosuppressive agents are desirable.
[0016] The modulation of signal transduction, proliferation,
apoptosis, morphological change, metastasis, and migration in
mammalian cells is desirable, for example for the treatment of
cancer, such as immune dysfunction, and for immunosuppression.
Accordingly, compositions and methods for modulating these
processes in mammalian cells are desirable.
SUMMARY OF THE INVENTION
[0017] The present invention provides compositions and methods for
modulating proliferation, survival, migration, metastasis,
morphology, cytoskeletal organization and intracellular signal
transduction in mammalian cells. Nucleic acids encoding proteins
and proteins so encoded which are capable of modulating
proliferation, survival, metastasis, migration, morphology,
cytoskeletal organization and intracellular signal transduction in
mammalian cells are provided. Compositions and methods for the
treatment of disorders related to cell proliferation, survival,
morphology, metastasis, and migration are also provided.
Prophylactics and methods for the prevention of such disorders are
also provided. Also provided are compositions and methods for
diagnostic and prognostic determination of such disorders. Further
provided are assays for the identification of bioactive agents
capable of modulating signal transduction, proliferation, survival,
metastasis, morphology and migration in mammalian cells.
[0018] Accordingly, the present invention provides MINK3 nucleic
acids, including nucleic acids encoding MINK3 protein, which are
capable of modulating proliferation, survival, migration,
morphology, metastasis, cytoskeletal organization and intracellular
signal transduction in mammalian cells. Also provided herein are
MINK3 antisense nucleic acids which are capable of modulating
proliferation, survival, migration, morphology, metastasis,
cytoskeletal organization and intracellular signal transduction in
mammalian cells. Also provided herein are MINK3 proteins, including
dominant negative MINK3 proteins, which are capable of modulating
proliferation, survival, migration, metastasis, morphology,
cytoskeletal organization and intracellular signal transduction in
mammalian cells.
[0019] MINK (misshapen/NIKs-related kinase) proteins and nucleic
acids having homology to the MINK3 proteins and nucleic acids
described herein have been previously described (Ippeita et. al.,
FEBS Letters, 469:19-23, 2000). For example, MINK1 is a gck kinase
family member which is upregulated during brain development
(Ippeita et. al., FEBS Letters, 469:19-23, 2000).
[0020] In one aspect, the invention is directed to MINK3 proteins.
In another aspect, the invention is directed to recombinant MINK3
nucleic acids, including nucleic acids encoding MINK3 proteins. In
a further aspect, the invention is directed to recombinant MINK3
antisense nucleic acids comprising nucleic acid sequences
complementary to the nucleic acid sequences of MINK3 nucleic acids
or fragments thereof.
[0021] In a preferred embodiment of the invention, the MINK3
nucleic acid comprises a nucleic acid sequence selected from the
group consisting of the nucleic acid sequences set forth in SEQ ID
NOs:2, 4, and 6, or complements thereof.
[0022] In another preferred embodiment, the MINK3 nucleic acid
comprises a nucleic acid sequence having at least about 90%
identity, more preferably at least about 95% identity to a nucleic
acid sequence selected from the group consisting of the nucleic
acid sequences set forth in SEQ ID NOs:2, 4, and 6, or complements
thereof.
[0023] In another preferred embodiment, the MINK3 nucleic acid will
hybridize under high stringency conditions to a nucleic acid
comprising a nucleic acid sequence selected from the group
consisting of the nucleic acid sequences set forth in SEQ ID NOs:2,
4, and 6, or complements thereof.
[0024] In a preferred embodiment, the MINK3 antisense nucleic acid
comprises a nucleic acid sequence complementary to the nucleic acid
sequence set forth in SEQ ID NO: 2, more preferably to the nucleic
acid sequence set forth by a fragment of SEQ ID NO:2, more
preferably to the nucleic acid sequence set forth by nucleotides
2804-3187 in SEQ ID NO:2.
[0025] In a preferred embodiment, the MINK3 antisense nucleic acid
has a nucleic acid sequence that consists essentially of the
complement of the nucleic acid sequence set forth by nucleotides
2804-3187 in SEQ ID NO:2.
[0026] In a preferred embodiment, the MINK3 antisense nucleic acid
hybridizes to MINK3a and MINK1 nucleic acids.
[0027] In a preferred embodiment, the MINK3 nucleic acid comprises
a nucleic acid sequence encoding MINK3 protein. Preferably MINK3
protein so encoded will bind Nck.
[0028] In a preferred embodiment, the MINK3 nucleic acid comprises
a nucleic acid sequence encoding a MINK3 protein comprising an
amino acid sequence selected from the group consisting of the amino
acid sequences set forth in SEQ ID NOs:1, 3, and 5.
[0029] In a preferred embodiment, the MINK3 nucleic acid comprises
a nucleic acid sequence encoding a MINK3 protein comprising an
amino acid sequence having at least about 90% identity, more
preferably at least about 95% identity, to an amino acid sequence
selected from the group consisting of the amino acid sequences set
forth in SEQ ID NOs:1, 3, AND 5.
[0030] In one aspect, the present invention provides MINK3 proteins
encoded by MINK3 nucleic acids described herein.
[0031] In one aspect, the invention provides three isoforms of
MINK3 protein and nucleic acid, namely MINK3a, MINK3b and MINK3c,
comprising amino acid and nucleic acid sequences as described
herein. MINK3b and MINK3c nucleic acids have a frameshift relative
to MINK3a. As a consequence, MINK3b and MINK3c proteins are kinase
dead proteins.
[0032] In one aspect, the invention provides MINK3 antisense
nucleic acids. In a preferred embodiment, the MINK3 antisense
nucleic acid will inhibit growth factor-induced activation of an
extracellular signal response kinase (ERK), preferably EGF-induced
ERK activation.
[0033] In a preferred embodiment, the MINK3 antisense nucleic acid
will inhibit proliferation in a mammalian cell, preferably a cancer
cell.
[0034] In a preferred embodiment, the MINK3 antisense nucleic acid
will inhibit aberrant cell proliferation, for example as occurs in
cancer. Preferably such aberrant cell proliferation involves
aberrant ERK and/or JNK pathway activation.
[0035] In a preferred embodiment, the MINK3 antisense nucleic acid
will inhibit growth factor-dependent proliferation in a mammalian
cell. Preferably such growth factor-dependent proliferation is
EGF-dependent.
[0036] In a preferred embodiment, the MINK3 antisense nucleic acid
will inhibit phosphorylation of c-JUN N-terminal kinase (JNK)
and/or ERK.
[0037] In a preferred embodiment, the MINK3 antisense nucleic acid
will inhibit activation of JNK and/or ERK.
[0038] In a preferred embodiment, the MINK3 antisense nucleic acid
will inhibit the JNK signal transduction pathway and/or the ERK
signal transduction pathway in a mammalian cell. Preferably the
mammalian cell is a cancer cell and/or a lymphocyte.
[0039] In a preferred embodiment, the MINK3 antisense nucleic acid
will inhibit taxol-induced cleavage of the retinoblastoma protein
(Rb) and apoptosis in a mammalian cell. In a preferred embodiment,
the MINK3 antisense nucleic acid will promote survival in a
mammalian cell following exposure to taxol.
[0040] In a preferred embodiment, the MINK3 antisense nucleic acid
will inhibit the transcription promoting activity of AP-1 in a
mammalian cell. In a preferred embodiment, the MINK3 antisense
nucleic acid will inhibit transcriptional activation by one or more
AP-1 response elements.
[0041] In a preferred embodiment, a MINK3 inhibitor will inhibit
cellular migration and or metastasis, preferably in a transformed,
malignant, cancerous, or tumor cell.
[0042] In one embodiment, a MINK3 nucleic acid has an activity
opposite to that of a MINK3 antisense nucleic acid.
[0043] In another aspect of the invention, expression vectors are
provided. The expression vectors comprise one or more MINK3 nucleic
acids described herein operably linked to regulatory sequences
recognized by a host cell transformed with the nucleic acid.
Further provided herein are host cells comprising the vectors and
MINK3 nucleic acids provided herein. Moreover, provided herein are
processes for producing MINK3 protein comprising culturing a host
cell under conditions suitable for expression of the MINK3 protein.
In one embodiment, the process includes recovering the MINK3
protein.
[0044] In one aspect, the invention is directed to MINK3
proteins.
[0045] In a preferred embodiment of the invention, the MINK3
protein comprises an amino acid sequence selected from the group
consisting of the amino acid sequences set forth in SEQ ID NOs:1,
3, and 5.
[0046] In another preferred embodiment, the MINK3 protein comprises
an amino acid sequence having at least about 90% identity, more
preferably at least about 95% identity to an amino acid sequence
selected from the group consisting of the amino acid sequences set
forth in SEQ ID NOs:1, 3, and 5.
[0047] In a preferred embodiment, the MINK3 protein will bind to
NCK protein, such as Nck protein comprising the amino acid sequence
set forth at Genbank accession number AAD13752.
[0048] In another preferred embodiment, the MINK3 protein will
effect a change in morphology in a mammalian cell, preferably a
cancer cell. In a preferred embodiment, the morphology affecting
activity of MINK3 protein is MEK-dependent.
[0049] In a preferred embodiment, the MINK3 protein will disrupt
actin filaments in a mammalian cell, preferably a cancer cell.
[0050] In a preferred embodiment, the MINK3 protein will
phosphorylate JNK and/or ERK.
[0051] In a preferred embodiment, the MINK3 protein will activate
JNK and/or ERK.
[0052] In a preferred embodiment, the MINK3 protein will activate
the JNK signal transduction pathway and/or the ERK signal
transduction pathway in a mammalian cell.
[0053] In another preferred embodiment, the MINK3 protein will
induce cell cycle progression and proliferation in a mammalian
cell.
[0054] In a preferred embodiment, the MINK3 protein comprises a
germinal center kinase domain (GCK) (sometimes referred to herein,
and in the literature, as a "CNH" domain) as that set forth by
amino acids 994-1292 or 994-1290 in SEQ ID NO:1.
[0055] In a preferred embodiment, the MINK3 protein comprises a
catalytic serine/threonine kinase domain, as that set forth by
amino acids 25-289 in SEQ ID NO:1.
[0056] In a preferred embodiment, the MINK3 protein comprises a
catalytic tyrosine kinase domain, as that set forth by amino acids
26-286 in SEQ ID NO:1.
[0057] In a preferred embodiment, the MINK3 protein provided herein
comprises an ATP-binding domain, such as that set forth by amino
acids 32-54 in SEQ ID NO:1.
[0058] In one aspect, the invention is directed to dominant
negative MINK3 proteins. A dominant negative MINK3 protein will
antagonize at least one MINK3 protein activity.
[0059] In a preferred embodiment, the dominant negative MINK3
protein will inhibit the JNK signal transduction pathway and/or the
ERK signal transduction pathway in a mammalian cell. Preferably the
mammalian cell is a cancer cell.
[0060] In a preferred embodiment, the dominant negative MINK3
protein will inhibit growth factor-induced ERK activation in a
mammalian cell, preferably a cancer cell.
[0061] In a preferred embodiment, the dominant negative MINK3
protein will inhibit growth factor-dependent proliferation in a
mammalian cell.
[0062] In another preferred embodiment, the dominant negative MINK3
protein will inhibit cell cycle progression and proliferation in a
mammalian cell. Preferably the mammalian cell is a cancer cell
and/or a lymphocyte.
[0063] In a preferred embodiment, the dominant negative MINK3
protein is a kinase dead MINK3 protein variant as described
herein.
[0064] In a preferred embodiment, the dominant negative kinase dead
MINK3 protein variant has a mutation in an ATP-binding domain.
Preferably the non-mutant ATP-binding domain of the non-variant
MINK3 protein (non-variant with respect to ATP-binding domain)
comprises the amino acid sequence set forth by amino acids 32-54 in
SEQ ID NO:1. In a preferred embodiment, the dominant negative
kinase dead MINK3 protein variant has a substitution mutation in
the ATP binding domain at a position corresponding to K54 in SEQ ID
NO:1. In a preferred embodiment, expression of kinase dead MINK3
reduces invasion potential of a cell.
[0065] In one aspect, the invention is directed to methods for
screening candidate bioactive agents for an ability to bind to
MINK3 proteins. In a preferred embodiment, the methods comprise
combining a MINK3 protein and a candidate bioactive agent and
determining the binding of candidate bioactive agent to MINK3
protein.
[0066] In another aspect, the invention is directed to methods for
screening a candidate bioactive agent for an ability to interfere
with the binding of a MINK3 protein. In one embodiment, the
interference is between the binding of anti-MINK3 antibody and
MINK3 protein. In a preferred embodiment, the interference is
between the binding of a MINK3 protein and an Nck protein such as
an Nck protein comprising the amino acid sequence set forth at
Genbank accession number AAD13752. In one embodiment, such a method
comprises combining a MINK3 protein, a candidate bioactive agent
and an Nck protein, and determining the binding of the MINK3
protein to Nck protein in the presence of candidate bioactive
agent. Preferably, the binding of MINK3 to Nck is determined in the
presence and absence of candidate bioactive agent. If desired, the
MINK3 protein and the Nck protein can be combined first.
[0067] In another aspect, the invention is directed to methods for
screening candidate bioactive agents for an ability to modulate
MINK3 protein activity. In one embodiment, candidate bioactive
agents identified in these assays include small organic molecules,
peptides, cyclic peptides, nucleic acids, antibodies, antisense
nucleic acids, RNAi, and ribozymes. In a preferred embodiment, the
methods comprise combining a MINK3 protein and a candidate
bioactive agent and determining the effect of the candidate agent
on the activity of MINK3 protein. In a preferred embodiment, a
library of candidate bioactive agents is added to a plurality of
cells comprising a recombinant nucleic acid encoding a MINK3
protein and MINK3 protein activity is determined. Preferably, MINK3
protein activity is determined in the presence and absence of
candidate bioactive agent.
[0068] In one aspect, the invention is directed to methods for
screening for a bioactive agent capable of modulating JNK
phosphorylation and/or activation. In one aspect, the invention is
directed to methods for screening for a bioactive agent capable of
modulating the JNK signal transduction pathway. In a preferred
embodiment, the methods comprise contacting a candidate bioactive
agent to a mammalian cell comprising a recombinant MINK3 nucleic
acid encoding a MINK3 protein and a JNK protein and determining JNK
activity in the presence of candidate agent. In a preferred
embodiment, JNK activity is determined in the presence and absence
of candidate agent. The recombinant MINK3 nucleic acid is expressed
in said mammalian cell and will activate JNK protein in the absence
of candidate bioactive agent. In a preferred embodiment, the
encoded MINK3 protein comprises an amino acid sequence having at
least about 90% identity to an amino acid sequence selected from
the group consisting of the amino acid sequences set forth in SEQ
ID NOs:1, 3, and 5. A decrease in the activity of JNK protein in
the presence of candidate bioactive agent indicates that the
candidate bioactive agent is capable of modulating JNK
activity.
[0069] In one aspect, the invention is directed to methods for
screening for a bioactive agent capable of modulating ERK
phosphorylation and/or activation. In one aspect, the invention is
directed to methods for screening for a bioactive agent capable of
modulating the ERK signal transduction pathway. In a preferred
embodiment, the methods comprise contacting a candidate bioactive
agent to a mammalian cell comprising a recombinant MINK3 nucleic
acid encoding a MINK3 protein and a ERK protein and determining ERK
activity in the presence of candidate agent. In a preferred
embodiment, ERK activity is determined in the presence and absence
of candidate agent. The recombinant MINK3 nucleic acid is expressed
in said mammalian cell and will activate ERK protein in the absence
of candidate bioactive agent. In a preferred embodiment, the
encoded MINK3 protein comprises an amino acid sequence having at
least about 90% identity to an amino acid sequence selected from
the group consisting of the amino acid sequences set forth in SEQ
ID NOs:1, 3, and 5. A decrease in the activity of ERK protein in
the presence of candidate bioactive agent indicates that the
candidate bioactive agent is capable of modulating ERK
activity.
[0070] In a preferred embodiment, the methods comprise contacting a
mammalian cell with a growth factor which will activate JNK and/or
ERK. In a preferred embodiment, the growth factor used in epidermal
growth factor (EGF).
[0071] In one aspect, the invention is directed to methods for
screening candidate bioactive agents for an ability to modulate
proliferation, survival, migration, metastasis, morphology,
cytoskeletal organization and intracellular signal transduction in
mammalian cells. Such cells are preferably cancer cells and/or
lymphocytes. In a preferred embodiment, the method involves
screening for a bioactive agent capable of binding to MINK3 protein
using assays provided herein. In another preferred embodiment, the
method involves screening for a bioactive agent capable of
modulating MINK3 binding using assays provided herein. In another
preferred embodiment, the method involves screening for a bioactive
agent capable of modulating MINK3 activity using assays provided
herein.
[0072] In a preferred embodiment the methods comprise combining a
MINK3 protein, a candidate bioactive agent and a cell or a
population of cells and determining the effect on the cell in the
presence and absence of candidate agent.
[0073] In a preferred embodiment, the methods comprise introducing
a recombinant MINK3 nucleic acid into a host cell capable of
expressing the nucleic acid, contacting the cell with a candidate
bioactive agent, and determining the effect on the cell in the
presence and absence of candidate bioactive agent. In another
preferred embodiment, a library of candidate bioactive agents is
added to a plurality of cells comprising a recombinant nucleic acid
encoding a MINK3 protein.
[0074] A MINK3 protein used in screening methods provided herein
may be recombinant, isolated or cell-free as in a cell lysate.
[0075] Preferred candidate bioactive agents for use in screening
methods provided herein include small molecule chemical compounds,
peptides, cyclic peptides, nucleic acids, antibodies, antisense
nucleic acids, RNAi, and ribozymes.
[0076] In another aspect, the invention provides compositions and
methods for diagnostic and prognostic determination of disorders
involving MINK3 dysfunction and/or dysregulation. Without being
bound by theory, such disorders involve the dysregulation of MINK3
gene expression, aberrant MINK3 gene structure and/or modification,
the dysregulation and/or dysfunction of MINK3 protein, and aberrant
MINK3 protein structure and/or modification.
[0077] Further provided herein are compositions and methods for
prophylaxis and therapeutic treatment of disorders related to
and/or involving MINK3 dysfunction or dysregulation. In a preferred
embodiment, such disorders include cancer, e.g., melanoma, breast,
ovarian, lung, gastrointestinal and colon, prostate, and leukemia
and lymphomas, e.g., multiple myeloma. In addition, such
compositions are useful for treating noncancerous disease states
caused by pathologically proliferating cells such as thyroid
hyperplasia (Grave's disease), psoriasis, benign prostatic
hypertrophy, neurofibromas, atherosclerosis, restenosis, and other
vasoproliferative disease. In a preferred embodiment, such
disorders involve dysfunction or dysregulation of leukocyte
function, preferably lymphocyte function.
[0078] In one aspect, the present invention provides isolated
polypeptides which specifically bind to a MINK3 protein as
described herein. In a preferred embodiment, the isolated peptide
is an anti-MINK3 antibody. In a further preferred embodiment, the
isolated peptide is an anti-MINK3 monoclonal antibody. In a
preferred embodiment, the anti-MINK3 antibody will reduce or
eliminate the biological function of MINK3 protein.
[0079] In a preferred embodiment, the present invention provides
MINK3 proteins and nucleic acids, as well as agents that bind to
them and/or modulate their activity, including and preferably small
molecule chemical compositions as discussed herein, which are
useful in the treatment of acute and chronic inflammatory diseases
and autoimmune diseases, as well as in the treatment of a host
receiving a transplant, as well as diseases characterized by
immunodeficiency.
[0080] The dysregulation of mechanisms of programmed cell death can
lead to cancer, particularly in lymphocytes (Chao et al., Ann. Rev.
Immunol. 16:395-419, 1998). For example, overexpression of Bcl-2,
which is involved in normal cell survival through the inhibition of
apoptosis, is thought to be responsible for the survival of
excessive numbers of lymphocytes in a form of lymphoma.
[0081] Without being bound by theory, the present invention
provides MINK3 proteins and nucleic acids, as well as agents that
bind to them and/or modulate their activity, including and
preferably small molecule chemical compositions as discussed
herein, which are useful in the modulation of T cell and B cell
survival and apoptosis.
[0082] Other aspects of the invention will become apparent to the
skilled artisan by the following description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0083] FIG. 1 shows the nucleic acid sequence and amino acid
sequence of MINK3a, MINK3b, and MINK3c. SEQ ID NOs:2 and 1 depict
the nucleic acid and amino acid sequences of MINK3a, respectively.
SEQ ID NOs:4 and 3 depict the nucleic acid and amino acid sequences
of MINK3b, respectively. SEQ ID NOs:6 and 5 depict the nucleic acid
and amino acid sequences of MINK3c, respectively.
[0084] FIG. 2 schematically describes the structure of MINK1 and
MINK3 isoforms a and b. The kinase domain, intermediate region, and
GCK (GCKH) domain are identified, as is the PxxPxR motif which
interacts with the SH3 domain of Nck. The relative position of a
MINK3 antisense nucleic acid ("MINK3 as (TR5)") is also shown. It
is recognized that the MINK3 antisense nucleic acid described is
directed to MINK1 as well. It is also recognized that MINK3b and
MINK3c have a frameshift relative to MINK3a; consequently MINK3b
and MINK3c proteins comprising MINK3b and MINK3c amino acid
sequence are kinase dead MINK3 proteins.
[0085] MINK3 antisense nucleic acid is herein equivalently referred
to as antisense MINK3 nucleic acid, antisense MINK nucleic acid,
MINK antisense, MINK antisense nucleic acid, and grammatical
equivalents thereof.
[0086] FIG. 3 graphically depicts the effects of MINK3a antisense
nucleic acid on AP-1 modulated transcriptional activity in 293
cells. MEKK1 means MEK kinase-1.
[0087] FIG. 4 schematically describes signal transduction pathways
mediating the response of mammalian cells to taxol.
[0088] FIGS. 5 and 6 schematically describes the signal
transduction pathways that propagate signals initiated by
cytokines, mutagens, and cellular stress.
[0089] FIG. 7 graphically demonstrates the ability of MINK3
antisense nucleic acid to inhibit taxol-induced cell death in Hela
cells.
[0090] FIG. 8 graphically demonstrates the ability of MINK3a to
inhibit proliferation of the human tumor cell line A549 in low
serum.
[0091] FIG. 9 graphically demonstrates the ability of MINK3
antisense nucleic acid to inhibit EGF-induced ERK-mediated
transcriptional activation.
[0092] FIG. 10 shows Western blots for retinoblastoma "Rb" and
cleaved Rb and Bcl-2 protein from cells expressing (1) Bcl2; (2)
MINK3 antisense nucleic acid "TR5"; (3) GFP; and (4), (5) cells
transfected with empty vector; in the presence and absence of
taxol.
[0093] FIG. 11 shows Northern blot of MINK3 mRNA in human tissue
samples.
[0094] FIG. 12 shows Northern blot of MINK3 mRNA in tumor cell
lines.
[0095] FIG. 13 shows JNK kinase assay using GST-cJUN as substrate,
and ERK kinase assay using MBP as substrate, done on extracts from
cells transfected with empty vector "pCDNA"; MINK3a, MINK3b or
MEKK1. Western blots "WB" are also done for JNK and ERK
proteins.
[0096] FIG. 14 shows MDA-MB-231 human tumor cells expressing
recombinant MINK3a, MINK3b, antisense MINK nucleic acid, or
GFP.
[0097] FIG. 15 shows stably transfected MDA-MB-231 cells expressing
recombinant MINK3a "MINK3a", and MDA-MB-231 cells alone
"MDA-MB-231", and stably transfected MDA-MB-231 cells expressing
recombinant MINK3a treated with the MEK inhibitor PD98059.
[0098] FIG. 16 shows that expression of kinase dead mutant of
Mink3a in HT1080 cell (human fibrosarcoma cell line) greatly
reduced its invasion potential comparing with GFP control and
wild-type Mink3.
DETAILED DESCRIPTION OF THE INVENTION
[0099] The present invention provides compositions and methods for
modulating proliferation, survival, migration, morphology,
metastasis, cytoskeletal organization and intracellular signal
transduction in mammalian cells. Nucleic acids encoding proteins
and proteins so encoded which are capable of modulating
proliferation, survival, migration, morphology, cytoskeletal
organization and intracellular signal transduction in mammalian
cells are provided. Compositions and methods for the treatment of
disorders related to cell proliferation, survival, morphology and
migration are also provided. Prophylactics and methods for the
prevention of such disorders are also provided. Also provided are
compositions and methods for diagnostic and prognostic
determination of such disorders. Further provided are assays for
the identification of bioactive agents capable of modulating signal
transduction, proliferation, survival, morphology and migration in
mammalian cells.
[0100] The present invention provides MINK3 nucleic acids,
including nucleic acids encoding MINK3 protein, which are capable
of modulating proliferation, survival, migration, metastasis,
morphology, cytoskeletal organization and intracellular signal
transduction in mammalian cells. Also provided herein are MINK3
antisense nucleic acids which are capable of modulating
proliferation, survival, migration, morphology, cytoskeletal
organization and intracellular signal transduction in mammalian
cells. Also provided herein are MINK3 proteins, including dominant
negative MINK3 proteins, which are capable of modulating
proliferation, survival, migration, morphology, cytoskeletal
organization and intracellular signal transduction in mammalian
cells.
[0101] MINK3 proteins of the present invention include proteins,
polypeptides, and peptides. Among the MINK3 proteins included
herein are dominant negative isoforms of MINK3 proteins which will
inhibit the activity of non-mutant MINK3 proteins in the presence
thereof.
[0102] In one embodiment, a MINK3 protein has one or more of the
following characteristics (MINK3 bioactivities): binding to Nck
(for example, an Nck protein comprising the amino acid sequence set
forth at Genbank accession no. AAD13752); kinase activity directed
at JNK, preferably JNK2; kinase activity directed at ERK,
preferably ERK1; an ability to activate JNK and/or ERK, preferably
JNK2 and ERK1; an ability to activate JNK and/or ERK signal
transduction pathways in mammalian cells; an ability to effect a
change in cell morphology in mammalian cells, preferably cancer
cells; an ability to disrupt F-actin in mammalian cells, preferably
cancer cells.
[0103] In a preferred embodiment, MINK3 protein binds to Nck
protein via the PxxPxR amino acid motif in MINK3 protein. In a
preferred embodiment, MINK3 protein binds to Nck in mammalian
cells. IN one embodiment, MINK3 binds to Nck in tumor cells, for
example 293 cells.
[0104] Also provided herein are MINK3 dominant negative proteins
which inhibit at least one MINK3 protein activity. In a preferred
embodiment, the MINK3 dominant negative protein has one or more of
the following characteristics (MINK3 dominant negative activities):
an ability to bind Nck protein; an inability to bind Nck protein;
an ability to inhibit growth factor-induced ERK activation,
preferably EGF-induced ERK activation; an ability to inhibit
proliferation in a mammalian cell, preferably a cancer cell; an
ability to inhibit growth factor-induced proliferation in a
mammalian cell, preferably EGF-induced proliferation; an ability to
inhibit growth factor-dependent proliferation in a mammalian cell,
preferably EGF-dependent proliferation; an ability to inhibit
aberrant cell proliferation, preferably involving aberrant JNK
and/or ERK activation, preferably in cancer cells; an ability to
inhibit phosphorylation of c-JUN N-terminal kinase (JNK) and/or
ERK, preferably JNK2 and/or ERK1; an ability to inhibit activation
of JNK and/or ERK, preferably JNK2 and/or ERK1; an ability to
inhibit the JNK signal transduction pathway and/or the ERK signal
transduction pathway in a mammalian cell, preferably a cancer cell,
preferably a lymphocyte; an ability to inhibit taxol-induced
cleavage of Rb and apoptosis in a mammalian cell; an ability to
promote survival in a mammalian cell following exposure to taxol;
an ability to inhibit the transcription promoting activity of AP-1
in a mammalian cell; an ability to inhibit transcriptional
activation by one or more AP-1 response elements.
[0105] In a preferred embodiment, the MINK3 protein has one or more
of the MINK3 bioactivities described herein. In other embodiments
where particular bioactivities are not required, a MINK3 protein
does not include all bioactivities described herein for MINK3
proteins.
[0106] It has been reported that the adaptor protein Nck links
receptor tyrosine kinases with the serine-threonine kinase Pak1.
Nck is an adaptor protein composed of a single SH2 domain and three
SH3 domains. Upon growth factor stimulation, Nck is recruited to
receptor tyrosine kinases via its SH2 domain, probably initiating
one or more signaling cascades. Galisteo, et al., J Biol Chem,
271(35):20997-1000 (1996). Also see, Chen, et al., J Biol Chem.,
273(39):25171-8 (1998) which reports on Nck family genes,
chromosomal localization and expression.
[0107] In one embodiment, MINK3 nucleic acids or MINK3 proteins are
initially identified by substantial nucleic acid and/or amino acid
sequence identity or similarity to sequences provided herein. In a
preferred embodiment, MINK3 nucleic acids or MINK3 proteins have
sequence identity or similarity to the sequences provided herein as
described below and one or more of the MINK3 protein bioactivities
(or MINK3 dominant negative activities or MINK3 antisense nucleic
acid activities) as described herein. Such sequence identity or
similarity can be based upon the overall nucleic acid or amino acid
sequence.
[0108] In a preferred embodiment, a protein is a "MINK3 protein" as
defined herein if it comprises an amino acid sequence having at
least about 80%, more preferably at least about 85%, more
preferably at least about 90%, more preferably at least about 95%,
more preferably at least about 98% identity to an amino acid
sequence selected from the group consisting of the amino acid
sequences set forth in SEQ ID NOs:1, 3, and 5.
[0109] In a preferred embodiment, the MINK3 protein comprises an
amino acid sequence selected from the group consisting of the amino
acid sequences set forth in SEQ ID NOs:1, 3, and 5.
[0110] In another preferred embodiment, a MINK3 protein has an
overall sequence similarity to an amino acid sequence selected from
the group consisting of the amino acid sequences set forth in SEQ
ID NOs:1, 3, and 5 of greater than at least about 80%, more
preferably at least about 85%, more preferably at least about 90%,
more preferably at least about 95%, more preferably at least about
98%, more preferably 100%.
[0111] In another preferred embodiment, a MINK 3 protein provided
herein comprises a GCK domain comprising an amino acid sequence
having at least about 80%, more preferably at least about 85%, more
preferably at least about 90%, more preferably at least about 95%,
more preferably at least about 98%, more preferably about 99%, more
preferably 100% identity to the amino acid sequence set forth by
amino acids 994-1292 or 994-1290 in SEQ ID NO:1.
[0112] In another preferred embodiment, a MINK 3 protein provided
herein comprises a catalytic serine/threonine kinase domain
comprising an amino acid sequence having at least about 80%, more
preferably at least about 85%, more preferably at least about 90%,
more preferably at least about 95%, more preferably at least about
98%, more preferably about 99%, more preferably 100% identity to
the amino acid sequence set forth by amino acids 25-289 in SEQ ID
NO:1.
[0113] In another preferred embodiment, a MINK 3 protein provided
herein comprises a catalytic tyrosine kinase domain comprising an
amino acid sequence having at least about 80%, more preferably at
least about 85%, more preferably at least about 90%, more
preferably at least about 95%, more preferably at least about 98%,
more preferably about 99%, more preferably 100% identity to the
amino acid sequence set forth by amino acids 26-286 in SEQ ID
NO:1.
[0114] In another preferred embodiment, a MINK 3 protein provided
herein comprises an ATP binding domain comprising an amino acid
sequence having at least about 80%, more preferably at least about
85%, more preferably at least about 90%, more preferably at least
about 95%, more preferably at least about 98%, more preferably
about 99%, more preferably 100% identity to the amino acid sequence
set forth by amino acids 32-54 in SEQ ID NO:1.
[0115] As is known in the art, a number of different programs can
be used to identify whether a protein (or nucleic acid as discussed
below) has sequence identity or similarity to a known sequence.
Sequence identity and/or similarity is determined using standard
techniques known in the art, including, but not limited to, the
local sequence identity algorithm of Smith, et al., Adv. Appl.
Math. 2:482 (1981), by the sequence identity alignment algorithm of
Needleman, et al., J. Mol. Biol., 48:443 (1970), by the search for
similarity method of Pearson, et al., PNAS USA, 85:2444 (1988), by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Drive, Madison, Wis.), the
Best Fit sequence program described by Devereux, et al., Nucl. Acid
Res., 12:387-395 (1984), preferably using the default settings, or
by inspection. Preferably, percent identity is calculated by FastDB
based upon the following parameters: mismatch penalty of 1; gap
penalty of 1; gap size penalty of 0.33; and joining penalty of 30,
"Current Methods in Sequence Comparison and Analysis,"
Macromolecule Sequencing and Synthesis, Selected Methods and
Applications, pp 127-149 (1988), Alan R. Liss, Inc.
[0116] An example of a useful algorithm is PILEUP. PILEUP creates a
multiple sequence alignment from a group of related sequences using
progressive, pairwise alignments. It can also plot a tree showing
the clustering relationships used to create the alignment. PILEUP
uses a simplification of the progressive alignment method of Feng,
et al., J. Mol. Evol., 35:351-360 (1987); the method is similar to
that described by Higgins, et al., CABIOS, 5:151-153 (1989). Useful
PILEUP parameters including a default gap weight of 3.00, a default
gap length weight of 0.10, and weighted end gaps.
[0117] Another example of a useful algorithm is the BLAST
algorithm, described in Altschul, et al., J. Mol. Biol.,
215:403-410, (1990) and Karlin, et al., PNAS USA, 90:5873-5787
(1993). A particularly useful BLAST program is the WU-BLAST-2
program which was obtained from Altschul, et al., Methods in
Enzymology, 266:460-480 (1996). WU-BLAST-2 uses several search
parameters, most of which are set to the default values. The
adjustable parameters are set with the following values: overlap
span=1, overlap fraction=0.125, word threshold (T)=11. The HSP S
and HSP S2 parameters are dynamic values and are established by the
program itself depending upon the composition of the particular
sequence and composition of the particular database against which
the sequence of interest is being searched; however, the values may
be adjusted to increase sensitivity.
[0118] An additional useful algorithm is gapped BLAST as reported
by Altschul, et al., Nucleic Acids Res., 25:3389-3402. Gapped BLAST
uses BLOSUM-62 substitution scores; threshold T parameter set to 9;
the two-hit method to trigger ungapped extensions; charges gap
lengths of k a cost of 10+k; Xu set to 16, and Xg set to 40 for
database search stage and to 67 for the output stage of the
algorithms. Gapped alignments are triggered by a score
corresponding to .about.22 bits.
[0119] A % amino acid sequence identity value is determined by the
number of matching identical residues divided by the total number
of residues of the "longer" sequence in the aligned region. The
"longer" sequence is the one having the most actual residues in the
aligned region (gaps introduced by WU-Blast-2 to maximize the
alignment score are ignored).
[0120] In a similar manner, "percent (%) nucleic acid sequence
identity" with respect to the coding sequence of the polypeptides
identified herein is defined as the percentage of nucleotide
residues in a candidate sequence that are identical with the
nucleotide residues in the coding sequence of the MINK3 protein. A
preferred method utilizes the BLASTN module of WU-BLAST-2 set to
the default parameters, with overlap span and overlap fraction set
to 1 and 0.125, respectively.
[0121] The alignment may include the introduction of gaps in the
sequences to be aligned. In addition, for sequences which contain
either more or fewer amino acids than a protein comprising an amino
acid sequence selected from the group consisting of the amino acid
sequences set forth in SEQ ID NOs:1, 3, and 5, it is understood
that in one embodiment, the percentage of sequence identity will be
determined based on the number of identical amino acids in relation
to the total number of amino acids. Thus, for example, sequence
identity of sequences shorter than that shown in SEQ ID NO:1, 3,
and 5, as discussed below, will be determined using the number of
amino acids in the shorter sequence, in one embodiment. In percent
identity calculations relative weight is not assigned to various
manifestations of sequence variation, such as, insertions,
deletions, substitutions, etc.
[0122] In one embodiment, only identities are scored positively
(+1) and all forms of sequence variation including gaps are
assigned a value of "0", which obviates the need for a weighted
scale or parameters as described below for sequence similarity
calculations. Percent sequence identity can be calculated, for
example, by dividing the number of matching identical residues by
the total number of residues of the "shorter" sequence in the
aligned region and multiplying by 100. The "longer" sequence is the
one having the most actual residues in the aligned region.
[0123] As will be appreciated by those skilled in the art, the
sequences of the present invention may contain sequencing errors.
That is, there may be incorrect nucleosides, frameshifts, unknown
nucleosides, or other types of sequencing errors in any of the
sequences; however, the correct sequences will fall within the
homology and stringency definitions herein.
[0124] MINK3 proteins of the present invention may be shorter or
longer than the amino acid sequences encoded by the nucleic acid
sequences set forth in SEQ ID NOs:2, 4, and 6. Thus, in a preferred
embodiment, included within the definition of MINK3 proteins are
portions or fragments of the amino acid sequences encoded by the
nucleic acid sequences provided herein. In one embodiment herein,
fragments of MINK3 proteins are considered MINK3 proteins if a)
they share at least one antigenic epitope; b) have at least the
indicated sequence identity; c) and preferably have MINK3
biological activity as further defined herein. In some cases, where
the sequence is used diagnostically, that is, when the presence or
absence of MINK3 nucleic acid is determined, only the indicated
sequence identity is required. The nucleic acids of the present
invention may also be shorter or longer than the sequences set
forth in SEQ ID NOs:2, 4, and 6. The nucleic acid fragments include
any portion of the nucleic acids provided herein which have a
sequence not exactly previously identified; fragments having
sequences with the indicated sequence identity to that portion not
previously identified are provided in an embodiment herein.
[0125] In addition, as is more fully outlined below, MINK3 proteins
can be made that are longer than those consisting essentially of an
amino acid sequence selected from the group consisting of the amino
acid sequences set forth in SEQ ID NOs:1, 3, and 5; for example, by
the addition of epitope or purification tags, the addition of other
fusion sequences, or the elucidation of additional coding and
non-coding sequences. As described below, the fusion of a MINK3
peptide to a fluorescent peptide, such as Green Fluorescent Peptide
(GFP), is particularly preferred.
[0126] MINK3 proteins may also be identified as encoded by MINK3
nucleic acids which hybridize to a nucleic acid comprising a
nucleic acid sequence selected from the group consisting of the
nucleic acid sequences set forth in SEQ ID NOs:2, 4, and 6.
Hybridization conditions are further described below.
[0127] In a preferred embodiment, when a MINK3 protein is to be
used to generate antibodies, a MINK3 protein must share at least
one epitope or determinant with the full length protein. By
"epitope" or "determinant" herein is meant a portion of a protein
which will generate and/or bind an antibody. Thus, in most
instances, antibodies made to a smaller MINK3 protein will be able
to bind to the full length protein. In a preferred embodiment, the
epitope is unique; that is, antibodies generated to a unique
epitope show little or no cross-reactivity. The term "antibody"
includes antibody fragments, as are known in the art, including Fab
Fab2, single chain antibodies (Fv for example), chimeric
antibodies, etc., either produced by the modification of whole
antibodies or those synthesized de novo using recombinant DNA
technologies.
[0128] In a preferred embodiment, the antibodies to a MINK3 protein
are capable of reducing or eliminating the biological function of
the MINK3 proteins described herein, as is described below. That
is, the addition of anti-MINK3 protein antibodies (either
polyclonal or preferably monoclonal) to MINK3 proteins (or cells
containing MINK3 proteins) may reduce or eliminate one or more
MINK3 bioactivities as described herein. Generally, at least a 25%
decrease in activity is preferred, with at least about 50% being
particularly preferred and about a 95-100% decrease being
especially preferred.
[0129] The MINK3 antibodies of the invention specifically bind to
MINK3 proteins. By "specifically bind" herein is meant that the
antibodies bind to the protein with a binding constant in the range
of at least 10-4-10-6 M-1, with a preferred range being 10-7-10-9
M-1. Antibodies are further described below.
[0130] In the case of the nucleic acid, the overall sequence
identity of the nucleic acid sequence is commensurate with amino
acid sequence identity but takes into account the degeneracy in the
genetic code and codon bias of different organisms. Accordingly,
the nucleic acid sequence identity may be either lower or higher
than that of the protein sequence. Thus the sequence identity of
the nucleic acid sequence as compared to a nucleic acid sequence
selected from the group of nucleic acid sequences set forth in SEQ
ID NOs:2, 4, and 6 is preferably greater than about 80%, more
preferably greater than about 85%, more preferably greater than
about 90%, more preferably greater than about 95%, more preferably
greater than about 98%, more preferably about 99%, more preferably
100%.
[0131] In a preferred embodiment, a MINK3 nucleic acid encodes a
MINK3 protein. As will be appreciated by those in the art, due to
the degeneracy of the genetic code, an extremely large number of
nucleic acids may be made, all of which encode the MINK3 proteins
of the present invention. Thus, having identified a particular
amino acid sequence, those skilled in the art could make any number
of different nucleic acids, by simply modifying the sequence of one
or more codons in a way which does not change the amino acid
sequence of the MINK3 protein.
[0132] In one embodiment, the nucleic acid is determined through
hybridization studies. Thus, for example, nucleic acids which
hybridize under high stringency conditions to a nucleic acid
comprising a nucleic acid sequence selected from the group
consisting of the nucleic acid sequences set forth in SEQ ID NOs:2,
4, and 6, or complements thereof, are considered MINK3 nucleic
acids. High stringency conditions, including washing conditions,
are known in the art; see for example Maniatis, et al., Molecular
Cloning: A Laboratory Manual, 2d Edition, 1989, and Current
Protocols in Molecular Biology, eds. F. Ausubel et al., New York,
Greene Pub. Associates & Wiley Interscience, 1988; both of
which are hereby incorporated by reference. Stringent conditions
are sequence-dependent and will be different in different
circumstances. Longer sequences hybridize specifically at higher
temperatures. An extensive guide to the hybridization of nucleic
acids is found in Tijssen, Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, "Overview of
principles of hybridization and the strategy of nucleic acid
assays" (1993). Generally, stringent conditions are selected to be
about 5-10.degree. C. lower than the thermal melting point (Tm) for
the specific sequence at a defined ionic strength pH. The Tm is the
temperature (under defined ionic strength, pH and nucleic acid
concentration) at which 50% of the probes complementary to the
target hybridize to the target sequence at equilibrium (as the
target sequences are present in excess, at Tm, 50% of the probes
are occupied at equilibrium). Stringent conditions will be those in
which the salt concentration is less than about 1.0 M sodium ion,
typically about 0.01 to 1.0 M sodium ion concentration (or other
salts) at pH 7.0 to 8.3 and the temperature is at least about
30.degree. C. for short probes (e.g. 10 to 50 nucleotides) and at
least about 60.degree. C. for long probes (e.g. greater than 50
nucleotides). Stringent conditions may also be achieved with the
addition of destabilizing agents such as formamide. Wash conditions
for stringent hybridization typically involve a lower sodium (or
other salt) concentration, and a lower temperature, than that used
in the hybridization step, as discussed in Maniatis (supra),
Ausubel (supra) and Tijssen (supra).
[0133] Stringent conditions may also be achieved with the addition
of destabilizing agents such as formamide. For selective or
specific hybridization, a positive signal is at least two times
background, preferably 10 times background hybridization. Exemplary
stringent hybridization conditions can be as following: 50%
formamide, 5.times.SSC, and 1% SDS, incubating at 42.degree. C.,
or, 5.times.SSC, 1% SDS, incubating at 65.degree. C., with wash in
0.2.times.SSC, and 0.1% SDS at 65.degree. C. Those of ordinary
skill will readily recognize that alternative hybridization and
wash conditions can be utilized to provide conditions of similar
stringency.
[0134] In another embodiment, less stringent hybridization
conditions are used; for example, moderate or low stringency
conditions may be used, as are known in the art; see Maniatis and
Ausubel, (supra), and Tijssen (supra).
[0135] Exemplary moderately stringent hybridization conditions
include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1%
SDS at 37.degree. C., and a wash in 1.times.SSC at 45.degree. C. A
positive hybridization is at least twice background. Those of
ordinary skill will readily recognize that alternative
hybridization and wash conditions can be utilized to provide
conditions of similar stringency.
[0136] For PCR, a temperature of about 36.degree. C. is typical for
low stringency amplification, although annealing temperatures may
vary between about 32.degree. C. and 48.degree. C. depending on
primer length. For high stringency PCR amplification, a temperature
of about 62.degree. C. is typical, although high stringency
annealing temperatures can range from about 50.degree. C. to about
65.quadrature.C, depending on the primer length and specificity.
Typical cycle conditions for both high and low stringency
amplifications include a denaturation phase of 90.degree.
C.-95.degree. C. for 30 sec-2 min., an annealing phase lasting 30
sec.-2 min., and an extension phase of about 72.degree. C. for 1-2
min. Protocols and guidelines for low and high stringency
amplification reactions are provided, e.g., in Innis et al. (1990)
PCR Protocols, A Guide to Methods and Applications, Academic Press,
Inc. N.Y.).
[0137] By "disorder associated with cellular proliferation" or
"disease associated with cellular proliferation" herein is meant a
disease state which is marked by either an excess or a deficit of
cellular proliferation or apoptosis. Such disorders associated with
increased cellular proliferation include, but are not limited to,
cancer and non-cancerous pathological proliferation.
[0138] The MINK3 proteins and nucleic acids of the present
invention are preferably recombinant. As used herein and further
defined below, "nucleic acid" may refer to either DNA or RNA, or
molecules which contain both deoxy- and ribonucleotides. The
nucleic acids include genomic DNA, cDNA and oligonucleotides
including sense and anti-sense nucleic acids. Such nucleic acids
may also contain modifications in the ribose-phosphate backbone to
increase stability and half life of such molecules in physiological
environments.
[0139] The nucleic acid may be double stranded, single stranded, or
contain portions of both double stranded or single stranded
sequence. As will be appreciated by those in the art, the depiction
of a single strand ("Watson") also defines the sequence of the
other strand ("Crick"); thus the sequences depicted in SEQ ID
NOs:2, 4, and 6 also include complements thereof. By the term
"recombinant nucleic acid" herein is meant nucleic acid, originally
formed in vitro, in general, by the manipulation of nucleic acid by
endonucleases, in a form not normally found in nature. Thus an
isolated MINK3 nucleic acid, in a linear form, or an expression
vector formed in vitro by ligating DNA molecules that are not
normally joined, are both considered recombinant for the purposes
of this invention. It is understood that once a recombinant nucleic
acid is made and reintroduced into a host cell or organism, it will
replicate non-recombinantly, i.e. using the in vivo cellular
machinery of the host cell rather than in vitro manipulations;
however, such nucleic acids, once produced recombinantly, although
subsequently replicated non-recombinantly, are still considered
recombinant for the purposes of the invention.
[0140] Similarly, a "recombinant protein" is a protein made using
recombinant techniques, i.e. through the expression of a
recombinant nucleic acid as depicted above. A recombinant protein
is distinguished from naturally occurring protein by at least one
or more characteristics. For example, the protein may be isolated
or purified away from some or all of the proteins and compounds
with which it is normally associated in its wild type host, and
thus may be substantially pure. For example, an isolated protein is
unaccompanied by at least some of the material with which it is
normally associated in its natural state, preferably constituting
at least about 0.5%, more preferably at least about 5% by weight of
the total protein in a given sample. A substantially pure protein
comprises at least about 75% by weight of the total protein, with
at least about 80% being preferred, and at least about 90% being
particularly preferred. The definition includes the production of a
MINK3 protein from one organism in a different organism or host
cell. Alternatively, the protein may be made at a significantly
higher concentration than is normally seen, through the use of a
inducible promoter or high expression promoter, such that the
protein is made at increased concentration levels. Alternatively,
the protein may be in a form not normally found in nature, as in
the addition of an epitope tag or amino acid substitutions,
insertions and deletions, as discussed below.
[0141] In one embodiment, the present invention provides MINK3
protein variants. These variants fall into one or more of three
classes: substitutional, insertional or deletional variants. These
variants ordinarily are prepared by site specific mutagenesis of
nucleotides in the DNA encoding a MINK3 protein, using cassette or
PCR mutagenesis or other techniques well known in the art, to
produce DNA encoding the variant, and thereafter expressing the DNA
in recombinant cell culture as outlined above. However, variant
MINK3 protein fragments having up to about 100-150 residues may be
prepared by in vitro synthesis using established techniques. Amino
acid sequence variants are characterized by the predetermined
nature of the variation, a feature that sets them apart from
naturally occurring allelic or interspecies variation of the MINK3
protein amino acid sequence. The variants typically exhibit the
same qualitative biological activity as the naturally occurring
analogue, although variants can also be selected which have
modified characteristics as will be more fully outlined below.
[0142] While the site or region for introducing an amino acid
sequence variation is predetermined, the mutation per se need not
be predetermined. For example, in order to optimize the performance
of a mutation at a given site, random mutagenesis may be conducted
at the target codon or region and the expressed MINK3 variants
screened for the optimal combination of desired activity.
Techniques for making substitution mutations at predetermined sites
in DNA having a known sequence are well known, for example, M13
primer mutagenesis and PCR mutagenesis. Screening of the mutants is
done using assays of MINK3 protein activities.
[0143] Amino acid substitutions are typically of single residues;
insertions usually will be on the order of from about 1 to 20 amino
acids, although considerably larger insertions may be tolerated.
Deletions range from about 1 to about 20 residues, although in some
cases deletions may be much larger.
[0144] Substitutions, deletions, insertions or any combination
thereof may be used to arrive at a final derivative. Generally
these changes are done on a few amino acids to minimize the
alteration of the molecule. However, larger changes may be
tolerated in certain circumstances. When small alterations in the
characteristics of the MINK3 protein are desired, substitutions are
generally made in accordance with the following chart:
TABLE-US-00001 CHART I Original Residue Exemplary Substitutions Ala
Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro
His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu,
Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile,
Leu
[0145] Substantial changes in function or immunological identity
are made by selecting substitutions that are less conservative than
those shown in Chart I. For example, substitutions may be made
which more significantly affect: the structure of the polypeptide
backbone in the area of the alteration, for example the
alpha-helical or beta-sheet structure; the charge or hydrophobicity
of the molecule at the target site; or the bulk of the side chain.
The substitutions which in general are expected to produce the
greatest changes in the polypeptide's properties are those in which
(a) a hydrophilic residue, e.g. seryl or threonyl, is substituted
for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,
phenylalanyl, valyl or alanyl; (b) a cysteine or proline is
substituted for (or by) any other residue; (c) a residue having an
electropositive side chain, e.g. lysyl, arginyl, or histidyl, is
substituted for (or by) an electronegative residue, e.g. glutamyl
or aspartyl; or (d) a residue having a bulky side chain, e.g.
phenylalanine, is substituted for (or by) one not having a side
chain, e.g. glycine.
[0146] The variants typically exhibit the same qualitative
biological activity and will elicit the same immune response as the
naturally-occurring analogue, although variants also are selected
to modify the characteristics of the MINK3 proteins as needed.
Alternatively, the variant may be designed such that the biological
activity of the MINK3 protein is altered. For example,
glycosylation sites may be altered or removed.
[0147] In one aspect the invention provides dominant negative MINK3
proteins which inhibit at least one MINK3 protein activity, as
described herein. In a preferred embodiment, the dominant negative
MINK3 protein is a kinase dead MINK3 protein. In a preferred
embodiment, the dominant negative kinase dead MINK3 protein variant
has a mutation in an ATP-binding domain. Preferably the non-mutant
ATP-binding domain of the non-variant MINK3 protein (non-variant
with respect to ATP-binding domain) comprises the amino acid
sequence set forth by amino acids 32-54 in SEQ ID NO:1. In a
preferred embodiment, the dominant negative kinase dead MINK3
protein variant has a substitution mutation in the ATP binding
domain at a position corresponding to K54 in SEQ ID NO:1.
[0148] In a preferred embodiment, the dominant negative MINK3
protein has MINK3 antisense nucleic acid activity.
[0149] Covalent modifications of MINK3 polypeptides are included
within the scope of this invention. One type of covalent
modification includes reacting targeted amino acid residues of a
MINK3 polypeptide with an organic derivatizing agent that is
capable of reacting with selected side chains or the N- or
C-terminal residues of a MINK3 polypeptide. Derivatization with
bifunctional agents is useful, for instance, for crosslinking MINK3
to a water-insoluble support matrix or surface for use in the
method for purifying anti-MINK3 antibodies or screening assays, as
is more fully described below. Commonly used crosslinking agents
include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate.
[0150] Other modifications include deamidation of glutaminyl and
asparaginyl residues to the corresponding glutamyl and aspartyl
residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the "-amino groups of lysine, arginine, and
histidine side chains (T. E. Creighton, Proteins: Structure and
Molecular Properties, W.H. Freeman & Co., San Francisco, pp.
79-86 (1983)), acetylation of the N-terminal amine, and amidation
of any C-terminal carboxyl group.
[0151] Another type of covalent modification of the MINK3
polypeptide included within the scope of this invention comprises
altering the native glycosylation pattern of the polypeptide.
"Altering the native glycosylation pattern" is intended for
purposes herein to mean deleting one or more carbohydrate moieties
found in native sequence MINK3 polypeptide, and/or adding one or
more glycosylation sites that are not present in the native
sequence MINK3 polypeptide.
[0152] Addition of glycosylation sites to MINK3 polypeptides may be
accomplished by altering the amino acid sequence thereof. The
alteration may be made, for example, by the addition of, or
substitution by, one or more serine or threonine residues to the
native sequence MINK3 polypeptide (for O-linked glycosylation
sites). The MINK3 amino acid sequence may optionally be altered
through changes at the DNA level, particularly by mutating the DNA
encoding the MINK3 polypeptide at preselected bases such that
codons are generated that will translate into the desired amino
acids.
[0153] Another means of increasing the number of carbohydrate
moieties on the MINK3 polypeptide is by chemical or enzymatic
coupling of glycosides to the polypeptide. Such methods are
described in the art, e.g., in WO 87/05330 published 11 Sep. 1987,
and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306
(1981).
[0154] Removal of carbohydrate moieties present on the MINK3
polypeptide may be accomplished chemically or enzymatically or by
mutational substitution of codons encoding for amino acid residues
that serve as targets for glycosylation. Chemical deglycosylation
techniques are known in the art and described, for instance, by
Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by
Edge, et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of
carbohydrate moieties on polypeptides can be achieved by the use of
a variety of endo- and exo-glycosidases as described by Thotakura,
et al., Meth. Enzymol., 138:350 (1987).
[0155] Another type of covalent modification comprises linking the
MINK3 polypeptide to one of a variety of nonproteinaceous polymers,
e.g., polyethylene glycol, polypropylene glycol, or
polyoxyalkylenes, in the manner set forth in U.S. Pat. No.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
[0156] MINK3 polypeptides of the present invention may also be
modified in a way to form chimeric molecules comprising a MINK3
polypeptide fused to another, heterologous polypeptide or amino
acid sequence. In one embodiment, such a chimeric molecule
comprises a fusion of a MINK3 polypeptide with a tag polypeptide
which provides an epitope to which an anti-tag antibody can
selectively bind. The epitope tag is generally placed at the amino-
or carboxyl-terminus of the MINK3 polypeptide. The presence of such
epitope-tagged forms of a MINK3 polypeptide can be detected using
an antibody against the tag polypeptide. Also, provision of the
epitope tag enables the MINK3 polypeptide to be readily purified by
affinity purification using an anti-tag antibody or another type of
affinity matrix that binds to the epitope tag. In an alternative
embodiment, the chimeric molecule may comprise a fusion of a MINK3
polypeptide with an immunoglobulin or a particular region of an
immunoglobulin. For a bivalent form of the chimeric molecule, such
a fusion could be to the Fc region of an IgG molecule as discussed
further below.
[0157] Various tag polypeptides and their respective antibodies are
well known in the art. Examples include poly-histidine (poly-his)
or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag
polypeptide and its antibody 12CA5 (Field, et al., Mol. Cell.
Biol., 8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10,
G4, B7 and 9E10 antibodies thereto (Evan, et al., Molecular and
Cellular Biology, 5:3610-3616 (1985)); and the Herpes Simplex virus
glycoprotein D (gD) tag and its antibody (Paborsky, et al., Protein
Engineering, 3(6):547-553 (1990)). Other tag polypeptides include
the Flag-peptide (Hopp, et al., BioTechnology, 6:1204-1210 (1988));
the KT3 epitope peptide (Martin, et al., Science, 255:192-194
(1992)); tubulin epitope peptide (Skinner, et al., J. Biol. Chem.,
266:15163-15166 (1991)); and the T7 gene 10 protein peptide tag
(Lutz-Freyermuth, et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397
(1990)).
[0158] In an embodiment herein, MINK3 genes from other organisms
are cloned and expressed as outlined below. Thus, probe or
degenerate polymerase chain reaction (PCR) primer sequences may be
used to find other related MINK3 proteins from humans or other
organisms. As will be appreciated by those in the art, particularly
useful probe and/or PCR primer sequences include the unique areas
of the MINK3 nucleic acid sequence. As is generally known in the
art, preferred PCR primers are from about 15 to about 35
nucleotides in length, with from about 20 to about 30 being
preferred, and may contain inosine as needed. The conditions for
the PCR reaction are well known in the art. It is therefore also
understood that provided along with the sequences in the sequences
listed herein are portions of those sequences, wherein unique
portions of 15 nucleotides or more are particularly preferred. The
skilled artisan can routinely synthesize or cut a nucleotide
sequence to the desired length.
[0159] Once isolated from its natural source, e.g., contained
within a plasmid or other vector or excised therefrom as a linear
nucleic acid segment, the recombinant MINK3 nucleic acid can be
further-used as a probe to identify and isolate other MINK3 nucleic
acids. It can also be used as a "precursor" nucleic acid to make
modified or variant MINK3 nucleic acids and proteins.
[0160] Using the nucleic acids of the present invention which
encode a MINK3 protein, a variety of expression vectors are made.
The expression vectors may be either self-replicating
extrachromosomal vectors or vectors which integrate into a host
genome. Generally, these expression vectors include transcriptional
and translational regulatory nucleic acid operably linked to the
nucleic acid encoding the MINK3 protein. The term "control
sequences" refers to DNA sequences necessary for the expression of
an operably linked coding sequence in a particular host organism.
The control sequences that are suitable for prokaryotes, for
example, include a promoter, optionally an operator sequence, and a
ribosome binding site. Eukaryotic cells are known to utilize
promoters, polyadenylation signals, and enhancers.
[0161] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. As another example, operably linked refers
to DNA sequences linked so as to be contiguous, and, in the case of
a secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice. The transcriptional and
translational regulatory nucleic acid will generally be appropriate
to the host cell used to express the MINK3 protein; for example,
transcriptional and translational regulatory nucleic acid sequences
from Bacillus are preferably used to express the MINK3 protein in
Bacillus. Numerous types of appropriate expression vectors, and
suitable regulatory sequences are known in the art for a variety of
host cells.
[0162] In general, the transcriptional and translational regulatory
sequences may include, but are not limited to, promoter sequences,
ribosomal binding sites, transcriptional start and stop sequences,
translational start and stop sequences, and enhancer or activator
sequences. In a preferred embodiment, the regulatory sequences
include a promoter and transcriptional start and stop
sequences.
[0163] Promoter sequences encode either constitutive or inducible
promoters. The promoters may be either naturally occurring
promoters or hybrid promoters. Hybrid promoters, which combine
elements of more than one promoter, are also known in the art, and
are useful in the present invention.
[0164] In addition, the expression vector may comprise additional
elements. For example, the expression vector may have two
replication systems, thus allowing it to be maintained in two
organisms, for example in mammalian or insect cells for expression
and in a procaryotic host for cloning and amplification.
Furthermore, for integrating expression vectors, the expression
vector contains at least one sequence homologous to the host cell
genome, and preferably two homologous sequences which flank the
expression construct. The integrating vector may be directed to a
specific locus in the host cell by selecting the appropriate
homologous sequence for inclusion in the vector. Constructs for
integrating vectors are well known in the art.
[0165] In addition, in a preferred embodiment, the expression
vector contains a selectable marker gene to allow the selection of
transformed host cells. Selection genes are well known in the art
and will vary with the host cell used.
[0166] A preferred expression vector system is a retroviral vector
system such as is generally described in PCT/US97/01019 and
PCT/US97/01048, both of which are hereby expressly incorporated by
reference.
[0167] MINK3 proteins of the present invention are produced by
culturing a host cell transformed with an expression vector
containing nucleic acid encoding a MINK3 protein, under the
appropriate conditions to induce or cause expression of the MINK3
protein. The conditions appropriate for MINK3 protein expression
will vary with the choice of the expression vector and the host
cell, and will be easily ascertained by one skilled in the art
through routine experimentation. For example, the use of
constitutive promoters in the expression vector will require
optimizing the growth and proliferation of the host cell, while the
use of an inducible promoter requires the appropriate growth
conditions for induction. In addition, in some embodiments, the
timing of the harvest is important. For example, the baculoviral
systems used in insect cell expression are lytic viruses, and thus
harvest time selection can be crucial for product yield.
[0168] Appropriate host cells include yeast, bacteria,
archebacteria, fungi, and insect and animal cells, including
mammalian cells. Of particular interest are Drosophila melanogaster
cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus
subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO,
COS, and HeLa cells, fibroblasts, Schwanoma cell lines,
immortalized mammalian myeloid and lymphoid cell lines, and human
tumor lines, including and preferably MDA-MB-231 cells.
[0169] In a preferred embodiment, the MINK3 proteins are expressed
in mammalian cells. Mammalian expression systems are also known in
the art, and include retroviral systems. A mammalian promoter is
any DNA sequence capable of binding mammalian RNA polymerase and
initiating the downstream (3') transcription of a coding sequence
for MINK3 protein into mRNA. A promoter will have a transcription
initiating region, which is usually placed proximal to the 5' end
of the coding sequence, and a TATA box, using a located 25-30 base
pairs upstream of the transcription initiation site. The TATA box
is thought to direct RNA polymerase II to begin RNA synthesis at
the correct site. A mammalian promoter will also contain an
upstream promoter element (enhancer element), typically located
within 100 to 200 base pairs upstream of the TATA box. An upstream
promoter element determines the rate at which transcription is
initiated and can act in either orientation. Of particular use as
mammalian promoters are the promoters from mammalian viral genes,
since the viral genes are often highly expressed and have a broad
host range. Examples include the SV40 early promoter, mouse mammary
tumor virus LTR promoter, adenovirus major late promoter, herpes
simplex virus promoter, and the CMV promoter.
[0170] Typically, transcription termination and polyadenylation
sequences recognized by mammalian cells are regulatory regions
located 3' to the translation stop codon and thus, together with
the promoter elements, flank the coding sequence. The 3' terminus
of the mature mRNA is formed by site-specific post-translational
cleavage and polyadenylation. Examples of transcription terminator
and polyadenlytion signals include those derived form SV40.
[0171] The methods of introducing exogenous nucleic acid into
mammalian hosts, as well as other hosts, is well known in the art,
and will vary with the host cell used. Techniques include
dextran-mediated transfection, calcium phosphate precipitation,
polybrene mediated transfection, protoplast fusion,
electroporation, viral infection, encapsulation of the
polynucleotide(s) in liposomes, and direct microinjection of the
DNA into nuclei.
[0172] In a preferred embodiment, MINK3 proteins are expressed in
bacterial systems. Bacterial expression systems are well known in
the art.
[0173] A suitable bacterial promoter is any nucleic acid sequence
capable of binding bacterial RNA polymerase and initiating the
downstream (3') transcription of the coding sequence of MINK3
protein into mRNA. A bacterial promoter has a transcription
initiation region which is usually placed proximal to the 5' end of
the coding sequence. This transcription initiation region typically
includes an RNA polymerase binding site and a transcription
initiation site. Sequences encoding metabolic pathway enzymes
provide particularly useful promoter sequences. Examples include
promoter sequences derived from sugar metabolizing enzymes, such as
galactose, lactose and maltose, and sequences derived from
biosynthetic enzymes such as tryptophan. Promoters from
bacteriophage may also be used and are known in the art. In
addition, synthetic promoters and hybrid promoters are also useful;
for example, the tac promoter is a hybrid of the trp and lac
promoter sequences. Furthermore, a bacterial promoter can include
naturally occurring promoters of non-bacterial origin that have the
ability to bind bacterial RNA polymerase and initiate
transcription.
[0174] In addition to a functioning promoter sequence, an efficient
ribosome binding site is desirable. In E. coli, the ribosome
binding site is called the Shine-Delgarno (SD) sequence and
includes an initiation codon and a sequence 3-9 nucleotides in
length located 3-11 nucleotides upstream of the initiation
codon.
[0175] The expression vector may also include a signal peptide
sequence that provides for secretion of the MINK3 protein in
bacteria. The signal sequence typically encodes a signal peptide
comprised of hydrophobic amino acids which direct the secretion of
the protein from the cell, as is well known in the art. The protein
is either secreted into the growth media (gram-positive bacteria)
or into the periplasmic space, located between the inner and outer
membrane of the cell (gram-negative bacteria).
[0176] The bacterial expression vector may also include a
selectable marker gene to allow for the selection of bacterial
strains that have been transformed. Suitable selection genes
include genes which render the bacteria resistant to drugs such as
ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and
tetracycline. Selectable markers also include biosynthetic genes,
such as those in the histidine, tryptophan and leucine biosynthetic
pathways.
[0177] These components are assembled into expression vectors.
Expression vectors for bacteria are well known in the art, and
include vectors for Bacillus subtilis, E. coli, Streptococcus
cremoris, and Streptococcus lividans, among others.
[0178] The bacterial expression vectors are transformed into
bacterial host cells using techniques well known in the art, such
as calcium chloride treatment, electroporation, and others.
[0179] In one embodiment, MINK3 proteins are produced in insect
cells. Expression vectors for the transformation of insect cells,
and in particular, baculovirus-based expression vectors, are well
known in the art.
[0180] In a preferred embodiment, MINK3 protein is produced in
yeast cells. Yeast expression systems are well known in the art,
and include expression vectors for Saccharomyces cerevisiae,
Candida albicans and C. maltosa, Hansenula polymorpha,
Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P.
pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
Preferred promoter sequences for expression in yeast include the
inducible GAL1,10 promoter, the promoters from alcohol
dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase,
glyceraldehyde-3-phosphate-dehydrogenase, hexokinase,
phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase,
and the acid phosphatase gene. Yeast selectable markers include
ADE2, HIS4, LEU2, TRP1, and ALG7, which confers resistance to
tunicamycin; the neomycin phosphotransferase gene, which confers
resistance to G418; and the CUP1 gene, which allows yeast to grow
in the presence of copper ions.
[0181] The MINK3 protein may also be made as a fusion protein,
using techniques well known in the art. Thus, for example, for the
creation of monoclonal antibodies, if the desired epitope is small,
the MINK3 protein may be fused to a carrier protein to form an
immunogen. Alternatively, the MINK3 protein may be made as a fusion
protein to increase expression, or for other reasons. For example,
when the MINK3 protein is a MINK3 peptide, the nucleic acid
encoding the peptide may be linked to other nucleic acid for
expression purposes. Similarly, MINK3 proteins of the invention can
be linked to protein labels, such as green fluorescent protein
(GFP), red fluorescent protein (RFP), blue fluorescent protein
(BFP), yellow fluorescent protein (YFP), etc.
[0182] In one embodiment, the MINK3 nucleic acids, proteins and
antibodies of the invention are labeled. By "labeled" herein is
meant that a compound has at least one element, isotope or chemical
compound attached to enable the detection of the compound. In
general, labels fall into three classes: a) isotopic labels, which
may be radioactive or heavy isotopes; b) immune labels, which may
be antibodies or antigens; and c) colored or fluorescent dyes. The
labels may be incorporated into the compound at any position.
[0183] In a preferred embodiment, the MINK3 protein is purified or
isolated after expression. MINK3 proteins may be isolated or
purified in a variety of ways known to those skilled in the art
depending on what other components are present in the sample.
Standard purification methods include electrophoretic, molecular,
immunological and chromatographic techniques, including ion
exchange, hydrophobic, affinity, and reverse-phase HPLC
chromatography, and chromatofocusing. For example, the MINK3
protein may be purified using a standard anti-MINK3 antibody
column. Ultrafiltration and diafiltration techniques, in
conjunction with protein concentration, are also useful. For
general guidance in suitable purification techniques, see Scopes,
R., Protein Purification, Springer-Verlag, NY (1982). The degree of
purification necessary will vary depending on the use of the MINK3
protein. In some instances no purification will be necessary.
[0184] Once expressed and purified if necessary, the MINK3 proteins
and nucleic acids are useful in a number of applications.
[0185] The nucleotide sequences (or their complement) encoding
MINK3 proteins have various applications in the art of molecular
biology, including uses as hybridization probes, in chromosome and
gene mapping and in the generation of anti-sense RNA and DNA. MINK3
nucleic acids are also useful for the preparation of MINK3 proteins
by the recombinant techniques described herein.
[0186] Full-length native sequence MINK3 genes, or portions
thereof, may be used as hybridization probes for a cDNA library to
isolate other genes (for instance, those encoding
naturally-occurring variants of MINK3 protein or MINK3 protein from
other species) which have a desired sequence identity to the MINK3
protein coding sequence. Optionally, the length of the probes will
be about 20 to about 50 bases. The hybridization probes may be
derived from the nucleotide sequences herein or from genomic
sequences including promoters, enhancer elements and introns of
native sequences as provided herein. By way of example, a screening
method will comprise isolating the coding region of the MINK3
protein gene using the known DNA sequence to synthesize a selected
probe of about 40 bases. Hybridization probes may be labeled by a
variety of labels, including radionucleotides such as 32P or 35S,
or enzymatic labels such as alkaline phosphatase coupled to the
probe via avidin/biotin coupling systems. Labeled probes having a
sequence complementary to that of the MINK3 protein gene of the
present invention can be used to screen libraries of human cDNA,
genomic DNA or mRNA to determine which members of such libraries
the probe hybridizes.
[0187] Nucleotide sequences encoding a MINK3 protein can also be
used to construct hybridization probes for mapping the gene which
encodes that MINK3 protein and for the genetic analysis of
individuals with genetic disorders. The nucleotide sequences
provided herein may be mapped to a chromosome and specific regions
of a chromosome using known techniques, such as in situ
hybridization, linkage analysis against known chromosomal markers,
and hybridization screening with libraries.
[0188] The isolation of mRNA comprises isolating total cellular RNA
by disrupting a cell and performing differential centrifugation.
Once the total RNA is isolated, mRNA is isolated by making use of
the adenine nucleotide residues known to those skilled in the art
as a poly (A) tail found on virtually every eukaryotic mRNA
molecule at the 3' end thereof. Oligonucleotides composed of only
deoxythymidine (oligo(dT)) are linked to cellulose and the
oligo(dT)-cellulose packed into small columns. When a preparation
of total cellular RNA is passed through such a column, the mRNA
molecules bind to the oligo(dT) by the poly (A) tails while the
rest of the RNA flows through the column. The bound mRNAs are then
eluted from the column and collected.
[0189] Nucleic acids which encode MINK3 protein or its modified
forms can also be used to generate either transgenic animals or
"knock out" animals which, in turn, are useful in the development
and screening of therapeutically useful reagents. A transgenic
animal (e.g., a mouse or rat) is an animal having cells that
contain a transgene, which transgene was introduced into the animal
or an ancestor of the animal at a prenatal, e.g., an embryonic
stage. A transgene is a DNA which is integrated into the genome of
a cell from which a transgenic animal develops. In one embodiment,
cDNA encoding a MINK3 protein can be used to clone genomic DNA
encoding a MINK3 protein in accordance with established techniques
and the genomic sequences used to generate transgenic animals that
contain cells which express the desired DNA. Methods for generating
transgenic animals, particularly animals such as mice or rats, have
become conventional in the art and are described, for example, in
U.S. Pat. Nos. 4,736,866 and 4,870,009. Typically, particular cells
would be targeted for the MINK3 protein transgene incorporation
with tissue-specific enhancers. Transgenic animals that include a
copy of a transgene encoding a MINK3 protein introduced into the
germ line of the animal at an embryonic stage can be used to
examine the effect of increased expression of the desired nucleic
acid. Such animals can be used as tester animals for reagents
thought to confer protection from, for example, pathological
conditions associated with its overexpression. In accordance with
this facet of the invention, an animal is treated with the reagent
and a reduced incidence of the pathological condition, compared to
untreated animals bearing the transgene, would indicate a potential
therapeutic intervention for the pathological condition.
[0190] Alternatively, non-human homologues of the MINK3 protein can
be used to construct a MINK3 protein "knock out" animal which has a
defective or altered gene encoding a MINK3 protein as a result of
homologous recombination between the endogenous gene encoding a
MINK3 protein and altered genomic DNA encoding a MINK3 protein
introduced into an embryonic cell of the animal. For example, cDNA
encoding a MINK3 protein can be used to clone genomic DNA encoding
a MINK3 protein in accordance with established techniques. A
portion of the genomic DNA encoding a MINK3 protein can be deleted
or replaced with another gene, such as a gene encoding a selectable
marker which can be used to monitor integration. Typically, several
kilobases of unaltered flanking DNA (both at the 5' and 3' ends)
are included in the vector (see e.g., Thomas, et al., Cell, 51:503
(1987) for a description of homologous recombination vectors). The
vector is introduced into an embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced DNA has
homologously recombined with the endogenous DNA are selected (see
e.g., Li, et al., Cell, 69:915 (1992)). The selected cells are then
injected into a blastocyst of an animal (e.g., a mouse or rat) to
form aggregation chimeras (see e.g., Bradley, in Teratocarcinomas
and Embryonic Stem Cells: A Practical Approach, E. J. Robertson,
ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then
be implanted into a suitable pseudopregnant female foster animal
and the embryo brought to term to create a "knock out" animal.
Progeny harboring the homologously recombined DNA in their germ
cells can be identified by standard techniques and used to breed
animals in which all cells of the animal contain the homologously
recombined DNA. Knockout animals can be characterized for instance,
for their ability to defend against certain pathological conditions
and for their development of pathological conditions due to absence
of the MINK3 protein.
[0191] It is understood that the models described herein can be
varied. For example, "knock-in" models can be formed, or the models
can be cell-based rather than animal models.
[0192] Nucleic acids encoding MINK3 polypeptides, antagonists or
agonists may also be used in gene therapy. In gene therapy
applications, genes are introduced into cells in order to achieve
in vivo synthesis of a therapeutically effective genetic product,
for example for replacement of a defective gene. "Gene therapy"
includes both conventional gene therapy where a lasting effect is
achieved by a single treatment, and the administration of gene
therapeutic agents, which involves the one time or repeated
administration of a therapeutically effective DNA or mRNA.
Antisense RNAs and DNAs can be used as therapeutic agents for
blocking the expression of certain genes in vivo. It has already
been shown that short antisense oligonucleotides can be imported
into cells where they act as inhibitors, despite their low
intracellular concentrations caused by their restricted uptake by
the cell membrane. (Zamecnik, et al., Proc. Natl. Acad. Sci. USA,
83:4143-4146 (1986)). The oligonucleotides can be modified to
enhance their uptake, e.g. by substituting their negatively charged
phosphodiester groups by uncharged groups.
[0193] In one aspect, the present invention provides antisense
MINK3 nucleic acids. In a preferred embodiment, the antisense MINK3
nucleic acid comprises a nucleic acid sequence complementary to the
nucleic acid sequence set forth by nucleotides 2804-3187 in SEQ ID
NO:2. In another preferred embodiment, the present invention
provides antisense MINK3 nucleic acids consisting essentially of a
nucleic acid sequence complementary to the nucleic acid sequence
set forth by nucleotides 2804-3187 in SEQ ID NO:2.
[0194] In a preferred embodiment, the MINK3 antisense nucleic acid
will hybridize to MINK3 and MINK1 nucleic acids.
[0195] In a preferred embodiment, the MINK3 antisense nucleic acid
inhibits one or more MINK3 protein activities. In another preferred
embodiment, the MINK3 antisense nucleic acid inhibits more than one
MINK3 protein activity. In a further preferred embodiment, the
MINK3 antisense nucleic acid inhibits all MINK3 protein activities.
In a preferred embodiment, the MINK3 antisense nucleic acid has an
activity shared by dominant negative MINK3 protein.
[0196] In a preferred embodiment, such antisense MINK3 nucleic
acids are capable of inhibiting taxol-induced death in mammalian
cells. In a preferred embodiment, such antisense MINK3 nucleic
acids are capable of inhibiting taxol-induced cleavage of Rb. In a
preferred embodiment, such antisense MINK3 nucleic acids are
capable of promoting survival in mammalian cells following exposure
to taxol.
[0197] In a preferred embodiment, such antisense MINK3 nucleic
acids are capable of inhibiting the transcription promoting
activity of AP-1 in mammalian cells. AP-1 is well known in the art.
AP-1 means activation complex-1 and refers to a complex of the
known transcription factors c-JUN and c-FOS. In a preferred
embodiment, such MINK3 nucleic acids are capable of inhibiting
transcriptional induction by an AP-1 response element. AP-1
response elements are well known in the art.
[0198] In a preferred embodiment, such antisense MINK3 nucleic
acids are capable of inhibiting growth factor-induced ERK activity
in mammalian cells, preferably EGF-induced ERK activity, preferably
in cancer cells. In a preferred embodiment, such antisense MINK3
nucleic acids are capable of inhibiting growth factor-induced
proliferation in mammalian cells, preferably EGF-induced
proliferation, preferably in cancer cells. In a preferred
embodiment, such antisense MINK3 nucleic acids are capable of
inhibiting growth factor-dependent proliferation in mammalian
cells, preferably cancer cells.
[0199] In a preferred embodiment, such antisense MINK3 nucleic
acids are capable of inhibiting aberrant cell proliferation
involving aberrant activation of the ERK and/or JNK signal
transduction pathways.
[0200] There are a variety of techniques available for introducing
nucleic acids into viable cells. The techniques vary depending upon
whether the nucleic acid is transferred into cultured cells in
vitro, or in vivo in the cells of the intended host. Techniques
suitable for the transfer of nucleic acid into mammalian cells in
vitro include the use of liposomes, electroporation,
microinjection, cell fusion, DEAE-dextran, the calcium phosphate
precipitation method, etc. The currently preferred in vivo gene
transfer techniques include transfection with viral (typically
retroviral) vectors and viral coat protein-liposome mediated
transfection (Dzau, et al., Trends in Biotechnology, 11:205-210
(1993)). In some situations it is desirable to provide the nucleic
acid source with an agent that targets the target cells, such as an
antibody specific for a cell surface membrane protein or the target
cell, a ligand for a receptor on the target cell, etc. Where
liposomes are employed, proteins which bind to a cell surface
membrane protein associated with endocytosis may be used for
targeting and/or to facilitate uptake, e.g. capsid proteins or
fragments thereof tropic for a particular cell type, antibodies for
proteins which undergo internalization in cycling, proteins that
target intracellular localization and enhance intracellular
half-life. The technique of receptor-mediated endocytosis is
described, for example, by Wu, et al., J. Biol. Chem.,
262:4429-4432 (1987); and Wagner, et al., Proc. Natl. Acad. Sci.
USA, 87:3410-3414 (1990). For review of gene marking and gene
therapy protocols see Anderson, et al., Science 256:808-813
(1992).
[0201] In a preferred embodiment, the MINK3 proteins, nucleic
acids, variants, modified proteins, cells and/or transgenics
containing the said nucleic acids or proteins are used in screening
assays. Identification of MINK3 proteins provided herein permits
the design of drug screening assays for compounds that bind or
interfere with the binding to the MINK3 protein and for compounds
which modulate MINK3 protein activity.
[0202] The assays described herein preferably utilize the human
MINK3 protein, although other mammalian proteins may also be used,
including rodents (mice, rats, hamsters, guinea pigs, etc.), farm
animals (cows, sheep, pigs, horses, etc.) and primates. These
latter embodiments may be preferred in the development of animal
models of human disease. In some embodiments, as outlined herein,
variant or derivative MINK3 proteins may be used, including
deletion MINK3 proteins as outlined above.
[0203] In a preferred embodiment, the methods comprise combining a
MINK3 protein and a candidate bioactive agent, and determining the
binding of the candidate agent to the MINK3 protein. In other
embodiments, further discussed below, binding interference or
bioactivity is determined.
[0204] The term "candidate bioactive agent" or "exogenous compound"
or "test compound" or "drug candidate" or "modulator" or
grammatical equivalents as used herein describes any molecule,
e.g., protein, small organic molecule (small molecule chemical
compound), carbohydrates (including polysaccharides),
polynucleotide, lipids, antisense molecules, RNAi, ribozymes,
peptides, oligopeptides (e.g., from about 5 to about 25 amino acids
in length, preferably from about 10 to 20 or 12 to 18 amino acids
in length, preferably 12, 15, or 18 amino acids in length), cyclic
peptides, etc. Small molecule chemical or organic compositions are
preferred. Generally a plurality of assay mixtures are run in
parallel with different agent concentrations to obtain a
differential response to the various concentrations. Typically, one
of these concentrations serves as a negative control, i.e., at zero
concentration or below the level of detection. The test compound
can be in the form of a library of test compounds, such as a
combinatorial or randomized library that provides a sufficient
range of diversity. Test compounds are optionally linked to a
fusion partner, e.g., targeting compounds, rescue compounds,
dimerization compounds, stabilizing compounds, addressable
compounds, and other functional moieties. Conventionally, new
chemical entities with useful properties are generated by
identifying a test compound (called a "lead compound") with some
desirable property or activity, e.g., inhibiting activity, creating
variants of the lead compound, and evaluating the property and
activity of those variant compounds. Often, high throughput
screening (HTS) methods are employed for such an analysis.
[0205] Candidate agents encompass numerous chemical classes, though
typically they are organic molecules, preferably small organic
compounds having a molecular weight of more than 100 and less than
about 2,500 daltons, preferably less than about 2000 daltons,
preferably less than about 1800 daltons, preferably less than about
1700 daltons, preferably less than about 1600 daltons, preferably
less than about 1500 daltons, preferably less than about 1400
daltons, preferably less than about 1300 daltons, preferably less
than about 1200 daltons, preferably less than about 1100 daltons,
preferably less than about 1000 daltons. Candidate agents comprise
functional groups necessary for structural interaction with
proteins, particularly hydrogen bonding, and typically include at
least an amine, carbonyl, hydroxyl or carboxyl group, preferably at
least two of the functional chemical groups. The candidate agents
often comprise cyclical carbon or heterocyclic structures and/or
aromatic or polyaromatic structures substituted with one or more of
the above functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof. Particularly preferred are peptides.
[0206] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides. Alternatively, libraries
of natural compounds in the form of bacterial, fungal, plant and
animal extracts are available or readily produced. Additionally,
natural or synthetically produced libraries and compounds are
readily modified through conventional chemical, physical and
biochemical means. Known pharmacological agents may be subjected to
directed or random chemical modifications, such as acylation,
alkylation, esterification, amidification to produce structural
analogs.
[0207] In a preferred embodiment, a library of different candidate
bioactive agents are used. Preferably, the library should provide a
sufficiently structurally diverse population of randomized agents
to effect a probabilistically sufficient range of diversity to
allow binding to a particular target. Accordingly, an interaction
library should be large enough so that at least one of its members
will have a structure that gives it affinity for the target.
Although it is difficult to gauge the required absolute size of an
interaction library, nature provides a hint with the immune
response: a diversity of 107-108 different antibodies provides at
least one combination with sufficient affinity to interact with
most potential antigens faced by an organism. Published in vitro
selection techniques have also shown that a library size of 107 to
108 is sufficient to find structures with affinity for the target.
A library of all combinations of a peptide 7 to 20 amino acids in
length, such as generally proposed herein, has the potential to
code for 207 (109) to 2020. Thus, with libraries of 107 to 108
different molecules the present methods allow a "working" subset of
a theoretically complete interaction library for 7 amino acids, and
a subset of shapes for the 2020 library. Thus, in a preferred
embodiment, at least 106, preferably at least 107, more preferably
at least 108 and most preferably at least 109 different sequences
are simultaneously analyzed in the subject methods. Preferred
methods maximize library size and diversity.
[0208] In a preferred embodiment, the candidate bioactive agents
are proteins. By "protein" herein is meant at least two covalently
attached amino acids, which includes proteins, polypeptides,
oligopeptides and peptides. The protein may be made up of naturally
occurring amino acids and peptide bonds, or synthetic
peptidomimetic structures. Thus "amino acid", or "peptide residue",
as used herein means both naturally occurring and synthetic amino
acids. For example, homo-phenylalanine, citrulline and noreleucine
are considered amino acids for the purposes of the invention.
"Amino acid" also includes imino acid residues such as proline and
hydroxyproline. The side chains may be in either the (R) or the (S)
configuration. In the preferred embodiment, the amino acids are in
the (S) or L-configuration. If non-naturally occurring side chains
are used, non-amino acid substituents may be used, for example to
prevent or retard in vivo degradations. Chemical blocking groups or
other chemical substituents may also be added.
[0209] In a preferred embodiment, the candidate bioactive agents
are naturally occurring proteins or fragments of naturally
occurring proteins. Thus, for example, cellular extracts containing
proteins, or random or directed digests of proteinaceous cellular
extracts, may be used. In this way libraries of procaryotic and
eukaryotic proteins may be made for screening in the systems
described herein. Particularly preferred in this embodiment are
libraries of bacterial, fungal, viral, and mammalian proteins, with
the latter being preferred, and human proteins being especially
preferred.
[0210] In a preferred embodiment, the candidate bioactive agents
are peptides of from about 5 to about 30 amino acids, with from
about 5 to about 20 amino acids being preferred, and from about 7
to about 15 being particularly preferred. The peptides may be
digests of naturally occurring proteins as is outlined above,
random peptides, or "biased" random peptides. By "randomized" or
grammatical equivalents herein is meant that each nucleic acid and
peptide consists of essentially random nucleotides and amino acids,
respectively. Since generally these random peptides (or nucleic
acids, discussed below) are chemically synthesized, they may
incorporate any nucleotide or amino acid at any position. The
synthetic process can be designed to generate randomized proteins
or nucleic acids, to allow the formation of all or most of the
possible combinations over the length of the sequence, thus forming
a library of randomized candidate bioactive proteinaceous
agents.
[0211] In one embodiment, the library is fully randomized, with no
sequence preferences or constants at any position. In a preferred
embodiment, the library is biased. That is, some positions within
the sequence are either held constant, or are selected from a
limited number of possibilities. For example, in a preferred
embodiment, the nucleotides or amino acid residues are randomized
within a defined class, for example, of hydrophobic amino acids,
hydrophilic residues, sterically biased (either small or large)
residues, towards the creation of cysteines, for cross-linking,
prolines for SH-3 domains, serines, threonines, tyrosines or
histidines for phosphorylation sites, etc., or to purines, etc.
[0212] In a preferred embodiment, the candidate bioactive agents
are nucleic acids. By "nucleic acid" or "oligonucleotide" or
grammatical equivalents herein means at least two nucleotides
covalently linked together. A nucleic acid of the present invention
will generally contain phosphodiester bonds, although in some
cases, as outlined below, nucleic acid analogs are included that
may have alternate backbones, comprising, for example,
phosphoramide (Beaucage, et al., Tetrahedron, 49(10):1925 (1993)
and references therein; Letsinger, J. Org. Chem., 35:3800 (1970);
Sprinzl, et al., Eur. J. Biochem., 81:579 (1977); Letsinger, et
al., Nucl. Acids Res., 14:3487 (1986); Sawai, et al., Chem. Lett.,
805 (1984), Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988);
and Pauwels, et al., Chemica Scripta, 26:141 (1986)),
phosphorothioate (Mag, et al., Nucleic Acids Res., 19:1437 (1991);
and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu, et al., J.
Am. Chem. Soc., 111:2321 (1989)), O-methylphosphoroamidite linkages
(see Eckstein, Oligonucleotides and Analogues: A Practical
Approach, Oxford University Press), and peptide nucleic acid
backbones and linkages (see Egholm, J. Am. Chem. Soc., 114:1895
(1992); Meier, et al., Chem. Int. Ed. Engl., 31:1008 (1992);
Nielsen, Nature, 365:566 (1993); Carlsson, et al., Nature, 380:207
(1996), all of which are incorporated by reference)). Other analog
nucleic acids include those with positive backbones (Denpcy, et
al., Proc. Natl. Acad. Sci. USA, 92:6097 (1995)); non-ionic
backbones (U.S. Pat. Nos. 5,386,023; 5,637,684; 5,602,240;
5,216,141; and 4,469,863; Kiedrowshi, et al., Angew. Chem. Intl.
Ed. English, 30:423 (1991); Letsinger, et al., J. Am. Chem. Soc.,
110:4470 (1988); Letsinger, et al., Nucleoside & Nucleotide,
13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580,
"Carbohydrate Modifications in Antisense Research", Ed. Y. S.
Sanghui and P. Dan Cook; Mesmaeker, et al., Bioorganic &
Medicinal Chem. Lett., 4:395 (1994); Jeffs, et al., J. Biomolecular
NMR, 34:17 (1994); Tetrahedron Lett., 37:743 (1996)) and non-ribose
backbones, including those described in U.S. Pat. Nos. 5,235,033
and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,
"Carbohydrate Modifications in Antisense Research", Ed. Y. S.
Sanghui and P. Dan Cook. Nucleic acids containing one or more
carbocyclic sugars are also included within the definition of
nucleic acids (see Jenkins, et al., Chem. Soc. Rev., pp. 169-176
(1995)). Several nucleic acid analogs are described in Rawls, C
& E News, Jun. 2, 1997, page 35. All of these references are
hereby expressly incorporated by reference. These modifications of
the ribose-phosphate backbone may be done to facilitate the
addition of additional moieties such as labels, or to increase the
stability and half-life of such molecules in physiological
environments. In addition, mixtures of naturally occurring nucleic
acids and analogs can be made. Alternatively, mixtures of different
nucleic acid analogs, and mixtures of naturally occurring nucleic
acids and analogs may be made. The nucleic acids may be single
stranded or double stranded, as specified, or contain portions of
both double stranded or single stranded sequence. The nucleic acid
may be DNA, both genomic and cDNA, RNA or a hybrid, where the
nucleic acid contains any combination of deoxyribo- and
ribo-nucleotides, and any combination of bases, including uracil,
adenine, thymine, cytosine, guanine, inosine, xathanine
hypoxathanine, isocytosine, isoguanine, etc.
[0213] As described above generally for proteins, nucleic acid
candidate bioactive agents may be naturally occurring nucleic
acids, random nucleic acids, or "biased" random nucleic acids. For
example, digests of procaryotic or eukaryotic genomes may be used
as is outlined above for proteins.
[0214] In a preferred embodiment, the candidate bioactive agents
are organic chemical moieties, a wide variety of which are
available in the literature.
[0215] In a preferred embodiment, the candidate bioactive agents
are linked to a fusion partner. By "fusion partner" or "functional
group" herein is meant a sequence that is associated with the
candidate bioactive agent, that confers upon all members of the
library in that class a common function or ability. Fusion partners
can be heterologous (i.e. not native to the host cell), or
synthetic (not native to any cell). Suitable fusion partners
include, but are not limited to: a) presentation structures, which
provide the candidate bioactive agents in a conformationally
restricted or stable form; b) targeting sequences, which allow the
localization of the candidate bioactive agent into a subcellular or
extracellular compartment; c) rescue sequences which allow the
purification or isolation of either the candidate bioactive agents
or the nucleic acids encoding them; d) stability sequences, which
confer stability or protection from degradation to the candidate
bioactive agent or the nucleic acid encoding it, for example
resistance to proteolytic degradation; e) dimerization sequences,
to allow for peptide dimerization; or f) any combination of a), b),
c), d), and e), as well as linker sequences as needed.
[0216] In one embodiment, the screening methods described herein
make use of portions of MINK3 proteins, or MINK3 protein fragments.
In a preferred embodiment, MINK3 protein fragments having at least
one MINK3 bioactivity as described herein are used. MINK3
bioactivity includes binding activity to Nck, modulation of
phosphorylation of JNK and/or ERK, modulation of JNK and/or ERK
phosphorylation, modulation of JNK and/or ERK activation,
modulation of JNK and/or ERK signal transduction, inhibition of
taxol-induced Rb cleavage and apoptosis, morphological change,
modulation of cell proliferation, and disruption of F-actin. In
some embodiments, the assays described herein utilize isolated
MINK3 proteins. In other embodiments, cells comprising MINK3
proteins are used. In other embodiments, MINK3 proteins that are
cell-free but not isolated, as in a cell lysate, are used.
[0217] Generally, in a preferred embodiment of the methods herein,
for example for binding assays, the MINK3 protein or the candidate
agent is non-diffusibly bound to an insoluble support having
isolated sample receiving areas (e.g. a microtiter plate, an array,
etc.). The insoluble supports may be made of any composition to
which the compositions can be bound, is readily separated from
soluble material, and is otherwise compatible with the overall
method of screening. The surface of such supports may be solid or
porous and of any convenient shape. Examples of suitable insoluble
supports include microtiter plates, arrays, membranes and beads.
These are typically made of glass, plastic (e.g., polystyrene),
polysaccharides, nylon or nitrocellulose, Teflon.TM., etc.
Microtiter plates and arrays are especially convenient because a
large number of assays can be carried out simultaneously, using
small amounts of reagents and samples. In some cases magnetic beads
and the like are included. The particular manner of binding of the
composition is not crucial so long as it is compatible with the
reagents and overall methods of the invention, maintains the
activity of the composition and is nondiffusable. Preferred methods
of binding include the use of antibodies (which do not sterically
block either the ligand binding site or activation sequence when
the protein is bound to the support), direct binding to "sticky" or
ionic supports, chemical crosslinking, the synthesis of the protein
or agent on the surface, etc. In some embodiments, Nck protein can
be used. Following binding of the protein or agent, excess unbound
material is removed by washing. The sample receiving areas may then
be blocked through incubation with bovine serum albumin (BSA),
casein or other innocuous protein or other moiety. Also included in
this invention are screening assays wherein solid supports are not
used; examples of such are described below.
[0218] In a preferred embodiment, the MINK3 protein is bound to the
support, and a candidate bioactive agent is added to the assay.
Alternatively, the candidate agent is bound to the support and the
MINK3 protein is added. Novel binding agents include specific
antibodies, non-natural binding agents identified in screens of
chemical libraries, peptide analogs, etc. Of particular interest
are screening assays for agents that have a low toxicity for human
cells. A wide variety of assays may be used for this purpose,
including labeled in vitro protein-protein binding assays,
electrophoretic mobility shift assays, immunoassays for protein
binding, functional assays (phosphorylation assays, etc.) and the
like.
[0219] The determination of the binding of the candidate bioactive
agent to the MINK3 protein may be done in a number of ways. In a
preferred embodiment, the candidate bioactive agent is labeled, and
binding determined directly. For example, this may be done by
attaching all or a portion of the MINK3 protein to a solid support,
adding a labeled candidate agent (for example a fluorescent label),
washing off excess reagent, and determining whether the label is
present on the solid support. Various blocking and washing steps
may be utilized as is known in the art.
[0220] By "labeled" herein is meant that the compound is either
directly or indirectly labeled with a label which provides a
detectable signal, e.g. radioisotope, fluorescers, enzyme,
antibodies, particles such as magnetic particles, chemiluminescers,
or specific binding molecules, etc. Specific binding molecules
include pairs, such as biotin and streptavidin, digoxin and
antidigoxin etc. For the specific binding members, the
complementary member would normally be labeled with a molecule
which provides for detection, in accordance with known procedures,
as outlined above. The label can directly or indirectly provide a
detectable signal.
[0221] In some embodiments, only one of the components is labeled.
For example, the proteins (or proteinaceous candidate agents) may
be labeled at tyrosine positions using 125I, or with fluorophores.
Alternatively, more than one component may be labeled with
different labels; using 125I for the proteins, for example, and a
fluorophor for the candidate agents.
[0222] In a preferred embodiment, the binding of the candidate
bioactive agent is determined through the use of competitive
binding assays. In this embodiment, the competitor is a binding
moiety known to bind to the target molecule (i.e. MINK3 protein),
such as an antibody, peptide, binding partner, ligand, etc. In a
preferred embodiment the competitor is Nck. Under certain
circumstances, there may be competitive binding as between the
bioactive agent and the binding moiety, with the binding moiety
displacing the bioactive agent. This assay can be used to determine
candidate agents which interfere with binding between MINK3
proteins and Nck. "Interference of binding" as used herein means
that native binding of the MINK3 protein differs in the presence of
the candidate agent. The binding can be eliminated or can be with a
reduced affinity. Therefore, in one embodiment, interference is
caused by, for example, a conformation change, rather than direct
competition for the native binding site.
[0223] In one embodiment, the candidate bioactive agent is labeled.
Either the candidate bioactive agent, or the competitor, or both,
is added first to the protein for a time sufficient to allow
binding, if present. Incubations may be performed at any
temperature which facilitates optimal activity, typically between 4
and 40.degree. C. Incubation periods are selected for optimum
activity, but may also be optimized to facilitate rapid high
through put screening. Typically between 0.1 and 1 hour will be
sufficient. Excess reagent is generally removed or washed away. The
second component is then added, and the presence or absence of the
labeled component is followed, to indicate binding.
[0224] In a preferred embodiment, the competitor is added first,
followed by the candidate bioactive agent. Displacement of the
competitor is an indication that the candidate bioactive agent is
binding to the MINK3 protein and thus is capable of binding to, and
potentially modulating, the activity of the MINK3 protein. In this
embodiment, either component can be labeled. Thus, for example, if
the competitor is labeled, the presence of label in the wash
solution indicates displacement by the agent. Alternatively, if the
candidate bioactive agent is labeled, the presence of the label on
the support indicates displacement.
[0225] In an alternative embodiment, the candidate bioactive agent
is added first, with incubation and washing, followed by the
competitor. The absence of binding by the competitor may indicate
that the bioactive agent is bound to the MINK3 protein with a
higher affinity. Thus, if the candidate bioactive agent is labeled,
the presence of the label on the support, coupled with a lack of
competitor binding, may indicate that the candidate agent is
capable of binding to the MINK3 protein.
[0226] A preferred embodiment utilizes differential screening to
identify drug candidates that bind to the native MINK3 protein, but
cannot bind to modified MINK3 proteins. The structure of the MINK3
protein may be modeled, and used in rational drug design to
synthesize agents that interact with that site. Drug candidates
that affect MINK3 bioactivity are also identified by screening
drugs for the ability to either enhance or reduce the activity of
the protein.
[0227] Positive controls and negative controls may be used in the
assays. Preferably all control and test samples are performed in at
least triplicate to obtain statistically significant results.
Incubation of all samples is for a time sufficient for the binding
of the agent to the protein. Following incubation, all samples are
washed free of non-specifically bound material and the amount of
bound, generally labeled agent determined. For example, where a
radiolabel is employed, the samples may be counted in a
scintillation counter to determine the amount of bound
compound.
[0228] A variety of other reagents may be included in the screening
assays. These include reagents like salts, neutral proteins, e.g.
albumin, detergents, etc which may be used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Also reagents that otherwise improve the efficiency
of the assay, such as protease inhibitors, nuclease inhibitors,
anti-microbial agents, etc., may be used. The mixture of components
may be added in any order that provides for the requisite
binding.
[0229] The proteins and nucleic acids provided herein can also be
used for screening purposes wherein the protein-protein
interactions of the MINK3 proteins can be identified. Genetic
systems have been described to detect protein-protein interactions.
The first work was done in yeast systems, namely the "yeast
two-hybrid" system. The basic system requires a protein-protein
interaction in order to turn on transcription of a reporter gene.
Subsequent work was done in mammalian cells. See Fields, et al.,
Nature, 340:245 (1989); Vasavada, et al., PNAS USA, 88:10686
(1991); Fearon, et al., PNAS USA, 89:7958 (1992); Dang, et al.,
Mol. Cell. Biol., 11:954 (1991); Chien, et al., PNAS USA, 88:9578
(1991); and U.S. Pat. Nos. 5,283,173, 5,667,973, 5,468,614,
5,525,490, and 5,637,463. a preferred system is described in Ser.
Nos. 09/050,863, filed Mar. 30, 1998 and 09/359,081 filed Jul. 22,
1999, entitled "Mammalian Protein Interaction Cloning System". For
use in conjunction with these systems, a particularly useful
shuttle vector is described in Ser. No. 09/133,944, filed Aug. 14,
1998, entitled "Shuttle Vectors".
[0230] In general, two nucleic acids are transformed into a cell,
where one is a "bait" such as the gene encoding a MINK3 protein or
a portion thereof, and the other encodes a test candidate. Only if
the two expression products bind to one another will an indicator,
such as a fluorescent protein, or a gene product required for
survival or growth, be expressed. Expression of the indicator
indicates when a test candidate binds to the MINK3 protein and can
be identified as an MINK3 protein. Using the same system and the
identified MINK3 proteins the reverse can be performed. Namely, the
MINK3 proteins provided herein can be used to identify new baits,
or agents which interact with MINK3 proteins. Additionally, the
two-hybrid system can be used wherein a test candidate is added in
addition to the bait and the MINK3 protein encoding nucleic acids
to determine agents which interfere with the bait, such as Nck, and
the MINK3 protein.
[0231] In one embodiment, a mammalian two-hybrid system is
preferred. Mammalian systems provide post-translational
modifications of proteins which may contribute significantly to
their ability to interact. In addition, a mammalian two-hybrid
system can be used in a wide variety of mammalian cell types to
mimic the regulation, induction, processing, etc. of specific
proteins within a particular cell type. For example, proteins
involved in a disease state (i.e., cancer, apoptosis related
disorders) could be tested in the relevant disease cells.
Similarly, for testing of random proteins, assaying them under the
relevant cellular conditions will give the highest positive
results. Furthermore, the mammalian cells can be tested under a
variety of experimental conditions that may affect intracellular
protein-protein interactions, such as in the presence of hormones,
drugs, growth factors and cytokines, radiation, chemotherapeutics,
cellular and chemical stimuli, etc., that may contribute to
conditions which can effect protein-protein interactions,
particularly those involved in cancer.
[0232] Assays involving binding such as the two-hybrid system may
take into account non-specific binding proteins (NSB).
[0233] Screening for agents that modulate the activity of MINK3
protein may also be done. In a preferred embodiment, methods for
screening for a bioactive agent capable of modulating the activity
of MINK3 protein comprise the steps of adding a candidate bioactive
agent to a sample of a MINK3 protein (or cells comprising a MINK3
protein) and determining an alteration in the biological activity
of the MINK3 protein. "Modulating the activity of a MINK3 protein"
includes an increase in activity, a decrease in activity, or a
change in the type or kind of activity present. Thus, in this
embodiment, the candidate agent may bind to MINK3 protein (although
this may not be necessary), and will alter at least one MINK3
biological or biochemical activity as described herein. The methods
include both in vitro screening methods, as are generally outlined
above, and in vivo screening of cells for alterations in the
presence, distribution, activity or amount of MINK3 protein.
[0234] In a preferred embodiment, the present invention sets forth
methods for screening for modulators of MINK3 activity. By "MINK3
activity" or "MINK3 protein activity" or grammatical equivalents
herein is meant at least one of the MINK3 protein's biological
activities, including, but not limited to, its ability to bind to
Nck, modulate the phosphorylation of JNK and/or ERK, modulate JNK
and/or ERK activity, modulate signal transduction via the JNK
and/or ERK pathways, modulate F-actin stability, phosphorylate
Gelsolin, inhibit taxol-induced RB cleavage and apoptosis, effect
changes in morphology or oppose such effective changes, modulate
cell proliferation, modulate growth factor-induced proliferation,
modulate growth factor-induced ERK activation, modulate AP-1
induced transcription, modulate transcription induced by AP-1
response elements. By "modulate" is meant increase, decrease, or
alter. In some embodiments, fragments of the MINK3 protein are
preferred, particularly fragments having one or more MINK3 protein
activities.
[0235] In a preferred embodiment, the activity of the MINK3 protein
is decreased; in another preferred embodiment, the activity of the
MINK3 protein is increased. Thus, bioactive agents that are
antagonists are preferred in some embodiments, and bioactive agents
that are agonists may be preferred in other embodiments. As used
herein, increased or overexpressed means an increase of at least
10%, more preferably 25-50%, more preferably 50%-75%, and more
preferably at least a 100% to 500% increase over the native state.
As used herein, decreased or underexpressed means a decrease of at
least 10%, more preferably 25-50%, more preferably 50%-75%, and
more preferably at least a 100% to 500% decrease over the native
state, i.e., compared to without administration of the MINK3
proteins, nucleic acids or candidate agents as described
herein.
[0236] In a preferred embodiment, the invention provides methods
for screening for bioactive agents capable of modulating the
activity of an MINK3 protein. The methods comprise adding a
candidate bioactive agent, as defined above, to a cell comprising a
MINK3 protein. Preferred cell types include almost any cell,
including HeLa cells, 293 cells, MDA-MB-231 cells and Phoenix
cells. The cells contain a recombinant nucleic acid that encodes an
MINK3 protein. In a preferred embodiment, a library of candidate
agents are tested on a plurality of cells comprising a nucleic acid
encoding a MINK3 protein.
[0237] The activity assays, such as having an effect on Nck
binding, cytoskeleton organization, JNK and/or ERK phosphorylation,
JNK and/or ERK activation, JNK and/or ERK signal transduction,
F-actin stability, cell proliferation, survival following taxol
treatment, Rb cleavage following taxol treatment, and growth
factor-induced ERK activation can be performed to confirm the
activity of MINK3 proteins which have already been identified by
their sequence identity/similarity to the sequences set forth in
SEQ ID NOs:1-6 or hybridization to the sequences set forth in SEQ
ID NOs:2, 4, and 6, as well as to further confirm the activity of
lead compounds identified as modulators of the MINK3 proteins
provided herein.
[0238] The components provided herein for the assays provided
herein may also be combined to form kits. The kits can be based on
the use of the protein and/or the nucleic acid encoding the MINK3
proteins. In one embodiment, other components are provided in the
kit. Such components include one or more of packaging,
instructions, antibodies, and labels. Additional assays such as
those used in diagnostics are further described below.
[0239] Using the activity and binding assays provided herein,
bioactive agents, preferably small molecule chemical compositions
as described herein, that may be used as pharmacological compounds
are identified. Compounds with pharmacological activity are able to
enhance or interfere with the activity of the MINK3 protein. The
compounds having the desired pharmacological activity may be
administered in a physiologically acceptable carrier to a host, as
further described below.
[0240] The present discovery relating to the role of MINK3 proteins
in cell proliferation thus provides methods and compositions for
inducing or preventing cell proliferation in cells. In a preferred
embodiment, the MINK3 proteins, and particularly MINK3 protein
fragments, are useful in the study or treatment of conditions which
are mediated by the MINK3 proteins, i.e., to diagnose, treat or
prevent MINK3 associated disorders. "MINK3 associated disorders" or
"disease states" include conditions involving both insufficient or
excessive cell proliferation, preferably cancer. In another
preferred embodiment, candidate bioactive agents, preferably small
molecule chemical compositions as described herein, are useful in
the study or treatment of conditions which are mediated by the
MINK3 proteins, i.e. to diagnose, treat or prevent MINK3 associated
disorders, including and preferably cancer.
[0241] Thus, in one embodiment, methods and compositions for the
modulation of proliferation in cells or organisms are provided. In
one embodiment, the methods comprise administering to a cell or
individual in need thereof, a MINK3 protein in a therapeutic
amount. Alternatively, an anti-MINK3 antibody that reduces or
eliminates the biological activity of the endogenous MINK3 protein
is administered. In another embodiment, a bioactive agent as
identified by the methods provided herein is administered.
Alternatively, the methods comprise administering to a cell or
individual a recombinant nucleic acid encoding an MINK3 protein. As
will be appreciated by those in the art, this may be accomplished
in any number of ways. In a preferred embodiment, the activity of
MINK3 is increased by increasing the amount of MINK3 in the cell,
for example by overexpressing the endogenous MINK3 or by
administering a gene encoding a MINK3 protein, using known
gene-therapy techniques, for example. In a preferred embodiment,
the gene therapy techniques include the incorporation of the
exogeneous gene using enhanced homologous recombination (EHR), for
example as described in PCT/US93/03868, hereby incorporated by
reference in its entirety.
[0242] In a preferred embodiment, MINK3 antisense nucleic acids are
administered to a cell or individual. In a preferred embodiment,
MINK3 antisense nucleic acid decreases the activity of MINK3 by
decreasing the amount of MINK3 mRNA and/or protein in the cell or
individual. In a preferred embodiment, such a MINK3 antisense
nucleic acid comprises the sequence complement of the nucleic acid
sequence set forth by nucleotides 2804-3187 in SEQ ID NO:2. In
another preferred embodiment, such a MINK3 antisense nucleic acid
consists essentially of the sequence complement of the nucleic acid
sequence set forth by nucleotides 2804-3187 in SEQ ID NO:2.
[0243] It appears that MINK3 is important in cell cycle regulation.
Without being bound by theory, the present invention provides
methods and compositions for the determination of cell cycle
disorders. In one embodiment, the invention provides methods for
identifying cells containing variant MINK3 genes comprising
determining all or part of the sequence of at least one endogenous
MINK3 gene in a cell. As will be appreciated by those in the art,
this may be done using any number of sequencing techniques. In a
preferred embodiment, the invention provides methods of identifying
the MINK3 genotype of an individual comprising determining all or
part of the sequence of at least one MINK3 gene of the individual.
This is generally done in at least one tissue of the individual,
and may include the evaluation of a number of tissues or different
samples of the same tissue. The method may include comparing the
sequence of the sequenced MINK3 gene to a known MINK3 gene, i.e. a
wild-type gene.
[0244] The sequence of all or part of the MINK3 gene can then be
compared to the sequence of a known MINK3 gene to determine if any
differences exist. This can be done using any number of known
sequence identity programs, such as Bestfit, etc. In a preferred
embodiment, the presence of a difference in the sequence between
the MINK3 gene of the patient and the known MINK3 gene is
indicative of a disease state or a propensity for a disease
state.
[0245] In one embodiment, methods for determining cell cycle
disorders comprise measuring the activity of MINK3 in a tissue from
the individual or patient, which may include a measurement of the
amount or specific activity of a MINK3 protein. This activity is
compared to the activity of MINK3 from either an unaffected second
individual or from an unaffected tissue from the first individual.
When these activities are different, the first individual may be at
risk for a cell cycle disorder such as cancer. In this way, for
example, monitoring of various disease conditions may be done by
monitoring the levels of protein or mRNA therefore, or by
monitoring protein activity. Similarly, expression levels and
activity levels may correlate to the prognosis.
[0246] In one aspect, the expression levels of MINK3 genes are
determined in different patient samples or cells for which either
diagnosis or prognosis information is desired. Gene expression
monitoring is done on genes encoding MINK3 proteins. In one aspect,
the expression levels of MINK3 genes are determined for different
cellular states, such as normal cells, cells undergoing apoptosis,
cells undergoing transformation, and cancer cells. Thus,
differential MINK3 gene expression between different cell states is
determined. By comparing MINK3 gene expression levels in cells in
different states, information including both up- and
down-regulation of MINK3 genes is obtained, which can be used in a
number of ways. For example, the evaluation of a particular
treatment regime may be evaluated: does a chemotherapeutic drug act
to improve the long-term prognosis in a particular patient, whereby
prognosis is determined based on MINK3 expression. Similarly,
diagnosis may be done or confirmed by comparing patient samples.
Furthermore, these gene expression levels allow screening of drug
candidates with an eye to mimicking or altering a particular
expression level. This may be done by making biochips comprising
sets of important MINK3 genes, such as those of the present
invention, which can then be used in these screens. These methods
can also be done on the protein basis; that is, protein expression
levels of the MINK3 proteins can be evaluated for diagnostic
purposes or to screen candidate agents. In addition, the MINK3
nucleic acid sequences can be administered for gene therapy
purposes, including the administration of antisense nucleic acids,
or the MINK3 proteins administered as therapeutic drugs.
[0247] "Differential expression," or grammatical equivalents as
used herein, refers to both qualitative as well as quantitative
differences in the genes' temporal and/or cellular expression
patterns within and among the cells. Thus, a differentially
expressed gene can qualitatively have its expression altered,
including an activation or inactivation, in, for example, normal
versus apoptotic cell. That is, genes may be turned on or turned
off in a particular state, relative to another state. As is
apparent to the skilled artisan, any comparison of two or more
states can be made. Such a qualitatively regulated gene will
exhibit an expression pattern within a state or cell type which is
detectable by standard techniques in one such state or cell type,
but is not detectable in both. Alternatively, the determination is
quantitative in that expression is increased or decreased; that is,
the expression of the gene is either upregulated, resulting in an
increased amount of transcript, or downregulated, resulting in a
decreased amount of transcript. The degree to which expression
differs need only be large enough to quantify via standard
characterization techniques as outlined below, such as by use of
Affymetrix GeneChip.TM. expression arrays, Lockhart, Nature
Biotechnology, 14:1675-1680 (1996), hereby expressly incorporated
by reference. Other techniques include, but are not limited to,
quantitative reverse transcriptase PCR, Northern analysis and RNase
protection.
[0248] MINK3 sequences bound to biochips include both nucleic acid
and amino acid sequences as defined herein. In a preferred
embodiment, nucleic acid probes to MINK3 nucleic acids (both the
nucleic acid sequences having the sequences outlined in SEQ ID
NOs:2, 4, and 6 and/or the complements thereof) are made. The
nucleic acid probes attached to the biochip are designed to be
substantially complementary to the MINK3 protein nucleic acids,
i.e. the target sequence (either the target sequence of the sample
or to other probe sequences, for example in sandwich assays), such
that hybridization of the target sequence and the probes of the
present invention occurs. As outlined below, this complementarity
need not be perfect; there may be any number of base pair
mismatches which will interfere with hybridization between the
target sequence and the single stranded nucleic acids of the
present invention. However, if the number of mutations is so great
that no hybridization can occur under even the least stringent of
hybridization conditions, the sequence is not a complementary
target sequence. Thus, by "substantially complementary" herein is
meant that the probes are sufficiently complementary to the target
sequences to hybridize under normal reaction conditions,
particularly high stringency conditions, as outlined herein.
[0249] A "nucleic acid probe" is generally single stranded but can
be partially single and partially double stranded. The strandedness
of the probe is dictated by the structure, composition, and
properties of the target sequence. In general, the nucleic acid
probes range from about 8 to about 100 bases long, with from about
10 to about 80 bases being preferred, and from about 30 to about 50
bases being particularly preferred. In some embodiments, much
longer nucleic acids can be used, up to hundreds of bases (e.g.,
whole genes).
[0250] As will be appreciated by those in the art, nucleic acids
can be attached or immobilized to a solid support in a wide variety
of ways. By "immobilized" and grammatical equivalents herein is
meant the association or binding between the nucleic acid probe and
the solid support is sufficient to be stable under the conditions
of binding, washing, analysis, and removal as outlined below. The
binding can be covalent or non-covalent. By "non-covalent binding"
and grammatical equivalents herein is meant one or more of either
electrostatic, hydrophilic, and hydrophobic interactions. Included
in non-covalent binding is the covalent attachment of a molecule,
such as, streptavidin to the support and the non-covalent binding
of the biotinylated probe to the streptavidin. By "covalent
binding" and grammatical equivalents herein is meant that the two
moieties, the solid support and the probe, are attached by at least
one bond, including sigma bonds, pi bonds and coordination bonds.
Covalent bonds can be formed directly between the probe and the
solid support or can be formed by a cross linker or by inclusion of
a specific reactive group on either the solid support or the probe
or both molecules. Immobilization may also involve a combination of
covalent and non-covalent interactions.
[0251] In general, the probes are attached to the biochip in a wide
variety of ways, as will be appreciated by those in the art. As
described herein, the nucleic acids can either be synthesized
first, with subsequent attachment to the biochip, or can be
directly synthesized on the biochip.
[0252] The biochip comprises a suitable solid substrate. By
"substrate" or "solid support" or other grammatical equivalents
herein is meant any material that can be modified to contain
discrete individual sites appropriate for the attachment or
association of the nucleic acid probes and is amenable to at least
one detection method. As will be appreciated by those in the art,
the number of possible substrates are very large, and include, but
are not limited to, glass and modified or functionalized glass,
plastics (including acrylics, polystyrene and copolymers of styrene
and other materials, polypropylene, polyethylene, polybutylene,
polyurethanes, TeflonJ, etc.), polysaccharides, nylon or
nitrocellulose, resins, silica or silica-based materials including
silicon and modified silicon, carbon, metals, inorganic glasses,
plastics, etc. In general, the substrates allow optical detection
and do not appreciably show fluorescence.
[0253] In a preferred embodiment, the surface of the biochip and
the probe may be derivatized with chemical functional groups for
subsequent attachment of the two. Thus, for example, the biochip is
derivatized with a chemical functional group including, but not
limited to, amino groups, carboxy groups, oxo groups and thiol
groups, with amino groups being particularly preferred. Using these
functional groups, the probes can be attached using functional
groups on the probes. For example, nucleic acids containing amino
groups can be attached to surfaces comprising amino groups, for
example using linkers as are known in the art; for example, homo-
or hetero-bifunctional linkers as are well known (see 1994 Pierce
Chemical Company catalog, technical section on cross-linkers, pages
155-200, incorporated herein by reference). In addition, in some
cases, additional linkers, such as alkyl groups (including
substituted and heteroalkyl groups) may be used.
[0254] In this embodiment, oligonucleotides, corresponding to the
nucleic acid probe, are synthesized as is known in the art, and
then attached to the surface of the solid support. As will be
appreciated by those skilled in the art, either the 5' or 3'
terminus may be attached to the solid support, or attachment may be
via an internal nucleoside.
[0255] In an additional embodiment, the immobilization to the solid
support may be very strong, yet non-covalent. For example,
biotinylated oligonucleotides can be made, which bind to surfaces
covalently coated with streptavidin, resulting in attachment.
[0256] Alternatively, the oligonucleotides may be synthesized on
the surface, as is known in the art. For example, photoactivation
techniques utilizing photopolymerization compounds and techniques
are used. In a preferred embodiment, the nucleic acids can be
synthesized in situ, using well known photolithographic techniques,
such as those described in WO 95/25116; WO 95/35505; U.S. Pat. Nos.
5,700,637 and 5,445,934; and references cited within, all of which
are expressly incorporated by reference; these methods of
attachment form the basis of the Affimetrix GeneChip.TM.
technology.
[0257] The present invention provides novel methods and
compositions for screening for compositions which modulate MINK3
bioactivities including Nck binding activity, as well as the
ability to modulate cytoskeleton organization, JNK and/or ERK
phosphorylation, JNK and/or ERK activation, JNK and/or ERK signal
transduction, F-actin stability, cell proliferation, survival
following taxol treatment, Rb cleavage following taxol treatment,
and growth factor-induced ERK activation. As above, this can be
done by screening for modulators of MINK3 gene expression or for
modulators of MINK3 protein activity. Gene expression and protein
activity may be evaluated on an individual gene and protein basis,
or by evaluating the effect of drug candidates on a gene expression
or protein expression profile. In a preferred embodiment, the
expression profiles are used, preferably in conjunction with high
throughput screening techniques to allow monitoring for expression
profile genes after treatment with a candidate agent.
[0258] A variety of assays my be used to evaluate the effects of
agents on MINK3 gene expression. In a preferred embodiment, assays
may be run on an individual gene or protein level. That is, having
identified the bioactivities of MINK3 described herein, candidate
bioactive agents may be screened for the ability to modulate MINK3
gene expression and MINK3 bioactivities. "Modulation" thus includes
both an increase and a decrease in gene expression or activity. The
preferred amount of modulation will depend on the original change
of the gene expression in normal versus tumor tissue, with changes
of at least 10%, preferably 50%, more preferably 100-300%, and in
some embodiments 300-1000% or greater. Thus, if a gene exhibits a 4
fold increase in tumor compared to normal tissue, a decrease of
about four fold is desired; a 10 fold decrease in tumor compared to
normal tissue makes a 10 fold increase in expression for a
candidate agent desirable, etc. Alternatively, where the MINK3
sequence has been altered but shows the same expression profile or
an altered expression profile, the protein will be detected as
outlined herein.
[0259] As will be appreciated by those in the art, this may be done
by evaluation at either the gene transcript or the protein level;
that is, the amount of gene expression may be monitored using
nucleic acid probes and the quantification of gene expression
levels, or, alternatively, the level of the gene product itself can
be monitored, for example through the use of antibodies to the
MINK3 protein and standard immunoassays. Alternatively, binding and
bioactivity assays with the protein may be done as outlined
herein.
[0260] In a preferred embodiment, gene expression monitoring is
done and a number of genes, i.e. an expression profile, are
monitored simultaneously, although multiple protein expression
monitoring can be done as well. For example, protein can be
monitored through the use of antibodies to the MINK3 protein and
standard immunoassays (ELISAs, etc.) or other techniques, including
mass spectroscopy assays, 2D gel electrophoresis assays, etc.
[0261] In this embodiment, the MINK3 nucleic acid probes are
attached to biochips as outlined herein for the detection and
quantification of MINK3 sequences in a particular cell.
[0262] In another method detection of the mRNA is performed in
situ. In this method permeabilized cells or tissue samples are
contacted with a detectably labeled nucleic acid probe for
sufficient time to allow the probe to hybridize with the target
mRNA. Following washing to remove the non-specifically bound probe,
the label is detected. For example a digoxygenin labeled riboprobe
(RNA probe) that is complementary to the mRNA encoding an MINK3
protein is detected by binding the digoxygenin with an
anti-digoxygenin secondary antibody and developed with nitro blue
tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.
[0263] In another preferred method, expression of MINK3 protein is
performed using in situ imaging techniques employing antibodies to
MINK3 proteins. In this method cells are contacted with from one to
many antibodies to the MINK3 protein(s). Following washing to
remove non-specific antibody binding, the presence of the antibody
or antibodies is detected. In one embodiment the antibody is
detected by incubating with a secondary antibody that contains a
detectable label. In another method the primary antibody to the
MINK3 protein(s) contains a detectable label. In another preferred
embodiment each one of multiple primary antibodies contains a
distinct and detectable label. This method finds particular use in
simultaneous screening for a plurality of MINK3 proteins. The label
may be detected in a luminometer which has the ability to detect
and distinguish emissions of different wavelengths. In addition, a
fluorescence activated cell sorter (FACS) can be used in this
method. As will be appreciated by one of ordinary skill in the art,
numerous other histological imaging techniques are useful in the
invention and the antibodies can be used in ELISA, immunoblotting
(Western blotting), immunoprecipitation, BIACORE technology, and
the like.
[0264] The present invention provides additional methods and
compositions for screening candidate bioactive agents for the
ability to modulate MINK3 bioactivities as described herein.
[0265] For example, candidate bioactive agents may be screened for
the ability to modulate transcriptional activation by the AP-1
protein complex. In one embodiment, such a method comprises the
steps of combining a mammalian cell comprising AP-1 protein complex
and a reporter gene fused to a transcriptional regulatory DNA
sequence comprising at least one AP-1 response element, and a
recombinant nucleic acid encoding a MINK3 protein, and a candidate
bioactive agent, and determining the level of reporter gene
expression in the presence and absence of candidate agent.
[0266] In a preferred embodiment, such a reporter gene is
luciferase. In a preferred embodiment, such a candidate agent is a
small molecule chemical compound. In a preferred embodiment, such a
mammalian cell is a HeLa cell, a 293 cell, a Phoenix cell, an
MDA-MD-231 cell, or an A549 cell.
[0267] As another example, candidate bioactive agents may be
screened for the ability to modulate growth factor-induced ERK
activation. In one embodiment, such a method comprises combining a
mammalian cell comprising a recombinant nucleic acid encoding a
MINK3 protein, and a candidate bioactive agent, and determining the
level of ERK activation in the presence and absence of candidate
agent.
[0268] ERK activation may be determined in several ways as will be
appreciated by those in the art. In a preferred embodiment, ERK1 is
immunoprecipitated from cell lysate using anti-ERK antibody, and
the immunoprecipitate is used in an in vitro kinase assay with
myelin basic protein (MBP) as a substrate. In vitro kinase assays
are known in the art. In such an in vitro kinase assay,
isotopically labeled ATP, preferably .gamma.P32-labeled, may be
used as a source of phosphate for the kinase assay. Briefly, MBP is
a substrate for activated ERK1. If activated ERK1 is present in the
immunoprecipitate, it will catalyze the transfer of a labeled
phosphate group to the substrate, MBP. The products of the in vitro
kinase assay may be separated by gel electrophoresis as is known in
the art. In this way, the MBP in the kinase assay mixture may be
separated from labeled ATP and other constituents in the assay
mixture. The amount of isotope incorporated by MBP, indicative of
the amount of ERK activity in the immunoprecipitate, may then be
determined using techniques known in the art.
[0269] In this way, the level of ERK activation in the presence and
absence of candidate agent may be determined and compared to
identify a candidate agent capable of either inhibiting ERK
activation, or inducing ERK activation.
[0270] In one embodiment, candidate agents are screened for the
ability to modulate ERK activation in response to growth factors.
In this embodiment, ERK activation may be determined as described
above. In a preferred embodiment, candidate agents are screened for
the ability to modulate ERK activation in response to epidermal
growth factor (EGF). In one embodiment, such a method comprises
exposing mammalian cells comprising the EGF receptor to EGF and
then determining ERK activation in the presence and absence of
candidate agent as described above.
[0271] In another embodiment, such a method comprises combining a
cell comprising ERK and MINK3 proteins, as well as comprising a
reporter gene under the control of an ERK responsive
transcriptional regulatory element (such as an ELK response
element, as is known in the art), and a candidate agent, and
determining the level of reporter gene expression in the presence
and absence of candidate agent.
[0272] As will be appreciated by those in the art, the in vitro
kinase assay described above may be used similarly to determine the
level of JNK activation. The present invention thus provides
methods for screening candidate bioactive agents for the ability to
modulate JNK activation. In a preferred embodiment, the level of
activation of JNK2 is determined in the presence and absence of
candidate agent. In such a method, JNK2 is immunoprecipitated with
anti-JNK antibody, and a glutathione-S-transferase:c-JUN fusion
protein serves as substrate for JNK in an in vitro kinase assay as
described above, wherein the c-JUN moiety is a substrate for
JNK.
[0273] As another example, candidate bioactive agents may be
screened for the ability to modulate JNK and/or ERK
phosphorylation. In one embodiment, such a method comprises the
steps of combining a candidate agent, and a cell comprising a
recombinant nucleic acid encoding a MINK3 protein, and ERK and/or
JNK proteins, and determining the level of phosphorylation of ERK
and/or JNK in the presence and absence of candidate agent. As will
be appreciated by those in the art, the determination of ERK and
JNK phosphorylation can be done in a number of ways. For example,
isotopically-labeled ATP may be added to the cells in order to
serve as a source of phosphate for the phosphorylation of JNK and
ERK. Following incubation, ERK and/or JNK may be immunoprecipitated
using appropriate antibodies as described above, and the
immunoprecipitates may be resolved by gel electrophoresis. The
amount of radioactive phosphate associated with JNK or ERK (at the
appropriate molecular weight) is then determined using techniques
known in the art.
[0274] Alternatively, such a method may comprise adding a candidate
agent to a MINK3 protein which may be isolated or cell-free as in a
cell lysate, and then adding an ERK and/or JNK protein, which may
be isolated or cell-free, and determining the level of
phosphorylation of ERK and/or JNK.
[0275] As another example, candidate bioactive agents may be
screened for the ability to modulate Rb cleavage in response to
exposure to taxol. In a preferred embodiment, such a method
comprises combining a mammalian cell comprising a recombinant
nucleic acid encoding a MINK3 protein, and Rb, and a candidate
agent, and exposing the cell to taxol, and determining the level of
Rb cleavage in response to taxol in the presence and absence of
candidate agent. The level of Rb cleavage may be determined by
immunoprecipitating Rb or a portion thereof from cell lysate using
an anti-Rb antibody. The immunoprecipitate may then be resolved
using gel electrophoresis. A western blot using anti-Rb antibody
may then be done to determine the molecular weight of the Rb
species in immunoprecipitate. The appearance of an Rb
immunoreactive band at a molecular weight lower than that of native
Rb indicates Rb cleavage has occurred. The relative amounts of
cleaved to native Rb determines the level of Rb cleavage in the
sample.
[0276] As another example, candidate bioactive agents may be
screened for the ability to modulate cell survival in response to
exposure to taxol. In a preferred embodiment, such a method
comprises combining a mammalian cell comprising a nucleic acid
encoding a MINK3 protein, and a candidate bioactive agent, exposing
the cell to taxol, and determining the level of cell survival in
the presence and absence of candidate agent. The level of cell
survival may be determined in many ways as will be appreciated by
those in the art. For example, enzymatic assays for mitochondrial
function, such as the MTT or XTT assays, may be used to determine
the level of respiration in cells, an indicator of cell survival.
Additionally, survival may be inferred from the absence of well
known indicators of apoptosis, including genomic DNA laddering.
[0277] As another example, candidate bioactive agents may be
screened for the ability to modulate proliferation in mammalian
cells. In a preferred embodiment, such a method comprises combining
a mammalian cell comprising a recombinant nucleic acid encoding a
MINK3 protein, and a candidate agent, and determining the
proliferation of the cell in the presence and absence of candidate
agent. As will be appreciated by those in the art, mammalian cell
proliferation may be determined in many ways. For example, cell
density in a sample of mammalian cells may be determined over time
by measuring the optical density of the sample, preferably at 490
nm. The density of the sample is indicative of the number of cells
in the sample, which is in turn indicative of the level of
proliferation in the cells of the sample. In a preferred
embodiment, A549 cells are used.
[0278] As another example, candidate bioactive agents may be
screened for the ability to modulate F-actin stability. In a
preferred embodiment, such a method comprises combining a mammalian
cell comprising a recombinant nucleic acid encoding a MINK3
protein, and a candidate agent, and determining the stability of
F-actin in the presence and absence of candidate agent. As will be
appreciated by those in the art, the stability of F-actin may be
determined in several ways. For example, the amount of actin in a
triton X-100 soluble fraction versus the amount of actin in a
triton X-100 insoluble fraction may be determined using by running
western blots with the fractions and using an anti-actin antibody
(Fu et al., JBC 274:30729-30737). After transfection, cells may be
lysed directly on a plate using 250 .mu.l Triton X-100 lysis buffer
(1% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl, pH 7.4) with
protease inhibitors. Cell lysates are centrifuged at 14,000 RPM for
10 min. Supernatant constitutes the Triton X-100 soluble fraction.
Pellets are washed once with 500 .mu.l Triton X-100 lysis buffer
and dissolved in 500 .mu.l of 1.times.SDS sample buffer. DNA is
sheared by sonication. This represents the Triton X-100 insoluble
fraction. Triton X-100 soluble and insoluble fractions derived from
the same number of cells are resolved on SDS-PAGE and blotted with
an anti-.beta.-actin mAb to determine the content of F- and
G-actin.
[0279] Alternatively, immunofluorescence using an anti-actin
antibody or labeled phalloidin may be done on whole cells to
visualize actin filaments in the cells.
[0280] As another example, candidate bioactive agents may be
screened for the ability to modulate cell morphology. In a
preferred embodiment, such a method comprises combining a mammalian
cell comprising a recombinant nucleic acid encoding a MINK3
protein, and a candidate agent, and determining cell morphology in
the presence and absence of candidate agent. As will be appreciated
by those in the art, cell morphology may be determined in many
ways. Light microscopy and a variety of cell stains known in the
art may be used to visualize cells and determine morphology.
[0281] In preferred embodiments, such a method uses Phoenix cells,
293 cells, or MDA-MB-231 cells.
[0282] In a preferred embodiment, the ability of a candidate agent
to modulate morphology is dependent on MEK activity. Dependence on
MEK activity can be determined through the use of the known MEK
inhibitor PD98059. Cell morphology can be determined as described
above, in the presence and absence of candidate agent, and further
in the presence and absence of the MEK inhibitor PD98059.
[0283] In one aspect, the invention is directed to methods for
screening for a bioactive agent capable of modulating JNK
phosphorylation and/or activation. In one aspect, the invention is
directed to methods for screening for a bioactive agent capable of
modulating the JNK signal transduction pathway. In a preferred
embodiment, the methods comprise contacting a candidate bioactive
agent to a mammalian cell comprising a recombinant MINK3 nucleic
acid encoding a MINK3 protein and a JNK protein and determining JNK
activity in the presence of candidate agent. In a preferred
embodiment, JNK activity is determined in the presence and absence
of candidate agent. The recombinant MINK3 nucleic acid is expressed
in said mammalian cell and will activate JNK protein in the absence
of candidate bioactive agent. In a preferred embodiment, the
encoded MINK3 protein comprises an amino acid sequence having at
least about 90% identity to an amino acid sequence selected from
the group consisting of the amino acid sequences set forth in SEQ
ID NOs:1, 3, and 5. A decrease in the activity of JNK protein in
the presence of candidate bioactive agent indicates that the
candidate bioactive agent is capable of modulating JNK
activity.
[0284] In one aspect, the invention is directed to methods for
screening for a bioactive agent capable of modulating ERK
phosphorylation and/or activation. In one aspect, the invention is
directed to methods for screening for a bioactive agent capable of
modulating the ERK signal transduction pathway. In a preferred
embodiment, the methods comprise contacting a candidate bioactive
agent to a mammalian cell comprising a recombinant MINK3 nucleic
acid encoding a MINK3 protein and a ERK protein and determining ERK
activity in the presence of candidate agent. In a preferred
embodiment, ERK activity is determined in the presence and absence
of candidate agent. The recombinant MINK3 nucleic acid is expressed
in said mammalian cell and will activate ERK protein in the absence
of candidate bioactive agent. In a preferred embodiment, the
encoded MINK3 protein comprises an amino acid sequence having at
least about 90% identity to an amino acid sequence selected from
the group consisting of the amino acid sequences set forth in SEQ
ID NOs:1, 3, and 5. A decrease in the activity of ERK protein in
the presence of candidate bioactive agent indicates that the
candidate bioactive agent is capable of modulating ERK
activity.
[0285] In a preferred embodiment, the methods comprise contacting a
mammalian cell with a growth factor which will activate JNK and/or
ERK. In a preferred embodiment, the growth factor used in epidermal
growth factor (EGF).
[0286] In one embodiment, the MINK3 proteins of the present
invention may be used to generate polyclonal and monoclonal
antibodies to MINK3 proteins, which are useful as described herein.
Similarly, the MINK3 proteins can be coupled, using standard
technology, to affinity chromatography columns. These columns may
then be used to purify MINK3 antibodies. In a preferred embodiment,
the antibodies are generated to epitopes unique to the MINK3
protein; that is, the antibodies show little or no cross-reactivity
to other proteins. These antibodies find use in a number of
applications. For example, the MINK3 antibodies may be coupled to
standard affinity chromatography columns and used to purify MINK3
proteins as further described below. The antibodies may also be
used as blocking polypeptides, as outlined above, since they will
specifically bind to the MINK3 protein.
[0287] The anti-MINK3 protein antibodies may comprise polyclonal
antibodies. Methods of preparing polyclonal antibodies are known to
the skilled artisan. Polyclonal antibodies can be raised in a
mammal, for example, by one or more injections of an immunizing
agent and, if desired, an adjuvant. Typically, the immunizing agent
and/or adjuvant will be injected in the mammal by multiple
subcutaneous or intraperitoneal injections. The immunizing agent
may include the MINK3 protein or a fusion protein thereof. It may
be useful to conjugate the immunizing agent to a protein known to
be immunogenic in the mammal being immunized. Examples of such
immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum albumin, bovine thyroglobulin, and soybean
trypsin inhibitor. Examples of adjuvants which may be employed
include Freund's complete adjuvant and MPL-TDM adjuvant
(monophosphoryl Lipid a, synthetic trehalose dicorynomycolate). The
immunization protocol may be selected by one skilled in the art
without undue experimentation.
[0288] The anti-MINK3 protein antibodies may, alternatively, be
monoclonal antibodies. Monoclonal antibodies may be prepared using
hybridoma methods, such as those described by Kohler, et al.,
Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or
other appropriate host animal, is typically immunized with an
immunizing agent to elicit lymphocytes that produce or are capable
of producing antibodies that will specifically bind to the
immunizing agent. Alternatively, the lymphocytes may be immunized
in vitro.
[0289] The immunizing agent will typically include the MINK3
protein or a fusion protein thereof. Generally, either peripheral
blood lymphocytes ("PBLs") are used if cells of human origin are
desired, or spleen cells or lymph node cells are used if non-human
mammalian sources are desired. The lymphocytes are then fused with
an immortalized cell line using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal
Antibodies: Principles and Practice, Academic Press, (1986) pp.
59-103). Immortalized cell lines are usually transformed mammalian
cells, particularly myeloma cells of rodent, bovine and human
origin. Usually, rat or mouse myeloma cell lines are employed. The
hybridoma cells may be cultured in a suitable culture medium that
preferably contains one or more substances that inhibit the growth
or survival of the unfused, immortalized cells. For example, if the
parental cells lack the enzyme hypoxanthine guanine phosphoribosyl
transferase (HGPRT or HPRT), the culture medium for the hybridomas
typically will include hypoxanthine, aminopterin, and thymidine
("HAT medium"), which substances prevent the growth of
HGPRT-deficient cells.
[0290] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Rockville, Md. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies (Kozbor, J.
Immunol., 133:3001 (1984); Brodeur, et al., Monoclonal Antibody
Production Techniques and Applications, Marcel Dekker, Inc., New
York, pp. 51-63 (1987)).
[0291] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against MINK3 protein. Preferably, the binding specificity
of monoclonal antibodies produced by the hybridoma cells is
determined by immunoprecipitation or by an in vitro binding assay,
such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay
(ELISA). Such techniques and assays are known in the art. The
binding affinity of the monoclonal antibody can, for example, be
determined by the Scatchard analysis of Munson, et al., Anal.
Biochem., 107:220 (1980).
[0292] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Supra). Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells may be
grown in vivo as ascites in a mammal.
[0293] The monoclonal antibodies secreted by the subclones may be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein a-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0294] The monoclonal antibodies may also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA. Once isolated, the DNA may be placed
into expression vectors, which are then transfected into host cells
such as simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also may be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences (U.S.
Pat. No. 4,816,567; Morrison, et al., supra) or by covalently
joining to the immunoglobulin coding sequence all or part of the
coding sequence for a non-immunoglobulin polypeptide. Such a
non-immunoglobulin polypeptide can be substituted for the constant
domains of an antibody of the invention, or can be substituted for
the variable domains of one antigen-combining site of an antibody
of the invention to create a chimeric bivalent antibody.
[0295] The antibodies may be monovalent antibodies. Methods for
preparing monovalent antibodies are well known in the art. For
example, one method involves recombinant expression of
immunoglobulin light chain and modified heavy chain. The heavy
chain is truncated generally at any point in the Fc region so as to
prevent heavy chain crosslinking. Alternatively, the relevant
cysteine residues are substituted with another amino acid residue
or are deleted so as to prevent crosslinking.
[0296] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art.
[0297] The anti-MINK3 protein antibodies of the invention may
further comprise humanized antibodies or human antibodies.
Humanized forms of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such
as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of
antibodies) which contain minimal sequence derived from non-human
immunoglobulin. Humanized antibodies include human immunoglobulins
(recipient antibody) in which residues from a complementary
determining region (CDR) of the recipient are replaced by residues
from a CDR of a non-human species (donor antibody) such as mouse,
rat or rabbit having the desired specificity, affinity and
capacity. In some instances, Fv framework residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Humanized antibodies may also comprise residues which are found
neither in the recipient antibody nor in the imported CDR or
framework sequences. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin (Jones, et
al., Nature, 321:522-525 (1986); Riechmann, et al., Nature,
332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596
(1992))
[0298] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones, et al.,
Nature, 321:522-525 (1986); Riechmann, et al., Nature, 332:323-327
(1988); Verhoeyen, et al., Science, 239:1534-1536 (1988)), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0299] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
(Hoogenboom, et al, J. Mol. Biol., 227:381 (1991); Marks, et al.,
J. Mol. Biol., 222:581 (1991)). The techniques of Cole, et al. and
Boerner, et al. are also available for the preparation of human
monoclonal antibodies (Cole, et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner, et al., J.
Immunol., 147(1):86-95 (1991)). Similarly, human antibodies can be
made by introducing of human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks, et al., Bio/Technology, 10:779-783 (1992);
Lonberg, et al., Nature, 368:856-859 (1994); Morrison, Nature,
368:812-13 (1994); Fishwild, et al., Nature Biotechnology,
14:845-51 (1996); Neuberger, Nature Biotechnology, 14:826 (1996);
Lonberg, et al., Intern. Rev. Immunol., 13:65-93 (1995).
[0300] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for the MINK3 protein, the other one is for any
other antigen, and preferably for a cell-surface protein or
receptor or receptor subunit.
[0301] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have
different specificities (Milstein, et al., Nature, 305:537-539
(1983)). Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published 13 May
1993, and in Traunecker, et al., EMBO J., 10:3655-3659 (1991).
[0302] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. For further details of generating bispecific
antibodies see, for example, Suresh, et al., Methods in Enzymology,
121:210 (1986).
[0303] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells (U.S.
Pat. No. 4,676,980), and for treatment of HIV infection (WO
91/00360; WO 92/200373; EP 03089). It is contemplated that the
antibodies may be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins may be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0304] The anti-MINK3 protein antibodies of the invention have
various utilities. For example, anti-MINK3 protein antibodies may
be used in diagnostic assays for an MINK3 protein, e.g., detecting
its expression in specific cells, tissues, or serum. Various
diagnostic assay techniques known in the art may be used, such as
competitive binding assays, direct or indirect sandwich assays and
immunoprecipitation assays conducted in either heterogeneous or
homogeneous phases (Zola, Monoclonal Antibodies: a Manual of
Techniques, CRC Press, Inc. pp. 147-158 (1987)). The antibodies
used in the diagnostic assays can be labeled with a detectable
moiety. The detectable moiety should be capable of producing,
either directly or indirectly, a detectable signal. For example,
the detectable moiety may be a radioisotope, such as 3H, 14C, 32P,
35S, or 125I, a fluorescent or chemiluminescent compound, such as
fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme,
such as alkaline phosphatase, beta-galactosidase or horseradish
peroxidase. Any method known in the art for conjugating the
antibody to the detectable moiety may be employed, including those
methods described by Hunter, et al., Nature, 144:945 (1962); David,
et al., Biochemistry, 13:1014 (1974); Pain, et al., J. Immunol.
Meth., 40:219 (1981); and Nygren, J. Histochem. and Cytochem.,
30:407 (1982).
[0305] Anti-MINK3 protein antibodies also are useful for the
affinity purification of MINK3 protein from recombinant cell
culture or natural sources. In this process, the antibodies against
MINK3 protein are immobilized on a suitable support, such a
Sephadex resin or filter paper, using methods well known in the
art. The immobilized antibody then is contacted with a sample
containing the MINK3 protein to be purified, and thereafter the
support is washed with a suitable solvent that will remove
substantially all the material in the sample except the MINK3
protein, which is bound to the immobilized antibody. Finally, the
support is washed with another suitable solvent that will release
the MINK3 protein from the antibody.
[0306] The anti-MINK3 protein antibodies may also be used in
treatment. In one embodiment, the genes encoding the antibodies are
provided, such that the antibodies bind to and modulate the MINK3
protein within the cell.
[0307] In one embodiment, a therapeutically effective dose of an
MINK3 protein, agonist or antagonist is administered to a patient.
By "therapeutically effective dose" herein is meant a dose that
produces the effects for which it is administered. The exact dose
will depend on the purpose of the treatment, and will be
ascertainable by one skilled in the art using known techniques. As
is known in the art, adjustments for MINK3 protein degradation, or
antagonist or agonist degradation or metabolism, as well as
systemic versus localized delivery, as well as the age, body
weight, general health, sex, diet, time of administration, drug
interaction and the severity of the condition may be necessary, and
will be ascertainable with routine experimentation by those skilled
in the art.
[0308] A "patient" for the purposes of the present invention
includes both humans and other animals, particularly mammals, and
organisms. Thus the methods are applicable to both human therapy
and veterinary applications. In the preferred embodiment the
patient is a mammal, and in the most preferred embodiment the
patient is human.
[0309] The administration of the MINK3 protein, agonist or
antagonist of the present invention can be done in a variety of
ways, including, but not limited to, orally, subcutaneously,
intravenously, intranasally, transdermally, intraperitoneally,
intramuscularly, intrapulmonary, vaginally, rectally, or
intraocularly. In some instances, for example, in the treatment of
wounds and inflammation, the composition may be directly applied as
a solution or spray. Depending upon the manner of introduction, the
compounds may be formulated in a variety of ways. The concentration
of therapeutically active compound in the formulation may vary from
about 0.1-100 wt. %.
[0310] The pharmaceutical compositions of the present invention
comprise an MINK3 protein, agonist or antagonist (including
antibodies and bioactive agents as described herein) in a form
suitable for administration to a patient. Small molecule chemical
compositions as described herein are especially preferred. In a
preferred embodiment, the pharmaceutical compositions are in a
water soluble form, such as being present as pharmaceutically
acceptable salts, which is meant to include both acid and base
addition salts. "Pharmaceutically acceptable acid addition salt"
refers to those salts that retain the biological effectiveness of
the free bases and that are not biologically or otherwise
undesirable, formed with inorganic acids such as hydrochloric acid,
hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and
the like, and organic acids such as acetic acid, propionic acid,
glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic
acid, succinic acid, fumaric acid, tartaric acid, citric acid,
benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,
ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the
like. "Pharmaceutically acceptable base addition salts" include
those derived from inorganic bases such as sodium, potassium,
lithium, ammonium, calcium, magnesium, iron, zinc, copper,
manganese, aluminum salts and the like. Particularly preferred are
the ammonium, potassium, sodium, calcium, and magnesium salts.
Salts derived from pharmaceutically acceptable organic non-toxic
bases include salts of primary, secondary, and tertiary amines,
substituted amines including naturally occurring substituted
amines, cyclic amines and basic ion exchange resins, such as
isopropylamine, trimethylamine, diethylamine, triethylamine,
tripropylamine, and ethanolamine.
[0311] The pharmaceutical compositions may also include one or more
of the following: carrier proteins such as serum albumin; buffers;
fillers such as microcrystalline cellulose, lactose, corn and other
starches; binding agents; sweeteners and other flavoring agents;
coloring agents; and polyethylene glycol. Additives are well known
in the art, and are used in a variety of formulations.
[0312] Combinations of the compositions may be administered.
Moreover, the compositions may be administered in combination with
other therapeutics, including growth factors or chemotherapeutics
and/or radiation. Targeting agents (i.e. ligands for receptors on
cancer cells) may also be combined with the compositions provided
herein.
[0313] Without being bound by theory, the pharmaceutical
compositions provided herein find use in the treatment and/or
prophylaxis of cancer, particularly as cancer involves dysregulated
cell proliferation, aberrant morphology, and aberrant migration as
in metastasis. Further cancer may involve aberrant JNK and/or ERK
phosphorylation, JNK and/or ERK activation, and JNK and/or ERK
signal transduction.
[0314] In one embodiment provided herein, the antibodies are used
for immunotherapy, thus, methods of immunotherapy are provided. By
"immunotherapy" is meant treatment of MINK3 protein related
disorders with an antibody raised against a MINK3 protein. As used
herein, immunotherapy can be passive or active. Passive
immunotherapy, as defined herein, is the passive transfer of
antibody to a recipient (patient). Active immunization is the
induction of antibody and/or T-cell responses in a recipient
(patient). Induction of an immune response can be the consequence
of providing the recipient with an MINK3 protein antigen to which
antibodies are raised. As appreciated by one of ordinary skill in
the art, the MINK3 protein antigen may be provided by injecting an
MINK3 protein against which antibodies are desired to be raised
into a recipient, or contacting the recipient with an MINK3 protein
nucleic acid, capable of expressing the MINK3 protein antigen,
under conditions for expression of the MINK3 protein antigen.
[0315] In a preferred embodiment, a therapeutic compound is
conjugated to an antibody, preferably an MINK3 protein antibody.
The therapeutic compound may be a cytotoxic agent. In this method,
targeting the cytotoxic agent to apoptotic cells or tumor tissue or
cells, results in a reduction in the number of afflicted cells,
thereby reducing symptoms associated with apoptosis, cancer MINK3
protein related disorders. Cytotoxic agents are numerous and varied
and include, but are not limited to, cytotoxic drugs or toxins or
active fragments of such toxins. Suitable toxins and their
corresponding fragments include diphtheria A chain, exotoxin A
chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin,
enomycin and the like. Cytotoxic agents also include radiochemicals
made by conjugating radioisotopes to antibodies raised against
MINK3 proteins, or binding of a radionuclide to a chelating agent
that has been covalently attached to the antibody.
[0316] In a preferred embodiment, MINK3 protein genes are
administered as DNA vaccines, either single nucleic acids or
combinations of MINK3 protein genes. Naked DNA vaccines are
generally known in the art; see Brower, Nature Biotechnology,
16:1304-1305 (1998). Methods for the use of nucleic acids as DNA
vaccines are well known to one of ordinary skill in the art, and
include placing an MINK3 protein gene or portion of an MINK3
protein nucleic acid under the control of a promoter for expression
in a patient. The MINK3 protein gene used for DNA vaccines can
encode full-length MINK3 proteins, but more preferably encodes
portions of the MINK3 proteins including peptides derived from the
MINK3 protein. In a preferred embodiment a patient is immunized
with a DNA vaccine comprising a plurality of nucleotide sequences
derived from a MINK3 protein gene. Similarly, it is possible to
immunize a patient with a plurality of MINK3 protein genes or
portions thereof, as defined herein. Without being bound by theory,
following expression of the polypeptide encoded by the DNA vaccine,
cytotoxic T-cells, helper T-cells and antibodies are induced which
recognize and destroy or eliminate cells expressing MINK3
proteins.
[0317] In a preferred embodiment, the DNA vaccines include a gene
encoding an adjuvant molecule with the DNA vaccine. Such adjuvant
molecules include cytokines that increase the immunogenic response
to the MINK3 protein encoded by the DNA vaccine. Additional or
alternative adjuvants are known to those of ordinary skill in the
art and find use in the invention.
EXAMPLES
[0318] The following examples serve to more fully describe the
manner of using the above-described invention, as well as to set
forth the best modes contemplated for carrying out various aspects
of the invention. It is understood that this example in no way
serve to limit the true scope of this invention, but rather are
presented for illustrative purposes. All references cited herein
are expressly incorporated by reference in their entirety.
Moreover, all sequences displayed, cited by reference or accession
number in the references are incorporated by reference herein.
Example 1
[0319] A MINK3 antisense nucleic acid complementary to a MINK3 cDNA
from a Jurkat cDNA library was identified in a functional screen
for nucleic acids capable of inhibiting cell death following
exposure of HeLa cells to taxol (data not shown). The antisense
nucleic acid comprises a nucleic acid sequence complimentary to
that set forth by nucleotides 2804-3187 in SEQ ID NO:2.
[0320] MINK3 antisense nucleic acid was used to clone and identify
three isoforms of MINK3 (MINK3a, MINK3b, MINK3c, set forth in SEQ
ID NOs:2, 4, and 6, respectively).
[0321] HeLa cells were transfected with expression vectors encoding
MINK3a, MINK3b, Bcl2, or MINK3 antisense nucleic acid complementary
to the nucleic acid sequence set forth by nucleotides 2804-3187 in
SEQ ID NO:2. As an additional control, cells were transfected with
empty vector.
[0322] Following transfection, cells were exposed to a range of
taxol concentrations and a mitochondrial respiration assay (XTT)
was performed on cell lysates to determine viable cells.
[0323] Both Bcl2 and MINK3 antisense nucleic acid had an inhibitory
effect on taxol-induced cell death, while MINK3a and MINK3b did not
appear to affect cell death (FIG. 7).
Example 2
[0324] Hela cells were transfected with expression vectors encoding
either Bcl2 or GFP, or an expression vector comprising a MINK3
antisense nucleic acid complementary to the nucleic acid sequence
set forth by nucleotides 2804-3187 in SEQ ID NO:2. As an additional
control, Hela cells were transfected with empty vector alone.
[0325] Following transfection, cells were exposed to taxol at a
concentration of 40 nm or 60 nm. Cells were collected following
taxol treatment and cell lysate was obtained. Western blots were
run on cell lysates using anti-RB antibody and anti-Bcl2 antibody.
As a control, anti-cdk2 antibody was used.
[0326] Rb immunodetection was done on lysates from cells exposed or
not exposed to taxol. In samples exposed to taxol, a faster
migrating Rb-immunoreactive band ("cleaved Rb") was detected in
addition to the normal Rb immunoreactive band. Cleaved Rb was not
detectable in cells transfected with Bcl2 and exposed to taxol. The
amount of cleaved Rb formed in response to taxol was dramatically
reduced in cells transfected with MINK3 antisense nucleic acid
complementary to the nucleic acid sequence set forth by nucleotides
2804-3187 in SEQ ID NO:2. GFP and empty expression vectors had no
effect on the formation of cleaved Rb in response to taxol (FIG.
10).
Example 3
[0327] 293 cells comprising a luciferase reporter gene fused to a
regulatory sequence comprising an AP1 element were transfected with
MINK3 antisense nucleic acid complementary to the nucleic acid
sequence set forth by nucleotides 2804-3187 in SEQ ID NO:2, or an
MEKK1 expression vector, or an empty expression vector.
[0328] Following transfection, luciferase activity was determined
using a luminometer, in order to determine the level of expression
of the reporter gene.
[0329] MEKK1 induced reporter gene expression, while MINK3
antisense nucleic acid inhibited the basal level of reporter gene
expression (FIG. 3).
Example 4
[0330] Northern blot analysis showed that MINK3 mRNA is expressed
at different levels in a number of human tissues, including spleen,
thymus, prostate, testis, ovary, small intestine, colon, PBL,
leukocytes, heart, brain, placenta, lung, liver, skeletal muscle,
kidney and pancreas (FIG. 11).
Example 5
[0331] Northern blot analysis showed that MINK3 mRNA is expressed
at different levels in a number of tumor cell lines, including
HL-60, HeLa S3, K-562, MOLT-4, Raji, SW480, A549, and G361 (FIG.
12).
Example 6
[0332] Cells were cotransfected with expression vectors encoding
MINK3a, MINK3b, or MEKK1. As a control, empty vector was used in
place of MINK3a, MINK3b, or MEKK1 expression vectors.
[0333] Cell lysates were collected, and JNK and ERK were
immunoprecipitated.
[0334] Kinase assays were done using JNK immunoprecipitate,
GST-cJUN as substrate, and isotopically labeled .gamma.-ATP.
[0335] The kinase assay mixture was resolved using gel
electrophoresis, and the level of isotope incorporated into
substrate was determined.
[0336] Kinase assays were also done using ERK immunoprecipitate,
MBP as substrate, and isotopically labeled .gamma.-ATP.
[0337] The kinase assay mixture was resolved using gel
electrophoresis, and the level of isotope incorporated into
substrate was determined.
[0338] As a control, western blots "WB" were performed on
immunoprecipitates from different samples to compare the amount of
JNK2 and ERK1 in each immunoprecipitate.
[0339] MINK3a, MINK3b, and MEKK1 induced JNK2 activation, as shown
by the increased phosphorylation of GST-cJUN in kinase assays.
[0340] MINK3a induced ERK1 activation, as shown by the increased
phosphorylation of MBP in kinase assays (FIG. 13).
Example 7
[0341] MINK3a interacts with Nck in a yeast two hybrid assay. (data
not shown).
Example 8
[0342] 293 cells were transfected with expression vectors encoding
MINK3a and Nck.
[0343] Following transfection, cells lysates were collected, and
immunoprecipitations were done.
[0344] Nck associates with MINK3a in 293 cells. (data not
shown)
Example 9
[0345] Phoenix cells were transfected with expression vectors
encoding GFP and either TNIK, TNIK kinase dead variant (TNIK kd),
MINK3a or MINK3b.
[0346] Fluorescence microscopy was done to visualize the morphology
of transfected cells.
[0347] MINK3a caused a morphological change in Phoenix cells. (data
not shown)
Example 10
[0348] MDA-MB-231 cells expressing GFP, MINK3a, MINK3b, or MINK3
antisense nucleic acid complementary to the nucleic acid sequence
set forth by nucleotides 2804-3187 in SEQ ID NO:2 were
generated.
[0349] Light microscopy was used to visualize cell morphology of
transfected cells. MINK3a caused a morphological change in
MDA-MB-231 cells.
[0350] When MINK3a expressing clone was treated with the MEK
inhibitor PD98059, the cells reverted to their normal morphology,
i.e. a morphology characteristic of cells not transfected with
MINK3a expression vector.
[0351] Thus, the morphological change induced by MINK3a appears to
be MEK-dependent (FIGS. 14 and 15).
Example 11
[0352] MDA-MB-231 cells were transfected with expression vectors
encoding either GFP, MINK3a, MINK3b, or empty expression
vector.
[0353] Immunofluorescence was done on cell cultures using a
fluorescently-labeled F-actin binding toxin. (data not shown)
Example 12
[0354] A549 cells were transfected with expression vector encoding
either GFP, MINK3a, MINK3b, or MINK3 antisense nucleic acid
complementary to the nucleic acid sequence set forth by nucleotides
2804-3187 in SEQ ID NO:2.
[0355] Following transfection, cells were grown in low serum (0.5%
fetal bovine serum) or high serum (10% fetal bovine serum) for four
days.
[0356] The optical density of the cultures was determined at 490 nm
in order to determine the cell density and compare proliferation
between cultures.
[0357] MINK3a inhibited proliferation of this tumor cell line in
low serum (FIG. 8).
Example 13
[0358] Cells comprising an EGF receptor and a luciferase reporter
gene fused to a regulatory DNA sequence responsive to ERK
activation were transfected with expression constructs encoding
MINK3a, MINK3b, TNIK, or MINK antisense nucleic acid complementary
to the nucleic acid sequence set forth by nucleotides 2804-3187 in
SEQ ID NO:2. As a control, cells were transfected with empty
vector.
[0359] Following transfection, cells were exposed to EGF. Following
exposure to EGF, luciferase activity was determined using a
luminometer, in order to determine reporter gene expression.
[0360] MINK3 antisense nucleic acid inhibited the basal level of
ERK-mediated transcription of the reporter gene, as well as
EGF-induced ERK-mediated expression of the reporter gene (FIG.
9).
Example 14
[0361] HT-1080 cells (Human fibrosarcoma cell line) were infected
with retrovirus expressing IGFP, Mink3a and Mink3aKD (a kinase dead
mutant with point mutation in kinase domain.
[0362] Invasion assay was performed in BioCoat Invasion chambers
pre-coated with Matrigel according to manufacturer's instructions
(BD Biosciences) in the presence and the absence of 100 ng/ml HGF
(hepatocyte growth factor) (see, e.g., Trusolino & Comoglio,
(2002) Nat Rev Cancer 2(4):289-300; Wells, (2000) Adv. Cancer Res.
78:31-101; Wells et al., (2002) Acta Oncol 41(2):124-30).
[0363] Expression of kinase dead mutant of Mink3a in HT1080 cell
(Human fibrosarcoma cell line) greatly reduced its invasion
potential comparing with GFP control and wild-type Mink3 (see FIG.
16). These results indicate that Mink3a is involved in regulation
of cellular migration and tumor and/or cancer metastasis.
Modulators of Mink3a therefore would be useful for treatment of
cellular migration and metastasis.
Methods:
[0364] Antibodies and cytokines--Antibodies used in this report
include: anti-HA mAb (Babco) and pAb (Santa Cruz Biotechnology);
anti-FLAG mAb (Sigma) and pAb (Santa Cruz); anti-Myc mAb (Babco);
anti-Traf2 pAb (Santa Cruz); anti-NCK mAb (Transduction Labs);
anti-.beta.-actin mAb (Sigma). TNF.alpha. was purchased from
Calbiochem.
[0365] Plasmid construction--Full length human MINK3 was cloned
into pCI (Promega) derived expression vector pYCI under the control
of the CMV promoter with an HA epitope tag (AYPYDVPDYA) (SEQ ID
NO:7) inserted on the N-terminus by PCR. A kinase mutant form of
MINK3 was constructed using the QuikChange mutagenesis kit
(Stratagene) with Oligos AGCTTGCAGCCATCAGGGTTATGGATGTCAC (SEQ ID
NO:8) and GTGACATCCATAACCTTGATGGCTGCAAGCT (SEQ ID NO:9) to change
the highly conserved lysine 54 in the kinase domain to arginine.
Full length human NCK was similarly cloned into pYCI with a FLAG
epitope tag at the N-terminus. Myc-JNK2 and Myc-ERK1 were
constructed in the pCR3.1 vector with a Myc epitope tag
(ASMEQKLISEEDLN) (SEQ ID NO:10) inserted on the N-terminus of JNK2
and ERK1, respectively. All the truncation mutants were constructed
by PCR.
[0366] Cell culture, transfection of Phoenix-A cells and
immunoprecipitation--Phoenix-A cells (derivatives of 293 cells)
(Coligan, et al., Current Protocols in Immunology Supplement),
31:10.28.1-10.28.17 (1999)) were grown in Dulbecco's modified
Eagle's medium MINK3plemented with 10% fetal bovine serum.
Transfection of Phoenix-A cells was performed using the standard
calcium phosphate method (Coligan, et al., Current Protocols in
Immunology Supplement), 31:10.28.1-10.28.17 (1999)). Either
4.times.105 cells in a 6-well plate or 3.times.106 cells in a 100
mm tissue culture dish were seeded 16 hours before transfection. 3
.mu.g of DNA was used in the transfection for each well of a 6-well
plate, and 10 .mu.g DNA was used for each 100 mm dish. Media was
changed 8 hours after transfection. Cells were lysed in lysis
buffer (1% NP-40, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl) with
protease inhibitors (Boehringer Mannheim) and analyzed 24 hours
after transfection. Cell lysates were cleared by centrifugation
(14,000 RPM.times.10 min). For immunoprecipitation studies, cell
lysates (2.times.106 cells/lane) were rotated with 2-3 .mu.g of
desired antibodies and 20 .mu.l 50% slurry of protein A Sepharose
(Pharmacia) for 1.5 hrs. Immune complexes were precipitated and the
pellets washed three times with lysis buffer. Washed precipitates
were subjected to SDS-PAGE analysis and Western blotting.
Supersignal and Supersignal West Duro substrates (Piers) were used
as detection systems for the Western blotting.
[0367] In vitro kinase assays--For the JNK in vitro kinase assay,
Myc-JNK2 was co-transfected into Phoenix-A cells with MINK3
mutants, Traf2 or MEKK as described above. 24 hours after
transfection, cells were lysed with lysis buffer MINK3plemented
with 20 mM .beta.-glycerophosphate, 1 mM NaF, 1 mM Na3VO4 and
protease inhibitors. Myc-JNK2 was precipitated from clarified cell
lysates with an anti-Myc mAb and the pellets were washed three
times with lysis buffer and two times with kinase buffer (20 mM
HEPES, pH 7.4, 10 mM MnCl2, 10 mM MgCl2, 20 mM
.beta.-glycerophosphate, 1 mM NaF, 1 mM Na3VO4, 0.5 mM DTT). For
the kinase reactions, immunoprecipitates were incubated with 1
.mu.g glutathione S-transferase (GST) c-Jun (1-79) (Santa Cruz
Biotechnology) in 20 .mu.l kinase buffer MINK3plemented with 1
.mu.M PKI peptide (Sigma), 10 .mu.M ATP, 5 .mu.Ci .gamma.-P32 ATP
for 20 minutes at 30.degree. C. Kinase reactions were stopped by
addition of 20 .mu.l 2.times.SDS sample buffer (Norvex), heated at
95.degree. C. for 5 minutes and then loaded onto SDS-PAGE. ERK and
p38 in vitro kinase assays were conducted in a similar fashion. For
ERK kinase assays, an anti-Myc mAb was used to immunoprecipitate
Myc-ERK1 and Myelin Basic Protein (MBP, Sigma) was used as an
exogenous substrate. For p38 kinase assays, an anti-FLAG mAb was
used to immunoprecipitate FLAG-p38 and GST-ATF2 (Santa Cruz) was
used as an exogenous substrate. For in vitro kinase assays on
MINK3, 3 .mu.g wild type HA-MINK3 or 3 .mu.g kinase mutant form of
HA-MINK3 was expressed in Phoenix-A cells and immunoprecipitated
with an anti-HA antibody. Immune complexes were subjected to kinase
assays as described above in the absence or presence of 0.5 .mu.g
Gelsolin as an exogenous substrate.
[0368] Fluorescent microscopy--Phoenix-A cells seeded in 6-well
plates were co-transfected with GFP and MINK3 constructs as
described above. 24 hours after transfection, cells were observed
using a Nikon Eclipse TE 300 fluorescent microscope. For detection
of apoptosis, Hoechst 33258 was added to transfected Phoenix-A
cells (final concentration 5 .mu.g/ml) and the cells were incubated
for 30 min at 37.degree. C. before microscopic observation.
[0369] To determine kinase activity, a putative kinase mutant form
of MINK3, designated as MINK3(KM), was constructed with a conserved
lysine (Lys-54) residue in the ATP binding pocket mutated to
arginine. An HA epitope tag was inserted on the N-terminal portion
of MINK3(WT) and MINK3(KM). Both proteins were transiently
expressed in Phoenix-A cells, and the expressed proteins were
subjected to immunoprecipitation and an in vitro kinase assay. A
strong phosphorylated band at 150 kD was detected in the MINK3(WT)
expressed lane, but not in the MINK3(KM) expressed lane.
Immunoblotting with an anti-HA antibody showed equal levels of
expression of both MINK3(WT) and MINK3(KM) at 150 kD. Therefore,
the phosphorylated band in the in vitro kinase assay represented
autophosphorylated MINK3, and the MINK3 (KM) mutant was deficient
in protein kinase activity.
[0370] Tissue distribution of MINK3--The expression pattern of the
MINK3 message was examined by human multi-tissue Northern blot.
Since MINK3 shared high homology with NIK, a probe corresponding to
nucleotides 1264-2427 of MINK3 was used to rule out any potential
cross-hybridization. This region shared only 40% amino acid
identity with NIK. Three major bands of sizes 6.5 kb, 7.5 kb and
9.5 kb were detected. Alternative splicing in the coding region
described above is unlikely to account for the size differences
among the three messages, since the largest isoform is only 273 bps
bigger than the smallest isoform. Alternative splicing in the
untranslated region or alternative usage of polyA sites could be
possible explanations. This phenomenon is not unique to MINK3. NIK
and HGK also have multiple message sizes. MINK3 is ubiquitously
expressed, with higher levels of message detected in heart, brain
and skeletal muscle. Interestingly, heart and skeletal muscle
predominantly expressed the 6.5 kb form; placenta, kidney and
pancreas predominantly expressed the 7.5 kb form; brain, lung and
liver expressed all three forms at a similar level. It is currently
unknown whether these messages have different functional roles.
[0371] Interaction of MINK3 with NCK--The interaction of MINK3 with
NCK was investigated in a similar fashion. Following transient
expression of HA-MINK3 in Phoenix-A cells, the cell lysates were
immunoprecipitated with an anti-HA antibody and blotted with an
anti-NCK antibody. Endogenous NCK specifically
co-immunoprecipitated with HA-MINK3. To map the domains on MINK3
required for this interaction, HA-tagged MINK3 mutants were
co-expressed with FLAG-tagged NCK and the HA-MINK3 mutants were
immunoprecipitated with an anti-HA antibody. The immune complexes
were then blotted with an anti-FLAG antibody. MINK3(WT), MINK3(N2),
MINK3(C1) and MINK3(M) were all able to associate with NCK,
suggesting that the intermediate domain is also sufficient for
MINK3 to bind NCK. Neither the GCKH domain nor the kinase domain
showed any detectable binding to NCK. Immunoblotting cell lysates
with anti-HA and anti-FLAG antibodies showed equivalent levels of
expression of the transfected proteins.
[0372] Activation of JNK2 by MINK3--We further examined whether
MINK3 was able to activate the JNK pathway. 1 .mu.g, 2 .mu.g or 3
.mu.g of MINK3 expression plasmid was co-transfected into Phoenix-A
cells with Myc-JNK2. 24 hours after transfection, Myc-JNK2 was
immunoprecipitated from cell lysates and its kinase activity
measured using GST-cJun(1-79) as a substrate. Co-transfection of
MINK3 enhanced JNK2 kinase activity in a dose dependent fashion.
When 3 .mu.g of MINK3 was transfected, JNK2 activity was enhanced
3-4 fold. A similar magnitude of JNK2 activation was observed when
cells were treated for 15 minutes with 100 ng/ml of TNF. Also
consistent with published result (Natoli, et al., Science,
275:200-203 (1997)), TRAF2 potently activated JNK2 activity. The
expression levels of Myc-JNK2 were controlled by immunoblotting
cell lysates with an anti-Myc antibody.
[0373] To determine whether MINK3 can also activate the ERK and p38
pathways, Myc-ERK1 and FLAG-p38 were co-transfected into Phoenix-A
cells with different doses of MINK3. The transfected kinases were
then immunoprecipitated from cell lysates and the kinase activities
measured using MBP and GST-ATF2 as exogenous substrates. In
contrast to JNK2, neither ERK1 nor p38 was activated by MINK3
overexpression, while co-transfection of MEKK1 potently activated
both kinases. In addition, MINK3 did not activate NF-.kappa.B (data
not shown).
[0374] To further investigate the mechanism of this activation, the
cohort of MINK3 mutants were co-transfected into Phoenix-A cells
with Myc-JNK2 and the ability of these mutants to up-regulate JNK2
kinase activity was examined by the in vitro kinase assay.
MINK3(WT), MINK3(KM), MINK3(C1) and MINK3(C2) were all able to
activate Myc-JNK2, while MINK3(N1), MINK3(N2), MINK3(M) were not.
This result suggested that the C-terminal GCKH region is both
necessary and sufficient for activation of the JNK pathway, while
the kinase domain is dispensable.
[0375] NIK was cloned by its ability to interact with the adapter
protein NCK. It associated with NCK SH3 domains via two PxxPxR
sequences in the intermediate domain, PCPPSR (aa 574-579) (SEQ ID
NO:1) and PRVPVR (aa 611-616) (SEQ ID NO:12). Both sequences were
required for efficient interaction (Su, et al., EMBO J.,
16:1279-1290 (1997)). Similar to NIK, MINK3 also interacted with
NCK via the intermediate domain. However, PCPPSR (SEQ ID NO:11) is
not conserved in MINK3. Instead, MINK3 contained two other PxxPxR
sequences, PNLPPR (aa 562-567) (SEQ ID NO:13) and PPLPTR (aa
647-652) (SEQ ID NO:14), in addition to the conserved PKVPQR (aa
670-675) (SEQ ID NO:15). MINK3 likely interacted with NCK through
the cooperative interaction with these three PxxPxR sequences. NCK
is an adapter protein involved in many growth factor receptor
mediated signal transduction pathways (McCarthy, Bioessays,
20:913-921 (1998)). It has been proposed that the NIK-NCK
interaction may recruit NIK to receptor or non-receptor tyrosine
kinases to regulate MEKK1 (Su, et al., EMBO J., 16:1279-1290
(1997)). MINK3 may be recruited in a similar fashion.
Sequence CWU 1
1
15 1 1312 PRT Homo sapiens MINK3a (misshapen/NIKs-related kinase
isoform 3a) 1 Met Gly Asp Pro Ala Pro Ala Arg Ser Leu Asp Asp Ile
Asp Leu Ser 1 5 10 15 Ala Leu Arg Asp Pro Ala Gly Ile Phe Glu Leu
Val Glu Val Val Gly 20 25 30 Asn Gly Thr Tyr Gly Gln Val Tyr Lys
Gly Arg His Val Lys Thr Gly 35 40 45 Gln Leu Ala Ala Ile Lys Val
Met Asp Val Thr Glu Asp Glu Glu Glu 50 55 60 Glu Ile Lys Gln Glu
Ile Asn Met Leu Lys Lys Tyr Ser His His Arg 65 70 75 80 Asn Ile Ala
Thr Tyr Tyr Gly Ala Phe Ile Lys Lys Ser Pro Pro Gly 85 90 95 Asn
Asp Asp Gln Leu Trp Leu Val Met Glu Phe Cys Gly Ala Gly Ser 100 105
110 Val Thr Asp Leu Val Lys Asn Thr Lys Gly Asn Ala Leu Lys Glu Asp
115 120 125 Cys Ile Ala Tyr Ile Cys Arg Glu Ile Leu Arg Gly Leu Ala
His Leu 130 135 140 His Ala His Lys Val Ile His Arg Asp Ile Lys Gly
Gln Asn Val Leu 145 150 155 160 Leu Thr Glu Asn Ala Glu Val Lys Leu
Val Asp Phe Gly Val Ser Ala 165 170 175 Gln Leu Asp Arg Thr Val Gly
Arg Arg Asn Thr Phe Ile Gly Thr Pro 180 185 190 Tyr Trp Met Ala Pro
Glu Val Ile Ala Cys Asp Glu Asn Pro Asp Ala 195 200 205 Thr Tyr Asp
Tyr Arg Ser Asp Ile Trp Ser Leu Gly Ile Thr Ala Ile 210 215 220 Glu
Met Ala Glu Gly Ala Pro Pro Leu Cys Asp Met His Pro Met Arg 225 230
235 240 Ala Leu Phe Leu Ile Pro Arg Asn Pro Pro Pro Arg Leu Lys Ser
Lys 245 250 255 Lys Trp Ser Lys Lys Phe Ile Asp Phe Ile Asp Thr Cys
Leu Ile Lys 260 265 270 Thr Tyr Leu Ser Arg Pro Pro Thr Glu Gln Leu
Leu Lys Phe Pro Phe 275 280 285 Ile Arg Asp Gln Pro Thr Glu Arg Gln
Val Arg Ile Gln Leu Lys Asp 290 295 300 His Ile Asp Arg Ser Arg Lys
Lys Arg Gly Glu Lys Glu Glu Thr Glu 305 310 315 320 Tyr Glu Tyr Ser
Gly Ser Glu Glu Glu Asp Asp Ser His Gly Glu Glu 325 330 335 Gly Glu
Pro Ser Ser Ile Met Asn Val Pro Gly Glu Ser Thr Leu Arg 340 345 350
Arg Glu Phe Leu Arg Leu Gln Gln Glu Asn Lys Ser Asn Ser Glu Ala 355
360 365 Leu Lys Gln Gln Gln Gln Leu Gln Gln Gln Gln Gln Arg Asp Pro
Glu 370 375 380 Ala His Ile Lys His Leu Leu His Gln Arg Gln Arg Arg
Ile Glu Glu 385 390 395 400 Gln Lys Glu Glu Arg Arg Arg Val Glu Glu
Gln Gln Arg Arg Glu Arg 405 410 415 Glu Gln Arg Lys Leu Gln Glu Lys
Glu Gln Gln Arg Arg Leu Glu Asp 420 425 430 Met Gln Ala Leu Arg Arg
Glu Glu Glu Arg Arg Gln Ala Glu Arg Glu 435 440 445 Gln Glu Tyr Lys
Arg Lys Gln Leu Glu Glu Gln Arg Gln Ser Glu Arg 450 455 460 Leu Gln
Arg Gln Leu Gln Gln Glu His Ala Tyr Leu Lys Ser Leu Gln 465 470 475
480 Gln Gln Gln Gln Gln Gln Gln Leu Gln Lys Gln Gln Gln Gln Gln Leu
485 490 495 Leu Pro Gly Asp Arg Lys Pro Leu Tyr His Tyr Gly Arg Gly
Met Asn 500 505 510 Pro Ala Asp Lys Pro Ala Trp Ala Arg Glu Val Glu
Glu Arg Thr Arg 515 520 525 Met Asn Lys Gln Gln Asn Ser Pro Leu Ala
Lys Ser Lys Pro Gly Ser 530 535 540 Thr Gly Pro Glu Pro Pro Ile Pro
Gln Ala Ser Pro Gly Pro Pro Gly 545 550 555 560 Pro Leu Ser Gln Thr
Pro Pro Met Gln Arg Pro Val Glu Pro Gln Glu 565 570 575 Gly Pro His
Lys Ser Leu Gln Asp Gln Pro Thr Arg Asn Leu Ala Ala 580 585 590 Phe
Pro Ala Ser His Asp Pro Asp Pro Ala Ile Pro Ala Pro Thr Ala 595 600
605 Thr Pro Ser Ala Arg Gly Ala Val Ile Arg Gln Asn Ser Asp Pro Thr
610 615 620 Ser Glu Gly Pro Gly Pro Ser Pro Asn Pro Pro Ala Trp Val
Arg Pro 625 630 635 640 Asp Asn Glu Ala Pro Pro Lys Val Pro Gln Arg
Thr Ser Ser Ile Ala 645 650 655 Thr Ala Leu Asn Thr Ser Gly Ala Gly
Gly Ser Arg Pro Ala Gln Ala 660 665 670 Val Arg Ala Arg Pro Arg Ser
Asn Ser Ala Trp Gln Ile Tyr Leu Gln 675 680 685 Arg Arg Ala Glu Arg
Gly Thr Pro Lys Pro Pro Gly Pro Pro Ala Gln 690 695 700 Pro Pro Gly
Pro Pro Asn Ala Ser Ser Asn Pro Asp Leu Arg Arg Ser 705 710 715 720
Asp Pro Gly Trp Glu Arg Ser Asp Ser Val Leu Pro Ala Ser His Gly 725
730 735 His Leu Pro Gln Ala Gly Ser Leu Glu Arg Asn Arg Val Gly Ala
Ser 740 745 750 Ser Lys Leu Asp Ser Ser Pro Val Leu Ser Pro Gly Asn
Lys Ala Lys 755 760 765 Pro Asp Asp His Arg Ser Arg Pro Gly Arg Pro
Ala Asp Phe Val Leu 770 775 780 Leu Lys Glu Arg Thr Leu Asp Glu Ala
Pro Arg Pro Pro Lys Lys Ala 785 790 795 800 Met Asp Tyr Ser Ser Ser
Ser Glu Glu Val Glu Ser Ser Glu Asp Asp 805 810 815 Glu Glu Glu Gly
Glu Gly Gly Pro Ala Glu Gly Ser Arg Asp Thr Pro 820 825 830 Gly Gly
Arg Ser Asp Gly Asp Thr Asp Ser Val Ser Thr Met Val Val 835 840 845
His Asp Val Glu Glu Ile Thr Gly Thr Gln Pro Pro Tyr Gly Gly Gly 850
855 860 Thr Met Val Val Gln Arg Thr Pro Glu Glu Glu Arg Asn Leu Leu
His 865 870 875 880 Ala Asp Ser Asn Gly Tyr Thr Asn Leu Pro Asp Val
Val Gln Pro Ser 885 890 895 His Ser Pro Thr Glu Asn Ser Lys Gly Gln
Ser Pro Pro Ser Lys Asp 900 905 910 Gly Ser Gly Asp Tyr Gln Ser Arg
Gly Leu Val Lys Ala Pro Gly Lys 915 920 925 Ser Ser Phe Thr Met Phe
Val Asp Leu Gly Ile Tyr Gln Pro Gly Gly 930 935 940 Ser Gly Asp Ser
Ile Pro Ile Thr Ala Leu Val Gly Gly Glu Gly Thr 945 950 955 960 Arg
Leu Asp Gln Leu Gln Tyr Asp Val Arg Lys Gly Ser Val Val Asn 965 970
975 Val Asn Pro Thr Asn Thr Arg Ala His Ser Glu Thr Pro Glu Ile Arg
980 985 990 Lys Tyr Lys Lys Arg Phe Asn Ser Glu Ile Leu Cys Ala Ala
Leu Trp 995 1000 1005 Gly Val Asn Leu Leu Val Gly Thr Glu Asn Gly
Leu Met Leu Leu Asp 1010 1015 1020 Arg Ser Gly Gln Gly Lys Val Tyr
Gly Leu Ile Gly Arg Arg Arg Phe 1025 1030 1035 1040 Gln Gln Met Asp
Val Leu Glu Gly Leu Asn Leu Leu Ile Thr Ile Ser 1045 1050 1055 Gly
Lys Arg Asn Lys Leu Arg Val Tyr Tyr Leu Ser Trp Leu Arg Asn 1060
1065 1070 Lys Ile Leu His Asn Asp Pro Glu Val Glu Lys Lys Gln Gly
Trp Thr 1075 1080 1085 Thr Val Gly Asp Met Glu Gly Cys Gly His Tyr
Arg Val Val Lys Tyr 1090 1095 1100 Glu Arg Ile Lys Phe Leu Val Ile
Ala Leu Lys Ser Ser Val Glu Val 1105 1110 1115 1120 Tyr Ala Trp Ala
Pro Lys Pro Tyr His Lys Phe Met Ala Phe Lys Ser 1125 1130 1135 Phe
Ala Asp Leu Pro His Arg Pro Leu Leu Val Asp Leu Thr Val Glu 1140
1145 1150 Glu Gly Gln Arg Leu Lys Val Ile Tyr Gly Ser Ser Ala Gly
Phe His 1155 1160 1165 Ala Val Asp Val Asp Ser Gly Asn Ser Tyr Asp
Ile Tyr Ile Pro Val 1170 1175 1180 His Ile Gln Ser Gln Ile Thr Pro
His Ala Ile Ile Phe Leu Pro Asn 1185 1190 1195 1200 Thr Asp Gly Met
Glu Met Leu Leu Cys Tyr Glu Asp Glu Gly Val Tyr 1205 1210 1215 Val
Asn Thr Tyr Gly Arg Ile Ile Lys Asp Val Val Leu Gln Trp Gly 1220
1225 1230 Glu Met Pro Thr Ser Val Ala Tyr Ile Cys Ser Asn Gln Ile
Met Gly 1235 1240 1245 Trp Gly Glu Lys Ala Ile Glu Ile Arg Ser Val
Glu Thr Gly His Leu 1250 1255 1260 Asp Gly Val Phe Met His Lys Arg
Ala Gln Arg Leu Lys Phe Leu Cys 1265 1270 1275 1280 Glu Arg Asn Asp
Lys Val Phe Phe Ala Ser Val Arg Ser Gly Gly Ser 1285 1290 1295 Ser
Gln Val Tyr Phe Met Thr Leu Asn Arg Asn Cys Ile Met Asn Trp 1300
1305 1310 2 3951 DNA Homo sapiens MINK3a (misshapen/NIKs-related
kinase isoform 3a) 2 gcccttatgg gcgacccagc ccccgcccgc agcctggacg
acatcgacct gtccgccctg 60 cgggaccctg ctgggatctt tgagcttgtg
gaggtggtcg gcaatggaac ctacggacag 120 gtgtacaagg gtcggcatgt
caagacgggg cagctggctg ccatcaaggt catggatgtc 180 acggaggacg
aggaggaaga gatcaaacag gagatcaaca tgctgaaaaa gtactctcac 240
caccgcaaca tcgccaccta ctacggagcc ttcatcaaga agagcccccc gggaaacgat
300 gaccagctct ggctggtgat ggagttctgt ggtgctggtt cagtgactga
cctggtaaag 360 aacacaaaag gcaacgccct gaaggaggac tgtatcgcct
atatctgcag ggagatcctc 420 aggggtctgg cccatctcca tgcccacaag
gtgatccatc gagacatcaa ggggcagaat 480 gtgctgctga cagagaatgc
tgaggtcaag ctagtggatt ttggggtgag tgctcagctg 540 gaccgcaccg
tgggcagacg gaacactttc attgggactc cctactggat ggctccagag 600
gtcatcgcct gtgatgagaa ccctgatgcc acctatgatt acaggagtga tatttggtct
660 ctaggaatca cagccatcga gatggcagag ggagcccccc ctctgtgtga
catgcacccc 720 atgcgagccc tcttcctcat tcctcggaac cctccgccca
ggctcaagtc caagaagtgg 780 tctaagaagt tcattgactt cattgacaca
tgtctcatca agacttacct gagccgccca 840 cccacggagc agctactgaa
gtttcccttc atccgggacc agcccacgga gcggcaggtc 900 cgcatccagc
ttaaggacca cattgaccga tcccggaaga agcggggtga gaaagaggag 960
acagaatatg agtacagcgg cagcgaggag gaagatgaca gccatggaga ggaaggagag
1020 ccaagctcca tcatgaacgt gcctggagag tcgactctac gccgggagtt
tctccggctc 1080 cagcaggaaa ataagagcaa ctcagaggct ttaaaacagc
agcagcagct gcagcagcag 1140 cagcagcgag accccgaggc acacatcaaa
cacctgctgc accagcggca gcggcgcata 1200 gaggagcaga aggaggagcg
gcgccgcgtg gaggagcaac agcggcggga gcgggagcag 1260 cggaagctgc
aggagaagga gcagcagcgg cggctggagg acatgcaggc tctgcggcgg 1320
gaggaggagc ggcggcaggc ggagcgtgag caggaataca agcggaagca gctggaggag
1380 cagcggcagt cagaacgtct ccagaggcag ctgcagcagg agcatgccta
cctcaagtcc 1440 ctgcagcagc agcaacagca gcagcagctt cagaaacagc
agcagcagca gctcctgcct 1500 ggggacagga agcccctgta ccattatggt
cggggcatga atcccgctga caaaccagcc 1560 tgggcccgag aggtagaaga
gagaacaagg atgaacaagc agcagaactc tcccttggcc 1620 aagagcaagc
caggcagcac ggggcctgag ccccccatcc cccaggcctc cccagggccc 1680
ccaggacccc tttcccagac tcctcctatg cagaggccgg tggagcccca ggagggaccg
1740 cacaagtccc tgcaggacca gcccacccga aacctggctg ccttcccagc
ctcccatgac 1800 cccgaccctg ccatccccgc acccactgcc acgcccagtg
cccgaggagc tgtcatccgc 1860 cagaattcag accccacctc tgaaggacct
ggccccagcc cgaatccccc agcctgggtc 1920 cgcccagata acgaggcccc
acccaaggtg cctcagagga cctcatctat cgccactgcc 1980 cttaacacca
gtggggccgg agggtcccgg ccagcccagg cagtccgtgc cagacctcgc 2040
agcaactccg cctggcaaat ctatctgcaa aggcgggcag agcggggcac cccaaagcct
2100 ccagggcccc ctgctcagcc ccctggcccg cccaacgcct ctagtaaccc
cgacctcagg 2160 aggagcgacc ctggctggga acgctcggac agcgtccttc
cagcctctca cgggcacctc 2220 ccccaggctg gctcactgga gcggaaccgc
gtgggagcct cctccaaact ggacagctcc 2280 cctgtgctct cccctgggaa
taaagccaag cccgacgacc accgctcacg gccaggccgg 2340 cccgcagact
ttgtgttgct gaaagagcgg actctggacg aggcccctcg gcctcccaag 2400
aaggccatgg actactcgtc gtccagcgag gaggtggaaa gcagtgagga cgacgaggag
2460 gaaggcgaag gcgggccagc agaggggagc agagataccc ctgggggccg
cagcgatggg 2520 gatacagaca gcgtcagcac catggtggtc cacgacgtcg
aggagatcac cgggacccag 2580 cccccatacg ggggcggcac catggtggtc
cagcgcaccc ctgaagagga gcggaacctg 2640 ctgcatgctg acagcaatgg
gtacacaaac ctgcctgacg tggtccagcc cagccactca 2700 cccaccgaga
acagcaaagg ccaaagccca ccctcgaagg atgggagtgg tgactaccag 2760
tctcgtgggc tggtaaaggc ccctggcaag agctcgttca cgatgtttgt ggatctaggg
2820 atctaccagc ctggaggcag tggggacagc atccccatca cagccctagt
gggtggagag 2880 ggcactcggc tcgaccagct gcagtacgac gtgaggaagg
gttctgtggt caacgtgaat 2940 cccaccaaca cccgggccca cagtgagacc
cctgagatcc ggaagtacaa gaagcgattc 3000 aactccgaga tcctctgtgc
agccctttgg ggggtcaacc tgctggtggg cacggagaac 3060 gggctgatgt
tgctggaccg aagtgggcag ggcaaggtgt atggactcat tgggcggcga 3120
cgcttccagc agatggatgt gctggagggg ctcaacctgc tcatcaccat ctcagggaaa
3180 aggaacaaac tgcgggtgta ttacctgtcc tggctccgga acaagattct
gcacaatgac 3240 ccagaagtgg agaagaagca gggctggacc accgtggggg
acatggaggg ctgcgggcac 3300 taccgtgttg tgaaatacga gcggattaag
ttcctggtca tcgccctcaa gagctccgtg 3360 gaggtgtatg cctgggcccc
caaaccctac cacaaattca tggccttcaa gtcctttgcc 3420 gacctccccc
accgccctct gctggtcgac ctgacagtag aggaggggca gcggctcaag 3480
gtcatctatg gctccagtgc tggcttccat gctgtggatg tcgactcggg gaacagctat
3540 gacatctaca tccctgtgca catccagagc cagatcacgc cccatgccat
catcttcctc 3600 cccaacaccg acggcatgga gatgctgctg tgctacgagg
acgagggtgt ctacgtcaac 3660 acgtacgggc gcatcattaa ggatgtggtg
ctgcagtggg gggagatgcc tacttctgtg 3720 gcctacatct gctccaacca
gataatgggc tggggtgaga aagccattga gatccgctct 3780 gtggagacgg
gccacctcga cggggtcttc atgcacaaac gagctcagag gctcaagttc 3840
ctgtgtgagc ggaatgacaa ggtgtttttt gcctcagtcc gctctggggg cagcagccaa
3900 gtttacttca tgactctgaa ccgtaactgc atcatgaact ggtgaaaggg c 3951
3 792 PRT Homo sapiens MINK3b (misshapen/NIKs-related kinase
isoform 3b) 3 Met Asp Val Thr Glu Asp Glu Glu Glu Glu Ile Lys Gln
Glu Ile Asn 1 5 10 15 Met Leu Lys Lys Tyr Ser His His Arg Asn Ile
Ala Thr Tyr Tyr Gly 20 25 30 Ala Phe Ile Lys Lys Ser Pro Pro Gly
Asn Asp Asp Gln Leu Trp Leu 35 40 45 Val Met Glu Phe Cys Gly Ala
Gly Ser Val Thr Asp Leu Val Lys Asn 50 55 60 Thr Lys Gly Asn Ala
Leu Lys Glu Asp Cys Ile Ala Tyr Ile Cys Arg 65 70 75 80 Glu Ile Leu
Arg Gly Leu Ala His Leu His Ala His Lys Val Ile His 85 90 95 Arg
Asp Ile Lys Gly Gln Asn Val Leu Leu Thr Glu Asn Ala Glu Val 100 105
110 Lys Leu Val Asp Phe Gly Val Ser Ala Gln Leu Asp Arg Thr Val Gly
115 120 125 Arg Arg Asn Thr Phe Ile Gly Thr Pro Tyr Trp Met Ala Pro
Glu Val 130 135 140 Ile Ala Cys Asp Glu Asn Pro Asp Ala Thr Tyr Asp
Tyr Arg Ser Asp 145 150 155 160 Ile Trp Ser Leu Gly Ile Thr Ala Ile
Glu Met Ala Glu Gly Ala Pro 165 170 175 Pro Leu Cys Asp Met His Pro
Met Arg Ala Leu Phe Leu Ile Pro Arg 180 185 190 Asn Pro Pro Pro Arg
Leu Lys Ser Lys Lys Trp Ser Lys Lys Phe Ile 195 200 205 Asp Phe Ile
Asp Thr Cys Leu Ile Lys Thr Tyr Leu Ser Arg Pro Pro 210 215 220 Thr
Glu Gln Leu Leu Lys Phe Pro Phe Ile Arg Asp Gln Pro Thr Glu 225 230
235 240 Arg Gln Val Arg Ile Gln Leu Lys Asp His Ile Asp Arg Ser Arg
Lys 245 250 255 Lys Arg Gly Glu Lys Glu Glu Thr Glu Tyr Glu Tyr Ser
Gly Ser Glu 260 265 270 Glu Glu Asp Asp Ser His Gly Glu Glu Gly Glu
Pro Ser Ser Ile Met 275 280 285 Asn Val Pro Gly Glu Ser Thr Leu Arg
Arg Glu Phe Leu Arg Leu Gln 290 295 300 Gln Glu Asn Lys Ser Asn Ser
Glu Ala Leu Lys Gln Gln Gln Gln Leu 305 310 315 320 Gln Gln Gln Gln
Gln Arg Asp Pro Glu Ala His Ile Lys His Leu Leu 325 330 335 His Gln
Arg Gln Arg Arg Ile Glu Glu Gln Lys Glu Glu Arg Arg Arg 340 345 350
Val Glu Glu Gln Gln Arg Arg Glu Arg Glu Gln Arg Lys Leu Gln Glu 355
360 365 Lys Glu Gln Gln Arg Arg Leu Glu Asp Met Gln Ala Leu Arg Arg
Glu 370 375 380 Glu Glu Arg Arg Gln Ala Glu Arg Glu Gln Glu Tyr Lys
Arg Lys Gln 385 390 395 400 Leu Glu Glu Gln Arg Gln Ser Glu Arg Leu
Gln Arg Gln Leu Gln Gln 405 410 415 Glu His Ala Tyr Leu Lys Ser Leu
Gln Gln Gln Gln Gln Gln Gln Gln 420 425 430 Leu Gln Lys Gln Gln Gln
Gln Gln Leu Leu Pro Gly Asp Arg Lys Pro 435 440 445 Leu Tyr His Tyr
Gly Arg Gly Met Asn Pro Ala Asp Lys Pro Ala Trp 450
455 460 Ala Arg Glu Val Glu Glu Arg Thr Arg Met Asn Lys Gln Gln Asn
Ser 465 470 475 480 Pro Leu Ala Lys Ser Lys Pro Gly Ser Thr Gly Pro
Glu Pro Pro Ile 485 490 495 Pro Gln Ala Ser Pro Gly Pro Pro Gly Pro
Leu Ser Gln Thr Pro Pro 500 505 510 Met Gln Arg Pro Val Glu Pro Gln
Glu Gly Pro His Lys Ser Leu Val 515 520 525 Ala His Arg Val Pro Leu
Lys Pro Tyr Ala Ala Pro Val Pro Arg Ser 530 535 540 Gln Ser Leu Gln
Asp Gln Pro Thr Arg Asn Leu Ala Ala Phe Pro Ala 545 550 555 560 Ser
His Asp Pro Asp Pro Ala Ile Pro Ala Pro Thr Ala Thr Pro Ser 565 570
575 Ala Arg Gly Ala Val Ile Arg Gln Asn Ser Asp Pro Thr Ser Glu Gly
580 585 590 Pro Gly Pro Ser Pro Asn Pro Pro Ala Trp Val Arg Pro Asp
Asn Glu 595 600 605 Ala Pro Pro Lys Val Pro Gln Arg Thr Ser Ser Ile
Ala Thr Ala Leu 610 615 620 Asn Thr Ser Gly Ala Gly Gly Ser Arg Pro
Ala Gln Ala Val Arg Ala 625 630 635 640 Arg Pro Arg Ser Asn Ser Ala
Trp Gln Ile Tyr Leu Gln Arg Arg Ala 645 650 655 Glu Arg Gly Thr Pro
Lys Pro Pro Gly Pro Pro Ala Gln Pro Pro Gly 660 665 670 Pro Pro Asn
Ala Ser Ser Asn Pro Asp Leu Arg Arg Ser Asp Pro Gly 675 680 685 Trp
Glu Arg Ser Asp Ser Val Leu Pro Ala Ser His Gly His Leu Pro 690 695
700 Gln Ala Gly Ser Leu Glu Arg Asn Arg Val Gly Ala Ser Ser Lys Leu
705 710 715 720 Asp Ser Ser Pro Val Leu Ser Pro Gly Asn Lys Ala Lys
Pro Asp Asp 725 730 735 His Arg Ser Arg Pro Gly Arg Pro Ala Val Ser
His Leu Val Ala Gly 740 745 750 Met Ala Cys Leu Ile Leu Val Trp Gly
Leu Ala Ser Gly Cys Trp Val 755 760 765 Ser Gly Val Gly Ser Pro Leu
Ile Tyr Arg Glu Gly Leu Trp Gly Trp 770 775 780 Arg Asp Trp Cys Phe
Ser Trp Cys 785 790 4 4414 DNA Homo sapiens MINK3b
(misshapen/NIKs-related kinase isoform 3b) 4 gcccttacca ttctggaagc
tccctagaat ctcctggaat gcttaatgga cctttccagc 60 accgaaattc
aagaattatg actcatcggt cagcagaaaa gaccctgctg ggatctttga 120
gcttgtggag gtggtcggca atggaaccta cggacaggtg tacaagggtc ggcatgtcaa
180 gacggggcag ctggctgcca tcaaggtcat ggatgtcacg gaggacgagg
aggaagagat 240 caaacaggag atcaacatgc tgaaaaagta ctctcaccac
cgcaacatcg ccacctacta 300 cggagccttc atcaagaaga gccccccggg
aaacgatgac cagctctggc tggtgatgga 360 gttctgtggt gctggttcag
tgactgacct ggtaaagaac acaaaaggca acgccctgaa 420 ggaggactgt
atcgcctata tctgcaggga gatcctcagg ggtctggccc atctccatgc 480
ccacaaggtg atccatcgag acatcaaggg gcagaatgtg ctgctgacag agaatgctga
540 ggtcaagcta gtggattttg gggtgagtgc tcagctggac cgcaccgtgg
gcagacggaa 600 cactttcatt gggactccct actggatggc tccagaggtc
atcgcctgtg atgagaaccc 660 tgatgccacc tatgattaca ggagtgatat
ttggtctcta ggaatcacag ccatcgagat 720 ggcagaggga gccccccctc
tgtgtgacat gcaccccatg cgagccctct tcctcattcc 780 tcggaaccct
ccgcccaggc tcaagtccaa gaagtggtct aagaagttca ttgacttcat 840
tgacacatgt ctcatcaaga cttacctgag ccgcccaccc acggagcagc tactgaagtt
900 tcccttcatc cgggaccagc ccacggagcg gcaggtccgc atccagctta
aggaccacat 960 tgaccgatcc cggaagaagc ggggtgagaa agaggagaca
gaatatgagt acagcggcag 1020 cgaggaggaa gatgacagcc atggagagga
aggagagcca agctccatca tgaacgtgcc 1080 tggagagtcg actctacgcc
gggagtttct ccggctccag caggaaaata agagcaactc 1140 agaggcttta
aaacagcagc agcagctgca gcagcagcag cagcgagacc ccgaggcaca 1200
catcaaacac ctgctgcacc agcggcagcg gcgcatagag gagcagaagg aggagcggcg
1260 ccgcgtggag gagcaacagc ggcgggagcg ggagcagcgg aagctgcagg
agaaggagca 1320 gcagcggcgg ctggaggaca tgcaggctct gcggcgggag
gaggagcggc ggcaggcgga 1380 gcgtgagcag gaatacaagc ggaagcagct
ggaggagcag cggcagtcag aacgtctcca 1440 gaggcagctg cagcaggagc
atgcctacct caagtccctg cagcagcagc aacagcagca 1500 gcagcttcag
aaacagcagc agcagcagct cctgcctggg gacaggaagc ccctgtacca 1560
ttatggtcgg ggcatgaatc ccgctgacaa accagcctgg gcccgagagg tagaagagag
1620 aacaaggatg aacaagcagc agaactctcc cttggccaag agcaagccag
gcagcacggg 1680 gcctgagccc cccatccccc aggcctcccc agggccccca
ggaccccttt cccagactcc 1740 tcctatgcag aggccggtgg agccccagga
gggaccgcac aagagcctgg tggcacaccg 1800 ggtcccactg aagccatatg
cagcacctgt accccgatcc cagtccctgc aggaccagcc 1860 cacccgaaac
ctggctgcct tcccagcctc ccatgacccc gaccctgcca tccccgcacc 1920
cactgccacg cccagtgccc gaggagctgt catccgccag aattcagacc ccacctctga
1980 aggacctggc cccagcccga atcccccagc ctgggtccgc ccagataacg
aggccccacc 2040 caaggtgcct cagaggacct catctatcgc cactgccctt
aacaccagtg gggccggagg 2100 gtcccggcca gcccaggcag tccgtgccag
acctcgcagc aactccgcct ggcaaatcta 2160 tctgcaaagg cgggcagagc
ggggcacccc aaagcctcca gggccccctg ctcagccccc 2220 tggcccgccc
aacgcctcta gtaaccccga cctcaggagg agcgaccctg gctgggaacg 2280
ctcggacagc gtccttccag cctctcacgg gcacctcccc caggctggct cactggagcg
2340 gaaccgcgtg ggagcctcct ccaaactgga cagctcccct gtgctctccc
ctgggaataa 2400 agccaagccc gacgaccacc gctcacggcc aggccggccc
gcagtgagtc acctggtggc 2460 aggcatggcc tgcctcatcc tggtttgggg
cttagcctca gggtgctggg tgtcaggggt 2520 ggggtctccg ctgatctacc
gagaagggct gtggggatgg agggactggt gcttctcatg 2580 gtgctaacct
ttcctaacct ctctcctaac ctctctccta acctctcttc tggctctttc 2640
ttcccctgcg gcccctccca gagctataag cgagcaattg gtgaggttag tgagatgggc
2700 ctgcttgtgg gagcccctcc tgtcgccctg ctggggcgtc ccggcaccct
ttgtctacct 2760 ccacccaggc ccagcttctc cctgcccctc acgtggctcc
tccctgcagg actttgtgtt 2820 gctgaaagag cggactctgg acgaggcccc
tcggcctccc aagaaggcca tggactactc 2880 gtcgtccagc gaggaggtgg
aaagcagtga ggacgacgag gaggaaggcg aaggcgggcc 2940 agcagagggg
agcagagata cccctggggg ccgcagcgat ggggatacag acagcgtcag 3000
caccatggtg gtccacgacg tcgaggagat caccgggacc cagcccccat acgggggcgg
3060 caccatggtg gtccagcgca cccctgaaga ggagcggaac ccgctgcatg
ctgacagcaa 3120 tgggtacaca aacctgcctg acgtggtcca gcccagccac
tcacccaccg agaacagcaa 3180 aggccaaagc ccaccctcga aggatgggag
tggtgactac cagtctcgtg ggctggtaaa 3240 ggcccctggc aagagctcgt
tcacgatgtt tgtggatcta gggatctacc agcctggagg 3300 cagtggggac
agcatcccca tcacagccct agtgggtgga gagggcactc ggctcgacca 3360
gctgcagtac gacgtgagga agggttctgt ggtcaacgtg aatcccacca acacccgggc
3420 ccacagtgag acccctgaga tccggaagta caagaagcga ttcaactccg
agatcctctg 3480 tgcagccctt tggggggtca acctgctggt gggcacggag
aacgggctga tgttgctgga 3540 ccgaagtggg caggacaagg tgtatggact
cattgggcga cgacgcttcc agcagatgga 3600 tgtgctggag gggctcaacc
tgctcatcac catctcaggg aaaaggaaca aactgcgggt 3660 gtattacctg
tcctggctcc ggaacaagat tctgcacaat gacccagaag tggagaagaa 3720
gcagggctgg accaccgtgg gggacatgga gggctgcggg cactaccgtg ttgtgaaata
3780 cgagcggatt aagttcctgg tcatcgccct caagagctcc gtggaggtgt
atgcctgggc 3840 ccccaaaccc taccacaaat tcatggcctt caagtccttt
gccgacctcc cccaccgccc 3900 tctgctggtc gacctgacag tagaggaggg
gcagcggctc aaggtcatct atggctccag 3960 tgctggcttc catgctgtgg
atgtcgactc ggggaacagc tatgacatct acatccctgt 4020 gcacatccag
agccagatca cgccccatgc catcatcttc ctccccaaca ccgacggcat 4080
ggagatgctg ctgtgctacg aggacgaggg tgtctacgtc aacacgtacg ggcgcatcat
4140 taaggatgtg gtgctgcagt ggggggagat gcctacttct gtggcctaca
tctgctccaa 4200 ccagataatg ggctggggtg agaaagccat tgagatccgc
tctgtggaga cgggccacct 4260 cgacggggtc ttcatgcaca aacgagctca
gaggctcaag ttcctgtgtg agcggaatga 4320 caaggtgttt tttgcctcag
tccgctctgg gggcagcagc caagtttact tcatgactct 4380 gaaccgtaac
tgcatcatga actggtgaaa gggc 4414 5 1276 PRT Homo sapiens MINK3c
(misshapen/NIKs-related kinase isoform 3c) 5 Met Asp Val Thr Glu
Asp Glu Glu Glu Glu Ile Lys Gln Glu Ile Asn 1 5 10 15 Met Leu Lys
Lys Tyr Ser His His Arg Asn Ile Ala Thr Tyr Tyr Gly 20 25 30 Ala
Phe Ile Lys Lys Ser Pro Pro Gly Asn Asp Asp Gln Leu Trp Leu 35 40
45 Val Met Glu Phe Cys Gly Ala Gly Ser Val Thr Asp Leu Val Lys Asn
50 55 60 Thr Lys Gly Asn Ala Leu Lys Glu Asp Cys Ile Ala Tyr Ile
Cys Arg 65 70 75 80 Glu Ile Leu Arg Gly Leu Ala His Leu His Ala His
Lys Val Ile His 85 90 95 Arg Asp Ile Lys Gly Gln Asn Val Leu Leu
Thr Glu Asn Ala Glu Val 100 105 110 Lys Leu Val Asp Phe Gly Val Ser
Ala Gln Leu Asp Arg Thr Val Gly 115 120 125 Arg Arg Asn Thr Phe Ile
Gly Thr Pro Tyr Trp Met Ala Pro Glu Val 130 135 140 Ile Ala Cys Asp
Glu Asn Pro Asp Ala Thr Tyr Asp Tyr Arg Ser Asp 145 150 155 160 Ile
Trp Ser Leu Gly Ile Thr Ala Ile Glu Met Ala Glu Gly Ala Pro 165 170
175 Pro Leu Cys Asp Met His Pro Met Arg Ala Leu Phe Leu Ile Pro Arg
180 185 190 Asn Pro Pro Pro Arg Leu Lys Ser Lys Lys Trp Ser Lys Lys
Phe Ile 195 200 205 Asp Phe Ile Asp Thr Cys Leu Ile Lys Thr Tyr Leu
Ser Arg Pro Pro 210 215 220 Thr Glu Gln Leu Leu Lys Phe Pro Phe Ile
Arg Asp Gln Pro Thr Glu 225 230 235 240 Arg Gln Val Arg Ile Gln Leu
Lys Asp His Ile Asp Arg Ser Arg Lys 245 250 255 Lys Arg Gly Glu Lys
Glu Glu Thr Glu Tyr Glu Tyr Ser Gly Ser Glu 260 265 270 Glu Glu Asp
Asp Ser His Gly Glu Glu Gly Glu Pro Ser Ser Ile Met 275 280 285 Asn
Val Pro Gly Glu Ser Thr Leu Arg Arg Glu Phe Leu Arg Leu Gln 290 295
300 Gln Glu Asn Lys Ser Asn Ser Glu Ala Leu Lys Gln Gln Gln Gln Leu
305 310 315 320 Gln Gln Gln Gln Gln Arg Asp Pro Glu Ala His Ile Lys
His Leu Leu 325 330 335 His Gln Arg Gln Arg Arg Ile Glu Glu Gln Lys
Glu Glu Arg Arg Arg 340 345 350 Val Glu Glu Gln Gln Arg Arg Gly Arg
Glu Gln Arg Lys Leu Gln Glu 355 360 365 Lys Glu Gln Gln Arg Arg Leu
Glu Asp Met Gln Ala Leu Arg Arg Glu 370 375 380 Glu Glu Arg Arg Gln
Ala Glu Arg Glu Gln Glu Tyr Lys Arg Lys Gln 385 390 395 400 Leu Glu
Glu Gln Arg Gln Ser Glu Arg Leu Gln Arg Gln Leu Gln Gln 405 410 415
Glu His Ala Tyr Leu Lys Ser Leu Gln Gln Gln Gln Gln Gln Gln Gln 420
425 430 Leu Gln Lys Gln Gln Gln Gln Gln Leu Leu Pro Gly Asp Arg Lys
Pro 435 440 445 Leu Tyr His Tyr Gly Arg Gly Met Asn Pro Ala Asp Lys
Pro Ala Trp 450 455 460 Ala Arg Glu Val Glu Glu Arg Thr Arg Met Asn
Lys Gln Gln Asn Ser 465 470 475 480 Pro Leu Ala Lys Ser Lys Pro Gly
Ser Thr Gly Pro Glu Pro Pro Ile 485 490 495 Pro Gln Ala Ser Pro Gly
Pro Pro Gly Pro Leu Ser Gln Thr Pro Pro 500 505 510 Met Gln Arg Pro
Val Glu Pro Gln Glu Gly Pro His Lys Ser Leu Val 515 520 525 Ala His
Arg Val Pro Leu Lys Pro Tyr Ala Ala Pro Val Pro Arg Ser 530 535 540
Gln Ser Leu Gln Asp Gln Pro Thr Arg Asn Leu Ala Ala Phe Pro Ala 545
550 555 560 Ser His Asp Pro Asp Pro Ala Ile Pro Ala Pro Thr Ala Thr
Pro Ser 565 570 575 Ala Arg Gly Ala Val Ile Arg Gln Asn Ser Asp Pro
Thr Ser Glu Gly 580 585 590 Pro Gly Pro Ser Pro Asn Pro Pro Ala Trp
Val Arg Pro Asp Asn Glu 595 600 605 Ala Pro Pro Lys Val Pro Gln Arg
Thr Ser Ser Ile Ala Thr Ala Leu 610 615 620 Asn Thr Ser Gly Ala Gly
Gly Ser Arg Pro Ala Gln Ala Val Arg Ala 625 630 635 640 Arg Pro Arg
Ser Asn Ser Ala Trp Gln Ile Tyr Leu Gln Arg Arg Ala 645 650 655 Glu
Arg Gly Thr Pro Lys Pro Pro Gly Pro Pro Ala Gln Pro Pro Gly 660 665
670 Pro Pro Asn Ala Ser Ser Asn Pro Asp Leu Arg Arg Ser Asp Pro Gly
675 680 685 Trp Glu Arg Ser Asp Ser Val Leu Pro Ala Ser His Gly His
Leu Pro 690 695 700 Gln Ala Gly Ser Leu Glu Arg Asn Arg Val Gly Ala
Ser Ser Lys Leu 705 710 715 720 Asp Ser Ser Pro Val Leu Ser Pro Gly
Asn Lys Ala Lys Pro Asp Asp 725 730 735 His Arg Ser Arg Pro Gly Arg
Pro Ala Asp Phe Val Leu Leu Lys Glu 740 745 750 Arg Thr Leu Asp Glu
Ala Pro Arg Pro Pro Lys Lys Ala Met Asp Tyr 755 760 765 Ser Ser Ser
Ser Glu Glu Val Glu Ser Ser Glu Asp Asp Glu Glu Glu 770 775 780 Gly
Glu Gly Gly Pro Ala Glu Gly Ser Arg Asp Thr Pro Gly Gly Arg 785 790
795 800 Asp Gly Asp Thr Asp Ser Val Ser Thr Met Val Val His Asp Val
Glu 805 810 815 Glu Ile Thr Gly Thr Gln Pro Pro Tyr Gly Gly Gly Thr
Met Val Val 820 825 830 Gln Arg Thr Pro Glu Glu Glu Arg Asn Leu Leu
His Ala Asp Ser Asn 835 840 845 Gly Tyr Thr Asn Leu Pro Asp Val Val
Gln Pro Ser His Ser Pro Thr 850 855 860 Glu Asn Ser Lys Gly Gln Ser
Pro Pro Ser Lys Asp Gly Ser Gly Asp 865 870 875 880 Tyr Gln Ser Arg
Gly Leu Val Lys Ala Pro Gly Lys Ser Ser Phe Thr 885 890 895 Met Phe
Val Asp Leu Gly Ile Tyr Gln Pro Gly Gly Ser Gly Asp Ser 900 905 910
Ile Pro Ile Thr Ala Leu Val Gly Gly Glu Gly Thr Arg Leu Asp Gln 915
920 925 Leu Gln Tyr Asp Val Arg Lys Gly Ser Val Val Asn Val Asn Pro
Thr 930 935 940 Asn Thr Arg Ala His Ser Glu Thr Pro Glu Ile Arg Lys
Tyr Lys Lys 945 950 955 960 Arg Phe Asn Ser Glu Ile Leu Cys Ala Ala
Leu Trp Gly Val Asn Leu 965 970 975 Leu Val Gly Thr Glu Asn Gly Leu
Met Leu Leu Asp Arg Ser Gly Gln 980 985 990 Gly Lys Val Tyr Gly Leu
Ile Gly Arg Arg Arg Phe Gln Gln Met Asp 995 1000 1005 Val Leu Glu
Gly Leu Asn Leu Leu Ile Thr Ile Ser Gly Lys Arg Asn 1010 1015 1020
Lys Leu Arg Val Tyr Tyr Leu Ser Trp Leu Arg Asn Lys Ile Leu His
1025 1030 1035 1040 Asn Asp Pro Glu Val Glu Lys Lys Gln Gly Trp Thr
Thr Val Gly Asp 1045 1050 1055 Met Glu Gly Cys Gly His Tyr Arg Val
Val Lys Tyr Glu Arg Ile Lys 1060 1065 1070 Phe Leu Val Ile Ala Leu
Lys Ser Ser Val Glu Val Tyr Ala Trp Ala 1075 1080 1085 Pro Lys Pro
Tyr His Lys Phe Met Ala Phe Lys Ser Phe Ala Asp Leu 1090 1095 1100
Pro His Arg Pro Leu Leu Val Asp Leu Thr Val Glu Glu Gly Gln Arg
1105 1110 1115 1120 Leu Lys Val Ile Tyr Gly Ser Ser Ala Gly Phe His
Ala Ala Asp Val 1125 1130 1135 Asp Ser Gly Asn Ser Tyr Asp Ile Tyr
Ile Pro Val His Ile Gln Ser 1140 1145 1150 Gln Ile Thr Pro His Ala
Ile Ile Phe Leu Pro Asn Thr Asp Gly Met 1155 1160 1165 Glu Met Leu
Leu Cys Tyr Glu Asp Glu Gly Val Tyr Val Asn Thr Tyr 1170 1175 1180
Gly Arg Ile Ile Lys Asp Val Val Leu Gln Trp Gly Glu Met Pro Thr
1185 1190 1195 1200 Ser Val Ala Tyr Ile Cys Ser Asn Gln Ile Met Gly
Trp Gly Glu Lys 1205 1210 1215 Ala Ile Glu Ile Arg Ser Val Glu Thr
Gly His Leu Asp Gly Val Phe 1220 1225 1230 Met His Lys Arg Ala Gln
Arg Leu Lys Phe Leu Cys Glu Arg Asn Asp 1235 1240 1245 Lys Val Phe
Phe Ala Ser Val Arg Ser Gly Gly Ser Ser Gln Val Tyr 1250 1255 1260
Phe Met Thr Leu Asn Arg Asn Cys Ile Met Asn Trp 1265 1270 1275 6
4033 DNA Homo sapiens MINK3c (misshapen/NIKs-related kinase isoform
3c) 6 accattctgg aagctcccta gaatctcctg gaatgcttaa tggacctttc
cagcaccgaa 60 attcaagaat tatgactcat cggtcagcag aaaagaccct
gctgggatct ttgagcttgt 120 ggaggtggtc ggcaatggaa cctacggaca
ggtgtacaag ggtcggcatg tcaagacggg 180 gcagctggct gccatcaagg
tcatggatgt cacggaggac gaggaggaag agatcaaaca 240 ggagatcaac
atgctgaaaa agtactctca ccaccgcaac atcgccacct actacggagc 300
cttcatcaag aagagccccc cgggaaacga tgaccagctc tggctggtga tggagttctg
360 tggtgctggt tcagtgactg acctggtaaa gaacacaaaa ggcaacgccc
tgaaggagga 420 ctgtatcgcc tatatctgca gggagatcct caggggtctg
gcccatctcc atgcccacaa 480 ggtgatccat cgagacatca aggggcagaa
tgtgctgctg acagagaatg ctgaggtcaa
540 gctagtggat tttggggtga gtgctcagct ggaccgcacc gtgggcagac
ggaacacttt 600 cattgggact ccctactgga tggctccaga ggtcatcgcc
tgtgatgaga accctgatgc 660 cacctatgat tacaggagtg atatttggtc
tctaggaatc acagccatcg agatggcaga 720 gggagccccc cctctgtgtg
acatgcaccc catgcgagcc ctcttcctca ttcctcggaa 780 ccctccgccc
aggctcaagt ccaagaagtg gtctaagaag ttcattgact tcattgacac 840
atgtctcatc aagacttacc tgagccgccc acccacggag cagctactga agtttccctt
900 catccgggac cagcccacgg agcggcaggt ccgcatccag cttaaggacc
acattgaccg 960 atcccggaag aagcggggtg agaaagagga gacagaatat
gagtacagcg gcagcgagga 1020 ggaagatgac agccatggag aggaaggaga
gccaagctcc atcatgaacg tgcctggaga 1080 gtcgactcta cgccgggagt
ttctccggct ccagcaggaa aataagagca actcagaggc 1140 tttaaaacag
cagcagcagc tgcagcagca gcagcagcga gaccccgagg cacacatcaa 1200
acacctgctg caccagcggc agcggcgcat agaggagcag aaggaggagc ggcgccgcgt
1260 ggaggagcaa cagcggcggg ggcgggagca gcggaagctg caggagaagg
agcagcagcg 1320 gcggctggag gacatgcagg ctctgcggcg ggaggaggag
cggcggcagg cggagcgtga 1380 gcaggaatac aagcggaagc agctggagga
gcagcggcag tcagaacgtc tccagaggca 1440 gctgcagcag gagcatgcct
acctcaagtc cctgcagcag cagcaacagc agcagcagct 1500 tcagaaacag
cagcagcagc agctcctgcc tggggacagg aagcccctgt accattatgg 1560
tcggggcatg aatcccgctg acaaaccagc ctgggcccga gaggtagaag agagaacaag
1620 gatgaacaag cagcagaact ctcccttggc caagagcaag ccaggcagca
cggggcctga 1680 gccccccatc ccccaggcct ccccagggcc cccaggaccc
ctttcccaga ctcctcctat 1740 gcagaggccg gtggagcccc aggagggacc
gcacaagagc ctggtggcac accgggtccc 1800 actgaagcca tatgcagcac
ctgtaccccg atcccagtcc ctgcaggacc agcccacccg 1860 aaacctggct
gccttcccag cctcccatga ccccgaccct gccatccccg cacccactgc 1920
cacgcccagt gcccgaggag ctgtcatccg ccagaattca gaccccacct ctgaaggacc
1980 tggccccagc ccgaatcccc cagcctgggt ccgcccagat aacgaggccc
cacccaaggt 2040 gcctcagagg acctcatcta tcgccactgc ccttaacacc
agtggggccg gagggtcccg 2100 gccagcccag gcagtccgtg ccagacctcg
cagcaactcc gcctggcaaa tctatctgca 2160 aaggcgggca gagcggggca
ccccaaagcc tccagggccc cctgctcagc cccctggccc 2220 gcccaacgcc
tctagtaacc ccgacctcag gaggagcgac cctggctggg aacgctcgga 2280
cagcgtcctt ccagcctctc acgggcacct cccccaggct ggctcactgg agcggaaccg
2340 cgtgggagcc tcctccaaac tggacagctc ccctgtgctc tcccctggga
ataaagccaa 2400 gcccgacgac caccgctcac ggccaggccg gcccgcagac
tttgtgttgc tgaaagagcg 2460 gactctggac gaggcccctc ggcctcccaa
gaaggccatg gactactcgt cgtccagcga 2520 ggaggtggaa agcagtgagg
acgacgagga ggaaggcgaa ggcgggccag cagaggggag 2580 cagagatacc
cctgggggcc gcgatgggga tacagacagc gtcagcacca tggtggtcca 2640
cgacgtcgag gagatcaccg ggacccagcc cccatacggg ggcggcacca tggtggtcca
2700 gcgcacccct gaagaggagc ggaacctgct gcatgctgac agcaatgggt
acacaaacct 2760 gcctgacgtg gtccagccca gccactcacc caccgagaac
agcaaaggcc aaagcccacc 2820 ctcgaaggat gggagtggtg actaccagtc
tcgtgggctg gtaaaggccc ctggcaagag 2880 ctcgttcacg atgtttgtgg
atctagggat ctaccagcct ggaggcagtg gggacagcat 2940 ccccatcaca
gccctagtgg gtggagaggg cactcggctc gaccagctgc agtacgacgt 3000
gaggaagggt tctgtggtca acgtgaatcc caccaacacc cgggcccaca gtgagacccc
3060 tgagatccgg aagtacaaga agcgattcaa ctccgagatc ctctgtgcag
ccctttgggg 3120 ggtcaacctg ctggtgggca cggagaacgg gctgatgttg
ctggaccgaa gtgggcaggg 3180 caaggtgtat ggactcattg ggcggcgacg
cttccagcag atggatgtgc tggaggggct 3240 caacctgctc atcaccatct
cagggaaaag gaacaaactg cgggtgtatt acctgtcctg 3300 gctccggaac
aagattctgc acaatgaccc agaagtggag aagaagcagg gctggaccac 3360
cgtgggggac atggagggct gcgggcacta ccgtgttgtg aaatacgagc ggattaagtt
3420 cctggtcatc gccctcaaga gctccgtgga ggtgtatgcc tgggccccca
aaccctacca 3480 caaattcatg gccttcaagt cctttgccga cctcccccac
cgccctctgc tggtcgacct 3540 gacagtagag gaggggcagc ggctcaaggt
catctatggc tccagtgctg gcttccatgc 3600 tgcggatgtc gactcgggga
acagctatga catctacatc cctgtgcaca tccagagcca 3660 gatcacgccc
catgccatca tcttcctccc caacaccgac ggcatggaga tgctgctgtg 3720
ctacgaggac gagggtgtct acgtcaacac gtacgggcgc atcattaagg atgtggtgct
3780 gcagtggggg gagatgccta cttctgtggc ctacatctgc tccaaccaga
taatgggctg 3840 gggtgagaaa gccattgaga tccgctctgt ggagacgggc
cacctcgacg gggtcttcat 3900 gcacaaacga gctcagaggc tcaagttcct
gtgtgagcgg aatgacaagg tgttttttgc 3960 ctcagtccgc tctgggggca
gcagccaagt ttacttcatg actctgaacc gtaactgcat 4020 catgaactgg tga
4033 7 10 PRT Artificial Sequence Description of Artificial
SequenceN-terminus flu HA epitope tag 7 Ala Tyr Pro Tyr Asp Val Pro
Asp Tyr Ala 1 5 10 8 31 DNA Artificial Sequence Description of
Artificial SequenceQuikChange mutagenesis kit oligo 8 agcttgcagc
catcagggtt atggatgtca c 31 9 31 DNA Artificial Sequence Description
of Artificial SequenceQuikChange mutagenesis kit oligo 9 gtgacatcca
taaccttgat ggctgcaagc t 31 10 14 PRT Artificial Sequence
Description of Artificial SequenceN-terminus Myc epitope tag 10 Ala
Ser Met Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn 1 5 10 11 6 PRT
Artificial Sequence Description of Artificial SequencePxxPxR motif
in intermediate domain of NIK 11 Pro Cys Pro Pro Ser Arg 1 5 12 6
PRT Artificial Sequence Description of Artificial SequencePxxPxR
motif in intermediate domain of NIK 12 Pro Arg Val Pro Val Arg 1 5
13 6 PRT Artificial Sequence Description of Artificial
Sequenceunconserved PxxPxR motif in intermediate domain of MINK3 13
Pro Asn Leu Pro Pro Arg 1 5 14 6 PRT Artificial Sequence
Description of Artificial Sequenceunconserved PxxPxR motif in
intermediate domain of MINK3 14 Pro Pro Leu Pro Thr Arg 1 5 15 6
PRT Artificial Sequence Description of Artificial Sequenceconserved
PxxPxR motif in intermediate domain of MINK3 15 Pro Lys Val Pro Gln
Arg 1 5
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