U.S. patent application number 10/870859 was filed with the patent office on 2004-11-25 for mitogen-activated protein kinase kinase mek6 and methods of use thereof.
This patent application is currently assigned to Signal Pharmaceuticals, Inc.. Invention is credited to Stein, Bernd, Yang, Maria X. H..
Application Number | 20040235112 10/870859 |
Document ID | / |
Family ID | 24303539 |
Filed Date | 2004-11-25 |
United States Patent
Application |
20040235112 |
Kind Code |
A1 |
Stein, Bernd ; et
al. |
November 25, 2004 |
Mitogen-activated protein kinase kinase MEK6 and methods of use
thereof
Abstract
Compositions and methods are provided for potentiating the
activity of the mitogen-activated protein kinase p38. In particular
the mitogen-activated protein kinase kinase MEK6, and variants
thereof that stimulate phosphorylation of p38 are provided. Such
compounds may be used, for example, for therapy of diseases
associated with the p38 cascade and to identify antibodies and
other agents that inhibit or activate signal transduction via
p38.
Inventors: |
Stein, Bernd; (San Diego,
CA) ; Yang, Maria X. H.; (San Diego, CA) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Assignee: |
Signal Pharmaceuticals,
Inc.
|
Family ID: |
24303539 |
Appl. No.: |
10/870859 |
Filed: |
June 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10870859 |
Jun 16, 2004 |
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09593288 |
Jun 13, 2000 |
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09593288 |
Jun 13, 2000 |
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08576240 |
Dec 20, 1995 |
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6074862 |
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Current U.S.
Class: |
435/69.1 ;
435/194; 435/320.1; 435/325; 530/388.26; 536/23.2 |
Current CPC
Class: |
C12N 9/1205 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
435/069.1 ;
435/194; 435/320.1; 435/325; 530/388.26; 536/023.2 |
International
Class: |
C12N 009/12; C07H
021/04 |
Claims
1. A An isolated polypeptide comprising the amino acid sequence
provided in SEQ ID NO:2.
2. A constitutively active variant of a polypeptide according to
claim 1.
3. A polypeptide comprising the amino acid sequence provided in SEQ
ID NO:2 modified at no more than 10% 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. An isolated DNA molecule comprising the nucleotide sequence
provided in SEQ ID NO: 1.
6. A recombinant expression vector comprising a DNA molecule
according to claim 4.
7. A host cell transformed or transfected with an expression vector
according to claim 6.
8. A method for phosphorylating p38 comprising contacting p38 with
a polypeptide according to either of claims 1 or 2, thereby
phosphorylating p38.
9. A method for activating a member of the p38 cascade in an
organism, comprising administering to an organism a polypeptide
according to either of claims 1 or 2, thereby activating a member
of the p38 cascade.
10. The method of claim 9 wherein the member of the p38 cascade is
p38.
11. A method for screening for an agent that inhibits signal
transduction via the p38 cascade, comprising: (a) contacting a
candidate agent with a polypeptide according to either of claims 1
or 2; and (b) subsequently measuring the ability of said
polypeptide to activate p38, and thereby evaluating the ability of
the compound to inhibit signal transduction via the p38
cascade.
12. A method for screening for an agent that stimulates signal
transduction via the p38 cascade, comprising: (a) contacting a
candidate agent with a polypeptide according to either of claims 1
or 2; and (b) subsequently measuring the ability of said
polypeptide to activate p38, and thereby evaluating the ability of
the compound to stimulate signal transduction via the p38
cascade.
13. A monoclonal antibody that binds to a polypeptide according to
either of claims 1 or 2.
14. A monoclonal antibody according to claim 13, wherein said
antibody inhibits the phosphorylation of p38 by said
polypeptide.
15. A method for treating a patient afflicted with a disease
associated with the p38 cascade, comprising administering to a
patient a compound that inhibits the phosphorylation of p38 by
MEK6.
16. The method of claim 15 wherein said compound is a monoclonal
antibody.
17. The method of claim 15 wherein said compound comprises a
nucleotide sequence.
18. A method for detecting MEK6 kinase activity in a sample,
comprising evaluating the ability of the sample to phosphorylate
p38, thereby detecting MEK6 kinase activity in the sample.
19. A kit for detecting MEK6 kinase activity in a sample,
comprising p38 in combination with MEK6 and a suitable buffer.
20. A method for identifying a composition which affects MEK6
kinase activity, comprising: (a) incubating the composition and
MEK6 kinase or polynucleotide encoding the kinase, wherein the step
of incubation is carried out under conditions and for a time
sufficient to allow the components to interact; and (b) measuring
the effect of the composition on MEK6 kinase or polynucleotide
encoding the kinase.
21. A method of treating an immunologically related disorder
associated with MEK6 kinase activity, comprising administering to a
subject having the disorder a therapeutically effective amount of a
compound which modulates MEK6 kinase activity.
22. The isolated polypeptide of claim 1 wherein said polypeptide
also comprises additional amino acid residues at the N-terminus or
C-terminus.
23. An isolated polypeptide comprising a variant of the amino acid
sequence provided in SEQ ID NO:2 that differs from SEQ ID NO:2 at
10% or less of the amino acid residues, wherein said polypeptide is
capable of phosphorylating a p38 substrate.
24. The isolated polypeptide of claim 23 wherein the ability of
said polypeptide to stimulate p38 phosphorylation is not
substantially diminished as compared to a polypeptide having the
amino acid sequence provided in SEQ ID NO:2.
25. The isolated polypeptide of claim 23 wherein said polypeptide
is constitutively active.
26. A purified antibody that binds to a polypeptide according to
any of claims 1, 22, 23, 24, or 25.
27. An antibody according to claim 26 which is a monoclonal
antibody.
28. An antibody according to claim 26 which is a polyclonal
antibody.
29. An antibody according to claim 26, wherein said antibody
inhibits the phosphorylation of p38 by said polypeptide.
30. A substantially pure human mitogen-activated protein kinase
kinase (MKK) polypeptide having serine, threonine, and tyrosine
kinase activity, and human mitogen-activated protein (MAP) kinase
p38 phosphorylating activity comprising the amino acid sequence of
SEQ ID NO:2.
31. A substantially pure human mitogen-activated protein kinase
kinase (MKK) polypeptide having human mitogen-activated protein
(MAP) kinase p38 phosphorylating activity comprising the amino acid
sequence of SEQ ID NO:2.
32. A substantially pure human mitogen-activated protein kinase
kinase (MKK) polypeptide comprising the amino acid sequence of SEQ
ID NO:2.
33. A purified antibody which specifically binds to a polypeptide
of claim 30.
34. A purified antibody which specifically binds to a polypeptide
of claim 31.
35. A purified antibody which specifically binds to a polypeptide
of claim 32.
36. A purified monoclonal antibody which specifically binds to a
polypeptide of claim 30.
37. A purified monoclonal antibody which specifically binds to a
polypeptide of claim 31.
38. A purified monoclonal antibody which specifically binds to a
polypeptide of claim 32.
39. A purified polyclonal antibody which specifically binds to a
polypeptide of claim 30.
40. A purified polyclonal antibody which specifically binds to a
polypeptide of claim 31.
41. A polyclonal antibody which specifically binds to a polypeptide
of claim 32.
Description
TECHNICAL FIELD
[0001] The present invention relates generally to compositions and
methods for modulating the activity of the mitogen-activated
protein kinases, including p38. The invention is more particularly
related to the mitogen-activated protein kinase kinase MEK6 and
variants thereof that stimulate phosphorylation and activation of
substrates, such as p38, and to the use of compounds, for example,
to activate p38 and to identify antibodies and other agents that
inhibit or activate signal transduction via the p38 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] Two MAPKKs capable of activating p38 in vitro have been
described (see Derijard et al., Science 267:683-685 (1995)). MKK3
appears to be specific for p38 (i.e., does not activate JNK or
ERK), while MKK4 activates both p38 and JNK. MKK3 and MKK4 also
stimulate the phosphorylation of p38 in certain cell Lines after
treatment with stimuli, such as ultraviolet radiation and NaCl.
However, a stronger and more specific in vivo stimulator of p38
phosphorylation would have greater utility in therapeutic
methods.
[0005] Accordingly, there is a need in the art for improved methods
for modulating p38 activity and related enzymes or kinases in vivo,
and for treating diseases associated with the p38 signal
transduction pathway. 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 for modulating the activity of the mitogen-activated
protein kinase (MAPK) p38. In one aspect, the present invention
provides polypeptides comprising the amino acid sequence provided
in SEQ D NO:2 or a variant thereof that differs only in
conservative substitutions and/or modifications at no more than 10%
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 10% 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 further aspects, the present invention provides methods
for phosphorylating p38, comprising contacting p38 with a
polypeptide as described above, and for activating a member of the
p38 cascade in an organism, comprising administering to an organism
a polypeptide as described above. In a related aspect, the present
invention provides methods for treating a patient afflicted with a
disease associated with the p38 cascade, comprising administering
to a patient a compound that promotes or inhibits the
phosphorylation of p38 by MEK6.
[0009] Methods are also provided for screening for agents that
inhibit or stimulate signal transduction via the p38 cascade. Such
methods comprise: (a) contacting a candidate agent with a
polypeptide as described above; and (b) subsequently measuring the
ability of the polypeptide to activate p38, in yet another aspect,
monoclonal antibodies that bind to a polypeptide as described above
are provided.
[0010] Within further aspects, the present invention provides
methods and kits for detecting MEK6 kinase activity in a sample.
The methods comprise evaluating the ability of the sample to
phosphorylate p38, thereby detecting MEK6 kinase activity in the
sample. The kits for detecting MEK6 kinase activity in a sample
comprise p38 in combination with a suitable buffer.
[0011] 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
[0012] FIG. 1 presents the nucleotide and primary amino acid
sequence of MEK6, as deduced from the sequence of cDNA clones
isolated from a human MOLT-4 cDNA library. For the amino acid
sequence, standard one-letter codes are utilized.
[0013] FIGS. 2A and 2B are autoradiograms that depict Northern blot
analyses of the expression of human MKK3 (FIG. 2A) and human MEK6
(FIG. 2B) mRNA in selected adult human tissues. The position of RNA
size markers in kb is shown on the left.
[0014] FIGS. 3A and 3B are autoradiograms that present the results
of kinase assays evaluating the substrate specificity of MEK6. FIG.
3A shows the level of autophosphorylation of the substrates GST
(lane 1), GST-JNK2 (lane 2), GST-p38 (lane 3) and His-ERK1[K52R]
(lane 4) and the level of phosphorylation by GST-MEK6 of the
substrates GST (lane 5), GST-JNK2) (lane 6), GST-p38 (lane 7) and
His-ERK1[K52R] (lane 8). FIG. 3B shows the results of a coupled
kinase assay in which purified GST or GST-MEK6 was incubated with
purified GST-JNK2, GST-p38 or GST in the, presence of ATP. The
proteins were isolated and washed, and then incubated with
GST-cJun(1-79) (lanes 1-3) or GST-ATF2 (lanes 4-6) in the presence
of [.gamma.-.sup.32P]ATP. Reactions were separated by SDS-PAGE and
visualized by autoradiography. The position of protein molecular
weight markers in kDa is shown on the left.
[0015] FIG. 4 is a graph depicting the relative levels of MEK6
kinase activity in HeLa cells transiently transfected with
epitope-tagged MEK6 and treated with anisomycin (50 ng/ml) or UV
(254 nm; 120 J/m.sup.2) for the times indicated. The relative level
of MEK6 activity in untreated cells was arbitrarily assigned to be
1.
[0016] FIG. 5 is an autoradiogram and graph presenting the relative
levels of MEK6 kinase activity in HeLa cells transiently
transfected with epitope-tagged MEK6 and activated for 40 min with
20 to 120 J/m.sup.2 UV (254 nm) as indicated. Reactions were
separated by SDS-PAGE and visualized by autoradiography. The
position of protein molecular weight markers in kDa is shown on the
left, MEK6 activity was quantitated with a phosphorimager and
ImageQuant software and is shown in the bar graph.
[0017] FIG. 6 is an autoradiogram depicting the relative levels of
MEK6 kinase activity in HeLa cells transiently transfected with
epitope-tagged MEK6 (lanes 1-8) or the empty expression vector
SR.alpha.3 (lanes 9-16) and treated for 45 min with Anisomycin (An,
50 ng/ml) or left untreated (ctrl) as indicated. The position of
protein molecular weight markers in kDa is illustrated on the left.
The position of p38, ATF2 and an unknown protein (*) is indicated
on the right.
[0018] FIGS. 7A and 7B are autoradiograms and graphs showing the
relative levels of MEK6 kinase activity in HeLa cells (FIG. 7A) or
COS cells (FIG. 7B) transiently transfected with epitope-tagged
MEK6 (lanes 1 to 12) or the empty expression vector SR.alpha.3
(lanes 13 to 16) and treated for 45 min with IL-1.beta. (10 ng/ml),
TNF-.alpha. (10 ng/ml), EGF (50 ng/ml), NGF (50 ng/ml), PMA (50
ng/ml). Anisomycin (50 ng/ml), Cycloheximide (CX, 50 ng/ml),
Arsenite (200 .mu.M), NaCl (200 .mu.m) or UV (254 nm; 120
J/m.sup.2) or cotransfected with 1000 ng CMV5-MEKK as indicated.
The position of protein molecular weight markers in kDa is
illustrated on the left. MEK6 activity depicted in the graphs was
quantitated with a phosphorimager and ImageQuant software.
[0019] FIG. 8 is an autoradiogram showing the relative levels of
MEK6 kinase activity in COS cells transiently transfected with
epitope-tagged MEK6 (lanes 1 to 7) or JNKK (lanes 8 to 12) and
increasing amounts of CMV5-MEK expression vector as indicated. The
position of protein molecular weight markers in kDa is illustrated
on the left The position of p38 and JNK2 is indicated on the
right.
DETAILED DESCRIPTION OF THE INVENTION
[0020] As noted above, the present invention is generally directed
to compositions and methods for modulating (i.e., stimulating or
inhibiting) the activity of the mitogen-activated protein kinase
(MAPK) p38. Compositions that activate p38 generally stimulate p38
phosphorylation. Such compositions include polypeptides comprising
the human mitogen-activated protein kinase kinase (MAPKK) MEK6, or
a variant thereof that retains the ability to stimulate p38
phosphorylation. Alternatively, compositions that activate p38 may
include nucleic acid sequences that encode MEK6 or a variant
thereof. Polypeptide variants within the scope of the present
invention differ from MEK6 in one or more conservative
substitutions and or modifications, at no more than 10% of the
amino acid residues in the native polypeptide, such that the
ability of the variant to stimulate p38 phosphorylation is not
substantially diminished. Conservative substitutions may be made in
non-critical and/or critical regions of the native protein.
Variants may also, or alternatively, contain other conservative
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] Compositions that stimulate p38 phosphorylation (thereby
activating p38) may also, or alternatively, include one or more
agents that stimulate MEK6 kinase activity. Such agents include,
but are not limited to, stress-inducing signals (e.g., UV, osmotic
shock, DNA-damaging agents), anisomycin, LPS, and cytokines, and
may be identified by combining a test compound with MEK6 in vitro
and evaluating the effect of the test compound on the MEK6 kinase
activity using, for example, a representative assay described
herein.
[0022] Compositions that inactivate p38 generally inhibit p38
phosphorylation. Such compositions may include one or more agents
that inhibit or block MEK6 activity, such as an antibody that
neutralizes MEK6, a competing peptide that represents the substrate
binding domain of MEK6 or the dual phosphorylation motif of the
MEK6 substrate, an antisense polynucleotide or ribozyme that
interferes with transcription and/or translation of MEK6, a
molecule that inactivates MEK6 by binding to the kinase, a molecule
that binds to the MEK6 substrate and prevents phosphorylation by
MEK6 or a molecule that prevents transfer of phosphate groups from
the kinase to the substrate. Alternatively, an agent that
inactivates p38 may inhibit the kinase activity of phosphorylated
p38.
[0023] Agents that inhibit MEK6 kinase activity may be identified
by combining a test compound with MEK6 in vitro and evaluating the
activity of the MEK6 using a MEK6 kinase assay. Agents that inhibit
the activity of phosphorylated p38 may similarly be identified by
combining a test compound with phosphorylated p38 and evaluating
the effect of the test compound on the p38 kinase activity using,
for example, one of the representative assays described herein.
[0024] DNA sequences encoding native MEK6 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 unstimulated Jurkat T cells may be screened using the
5' specific forward primer 5'-TTGTGCTCCCCTCCCCCATCAAA GGAA-3' and
an adapter-specific primer. The resulting 1.6 kb cDNA has the
sequence provided in SEQ ID NO:1. The encoded MEK6 polypeptide,
shown in SEQ ID NO:2, has a predicted size of 334 amino acids, with
a calculated molecular weight of 37.5 kD. MEK6 is 88% identical to
its closest homolog MKK3, and all relevant kinase subdomains are
conserved. As shown in FIG. 1, the most divergent regions are the
N-terminal region, with an additional 18 amino acids, and the
C-terminal region.
[0025] 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.
[0026] The DNA sequences expressed in this manner may encode MEK6,
or may encode portions or other variants of MEK6. DNA molecules
encoding variants of MEK6 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 10% of the amino acid residues may contain
substitutions or other modifications, and any such changes
preferably should not diminish the ability of the variant to
stimulate p38 phosphorylation. In general, modifications may be
more readily made in non-critical regions, which are regions of the
native sequence that do not change the properties of MEK6.
Non-critical regions may be identified by modifying the MEK6
sequence in a particular region and assaying the ability of the
resulting variant in a kinase assay, using p38 as a substrate, as
described herein.
[0027] As noted above, MEK6 may also be modified by the addition of
sequences at the N- and/or C-terminus. For example, epitopes such
as GST (glutathione-S-transferase), HA (hemagglutinin)-tag, FLAG
and Histidine-tag may be added using techniques well known to those
of ordinary skill in the art.
[0028] Modifications may also be made in critical regions of MEK6,
provided that the resulting variant retains the ability to
stimulate p38 phosphorylation. Critical regions include the ATP
binding site Lys.sup.69, and the dual phosphorylation motif
(Ser.sup.207, Thr.sup.211). The effect of any modification on the
ability of the variant to stimulate p38 phosphorylation may
generally be evaluated using any assay for MEK6 kinase activity,
such as the representative assays described herein.
[0029] Variants of MEK6 include constitutively active proteins. In
general, activation of MEK6 in vivo requires stimulation by
cytokines, UV, stress-inducing agents or osmotic shock.
Constitutively active variants display the ability to stimulate p38
phosphorylation in the absence of such stimulation. Such variants
may be identified using the representative in vivo assays for MEK6
kinase activity described herein. Preferred constitutively active
variants include polypeptides in which the phospho-acceptor amino
acids within the MEK6 dual phosphorylation motif (Ser.sup.207 and
Thr.sup.211) are replaced with negatively charged amino acids such
as glutamic acid or aspartic acid.
[0030] MEK6 may also be modified so as to render the protein
constitutively inactive (i.e., unable to phosphorylate p38 even
when stimulated as described above). For example, mutation of the
conserved lysine in kinase subdomain I has been found to render
MAPKKs inactive. Accordingly, a preferred constitutively inactive
variant contains a modification of Lys.sup.69 in kinase subdomain I
of MEK6. Other such modifications may be identified using the
representative assays described herein. Genes encoding 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.
[0031] 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.
[0032] The present invention also provides methods for detecting
the level of MEK6 in a sample, as well as for detecting MEK6 kinase
activity in a sample. The level of MEK6, or nucleic acid encoding
MEK6, may generally be determined using a reagent that binds to the
MEK6 protein, DNA or RNA. To detect nucleic acid encoding MEK6,
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
MEK6 cDNA sequence provided in SEQ ID NO:1. To detect MEK6 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 Thr.sup.211) are replaced with negatively charged amino
acids such as glutamic acid or aspartic acid.
[0033] MEK6 may also be modified so as to render the protein
constitutively inactive (i.e., unable to phosphorylate p38 even
when stimulated as described above). For example, mutation of the
conserved lysine in kinase subdomain I has been found to render
MAPKKs inactive. Accordingly, a preferred constitutively inactive
variant contains a modification of Lys.sup.69 in kinase subdomain I
of MEK6. Other such modifications may be identified using the
representative assays described herein Genes encoding 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.
[0034] 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.
[0035] The present invention also provides methods for detecting
the level of MEK6 in a sample, as well as for detecting MEK6 kinase
activity in a sample. The level of MEK6, or nucleic acid encoding
MEK6, may generally be determined using a reagent that binds to the
MEK6 protein, DNA or RNA. To detect nucleic acid encoding MEK6,
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
MEK6 cDNA sequence provided in SEQ ID NO:1. To detect MEK6 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, 1983. 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.
[0036] MEK6 kinase assays, for use in evaluating the polypeptide
variants and other agents discussed above, include any assays that
evaluate a compound's ability to phosphorylate p38, thereby
rendering the p38 active (i.e., capable of phosphorylating in vivo
substrates such as ATF2). p38 for use in such methods may be
endogenous, purified or recombinant, and may be prepared using any
of a variety of techniques that win be apparent to those of
ordinary skill in the art. For example, cDNA encoding p38 may be
cloned by PCR amplification from a suitable human cDNA library,
using primers based on the published sequence (Han et al., Science
265:808-811 (1994); Lee et al., Nature 372:739-746 (1994)). p38
cDNA may then be cloned into a bacterial expression vector and the
protein produced in bacteria, such as E. coli, using standard
techniques. The bacterial expression vector may, but need not,
include DNA encoding an epitope such as glutathione-S transferase
protein (GST) such that the recombinant protein contains the
epitope at the N- or C-terminus.
[0037] A MEK6 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 polypeptide variant of MEK6 may be incubated with p38 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 0.5 .mu.g, of
the variant and 1 .mu.g, recombinant p38, 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 may be quantitated
using techniques well known to those of ordinary skill in the art,
such as with a phosphorimager. To evaluate the substrate
specificity of polypeptide variants, a kinase assay may generally
be performed as described above except that other MAPK substrates
(i.e., JNK2 and/or ERK) are substituted for the p38.
[0038] To determine whether p38 phosphorylation results in
activation, a coupled in vitro kinase assay may be performed using
a substrate for p38, 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 267:339-393 (1995). Briefly, following
phosphorylation of p38 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 (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 buffer 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 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 MEK6 activity, a kinase assay may be performed
as described above, except that MEK6 (rather than a variant
thereof) is generally employed and the candidate modulating agent
is added to the incubation mixture. The candidate agent may be
preincubated with MEK6 kinase before addition of ATP and substrate.
Alternatively, the substrate may be preincubated with the candidate
agent before the addition of kinase. Further variations include
adding the candidate agent to a mixture of kinase and ATP before
the addition of substrate, or a mixture of substrate and ATP before
the addition of MEK6 kinase, respectively. 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
MEK6 kinase activity 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
MEK6 without the addition of the candidate agent.
[0040] MEK6 activity may also be measured in whole cells
transfected with a reporter gene whose expression is dependent upon
the activation of ATF2. For example, cells 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,
which may be achieved by the stimulation of MEK6 with an activator
or by cotransfection with an expression vector that produces a
constitutively active variant of MEK6. Candidate modulating agents
may be added to the system, as described below, to evaluate their
effect on MEK6 activity.
[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
MEK6 modulating agents may then be added to the system to evaluate
their effect on ATF2-specific gene activation.
[0042] In other aspects of the subject invention, methods for using
the above polypeptides to phosphorylate and activate p38, or
peptide derivatives thereof, are provided. p38 substrate for use in
such methods may be prepared as described above. In one embodiment,
p38 may be phosphorylated in vitro by incubating p38 with MEK6, or
a variant thereof, and ATP in a suitable buffer as described above
for 30 minutes at 30.degree. C. In general, the amounts of the
reaction components may range from about 0.1 .mu.g to about 10
.mu.g of MEK6 or a variant thereof, from about 0.1 .mu.g to about
10 .mu.g of recombinant p38, and from about 10 nM to about 500 nM
of ATP. Phosphorylated proteins may then be purified by binding to
GSH-sepharose and washing. The extent of p38 phosphorylation may
generally be monitored by adding [.gamma.-.sup.32P]ATP to a test
aliquot, and evaluating the level of p38 phosphorylation as
described above. The activity of the phosphorylated p38 may be
evaluated using a coupled in vitro kinase assay, as described
above.
[0043] Once activated in vitro, p38 may be used, for example, to
identify agents that inhibit the kinase activity of p38. Such
inhibitory agents, which may be antibodies or drugs, may be
identified using the coupled assay described above. Briefly, a
candidate agent may be included in the mixture of p38 and ATF2,
with or without pre-incubation with one or more components of the
mixture, 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.M. The effect of the agent on p38 kinase
activity may then be evaluated by quantitating the incorporation of
[.sup.32P]phosphate into ATF2, as described above, and comparing
the level of incorporation with that achieved using activated p38
without the addition of a candidate agent.
[0044] The above polypeptides and/or modulating agents may also be
used to phosphorylate, and thereby activate, p38 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 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 cascade include any disorder which is
etiologically linked to MEK6 kinase activity, including
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).
[0045] For administration to a patient, one or more polypeptides
and/or modulating agents are generally formulated as a
pharmaceutical composition, formulated as 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
ingredients. Any suitable carrier known to those of ordinary skill
in the art may be employed in the pharmaceutical compositions of
this invention. Representative carriers include physiological
saline solutions, gelatins 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.
[0046] Alternatively, a pharmaceutical composition may contain DNA
encoding a polypeptide as described above, such that MEK6 or a
variant thereof is generated in situ, in combination with a
physiologically acceptable carrier. In such pharmaceutical
compositions, the DNA 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
DNA sequences for expression in the patient (such as a suitable
promoter and terminating signal). The DNA may also be "naked," as
described, for example, in Ulmer et al., Science 259:1745-1749
(1993).
[0047] 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.
[0048] 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.
[0049] Another targeted delivery system for MEK6 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
sells. 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, 1983).
[0050] The targeting of liposomes can be classified based on
anatomical and mechanistic factors. Anatomical classification is
based on the level of selectivity, for example, organ-specific,
cell-specific, and 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.
[0051] Routes and frequency of administration and polypeptide,
modulating agent or nucleic acid 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 DNA that is sufficient to show improvement in the
symptoms of a patient afflicted with a disease associated with the
p38 cascade. Such improvement may be detected based on a
determination of relevant cytokine levels (e.g., IL-2, 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.
[0052] The MEK6 protein kinase described herein is also useful in a
screening method for identifying compounds or compositions which
affect the activity of the kinase. Thus, in another embodiment, the
invention provides methods for identifying a composition which
affects MEK6 activity comprising incubating the components, which
include the composition to be tested and the kinase or a
polynucleotide encoding the kinase, under conditions sufficient to
allow the components to interact, then subsequently measuring the
effect the composition has on kinase activity or on a
polynucleotide encoding the kinase. The observed effect on the
kinase may be either inhibitory or stimulatory. For example, the
increase or decrease of the kinase activity can be measured by
adding a radioactive compound to the mixture of components such as
.sup.32P-ATP, and observing radioactive incorporation into p38 or
other suitable substrates for MEK6, to determine whether the
compound inhibits or stimulates kinase activity. A polynucleotide
encoding the kinase may be inserted into an expression vector and
the effect of a composition on transcription of the kinase can be
measured, for example, by Northern blot analysis.
[0053] In another embodiment, the invention provides a method of
treating immunological-related cell proliferative diseases such as
osteoarthritis, ischemia, reperfusion injury, trauma, certain
cancers and viral disorders, and autoimune diseases such as
rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory
bowel disease, and other acute phase responses. Essentially, any
disorder which is etiologically linked to MEK6 kinase activity
would be considered susceptible to treatment.
[0054] Treatment includes administration of a composition or
compound which modulates MEK6 kinase activity. Such modulation
includes the suppression of expression of MEK6 when it is over
expressed, or augmentation of MEK6 expression when it is under
expressed. Modulation may also include suppression of
phosphorylation of p38 or related kinases.
[0055] As noted above, the present invention also encompasses
antibodies, which may be polyclonal or monoclonal, specific for
MEK6 and/or one or more variants thereof. Preferred antibodies are
those antibodies that inhibit or block MEK6 activity in vivo and
within a MEK6 kinase assay as described above. 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.
[0056] Monoclonal antibodies specific for MEK6 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.
[0057] 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.
[0058] Antibodies and other agents having a desired effect on MEK6
activity, as described above, may be administered to a patient
(either prophylactically or for treatment of an existing disease)
to modulate the activation of p38 in vivo. For example, an agent
that decreases MEK6 activity in vivo may be administered to prevent
or treat inflammation, autoimmune diseases, cancer or degenerative
diseases. In general, for administration to a patient, an antibody
or other agent is formulated as a pharmaceutical composition which
additionally comprises a physiologically acceptable carrier. Any
suitable carrier known to those of ordinary skill in the art may be
employed in the pharmaceutical compositions of this invention,
including the representative carriers described above.
[0059] A pharmaceutical composition may also, or alternatively,
contain DNA encoding an antibody or other agent as described above,
such that the active agent is generated in situ. In such
pharmaceutical compositions, the DNA may be introduced using any of
a variety of delivery systems known to those of ordinary skill in
the art, such as those described above. For administration of such
agents, routes, frequency and doses will vary from patient to
patient. In general, however, the pharmaceutical compositions may
be administered as described above. A suitable dose of such an
agent is an amount sufficient to show benefit in the patient based
on the criteria noted above.
[0060] In a related aspect of the present invention, kits for
detecting MEK6 and MEK6 kinase activity are provided. Such kits may
be designed for detecting the level of MEK6 or nucleic acid
encoding MEK6, or may detect phosphorylation of p38 in a direct
kinase assay or a coupled kinase assay, in which both the level of
phosphorylation and the kinase activity of p38 may be determined.
MEK6 and MEK6 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.
[0061] A kit for detecting the level of MEK6, or nucleic acid
encoding MEK6, typically contains a reagent that binds to the MEK6
protein, DNA or RNA. To detect nucleic acid encoding MEK6, the
reagent may be a nucleic acid probe or a PCR primer. To detect MEK6
protein, the reagent is typically an antibody. The kit also
contains 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.
[0062] A kit for detecting MEK6 kite activity based on measuring
the phosphorylation of p38 generally comprises p38 in combination
with a suitable buffer. A kit for detecting MEK6 kinase activity
based on detecting p38 activity generally comprises p38 in
combination with a suitable p38 substrate, such as ATF2.
Optionally, the kit may additionally comprise a suitable buffer
and/or material for purification of p38 after activation and before
combination with ATF2. Such kits may be employed in direct or
coupled MEK6 kinase assays, which may be performed as described
above.
[0063] In yet another aspect, MEK6 or a variant thereof may be used
to identify one or more native upstream Bases (i,e., kinases that
phosphorylate and activate MEK6 in vivo). MEK6 may be used in a
yeast two-hybrid system to identify proteins that interact with
MEK6. Alternatively, an expression library may be sequenced for
cDNAs that phosphorylate MEK6.
[0064] The following Examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1
Cloning and Sequencing cDNA Encoding MEK6
[0065] This Example illustrates the cloning of a cDNA molecule
encoding the human MAPKK MEK6.
[0066] 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 NUM
amino acid sequence (Derijard et al., Science 267:682-685 (1995))
as query using the BLAST e-mail server. The 223 bp EST sequence
F00521 displayed the highest similarity score. A reverse PCR primer
(5'-CACATCTTCACTTGACCGAGAGCA-3') directed against this sequence was
designed with the help of the program Oligo V.4.0 (National
Biosciences, Inc., Plymouth, Minn.).
[0067] PolyA+ RNA was prepared from unstimulated Jurkat T cells
using the Micro-Fast Track Kit (Invitrogen, San Diego, Calif.). One
.mu.g of this RNA was used to generate an adaptor-ligated cDNA
library that can be used for 5' and 3' RACE (Marathon cDNA
Amplification Kit, Clontech Laboratories, Palo Alto, Calif.). The
adaptor specific primer from the kit and the gene specific reverse
primer were used to PCR-amplify the 5' portion of MEK6. PCR
amplification was performed with a combination of Taq and Pwo
polymerases (Expand Long Template PCR System, Boehringer-Mannheim
Corp., Indianapolis, Ind.) in the presence of TaqStart antibody
(Clontech Laboratories, Palo Alto, Calif.). This mixture is
designed to produce high yield of long PCR fragments and
proof-reading function. All PCR amplifications were carried out in
0.2 ml Perkin-Elmer thin-wall Micro-Amp tubes and a Perkin-Elmer
model 2400 or 9600 thermocycler. The resulting 0.8 kb PCR fragment
was ligated into pGEM-T (Promega, Madison, Wis.) and sequenced (dye
terminator cycle sequencing) with an ABI 373 Automated Sequencer
(Applied Biosystems. Inc., Foster City, Calif.).
[0068] The sequence information from the 5' end of the partial MEK6
cDNA was used to design a forward PCR primer
(5'-TTGTGCTCCCCTCCCCCATCAAAGG AA-3') for 3' RACE. The gene specific
forward primer and the adaptor specific primer were used to
PCR-amplify the complete MEK6 cDNA from an adaptor-ligated MOLT-4
cDNA library. This library was generated using one .mu.g MOLT-4
polyA+ RNA (Clontech Laboratories, Palo Alto, Calif.) and the
Marathon cDNA Amplification Kit (Clontech Laboratories. Palo Alto,
Calif.). The 1.6 kb PCR fragment was ligated into pGEM-T (Promega
Madison, Wis.) and three clones were sequenced several times on
both strands with an ABI 373 Automated Sequencer. A BLAST search of
the NCBI Genbank database for related cDNAs revealed no similar
sequences. The 1.6 kb cDNA encodes a potential protein of 334 amino
acids with a calculated molecular weight of 37.5 kdalton.
[0069] The Bestfit program (Wisconsin Genetics Computer Group,
Madison, Wis.) was used for calculating the amino acid identities
between MEK6 and MKK3, its closest homolog. The MacVector program
(Kodak-IBI, Rochester, N.Y.) was used for aligning the amino acids
of MKK3 and MEK6. MEK6 has 88% amino acid identity with MKK3, and
all relevant kinase subdomains, the ATP acceptor site and
phosphorylation sites are conserved. The most divergent regions are
the N-terminal region, with an additional 18 amino acids,-and the
C-terminal region (FIG. 1).
Example 2
In vivo Expression of MEK6
[0070] This Example illustrates the expression of MEK6, as compared
to MKK3, in various human tissues.
[0071] Northern blots were performed using 2 .mu.g of poly+ 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
MKK3 probe (700 bp MKK3 cDNA fragment) or MEK6 probe (870 bp MEK6
cDNA 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.
[0072] The results, shown in FIGS. 2A and 2B, demonstrate that is
widely expressed in many adult human tissues with highest levels in
skeletal muscle and leukocytes (FIG. 2A). In contrast, MEK6 is
predominantly expressed in skeletal muscle and at lower levels in
heart and pancreas (FIG. 2B). No MEK6 was detected in spleen,
thymus, prostate, ovary, small intestine, colon or leukocyte. All
16 tissues analyzed expressed equal amounts of .beta.-actin mRNA.
Some of the tissues expressed an MEK6-related mRNA of about 4.2 kb,
which was not observed when MEK6 specific probe directed against
the 3' of MEK6 cDNA was used.
Example 3
Substrate Specificity of MEK6
[0073] This Example illustrates the kinase activity and substrate
specificity of MEK6, as compared to MKK3, in in vitro and in vivo
assays.
[0074] cDNAs encoding MEK6 and MKK3 were subcloned into a bacterial
GST-fusion protein expression vector. GST-MEK6 was constructed by
ligating a 1.3 kb DNA fent encoding amino acid 1 through the stop
codon of MEK6 with a serine to alanine substitution of amino acid 2
into pGEX-KG (Guan and Dixon, Ann Biochem 192:262-267 (1991)).
Similarly, GST-MKK3, GST-p38 and GST-JNK2 were constructed by
ligating the respective cDNA fragments encoding amino acid 1
through the stop codon into pGEX-KG. Human p38 cDNA (Genbank
accession number U10871) was cloned by PCR amplification of a
Jurkat cDNA library with primers against the 5' end
(5'-CCAACCATGGCTCAGGAGAG-3') and 3' end
(5'-CGGTACCTTCAGGACTCCATCT-3'- ) of the published human p38
sequence. Each strand of the PCR fragment was sequenced several
times with an ABI 373 Automated Sequencer. His-ERK1[K52R] was
prepared as described previously (Robbins et al., J. Bio. Chem.
268:5097-5106 (1993)).
[0075] We investigated the substrate specificity of MEK6 in an in
vitro kinase assay with bacterially expressed MAPK substrates
(GST-JNK2, GST-p38 and His-ERK1[K52R]). The assays were performed
as previously described (Derijard et al., Cell 76:1025-1037 (1994);
Lin et al., Science 268:286-290 (1995)) with minor modifications.
0.5 .mu.g recombinant kinase and 1 .mu.g recombinant substrate were
used, and the concentration of [.gamma.-.sup.32P]ATP was 50 nM.
Phosphorylated proteins were separated by SDS-PAGE on 10% gels and
then subjected to autoradiography. Incorporation of
[C.sup.32P]phosphate was quantitated with a phosphorimager and
ImageQuant software (Molecular Dynamics, Inc., Sunnyvale,
Calif.).
[0076] FIG. 3A shows the level of autophosphorylation of the
substrates GST (lane 1), GST-JNK2 (lane 2), GST-p38 (lane 3) and
His-ERK1[K52R] (lane 4) and the level of phosphorylation by GST
MEK6 of the substrates GST (lane 5), GST-JNK2 (lane 6), GST-p38
(lane 7) and His-ERK1[K52R] (lane 8). In each case, 1 .mu.g of the
purified recombinant substrate was used. Autophosphorylation of
MEK6 was very low compared to MKK3. JNK2 autophosphorylated,
whereas p38 and ERK1(K52R) did not. MEK6 efficiently phosphorylated
p38 but none of the other substrates (FIG. 3A), compare lanes 1 to
4 with 5 to 8), although in parallel experiments the
phosphorylation of JNK by JNKK has been observed (data not shown).
This indicates that MEK6 has a substrate selectivity for the p38 is
subgroup of MAPKs.
[0077] To determine whether phosphorylation of p38 is an activating
event we analyzed the phosphorylation of recombinant ATF2 (a
substrate for p38) in a coupled in vitro kinase assay. GST-ATF2 was
prepared as previously described (Gupta et al., Science 267:389-393
(1995). FIG. 3B shows the results of a coupled kinase assay in
which purified GST or GST-MEK6 (0.1-10 .mu.g) was incubated with
purified GST-JNK2 (lanes 1 and 2), GST-p38 (lanes 4 and 5) or GST
(lanes 3 and 6) (0.1-10 .mu.g) in the presence of JNKK buffer Lin
et al., Science 268:286-290 (1995)) and 100 .mu.M ATP. The proteins
were isolated by binding to GSH-sepharose and after washing with 20
mM HEPES (pH 7.6), 20 mM MgCl.sub.2, incubated with GST-cJun(1-79)
(lanes 1-3) or GST-ATF2 (lanes 4-6) (0.1-10 .mu.g), in the presence
of JNK buffer with 20 mM HEPES (pH 7.6), 20 mM MgCl.sub.2, and
[.gamma.-.sup.32P]ATP (10-500 nM). Reactions were separated by
SDS-PAGE and visualized by autoradiography.
[0078] MEK6 did not cause increased phosphorylation of Jun
(GST-Jun(1-79), prepared as described in Hibi et al., Genes and
Development 7:2135-2148 (1993)) either directly or in combination
with JNK2 (FIG. 3B, lanes 1 to 3). ATF2, however, was strongly
phosphorylated by p38 that has been activated by MEK6 (FIG. 3B,
lane 5). ATF2 was not directly phosphorylated by MEK6. These data
establish that MEK6 is a functional MAPKK in vitro and that MEK6
specifically phosphorylates p38, resulting in its activation.
[0079] Next, we examined whether MEK6 can activate p38 in vivo. An
expression vector encoding epitope-tagged MEK6
(3xHA-MEK6-SR.alpha.3) was constructed by replacing serine in
position 2 of MEK6 with alanine, adding sequence encoding three
copies of a 10 amino acid hemagglutinin (HA) epitope to the
N-terminus of MEK6 and ligating the resulting cDNA into SR.alpha.3.
HeLa cells, cultured in Dulbecco's modified Eagle medium
supplemented with 10% fetal calf serum, 500 mg/l L-glutamine, and
antibiotics, were transiently transfected with 3xHA-MEK6 using
calcium phosphate-mediated DNA precipitation (Graham and van der
Eb, Virology 52:456-467 (1973)). Twenty-four hours later cells were
stimulated with anisomycin (50 ng/mL) or UV (254 nm; 120 J/m.sup.2)
for 0-120 minutes. 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. 72:243-254 (1976)).
[0080] In an initial experiment we investigated the time course of
MEK6 activation by anisomycin and UV treatment of transfected
cells. Cell lysates were used in an immune complex kinase assay
with GST-p38 substrate, performed as described above except that 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 quantitated with a
phosphorimager and ImageQuant software. The relative level of MEK6
activity in untreated cells was arbitrarily assigned 1. The
presence of equal amounts of MEK6 in all kinase reactions was
confirmed by Western blot analysis (data not shown).
[0081] MEK6 activation by anisomycin as measured by its ability to
phosphorylate p38, was observed as early as 10 min after treatment
(FIG. 4). The activation was transient and peaked at 40 min after
treatment In contrast, activation by UV was delayed by about 10 to
15 min and declined only slowly after a peak at 60 min (FIG. 4).
Analysis of the UV dose response of MEK6 in HeLa cells revealed
that doses up to 120 J/m.sup.2 yielded increasing activity of MEK6
(FIG. 5).
[0082] To determine whether the increase in p38 phosphorylation by
activated MEK6 augments p38 kinase activity a coupled immune
complex kinase assay was performed. Epitope-tagged MEK6 was
isolated from anisomycin-treated HeLa cells (45 minutes; 50 ng/mL)
and subjected to two subsequent kinase reactions as described above
using recombinant p38, ATF2 and GST alone. In support of our in
vitro results, anisomycin treatment caused increased
phosphorylation of ATF2 only when MEK6 and p38 were present (FIG.
6, compare lanes 5, 6 with 7, 8). Similar results have been found
with MEK6 activated by UV treatment of cells (data not shown). No
inducible phosphorylation of p38 or ATF2 was observed in HeLa cells
transfected with the empty expression vector SR.alpha.3 (FIG. 6,
compare lanes 5, 6 with 13, 14). This clearly indicates that the
inducible phosphorylation of ATF2 depends on a kinase cascade
comprised of MEK6 and p38. Interestingly, p38 also phosphorylated
weakly a protein with a mobility slightly faster than ATF2
(indicated by * in FIG. 6). This phosphorylation event was slightly
augmented by anisomycin in the presence of MEK6 (FIG. 6, compare
lanes 3 and 4 with lanes 11 and 12). This protein was not observed
in in vitro kinase assays, and therefore is most likely a
contamination of the immunoprecipitation.
Example 4
Activation of MEK5 by Stress-Inducing Agents
[0083] This Example illustrates the response of MEK6 to a variety
of stimulators of the MAPK pathway.
[0084] To investigate the pattern of regulation of MEK6, cells were
transiently transfected with 3xHA-MEK6 (as described in Example 3)
arid treated with various stimulators of the MAPK pathway. In HeLa
cells strongest inducers of MEK6 wee UV, anisomycin and NaCl
followed b weak induction with IL-1.beta. (FIG. 7A). NGF and EGF,
two strong inducers of the ERK pathway, did not activate MEK6
although we noted the inducible phosphorylation of two lower
molecular weight bands (see discussion).
[0085] Similar experiments were performed in COS cells, which were
transfected by the DEAE-Dextran method (Kawai and Nishizawa, Mol.
Cell. Biol. 4:1172-1174 (1984)). These experiments showed a strong
induction of MEK6 by UV and to a lesser extent by anisomycin (FIG.
7B). MEK6 was present at equal levels in all kinase reactions as
determined by Western Blot analysis (data not shown).
[0086] These results demonstrate that MEK6 is strongly activated by
stress-inducing and DNA damaging agents, anisomycin, UV and also by
osmotic shock. Phorbol esters, NGF and EGF, strong stimulators of
the ERK pathway did not stimulate MEK6. Similarly, cycloheximide, a
stimulator of p54 kinase and of the ERK pathway, did not
significantly activate MEK6. Interestingly, we noted in our in vivo
kinase assays with lysates prepared from HeLa cells, but not from
COS cells, two bands of variable intensity that were stimulated by
NGF and EGF. These bands most likely represent contaminants of the
immunoprecipitation phosphorylated by ERK family members.
Example 5
MEK6 is not a Physiological Substrate for MEKK
[0087] This Example evaluates the ability of MEKK to phosphorylate
MEK6 as compared to its ability to phosphorylate JNKK.
[0088] MEKK has been described as a MAPKKK leading to the
phosphorylation and activation of JNKK (Lin et al., Science
268:286-290 (1995); Minden et al., Science 266:1719-1722 (1994);
Yan et al., Nature 372:798-800 (1994)). In an initial experiment,
HeLa (FIG. 7A) or COS (FIG. 7B) cells were transiently transfected
with epitope-tagged MEK6 (lanes 1 to 12) or the empty expression
vector SR.alpha.3 (lanes 13 to 16) and treated for 45 min with
IL-1.beta. (10 ng/ml), TNF-.alpha. (10 ng/ml). EGF (50 ng/ml), NGF
(50 ng/ml), PMA (50 ng/ml), Anisomycin (50 ng/ml), Cycloheximide
(CX, 50 ng/ml), Arsenite (200 .mu.M), NaCl (200 .mu.M), UV (254 nm;
120 J/m.sup.2) or cotransfected with 1000 ng CMV5-MEKK as
indicated. Cell lysates were used in an immune complex kinase assay
with GST-p38 substrate as described in Example 3 MEK6 activity was
quantitated with a phosphorimager and ImageQuant software. The
presence of equal amounts of MEK6 in all kinase reactions was
confirmed by Western blot analysis (data not shown).
[0089] With 1000 ng cotransfected expression vector for MEKK, we
observed stimulation of MEK6 activity in COS cells but not HeLa
cells (FIG. 7A, lane 12, FIG. 7B, lane 12). This prompted us to
examine more carefully whether MEKK is able to phosphorylate MEK6.
COS cells were transiently transfected with increasing amounts of
expression vector encoding MEKK in the presence of a constant
amount of expression vector encoding epitope-tagged MEK6 (FIG. 8,
lanes 1 to 7) or JNKK (FIG. 8, lanes 8 to 12), and increasing
amounts of CMV5-MEKK expression vector as indicated in FIG. 8. Cell
lysates were used in an immune complex kinase assay with GST-p38
(lanes 1 to 7) or GST-JNK2 (lanes 8 to 12) substrate as described
in Example 3. Kinase activity was quantitated with a phosphorimager
and ImageQuant software.
[0090] We observed strong JNKK activation in cells transfected with
as little as 125 ng of the MEKK expression vector. Comparable
amounts of MEK6 activation, however, were not observed until 1000
ng of the MEKK expression vector were cotransfected. These data
suggest that MEKK does not participate in the kinase cascade
consisting of MEK6 and p38.
[0091] 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
6 1 1002 DNA homo sapien CDS (1)...(1002) 1 atg tct cag tcg aaa ggc
aag aag cga aac cct ggc ctt aaa att cca 48 Met Ser Gln Ser Lys Gly
Lys Lys Arg Asn Pro Gly Leu Lys Ile Pro 1 5 10 15 aaa gaa gca ttt
gaa caa cct cag acc agt tcc aca cca cct cga gat 96 Lys Glu Ala Phe
Glu Gln Pro Gln Thr Ser Ser Thr Pro Pro Arg Asp 20 25 30 tta gac
tcc aag gct tgc att tct att gga aat cag aac ttt gag gtg 144 Leu Asp
Ser Lys Ala Cys Ile Ser Ile Gly Asn Gln Asn Phe Glu Val 35 40 45
aag gca gat gac ctg gag cct ata atg gaa ctg gga cga ggt gcg tac 192
Lys Ala Asp Asp Leu Glu Pro Ile Met Glu Leu Gly Arg Gly Ala Tyr 50
55 60 ggg gtg gtg gag aag atg cgg cac gtg ccc agc ggg cag atc atg
gca 240 Gly Val Val Glu Lys Met Arg His Val Pro Ser Gly Gln Ile Met
Ala 65 70 75 80 gtg aag cgg atc cga gcc aca gta aat agc cag gaa cag
aaa cgg cta 288 Val Lys Arg Ile Arg Ala Thr Val Asn Ser Gln Glu Gln
Lys Arg Leu 85 90 95 ctg atg gat ttg gat att tcc atg agg acg gtg
gac tgt cca ttc act 336 Leu Met Asp Leu Asp Ile Ser Met Arg Thr Val
Asp Cys Pro Phe Thr 100 105 110 gtc acc ttt tat ggc gca ctg ttt cgg
gag ggt gat gtg tgg atc tgc 384 Val Thr Phe Tyr Gly Ala Leu Phe Arg
Glu Gly Asp Val Trp Ile Cys 115 120 125 atg gag ctc atg gat aca tca
cta gat aaa ttc tac aaa caa gtt att 432 Met Glu Leu Met Asp Thr Ser
Leu Asp Lys Phe Tyr Lys Gln Val Ile 130 135 140 gat aaa ggc cag aca
att cca gag gac atc tta ggg aaa ata gca gtt 480 Asp Lys Gly Gln Thr
Ile Pro Glu Asp Ile Leu Gly Lys Ile Ala Val 145 150 155 160 tct att
gta aaa gca tta gaa cat tta cat agt aag ctg tct gtc att 528 Ser Ile
Val Lys Ala Leu Glu His Leu His Ser Lys Leu Ser Val Ile 165 170 175
cac aga gac gtc aag cct tct aat gta ctc atc aat gct ctc ggt caa 576
His Arg Asp Val Lys Pro Ser Asn Val Leu Ile Asn Ala Leu Gly Gln 180
185 190 gtg aag atg tgc gat ttt gga atc agt ggc tac ttg gtg gac tct
gtt 624 Val Lys Met Cys Asp Phe Gly Ile Ser Gly Tyr Leu Val Asp Ser
Val 195 200 205 gct aaa aca att gat gca ggt tgc aaa cca tac atg gcc
cct gaa aga 672 Ala Lys Thr Ile Asp Ala Gly Cys Lys Pro Tyr Met Ala
Pro Glu Arg 210 215 220 ata aac cca gag ctc aac cag aag gga tac agt
gtg aag tct gac att 720 Ile Asn Pro Glu Leu Asn Gln Lys Gly Tyr Ser
Val Lys Ser Asp Ile 225 230 235 240 tgg agt ctg ggc atc acg atg att
gag ttg gcc atc ctt cga ttt ccc 768 Trp Ser Leu Gly Ile Thr Met Ile
Glu Leu Ala Ile Leu Arg Phe Pro 245 250 255 tat gat tca tgg gga act
cca ttt cag cag ctc aaa cag gtg gta gag 816 Tyr Asp Ser Trp Gly Thr
Pro Phe Gln Gln Leu Lys Gln Val Val Glu 260 265 270 gag cca tcg cca
caa ctc cca gca gac aag ttc tct gca gag ttt gtt 864 Glu Pro Ser Pro
Gln Leu Pro Ala Asp Lys Phe Ser Ala Glu Phe Val 275 280 285 gac ttt
acc tca cag tgc tta aag aag aat tcc aaa gaa cgg cct aca 912 Asp Phe
Thr Ser Gln Cys Leu Lys Lys Asn Ser Lys Glu Arg Pro Thr 290 295 300
tac cca gag cta atg caa cat cca ttt ttc acc cta cat gaa tcc aaa 960
Tyr Pro Glu Leu Met Gln His Pro Phe Phe Thr Leu His Glu Ser Lys 305
310 315 320 gga aca gat gtg gca tct ttt gta aaa ctg att ctt gga gac
1002 Gly Thr Asp Val Ala Ser Phe Val Lys Leu Ile Leu Gly Asp 325
330 2 333 PRT homo sapien 2 Ser Gln Ser Lys Gly Lys Lys Arg Asn Pro
Gly Leu Lys Ile Pro Lys 1 5 10 15 Glu Ala Phe Glu Gln Pro Gln Thr
Ser Ser Thr Pro Pro Arg Asp Leu 20 25 30 Asp Ser Lys Ala Cys Ile
Ser Ile Gly Asn Gln Asn Phe Glu Val Lys 35 40 45 Ala Asp Asp Leu
Glu Pro Ile Met Glu Leu Gly Arg Gly Ala Tyr Gly 50 55 60 Val Val
Glu Lys Met Arg His Val Pro Ser Gly Gln Ile Met Ala Val 65 70 75 80
Lys Arg Ile Arg Ala Thr Val Asn Ser Gln Glu Gln Lys Arg Leu Leu 85
90 95 Met Asp Leu Asp Ile Ser Met Arg Thr Val Asp Cys Pro Phe Thr
Val 100 105 110 Thr Phe Tyr Gly Ala Leu Phe Arg Glu Gly Asp Val Trp
Ile Cys Met 115 120 125 Glu Leu Met Asp Thr Ser Leu Asp Lys Phe Tyr
Lys Gln Val Ile Asp 130 135 140 Lys Gly Gln Thr Ile Pro Glu Asp Ile
Leu Gly Lys Ile Ala Val Ser 145 150 155 160 Ile Val Lys Ala Leu Glu
His Leu His Ser Lys Leu Ser Val Ile His 165 170 175 Arg Asp Val Lys
Pro Ser Asn Val Leu Ile Asn Ala Leu Gly Gln Val 180 185 190 Lys Met
Cys Asp Phe Gly Ile Ser Gly Tyr Leu Val Asp Ser Val Ala 195 200 205
Lys Thr Ile Asp Ala Gly Cys Lys Pro Tyr Met Ala Pro Glu Arg Ile 210
215 220 Asn Pro Glu Leu Asn Gln Lys Gly Tyr Ser Val Lys Ser Asp Ile
Trp 225 230 235 240 Ser Leu Gly Ile Thr Met Ile Glu Leu Ala Ile Leu
Arg Phe Pro Tyr 245 250 255 Asp Ser Trp Gly Thr Pro Phe Gln Gln Leu
Lys Gln Val Val Glu Glu 260 265 270 Pro Ser Pro Gln Leu Pro Ala Asp
Lys Phe Ser Ala Glu Phe Val Asp 275 280 285 Phe Thr Ser Gln Cys Leu
Lys Lys Asn Ser Lys Glu Arg Pro Thr Tyr 290 295 300 Pro Glu Leu Met
Gln His Pro Phe Phe Thr Leu His Glu Ser Lys Gly 305 310 315 320 Thr
Asp Val Ala Ser Phe Val Lys Leu Ile Leu Gly Asp 325 330 3 27 DNA
Artificial Sequence 5' specific forward primer 3 ttgtgctccc
ctcccccatc aaaggaa 27 4 24 DNA Artificial Sequence Reverse 5' PCR
primer 4 cacatcttca cttgaccgag agca 24 5 20 DNA Artificial Sequence
5' primers of human p38 5 ccaaccatgg ctcaggagag 20 6 22 DNA
Artificial Sequence 3' Primer of human p38 6 cggtaccttc aggactccat
ct 22
* * * * *