U.S. patent application number 10/198343 was filed with the patent office on 2003-03-20 for mitogen-activated protein kinase p38-2 and methods of use therefor.
This patent application is currently assigned to Signal Pharmaceuticals, Inc.. Invention is credited to Stein, Bernd, Yang, Maria X. H., Young, David B..
Application Number | 20030054528 10/198343 |
Document ID | / |
Family ID | 26968872 |
Filed Date | 2003-03-20 |
United States Patent
Application |
20030054528 |
Kind Code |
A1 |
Stein, Bernd ; et
al. |
March 20, 2003 |
Mitogen-activated protein kinase p38-2 and methods of use
therefor
Abstract
Compositions and methods are provided for the treatment of
diseases associated with mitogen activated protein kinase cascades.
In particular, the mitogen-activated protein kinase p38-2, and
polypeptide variants thereof that stimulate phosphorylation and
activation of substrates such as ATF2, are provided. The
polypeptides may be used, for example, to identify antibodies and
other agents that inhibit or activate signal transduction via the
p38-2 kinase cascade. Such polypeptides and agents may also be used
for the treatment of diseases associated with mitogen-activated
protein kinase cascades.
Inventors: |
Stein, Bernd; (San Diego,
CA) ; Yang, Maria X. H.; (San Diego, CA) ;
Young, David B.; (San Diego, CA) |
Correspondence
Address: |
PENNIE AND EDMONDS
1155 AVENUE OF THE AMERICAS
NEW YORK
NY
100362711
|
Assignee: |
Signal Pharmaceuticals,
Inc.
|
Family ID: |
26968872 |
Appl. No.: |
10/198343 |
Filed: |
July 18, 2002 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10198343 |
Jul 18, 2002 |
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09724768 |
Nov 28, 2000 |
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09724768 |
Nov 28, 2000 |
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09295029 |
Apr 20, 1999 |
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6444455 |
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09724768 |
Nov 28, 2000 |
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08651940 |
May 20, 1996 |
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5948885 |
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Current U.S.
Class: |
435/194 ;
435/252.3; 435/254.2; 435/320.1; 435/325; 435/348; 435/69.1;
536/23.2 |
Current CPC
Class: |
C12N 9/1205 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
435/194 ;
435/69.1; 435/320.1; 435/252.3; 435/254.2; 435/325; 435/348;
536/23.2 |
International
Class: |
C12N 009/12; C07H
021/04; C12P 021/02; C12N 005/06; C12N 001/21; C12N 001/18; C12N
015/74 |
Claims
1. A polypeptide comprising an amino acid sequence as recited in
SEQ ID NO:2, or a variant thereof that differs only in conservative
substitutions and/or modifications at no more than 25% of the amino
acid residues.
2. A constitutively active variant of a polypeptide according to
claim 1.
3. A polypeptide comprising the amino acid sequence recited in SEQ
ID NO:2, modified at no more than 25% of the amino acid residues,
such that said polypeptide is rendered constitutively inactive.
4. An isolated DNA molecule encoding a polypeptide according to any
of claims 1-3.
5. A DNA molecule according to claim 4, wherein the DNA molecule
comprises the nucleotide sequence provided in SEQ ID NO:1.
6. A recombinant expression vector comprising a DNA molecule
according to either of claims 4 or 5.
7. A host cell transformed or transfected with an expression vector
according to claim 6.
8. A host cell according to claim 7, wherein the host cell is
selected from the group consisting of bacteria, yeast, baculovirus
infected insect cells and mammalian cells.
9. A method for phosphorylating a substrate of p38-2, comprising
contacting a polypeptide according to either of claims 1 or 2 with
a substrate of p38-2, thereby phosphorylating the substrate of
p38-2.
10. The method of claim 9, wherein the substrate of p38-2 is
selected from the group consisting of ATF2, MAPKAP kinase 2 and
MAPKAP kinase.
11. A method for activating a substrate of p38-2 in a patient,
comprising administering to a patient a polypeptide according to
either of claims 1 or 2 in combination with a pharmaceutically
acceptable carrier, thereby activating a substrate of p38-2 in the
patient.
12. The method of claim 11, wherein the substrate of p38-2 is
selected form the group consisting of ATF2, MAPKAP kinase 2 and
MAPKAP kinase 3.
13. A method for screening for an agent that modulates signal
transduction via the p38-2 cascade, comprising: (a) contacting a
candidate agent with a polypeptide according to either of claims 1
or 2, wherein the step of contacting is carried out under
conditions and for a time sufficient to allow the candidate agent
and the polypeptide to interact; and (b) subsequently measuring the
ability of said candidate agent to modulate p38-2 activity, and
thereby evaluating the ability of the candidate agent to modulate
signal transduction via the p38 cascade.
14. A method for screening for an agent that modulates signal
transduction via the p38-2 cascade, comprising: (a) contacting a
candidate agent with a polynucleotide encoding a polypeptide
according to either of claims 1 or 2, wherein the step of
contacting is carried out under conditions and for a time
sufficient to allow generation of the polypeptide and interaction
between the polypeptide and the candidate agent; and (b)
subsequently measuring the ability of said candidate agent to
modulate p38-2 activity, and thereby evaluating the ability of the
candidate agent to modulate signal transduction via the p38-2
cascade.
15. An antibody that binds to a polypeptide according to either of
claims 1 or 2.
16. An antibody according to claim 15, wherein said antibody
inhibits the phosphorylation of substrate by said polypeptide.
17. A method for treating a disorder associated with the p38-2
cascade, comprising administering to a patient a therapeutically
effective amount of an agent that modulates signal transduction via
the p38-2 cascade.
18. A method for treating a patient afflicted with a disease
associated with the p38-2 cascade, comprising administering to a
patient an agent that inhibits p38-2 kinase activity.
19. A method for treating a patient afflicted with a disease
associated with the p38-2 cascade, comprising administering to a
patient an agent that inhibits phosphorylation of p38-2.
20. The method of any of claims 17-19, wherein said agent is a
monoclonal antibody.
21. The method of any of claims 17-19, wherein said agent comprises
a polynucleotide.
22. A method for detecting mitogen activated protein kinase kinase
activity in a sample, comprising evaluating the ability of the
sample to phosphorylate a polypeptide according to either of claims
1 or 2, thereby detecting mitogen activated protein kinase kinase
activity in the sample.
23. The method of claim 22, wherein the mitogen activated protein
kinase kinase is MEK6.
24. The method of claim 23, wherein the ability of the sample to
phosphorylate a polypeptide is evaluated using a coupled kinase
assay.
25. A kit for detecting mitogen activated protein kinase kinase
activity in a sample, comprising p38-2 in combination with a
suitable buffer.
Description
TECHNICAL FIELD
[0001] The present invention relates generally to compositions and
methods useful for the study of mitogen-activated protein kinase
cascades and for treating diseases associated with such pathways.
The invention is more particularly related to the mitogen-activated
protein kinase p38-2, and variants thereof that stimulate
phosphorylation and activation of substrates, such as ATF2. The
present invention is also related to the use of such polypeptides
to identify antibodies and other agents that inhibit or activate
signal transduction via the p38-2 kinase cascade.
BACKGROUND OF THE INVENTION
[0002] Mitogen-activated protein kinases (MAPKs) are members of
conserved signal transduction pathways that activate transcription
factors, translation factors and other target molecules in response
to a variety of extracellular signals. MAPKs are activated by
phosphorylation at a dual phosphorylation motif with the sequence
Thr-X-Tyr by mitogen-activated protein kinase kinases (MAPKKs). In
higher eukaryotes, the physiological role of MAPK signaling has
been correlated with cellular events such as proliferation,
oncogenesis, development and differentiation. Accordingly, the
ability to regulate signal transduction via these pathways could
lead to the development of treatments and preventive therapies for
human diseases associated with MAPK signaling, such as inflammatory
diseases, autoimmune diseases and cancer.
[0003] In mammalian cells, three parallel MAPK pathways have been
described. The best characterized pathway leads to the activation
of the extracellular-signal-regulated kinase (ERK). Less well
understood are the signal transduction pathways leading to the
activation of the cJun N-terminal kinase (JNK) and the p38 MAPK
(for reviews, see Davis, Trends Biochem. Sci. 19:470-473 (1994);
Cano and Mahadevan, Trends Biochem. Sci. 20:117-122(1995)). The
identification and characterization of members of these cascades is
critical for understanding the signal transduction pathways
involved and for developing methods for activating or inactivating
MAPKs in vivo.
[0004] Three MAPKKs capable of activating p38 in vitro have been
identified. MKK3 appears to be specific for p38 (i.e., does not
activate JNK or ERK), while MKK4 activates both p38 and JNK (see
Derijard et al., Science 267:682-685, 1995). The third MAPKK, MEK6,
appears to be a stronger and more specific in vivo stimulator of
p38 phosphorylation (see U.S. patent application Ser. No.
08/576,240). These proteins appear to have utility in therapeutic
methods for treating conditions associated with the p38 signal
transduction pathway. However, in order to precisely tailor such
therapeutic methods, and to gain an understanding of the pathways
involved, it would be advantageous to identify and characterize
other proteins that participate in this cascade and related MAP
kinase cascades.
[0005] Accordingly, there is a need in the art for improved methods
for modulating the activity of proteins involved in the MAP kinase
cascades, and for treating diseases associated with such cascades.
The present invention fulfills these needs and further provides
other related advantages.
SUMMARY OF THE INVENTION
[0006] Briefly stated, the present invention provides compositions
and methods employing the mitogen-activated protein kinase (MAPK)
p38-2, or a variant thereof. In one aspect, the present invention
provides polypeptides comprising the amino acid sequence provided
in SEQ ID NO:2 or a variant thereof that differs only in
conservative substitutions and/or modifications at no more than 25%
of the amino acid residues. Such variants include constitutively
active polypeptides. In a related aspect, polypeptides comprising
the amino acid sequence provided in SEQ ID NO:2 modified at no more
than 25% of the amino acid residues, such that the polypeptides are
rendered constitutively inactive, are provided.
[0007] In other aspects, isolated DNA molecules encoding
polypeptides as described above, as well as recombinant expression
vectors comprising such DNA molecules and host cells transformed or
transfected with such expression vectors, are provided.
[0008] In another aspect, the present invention provides methods
for phosphorylating a substrate of p38-2, comprising contacting a
polypeptide as described above with a substrate of p38-2, thereby
phosphorylating the substrate of p38-2. In a related aspect,
methods are provided for activating a substrate of p38-2 in a
patient, comprising administering to a patient a polypeptide as
described above in combination with a pharmaceutically acceptable
carrier, thereby activating a substrate of p38-2.
[0009] In further aspects, the present invention provides methods
for screening for an agent that modulates signal transduction via
the p38-2 cascade. In one embodiment the method comprises: (a)
contacting a candidate agent with a polypeptide as described above,
wherein the step of contacting is carried out under conditions and
for a time sufficient to allow the candidate agent and the
polypeptide to interact; and (b) subsequently measuring the ability
of the candidate agent to modulate p38-2 activity, and thereby
evaluating the ability of the candidate agent to modulate signal
transduction via the p38 cascade. In another embodiment, the method
comprises: (a) contacting a candidate agent with a polynucleotide
encoding a polypeptide as described above, wherein the step of
contacting is carried out under conditions and for a time
sufficient to allow generation of the polypeptide and interaction
between the polypeptide and the candidate agent; and (b)
subsequently measuring the ability of the candidate agent to
modulate p38-2 activity, and thereby evaluating the ability of the
candidate agent to modulate signal transduction via the p38-2
cascade.
[0010] In yet another aspect, the present invention provides
antibodies that bind to a polypeptide as described above.
[0011] In further aspects, methods are provided for treating a
disorder associated with the p38-2 cascade, comprising
administering to a patient a therapeutically effective amount of an
agent that modulates signal transduction via the p38-2 cascade. In
related aspects, methods are provided for treating a patient
afflicted with a disease associated with the p38-2 cascade,
comprising administering to a patient an agent that inhibits p38-2
kinase activity or that inhibits phosphorylation of p38-2.
[0012] In still further aspects, the present invention provides
methods and kits for detecting mitogen activated protein kinase
kinase activity in a sample. The methods comprise evaluating the
ability of the sample to phosphorylate a polypeptide as described
above, thereby detecting mitogen activated protein kinase kinase
activity in the sample. Kits comprise p38-2 in combination with a
suitable buffer.
[0013] These and other aspects of the present invention will become
apparent upon reference to the following detailed description and
attached drawings. All references disclosed herein are hereby
incorporated by reference in their entirety as if each was
incorporated individually.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 presents the primary amino acid sequence of p38-2,
and splice variants thereof, using standard one-letter codes.
[0015] FIGS. 2A and 2B are autoradiograms that depict Northern blot
analyses of the expression of human p38-2 (FIG. 2A) and p38 (FIG.
2B) mRNA in selected human tissues. The position of RNA size
markers in kb is shown on the left.
[0016] FIG. 3 is an autoradiogram that shows the size of in vitro
translated HA-tagged p38-2, as determined by SDS-PAGE. The position
of protein size markers in kDa is shown on the left.
[0017] FIG. 4 is an autoradiogram presenting the relative levels of
p38-2 kinase activity in COS cells transiently infected with
epitope tagged p38-2 (lanes 1 to 7) and treated for 45 minutes with
UV (250 nm, 120 J/m.sup.2; lane 2), anisomycin (50 ng/ml; lane 3),
or NaCl (200 .mu.M; lane 4), or cotransfected with 1000 ng of the
empty expression vector Sr.alpha.3 (lane 5), the expression vector
for the constitutively active mutant MEK6(DD) (lane 6) or the MAPK
TAK1.DELTA.N (lane 7).
[0018] FIG. 5 presents the nucleotide and amino acid sequence of
p38-2.
DETAILED DESCRIPTION OF THE INVENTION
[0019] As noted above, the present invention is generally directed
to compositions and methods for modulating (i.e., stimulating or
inhibiting) signal transduction via MAP kinase cascades. In
particular, the present invention is directed to compositions
comprising the MAP kinase p38-2 or a polypeptide variant thereof,
and to the use of such compositions for activating substrates of
p38-2 and for identifying modulators of p38-2 activity. As used
herein, the term "p38-2 polypeptide" encompasses the native p38-2
sequence, as well as variants thereof. The present invention also
encompasses compositions and methods for modulating p38-2 activity.
In general, compositions that inhibit p38-2 activity may inhibit
p38-2 phosphorylation, or may inhibit the ability of p38-2 to
phosphorylate a substrate. As used herein, the term "p38-2 cascade"
refers to any signal transduction pathway that involves p38-2, and
such a cascade may include any compound that modulates p38-2
activity or acts as a substrate for p38-2.
[0020] p38-2 polypeptide variants within the scope of the present
invention may contain one or more conservative substitutions and/or
modifications at no more than 25% of the amino acid residues in the
native polypeptide, such that the ability of the variant to
phosphorylate substrates (such as ATF2, MAPKAP kinase 2 and MAPKAP
kinase 3) is not substantially diminished. Preferably, a variant
contains substitutions and/or modifications at no more than 20% of
the amino acid residues, and more preferably at no more than 10% of
residues. Such substitutions, which are preferably conservative,
may be made in non-critical and/or critical regions of the native
protein. Variants may also, or alternatively, contain other
modifications, including the deletion or addition of amino acids
that have minimal influence on the activity of the polypeptide. In
particular, variants may contain additional amino acid sequences at
the amino and/or carboxy termini. Such sequences may be used, for
example, to facilitate purification or detection of the
polypeptide.
[0021] Substitutions and/or modifications may also be made at no
more than 25% (preferably at no more than 20%, and more preferably
at no more than 10%) of amino acid residues to render the
polypeptide constitutively active or inactive. Constitutively
active polypeptides display the ability to stimulate substrate
phosphorylation in the absence of stimulation, as described below.
Such variants may be identified using the representative assays for
p38-2 kinase activity described herein. Constitutively inactive
proteins are those which are unable to phosphorylate a substrate
even when stimulated as described below. Proteins modified so as to
be constitutively active or inactive may generally be used in
replacement therapy for treatment of a variety of disorders, as
discussed in more detail below.
[0022] DNA sequences encoding native p38-2 may be prepared by
amplification from a suitable human cDNA library, using polymerase
chain reaction (PCR) and methods well known to those of ordinary
skill in the art. For example, an adapter-ligated cDNA library
prepared from a cell line or tissue that expresses p38-2 (such as
skeletal muscle or heart) may be screened using a 5' specific
forward primer and an adapter-specific primer. The resulting 1.6 kb
cDNA has the sequence provided in SEQ ID NO:1 and FIG. 5. The
encoded p38-2 polypeptide, shown in SEQ ID NO:2 and FIG. 1, has a
predicted size of 364 amino acids, with a molecular weight of about
42 kD as determined by calculation and SDS-polyacrylamide gel
electrophoresis. p38-2 is 73% identical to its closest homolog p38
(see, e.g., Han et al., Science 265:808-811, 1994); Lee et al.,
Nature 372:739-746, 1994), and all kinase subdomains characteristic
for MAP kinase family members are conserved. Two alternate splice
variants of p38-2 have also been identified, with the sequences
provided in FIG. 1, as well as SEQ ID NO:3 and SEQ ID NO:4.
[0023] Polypeptides of the present invention may be prepared by
expression of recombinant DNA encoding the polypeptide in cultured
host cells. Preferably, the host cells are bacteria, yeast,
baculovirus-infected insect cells or mammalian cells. The
recombinant DNA may be cloned into any expression vector suitable
for use within the host cell, using techniques well known to those
of ordinary skill in the art. An expression vector may, but need
not, include DNA encoding an epitope, such that the recombinant
protein contains the epitope at the N- or C-terminus. Epitopes such
as glutathione-S transferase protein (GST), HA (hemagglutinin)-tag,
FLAG and Histidine-tag may be added using techniques well known to
those of ordinary skill in the art.
[0024] The DNA sequences expressed in this manner may encode p38-2,
or may encode alternate splice variants, portions or other variants
of p38-2, as well as constitutively inactive polypeptides. DNA
molecules encoding variants of p38-2 may generally be prepared
using standard mutagenesis techniques, such as
oligonucleotide-directed site-specific mutagenesis, and sections of
the DNA sequence may be removed to permit preparation of truncated
polypeptides. As noted above, up to 25% of the amino acid residues
may contain substitutions or other modifications. For variants of
p38-2, any such changes should not diminish the ability of the
variant to stimulate phosphorylation of substrates such as ATF2
(see, e.g., Gupta et al., Science 267:389-393, 1995), MAPKAP kinase
2 (see, e.g., Rouse et al., Cell 78:1027-1037, 1994 Ben Levy et
al., EMBO J.:14:5920-6930, 1995) or MAPKAP kinase 3 (see e.g.,
McLaughlin et al., J. Biol. Chem. 271:8488-8492, 1996). In general,
modifications may be more readily made in non-critical regions,
which are regions of the native sequence that do not substantially
change the properties of p38-2. Non-critical regions may be
identified by modifying the p38-2 sequence in a particular region
and assaying the ability of the resulting variant in a kinase
assay, using a suitable substrate, as described herein.
[0025] Modifications may also be made in critical regions of p38-2,
provided that the resulting variant substantially retains the
ability to stimulate substrate phosphorylation. The effect of any
modification on the ability of the variant to stimulate substrate
phosphorylation may generally be evaluated using any assay for
p38-2 kinase activity, such as the representative assays described
herein.
[0026] Expressed polypeptides of this invention are generally
isolated in substantially pure form. Preferably, the polypeptides
are isolated to a purity of at least 80% by weight, more preferably
to a purity of at least 95% by weight, and most preferably to a
purity of at least 99% by weight. In general, such purification may
be achieved using, for example, the standard techniques of ammonium
sulfate fractionation, SDS-PAGE electrophoresis, and affinity
chromatography. p38-2 polypeptides for use in the methods of the
present invention may be native, purified or recombinant.
[0027] In one aspect of the present invention, p38-2 polypeptides
may be used to identify modulating agents, which may be antibodies
or drugs, that inhibit or stimulate signal transduction via the
p38-2 cascade. Modulation includes the suppression of expression of
p38-2 when it is over expressed, or the augmentation of p38-2
expression when it is under expressed. Modulation may also include
suppression of phosphorylation of p38-2 or the stimulation or
inhibition of the ability of activated (i.e., phosphorylated) p38-2
to phosphorylate a substrate. For example, a modulating agent may
modulate the kinase activity of one or more MAPKKs, such as MEK6,
thereby stimulating or inhibiting p38-2 activation. Known
activators of MAPKKs include, but are not limited to,
stress-inducing signals (e.g., UV, osmotic shock, DNA-damaging
agents), anisomycin, LPS, and cytokines. Similarly, compositions
that inhibit p38-2 activity by inhibiting p38-2 phosphorylation may
include one or more agents that inhibit or block MAPKK activity,
such as an antibody that neutralizes a MAPKK, a competing peptide
that represents the substrate binding domain of a MAPKK or the dual
phosphorylation motif of p38-2, an antisense polynucleotide or
ribozyme that interferes with transcription and/or translation of a
MAPKK, a molecule that inactivates a MAPKK by binding to the
kinase, a molecule that binds to p38-2 and prevents phosphorylation
by a MAPKK or a molecule that prevents transfer of phosphate groups
from the kinase to the substrate.
[0028] In general, modulating agents may be identified by combining
a test compound with a p38-2 polypeptide (which may be activated or
constitutively active), or a polynucleotide encoding such a
polypeptide, in vitro or in vivo, and evaluating the effect of the
test compound on the p38-2 kinase activity using, for example, a
representative assay described herein. An increase or decrease in
kinase activity can be measured by adding a radioactive compound,
such as .sup.32P-ATP, to the mixture of components, and observing
radioactive incorporation into a suitable substrate for p38-2, to
determine whether the compound inhibits or stimulates kinase
activity. Briefly, a candidate agent may be included in a mixture
of active p38-2 polypeptide and substrate (such as ATF2), with or
without pre-incubation with one or more components of the mixture.
Activation of p38-2 may be achieved by any of a variety of means.
Typically, activation involves the addition of a MAP kinase kinase,
which may in turn be activated via stimulation as described above.
In general, a suitable amount of antibody or other agent for use in
such an assay ranges from about 0.1 .mu.M to about 10 .mu.l. The
effect of the agent on p38-2 kinase activity may then be evaluated
by quantitating the incorporation of [.sup.32P]phosphate into ATF2,
and comparing the level of incorporation with that achieved using
activated p38-2 without the addition of a candidate agent.
Alternatively, a polynucleotide encoding the kinase may be inserted
into an expression vector and the effect of a composition on
transcription of the kinase measured, for example, by Northern blot
analysis.
[0029] In another aspect of the present invention, a p38-2
polypeptide (which may be constitutively active) may be used for
phosphorylating and activating a substrate of p38-2. In one
embodiment, a substrate may be phosphorylated in vitro by
incubating a p38-2 polypeptide with a substrate and ATP in a
suitable buffer (described in more detail below) for 30 minutes at
30.degree. C. Any compound that can be phosphorylated by p38-2,
such as ATF2, MAPKAP kinase 2 and MAPKAP kinase 3 may be used as a
substrate. In general, the amounts of the reaction components may
range from about 0.1 .mu.g to about 10 .mu.g of p38-2 polypeptide,
from about 0.1 .mu.g to about 10 .mu.g of substrate, and from about
10 nM to about 500 nM of ATP. Phosphorylated substrate may then be
purified by binding to GSH-sepharose and washing. The extent of
substrate phosphorylation may generally be monitored by adding
[.gamma.-.sup.32P]ATP to a test aliquot, and evaluating the level
of substrate phosphorylation as described below.
[0030] One or more p38-2 polypeptides, modulating agents as
described above and/or polynucleotides encoding such polypeptides
and/or modulating agents may also be used to modulate p38-2
activity in a patient. As used herein, a "patient" may be any
mammal, including a human, and may be afflicted with a disease
associated with the p38-2 cascade or may be free of detectable
disease. Accordingly, the treatment may be of an existing disease
or may be prophylactic. Diseases associated with the p38-2 cascade
include any disorder which is etiologically linked to MAP kinase
activity, including cardiovascular disease, immune-related diseases
(e.g., inflammatory diseases, autoimmune diseases, malignant
cytokine production or endotoxic shock), cell growth-related
diseases (e.g., cancer, metabolic diseases, abnormal cell growth
and proliferation or cell cycle abnormalities) and cell
regeneration-related diseases (e.g., cancer, degenerative diseases,
trauma, environmental stress by heat, UV or chemicals or
abnormalities in development and differentiation). In particular,
the high expression of p38-2 in heart tissue suggests an important
role for p38-2 in cardiovascular diseases. Immunological-related
cell proliferative diseases appropriate for treatment with p38-2
polypeptides include osteoarthritis, ischemia, reperfusion injury,
trauma, certain cancers and viral disorders, and autoimmune
diseases such as rheumatoid arthritis, multiple sclerosis,
psoriasis, inflammatory bowel disease, and other acute phase
responses.
[0031] Treatment may include administration of a p38-2 polypeptide
and/or a compound which modulates p38-2 activity. For
administration to a patient, one or more polypeptides (and/or
modulating agents) are generally formulated as a pharmaceutical
composition. A pharmaceutical composition may be a sterile aqueous
or non-aqueous solution, suspension or emulsion, which additionally
comprises a physiologically acceptable carrier (i.e., a non-toxic
material that does not interfere with the activity of the active
ingredient). Any suitable carrier known to those of ordinary skill
in the art may be employed in the pharmaceutical compositions of
the present invention. Representative carriers include
physiological saline solutions, gelatin, water, alcohols, natural
or synthetic oils, saccharide solutions, glycols, injectable
organic esters such as ethyl oleate or a combination of such
materials. Optionally, a pharmaceutical composition may
additionally contain preservatives and/or other additives such as,
for example, antimicrobial agents, anti-oxidants, chelating agents
and/or inert gases, and/or other active ingredients. Alternatively,
a pharmaceutical composition may comprise a polynucleotide encoding
a p38-2 polypeptide, and/or modulating agent, such that the
polypeptide and/or modulating agent is generated in situ, in
combination with a physiologically acceptable carrier. In such
pharmaceutical compositions, the polynucleotide may be present
within any of a variety of delivery systems known to those of
ordinary skill in the art, including nucleic acid, bacterial and
viral expression systems, as well as colloidal dispersion systems,
including liposomes. Appropriate nucleic acid expression systems
contain the necessary polynucleotide sequences for expression in
the patient (such as a suitable promoter and terminating signal).
DNA may also be "naked," as described, for example, in Ulmer et
al., Science 259:1745-1749 (1993).
[0032] Various viral vectors that can be used to introduce a
nucleic acid sequence into the targeted patient's cells include,
but are not limited to, vaccinia or other pox virus, herpes virus,
retrovirus, or adenovirus. Techniques for incorporating DNA into
such vectors are well known to those of ordinary skill in the art.
Preferably, the retroviral vector is a derivative of a murine or
avian retrovirus including, but not limited to, Moloney murine
leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),
murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A
retroviral vector may additionally transfer or incorporate a gene
for a selectable marker (to aid in the identification or selection
of transduced cells) and/or a gene that encodes the ligand for a
receptor on a specific target cell (to render the vector target
specific). For example, retroviral vectors can be made target
specific by inserting a nucleotide sequence encoding a sugar, a
glycolipid, or a protein. Targeting may also be accomplished using
an antibody, by methods known to those of ordinary skill in the
art.
[0033] Viral vectors are typically non-pathogenic (defective),
replication competent viruses, which require assistance in order to
produce infectious vector particles. This assistance can be
provided, for example, by using helper cell lines that contain
plasmids that encode all of the structural genes of the retrovirus
under the control of regulatory sequences within the LTR, but that
are missing a nucleotide sequence which enables the packaging
mechanism to recognize an RNA transcript for encapsulation. Such
helper cell lines include (but are not limited to) .PSI.2, PA317
and PA12. A retroviral vector introduced into such cells can be
packaged and vector virion produced. The vector virions produced by
this method can then be used to infect a tissue cell line, such as
NIH 3T3 cells, to produce large quantities of chimeric retroviral
virions.
[0034] Another targeted delivery system for p38-2 polynucleotides
is a colloidal dispersion system. Colloidal dispersion systems
include macromolecule complexes, nanocapsules, microspheres, beads,
and lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles, and liposomes. A preferred colloidal system for use
as a delivery vehicle in vitro and in vivo is a liposome (i.e., an
artificial membrane vesicle). It has been shown that large
unilamellar vesicles (LUV), which range in size from 0.2-4.0 .mu.m
can encapsulate a substantial percentage of an aqueous buffer
containing large macromolecules. RNA, DNA and intact virions can be
encapsulated within the aqueous interior and be delivered to cells
in a biologically active form (Fraley, et al., Trends Biochem. Sci.
6:77, 1981). In addition to mammalian cells, liposomes have been
used for delivery of polynucleotides in plant, yeast and bacterial
cells. In order for a liposome to be an efficient gene transfer
vehicle, the following characteristics should be present: (1)
encapsulation of the genes of interest at high efficiency while not
compromising their biological activity; (2) preferential and
substantial binding to a target cell in comparison to non-target
cells; (3) delivery of the aqueous contents of the vesicle to the
target cell cytoplasm at high efficiency; and (4) accurate and
effective expression of genetic information (Mannino, et al.,
Biotechniques 6:882, 1988).
[0035] The targeting of liposomes can be classified based on
anatomical and mechanistic factors. Anatomical classification is
based on the level of selectivity and may be, for example,
organ-specific, cell-specific, and/or organelle-specific.
Mechanistic targeting can be distinguished based upon whether it is
passive or active. Passive targeting utilizes the natural tendency
of liposomes to distribute to cells of the reticuloendothelial
system (RES) in organs which contain sinusoidal capillaries. Active
targeting, on the other hand, involves alteration of the liposome
by coupling the liposome to a specific ligand such as a monoclonal
antibody, sugar, glycolipid, or protein, or by changing the
composition or size of the liposome in order to achieve targeting
to organs and cell types other than the naturally occurring sites
of localization.
[0036] Routes and frequency of administration, as well doses, will
vary from patient to patient. In general, the pharmaceutical
compositions may be administered intravenously, intraperitoneally,
intramuscularly, subcutaneously, intracavity or transdermally.
Between 1 and 6 doses may be administered daily. A suitable dose is
an amount of polypeptide or polynucleotide that is sufficient to
show improvement in the symptoms of a patient afflicted with a
disease associated with the p38-2 cascade. Such improvement may be
detected based on a determination of relevant cytokine levels
(e.g., IL-1, IL-6 and/or IL-8), by monitoring inflammatory
responses (e.g., edema, transplant rejection, hypersensitivity) or
through an improvement in clinical symptoms associated with the
disease, in general, the amount of polypeptide present in a dose,
or produced in situ by DNA present in a dose, ranges from about 1
.mu.g to about 250 .mu.g per kg of host, typically from about 1
.mu.g to about 60 .mu.g. Suitable dose sizes will vary with the
size of the patient, but will typically range from about 10 mL to
about 500 mL for 10-60 kg animal.
[0037] The present invention also provides methods for detecting
the level of mitogen activated protein kinase kinase (such as MEK6)
activity in a sample. The level of MAPKK activity may generally be
determined by evaluating the ability of the sample to phosphorylate
a p38-2 polypeptide, thereby rendering the p38-2 polypeptide active
(i.e., capable of phosphorylating in vivo substrates such as ATF2).
In one embodiment, a kinase assay may be performed substantially as
described in Derijard et al., Cell 76:1025-1037, 1994 and Lin et
al., Science 268:286-290, 1995, with minor modifications. Briefly,
a sample may be incubated with p38-2 and [.gamma.-.sup.32P]ATP in a
suitable buffer (such as 20 mM HEPES (pH 7.6), 5 mM MnCl.sub.2, 10
mM MgCl.sub.2, 1 mM dithiothreitol) for 30 minutes at 30.degree. C.
In general, approximately 1 .mu.g recombinant p38-2, with 50 nM
[.gamma.-.sup.32P]ATP, is sufficient. Proteins may then be
separated by SDS-PAGE on 10% gels and subjected to autoradiography.
Incorporation of [.sup.32P]phosphate into p38-2 may be quantitated
using techniques well known to those of ordinary skill in the art,
such as with a phosphorimager.
[0038] To determine whether p38-2 phosphorylation results in
activation, a coupled in vitro kinase assay may be performed using
a substrate for p38-2, such as ATF2, with or without an epitope
tag. ATF2 for use in such an assay may be prepared as described in
Gupta et al., Science 267:389-393, 1995. Briefly, following
phosphorylation of p38-2 as described above, isolation of the
protein by binding to GSH-sepharose and washing with 20 mM HEPES
(pH 7.6), 20 mM MgCl.sub.2, the p38-2 (0.1-10 .mu.g) may be
incubated with ATF2 (0.1-10 .mu.g) and [.gamma.-.sup.32P]ATP
(10-500 nM) in a buffer containing 20 mM HEPES (pH 7.6), 20 mM
MgCl.sub.2. It should be noted that alternative buffers may be used
and that buffer composition can vary without significant effects on
kinase activity. Reactions may be separated by SDS-PAGE, visualized
by autoradiography and quantitated using any of a variety of known
techniques. Activated p38-2 will be capable of phosphorylating ATF2
at a level of at least 5% above background using this assay.
[0039] To evaluate the effect of an antibody or other candidate
modulating agent on the level of signal transduction via the p38-2
cascade, a kinase assay may be performed as described above, except
that a MAPKK, such as MEK6 (rather than a sample) is generally
employed and the candidate modulating agent is added to the
incubation mixture. The candidate agent may be preincubated with
MAPKK before addition of ATP and p38-2 polypeptide. Alternatively,
the p38-2 may be preincubated with the candidate agent before the
addition of MAPKK. Further variations include adding the candidate
agent to a mixture of MAPKK and ATP before the addition of p38-2,
or to a mixture of p38-2 and ATP before the addition of MAPKK,
respectively. To identify modulators that inhibit the kinase
activity of activated p38-2, a constitutively active variant of
p38-2 may be employed. All these assays can further be modified by
removing the candidate agent after the initial preincubation step.
In general, a suitable amount of antibody or other candidate agent
for use in such an assay ranges from about 0.1 .mu.M to about 10
.mu.M. The effect of the agent on phosphorylation of p38-2 may then
be evaluated by quantitating the incorporation of
[.sup.32P]phosphate into p38, as described above, and comparing the
level of incorporation with that achieved using MAPKK without the
addition of the candidate agent.
[0040] p38-2 activity may also be measured in whole cells
transfected with a reporter gene whose expression is dependent upon
the activation of an appropriate substrate, such as ATF2. For
example, appropriate cells (i.e., cells that express p38-2) may be
transfected with an ATF2-dependent promoter linked to a reporter
gene such as luciferase. In such a system, expression of the
luciferase gene (which may be readily detected using methods well
known to those of ordinary skill in the art) depends upon
activation of ATF2 by p38-2, which may be achieved by the
stimulation of MAPKK with an activator or by cotransfection with an
expression vector that produces a constitutively active variant of
MAPKK, such as MEK6. Candidate modulating agents may be added to
the system, as described above, to evaluate their effect on the
p38-2 cascade.
[0041] Alternatively, a whole cell system may employ only the
transactivation domain of ATF2 fused to a suitable DNA binding
domain, such as GHF-1 or GAL4. The reporter system may then
comprise the GH-luciferase or GAL4-luciferase plasmid Candidate
modulating agents may then be added to the system to evaluate their
effect on ATF2-specific gene activation.
[0042] The present invention also provides methods for detecting
the level of p38-2 polypeptide in a sample. The level of p38-2, or
nucleic acid encoding p38-2, may generally be determined using a
reagent that binds to p38-2, or to DNA or RNA encoding p38-2. To
detect nucleic acid encoding p38-2, standard hybridization and/or
PCR techniques may be employed using a nucleic acid probe or a PCR
primer. Suitable probes and primers may be designed by those of
ordinary skill in the art based on the p38-2 cDNA sequence provided
in SEQ ID NO:1. To detect p38-2 protein, the reagent is typically
an antibody, which may be prepared as described below. There are a
variety of assay formats known to those of ordinary skill in the
art for using an antibody to detect a polypeptide in a sample. See,
e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, 1988. For example, the antibody may be
immobilized on a solid support such that it can bind to and remove
the polypeptide from the sample. The bound polypeptide may then be
detected using a second antibody that binds to the antibody/peptide
complex and contains a detectable reporter group. Alternatively, a
competitive assay, may be utilized, in which polypeptide that binds
to the immobilized antibody is labeled with a reporter group and
allowed to bind to the immobilized antibody after incubation of the
antibody with the sample. The extent to which components of the
sample inhibit the binding of the labeled polypeptide to the
antibody is indicative of the level of polypeptide within the
sample. Suitable reporter groups for use in these methods include,
but are not limited to, enzymes (e.g., horseradish peroxidase),
substrates, cofactors, inhibitors, dyes, radionuclides, luminescent
groups, fluorescent groups and biotin.
[0043] Antibodies encompassed by the present invention may be
polyclonal or monoclonal, and may be specific for p38-2 and/or one
or more variants thereof Preferred antibodies are those antibodies
that inhibit or block p38-2 activity in vivo and within an in vitro
assay, as described above. As noted above, antibodies and other
agents having a desired effect on p38-2 activity, may be
administered to a patient (either prophylactically or for treatment
of an existing disease) to modulate the activation of p38-2 in
vivo.
[0044] Antibodies may be prepared by any of a variety of techniques
known to those of ordinary skill in the art (see, e.g., Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, 1988). In one such technique, an immunogen comprising
the polypeptide is initially injected into a suitable animal (e.g.,
mice, rats, rabbits, sheep and goats), preferably according to a
predetermined schedule incorporating one or more booster
immunizations, and the animals are bled periodically. Polyclonal
antibodies specific for the polypeptide may then be purified from
such antisera by, for example, affinity chromatography using the
polypeptide coupled to a suitable solid support.
[0045] Monoclonal antibodies specific for p38-2 or a variant
thereof may be prepared, for example, using the technique of Kohler
and Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvements
thereto. Briefly, these methods involve the preparation of immortal
cell lines capable of producing antibodies having the desired
specificity (i.e., reactivity with the polypeptide of interest).
Such cell lines may be produced, for example, from spleen cells
obtained from an animal immunized as described above. The spleen
cells are then immortalized by, for example, fusion with a myeloma
cell fusion partner, preferably one that is syngeneic with the
immunized animal. For example, the spleen cells and myeloma cells
may be combined with a nonionic detergent for a few minutes and
then plated at low density on a selective medium that supports the
growth of hybrid cells, but not myeloma cells. A preferred
selection technique uses HAT (hypoxanthine, aminopterin, thymidine)
selection. After a sufficient time, usually about 1 to 2 weeks,
colonies of hybrids are observed. Single colonies are selected and
tested for binding activity against the polypeptide. Hybridomas
having high reactivity and specificity are preferred.
[0046] Monoclonal antibodies may be isolated from the supernatants
of growing hybridoma colonies. In addition, various techniques may
be employed to enhance the yield, such as injection of the
hybridoma cell line into the peritoneal cavity of a suitable
vertebrate host, such as a mouse. Monoclonal antibodies may then be
harvested from the ascites fluid or the blood. Contaminants may be
removed from the antibodies by conventional techniques, such as
chromatography, gel filtration, precipitation, and extraction.
[0047] In a related aspect of the present invention, kits for
detecting p38-2 and p38-2 kinase activity, as well as MAPKK kinase
activity, are provided. Such kits may be designed for detecting the
level of p38-2 or nucleic acid encoding p38-2, or may detect kinase
activity of p38-2 or MAPKK in a direct kinase assay or a coupled
kinase assay, in which both the level of phosphorylation and the
kinase activity of p38-2 may be determined. MAPKK or p38-2 kinase
activity may be detected in any of a variety of samples, such as
eukaryotic cells, bacteria, viruses, extracts prepared from such
organisms and fluids found within living organisms. In general, the
kits of the present invention comprise one or more containers
enclosing elements, such as reagents or buffers, to be used in the
assay.
[0048] A kit for detecting the level of p38-2, or nucleic acid
encoding p38-2, typically contains a reagent that binds to the
p38-2 protein, DNA or RNA. To detect nucleic acid encoding p38-2,
the reagent may be a nucleic acid probe or a PCR primer. To detect
p38-2 protein, the reagent is typically an antibody. Such kits also
contain a reporter group suitable for direct or indirect detection
of the reagent (i.e., the reporter group may be covalently bound to
the reagent or may be bound to a second molecule, such as Protein
A, Protein G, immunoglobulin or lectin, which is itself capable of
binding to the reagent). Suitable reporter groups include, but are
not limited to, enzymes (e.g., horseradish peroxidase), substrates,
cofactors, inhibitors, dyes, radionuclides, luminescent groups,
fluorescent groups and biotin. Such reporter groups may be used to
directly or indirectly detect binding of the reagent to a sample
component using standard methods known to those of ordinary skill
in the art.
[0049] Kits for detecting p38-2 activity typically comprise a p38-2
substrate in combination with a suitable buffer. p38-2 activity may
be specifically detected by performing an immunoprecipitation step
with a p38-2-specific antibody prior to performing a kinase assay
as described above. Alternatively, the substrate provided may be a
substrate that is phosphorylated only by p38-2 (i.e., is not
phosphorylated by p38). A kit for detecting MAPKK kinase activity
based on measuring the phosphorylation of p38-2 generally comprises
a p38-2 polypeptide in combination with a suitable buffer. A kit
for detecting MAPKK kinase activity based on detecting p38-2
activity generally comprises a p38-2 polypeptide in combination
with a suitable p38-2 substrate, such as ATF2. Optionally, a kit
may additionally comprise a suitable buffer and/or material for
purification of p38 after activation and before combination with
ATF2. Other reagents for use in detecting phosphorylation of p38-2
and/or kinase activity may also be provided. Such kits may be
employed in direct or coupled kinase assays, which may be performed
as described above.
[0050] In yet another aspect, p38-2 or a variant thereof may be
used to identify one or more native upstream kinases (i.e., kinases
that phosphorylate and activate p38-2 in vivo). A p38-2 polypeptide
may be used in a yeast two-hybrid system to identify proteins that
interact with p38-2. Alternatively, an expression library may be
sequenced for cDNAs that phosphorylate p38-2.
[0051] The following Examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1
Cloning and Sequencing cDNA Encoding 138-2
[0052] This Example illustrates the cloning of a cDNA molecule
encoding the human MAPK p38-2.
[0053] The Expressed Sequence Tags (EST) subdivision of the
National Center for Biotechnology Information (NCBI) Genbank
databank was searched with the tblastn program and the human p38
amino acid sequence (Han et al., Science 265:808-811, 1994; Lee et
al., Nature 372:739-746, 1994) as query using the BLAST e-mail
server. The EST sequence R72598 from a breast cDNA library
displayed the highest similarity score. A clone corresponding to
the EST sequence R72598 was obtained from Research Genetics Inc.,
(Huntsville, Ala.), and the insert size was determined to be about
0.9 kb. Sequencing revealed that this clone encodes the 5' portion
of a previously unknown gene and that the 3' end with the polyA
tail was missing. The 3' portion was obtained from a skeletal
muscle cDNA library by RACE PCR using a gene specific forward
primer and an adapter-based reverse primer. The complete cDNA was
obtained by fusion ligation of the 5 portion and the 3' portion
using a common KpnI site into pBluescript (Stratagene, La Jolla,
Calif.), and verified by miniprep analysis.
[0054] Full length clones with and without an intron were
identified. The sequences were obtained using dye terminator cycle
sequencing with an ABI 373 Automated Sequencer (Applied Biosystems,
Inc., Foster City, Calif.), and the sequence of the full length
clone without intron is shown in SEQ ID NO: 1 and FIG. 5.
Example 2
In vivo Expression of p38-2
[0055] This Example illustrates the expression of p38-2, as
compared to p38, in various human tissues.
[0056] Northern blots were performed using 2 .mu.g of polyA.sup.+
RNA isolated from 16 different adult human tissues, fractionated by
denaturing formaldehyde 1.2% agarose gel electrophoresis, and
transferred onto a charge-modified nylon membrane (Clontech
Laboratories, Palo Alto, Calif.). The blots were hybridized to a
p38-2 probe (900 bp p38-2 fragment) or p38 probe (850 bp p38
fragment) using ExpressHyb (Clontech Laboratories, Palo Alto,
Calif.) according to the manufacturer's instructions. Both probes
were prepared by labeling the cDNA with [.alpha.-.sup.32P]dCTP
(NEN, Boston, Mass.) by random priming (Stratagene, La Jolla,
Calif.). For control purposes, the blots were also hybridized to a
radiolabeled .beta.-actin probe.
[0057] The results, shown in FIGS. 2A and 2B, demonstrate that
p38-2 is widely expressed in many adult human tissues, with highest
levels in heart and skeletal muscle (FIG. 2A). In contrast, p38 is
predominantly expressed in skeletal muscle only (FIG. 2B). In
addition, expression of p38-2 is higher than p38 in heart and
testis, whereas expression of p38 is higher than p38-2 in placenta
and small intestine. All 16 tissues analyzed expressed equal
amounts of .beta.-actin mRNA (not shown).
Example 3
Preparation of p38-2
[0058] This Example illustrates the in vitro translation of
HA-tagged p38-2.
[0059] HA tagged p38-2 was in vitro transcribed and translated
using the Promega TNT Coupled Reticulocyte Lysate System (Promega,
Madison, Wis.) in the presence of 35S-methionine using SP6
polymerase and the template DNA 3.times.HA-p38-2-SR.alpha.3).
Radioactive, in vitro-translated proteins were separated by
SDS-PAGE and visualized by autoradiography (FIG. 3).
[0060] These results demonstrate that the molecular weight of the
epitope tagged p38-2 is approximately 42 kDa.
Example 4
Activation of p38-2 by Stress-Inducing Agents
[0061] This Example illustrates the activation of p38-2 a variety
of stimulators of the MAPK pathway.
[0062] An expression vector encoding epitope-tagged p38-2
(3.times.HA-p38-2-SR.alpha.3) was constructed by adding sequence
encoding three copies of a 10 amino acid hemagglutinin (HA) epitope
to the N-terminus of p38-2 and ligating the resulting cDNA into the
expression vector SR.alpha.3. To investigate the pattern of
regulation of p38-2, COS cells were transiently transfected with
HA-p38-2-SR.alpha.3 (as described above) by the DEAE-Dextran method
(Kawai and Nishizawa, Mol. Cell. Biol. 4:1172-1174, 1984). These
cells were then treated with various stimulators of the MAPK
pathway (i.e., treated for 45 minutes with UV (250 nm, 120
J/m.sup.2), anisomycin (50 ng/ml) or NaCl (200 .mu.M) or
cotransfected with 1000 ng of the empty expression vector
Sr.alpha.3, the expression vector for the constitutively active
mutant MEK6(DD) or the MAPK TAK1.DELTA.N).
[0063] Following treatment, cell lysates were prepared by
solubilization in lysis buffer as described (Derijard et al., Cell
76:1025-1037, 1994), and protein concentration of lysates was
determined by Bradford assay (Bradford, Ann. Biochem. 72:248-254,
1976). Cell lysates were used in an immune complex kinase assay
with GST-ATF2 substrate, prepared as previously described (Gupta et
al., Science 267:389-39, 1995). The assay was generally performed
as described previously (Derijard et al., Cell 76:1025-1037, 1994;
Lin et al., Science 268:286-290, 1995) with minor modifications.
The concentration of [.gamma.-.sup.32P]ATP was 50 nM, and 30 .mu.g
cell lysate was immunoprecipitated for 2 hours with the anti-HA
antibody 12CA5 (Boehringer-Mannheim Corp., Indianapolis, Ind.) and
then incubated with 1 .mu.g of recombinant substrate. Reactions
were separated by SDS-PAGE, and the results are presented in FIG.
4.
[0064] These experiments showed a strong induction of p38-2 by UV
(254 nm; 120 J/m.sup.2 for 45 minutes), anisomycin (50 ng/mL for 45
minutes) and MEK6(DD), a constitutively active variant of MEK6
(FIG. 4). No increase in p38-2 activity was observed when the cells
were treated with NaCl, expression vector alone or the MAP kinase
TAK1.DELTA.N (see Yamaguchi et al., Science 270:2008-2011, 1995).
This clearly indicates that the inducible phosphorylation of ATF2
depends on a kinase cascade comprised of MEK6 and p38-2.
[0065] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for the purpose of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Sequence CWU 1
1
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