Note: Descriptions are shown in the official language in which they were submitted.
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Description
MITOGEN-ACTIVATED PROTEIN KIN~SE p38-2
AND METHODS OF USE THEREFOR
S
Technical Field
The present invention relates generally to compositions and methods
useful for the study of mitogen-activated protein kinase c~cA.(les and for treating
10 conditions associated with such cA~c:~es. The invention is more particularly related to
a mitogen-activated protein kinase p38-2, and variants thereof that may be activated by
bradykinin to 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 signal transduction via the p38-2 kinase
15 cascade. Such agents may be used, for exarnple, to reduce pain sensations.
Background of the Invention
Mitogen-activated protein kinases (MAPKs) are members of conserved
signal transduction pathways that activate transcription factors translation factors and
20 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-
~yr by mitogen-activated protein kinase kinases (MAPKKs). In higher eukaryotes, the
physiological role of MAPK ,signA1ing has been correlated with cellular events such as
proliferation, oncogenesis, development and differentiation. Accordingly, the ability to
25 regulate signal transduction via these pathways could lead to the development of
treatments and preventive therapies for human diseases associated with MAPK
sign~ling, such as inflAmm~tory diseases, autoimmune diseases and cancer.
In m~mmAli~n cells, three parallel MAPK pathways have been
described. The best characterized pathway leads to the activation of the extracellular-
30 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
.. . ..
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Mahadevan, Trends Biochem. Sci. 20: 11 7- 1 22, 1 995). The identification and
characterization of members of these c~c~des is critical for underst~n~ing the signal
transduction pathways involved and for developing methods for activating or
inactivating MAPKs in vivo.
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 Serial Number
10 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 under~t~n~ling of the pathways
involved, it would be advantageous to identify and characterize other proteins that
participate in this cascade and related MAP kinase c~c~-les.
Accordingly, there is a need in the art for improved methods for
modulating the activity of proteins involved in the MAP kinase ca~ç~es, and for
treating conditions associated with such c~c~des. The present invention fulfills these
needs and further provides other related advantages.
20 Summary of the Invention
Briefly stated, the present invention provides compositions and methods
employing a mitogen-activated protein kinase (MAPK) p38-2, or a variant thereof. In
one aspect, the present invention provides polypeptides capable of activating a substrate
of p38-2. The polypeptides may comprise an amino acid sequence recited in SEQ ID25 NO:2, or a variant thereof. In another such aspect, the polypeptide is selectively
activated by bradykinin.
The present invention also provides a polypeptide comprising the amino
acid sequence recited in SEQ ID NO:2, modified at no more than 25% of the arninoacid resi~lue.~, such that said polypeptide is rendered constitutively inactive.In related aspects, the present invention provides isolated DNA
molecules encoding polypeptides as described above, as well as recombinant
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expression vectors comprising such DNA molecules and host cells transformed or
transfected with such expression vectors.
In another aspect, the present invention provides methods for
phosphorylating a substrate of p38-2, comprising contacting a polypeptide as described
S 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 ~(lmini~tering to a patient a polypeptide as described
above in combination with a pharmaceutically acceptable carrier, thereby activating a
substrate of p38-2.
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 kinase activity of
said polypeptide.
Within another embodiment, the method comprises: (a) contacting a
candidate agent with a polynucleotide encoding a polypeptide according to either of
claims I or 4, 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.
In yet another aspect, the present invention provides antibodies that bind
to a polypeptide as described above.
In further aspects, methods are provided for treating a condition
associated with the p38-2 cascade, comprising ~lmini~tering to a patient a
therapeutically effective amount of an agent that modulates signal transduction via the
p38-2 cascade. Such an agent may modulate p38-2 kinase activity and/or may
modulate phosphorylation of p38-2. Within one embodiment, such methods may
reduce a pain sensation in a patient.
.. . ..
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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.
These and other aspects of the present invention will become ~parenl
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 Drawin~s
Figure 1 presents the primary amino acid sequence of p38-2, and splice
variants thereof, using standard one-letter codes.
Figures 2A and 2B are autoradiograms that depict Northem blot
15 analyses of the expression of human p38-2 (Figure 2A) and p38 (Figure 2B) mRNA in
selected human tissues. The position of RNA size markers in kb is shown on the left.
Figure 3 is an autoradiogram that shows the size of in vitro tr~n~!~tt-d
HA-tagged p38-2, as dett?rminP~I by SDS-PAGE. The position of protein size markers
in kDa is shown on the left.
Figure 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/m2; lane 2), anisomycin (50ng/ml; lane 3), or NaCl (200 IlM; lane 4), or cotransfected with 1000 ng of the empty
expression vector Sr(x3 (lane 5), the expression vector for the constitutively active
25 mutant MEK6(DD) (lane 6) or the MAPK TAKl ~N (lane 7).
Figure S presents the nucleotide and amino acid sequence of a native
p38-2 polypeptide.
Figure 6 is an immllnoblot showing the levels of p38 and BRK detected
in NG108-15 cells with polyclonal antibodies. The endogenous p38 and BRK proteins
are shown in the lanes identifled as lysate. In the left panel, antibodies were raised
against the full length p38 protein, and recombinant p38 was used as a standard to
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identify the species that migrates as a band of 38 kD. In the right panel, antibodies
were raised against a unique small peptide derived from BRK, and BRK protein
generated by in vitro transcription and translation in the present of 3sS-methionine
migrates as a band of about 40 kD.
S Figure 7 is an autoradiogram showin~ the results of an
immunoprecipitation assay to evaluate the level of BRK in NG108-15 cells following
treatment with bradykinin. Lanes 1-5 show the levels in untreated cells at 1, 2, 5, 15
and 30 minutes, respectively. Lanes 6-10 show the levels in cells treated with 1 ~lM
bradykinin for 1, 2, 5, 15 and 30 minutes, respectively.
Figures 8A-D show the leakage- and capacitance-subtracted current
traces observed in NG108-15 cells in the presence and absence of inhibitors.
Sequential responses to bradykinin (Figure 8A and C) or Leu-Enk (Figure 8B and D) in
cells dialyzed with 20 ,uM SB203580 (Figures 8A and B) or SKF106978 (Figures 8C
and D) are shown. The lCa v was activated by a 100 ms test comrnand to O mV~ applied
15 every 10 s from a holding potential of -90 mV (sampling to 10 kHz). Leakage- and
capacitance-subtracted current traces are displayed, showing ICa v before (con) and at the
peak of action by bradykinin (0.1 ~M). The continuous line marks the zero current.
Figure 8E and F present a summary of the responses to bradykinin (Figure 8E) andLeu-Enk (Figure 8F). Mean and standard deviations are displayed. The numbers on
20 the left of each bar indicate the number of cells studied.
Figures 9A and 9B show peak Ic,,v-voltage relations in two NG108-15
cells before (open circles) and during (closed circles) application of bradykinin (0.1
~M) after intracellular dialysis of SKF106978 (Figure 9 A; 20 IlM) or SB203580
(Figure 9B; 20 ~lM). Figure 9C presents the time course of the peak ICaV during
25 perfusion with SB203580 (20 IlM) and application of bradykinin and Leu-Enk (both at
0.1 ~M). Between ) and 20 minutes, lCaV was activated every 30 s. In the rem~ining
portion of the time course, ICav was activated every 10 s. Data were acquired at 10
kHz.
Figure 1 OA shows the activation of IK ~K by BK (0.1 ,uM) obtained from
30 two NG108-15 cells, after intrapipette dialysis with SB203580 (20 ~lM, panel Al) or
SKF106978 (20 ~LM, panel A2). Data were acquired at 100 Hz. Figure 1 OB is a graph
.. , . , , , . , _ .
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showing a summary of the IK BK responses to BK (0.1 ~M). The graph displays means
and standard deviations. Figure lOC presents a proposed pathway for the inhibition of
Ica v by BK
Detailed Description of the lnvention
As noted above, the present invention is generally directed to
compositions and methods for mo~ ting (i. e., stimulating or inhibiting) signal
transduction via MAP kinase c~c~des, and for treating conditions associated with such
cascades. In particular, the present invention is directed to compositions comprising a
10 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 native p38-2 sequences, as well
as variants thereof. Preferably, a p38-2 polypeptide is selectively activated bybradykinin. When active, a p38-2 polypeptide is generally capable of activating at least
15 one substrate of p38-2 (e.g, ATF-2, MAPKAP kinase 2, MAPKAP kinase 3, MNK1,
PH,AS-1 and/or Sapl-a). A substrate of a p38-2 polypeptide is said to be "activated" if
its biological or enzymatic activity increases by a statistically significant amount.
Variants of a native p38-2 sequence are modified such that the ability of the variant to
phosphorylate substrates is not substantially (limini~hed.
The present invention also encompasses compositions and methods for
mocl~ ting p38-2 activity. In general, compositions that inhibit p38-2 activity may
inhibit phosphorylation of p38-2, or may inhibit the ability of p38-2 to phosphorylate a
substrate. As used herein, the term "p38-2 cascade" refers to any signal kansduction
pathway that involves p38-2, and such a cascade may include any compound that
25 modulates p38-2 activity or acts as a substrate for p38-2.
p38-2 polypeptide variants within the scope ofthe present invention may
contain one or more substitutions and/or modifications, such that the ability of the
variant to phosphorylate substrates (such as ATF2, MAPKAP kinase 2 and MAPKAP
kinase 3) is not substantially ~limini~hed. In certain preferred embodiments, a variant
30 contains substitutions and/or modifications at no more than 25% of the arnino acid
residues, more preferably at no more than 20% of the amino acid residues and most
.
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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 minim~l influence on the activity of the
5 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.
As noted above, p38-2 polypeptides, including native p38-2 sequences
and variants thereof, may be selectively activated by bradykinin. Bradykinin (BK)
10 inhibits neurotransmitter voltage-dependent calcium currents (ICa v) In the
representative neuroblastoma-glioma cell line NG108-15 (ATCC Accession No. HB-
12317), BK inhibits ICav via the sequential action of at least two G proteins (G,3 and
Racl/Cdc42). It has been found, within the context of the present invention, that that
the inhibitory action of Racl/Cdc42 is mediated by p38-2.
The terrn "selectively activated," as used herein refers to a strong
stimulation (i.e., at least three fold) of kinase activity of a p38-2 polypeptide under
conditions that produce at most only a modest stimulation (i.e., between about 1.5 and 3
fold) of JNK and/or ERK activity and little or no measurable activation (i.e., less than
1.5 fold) of type I and/or type 3 p38 kinases. In general, the selective activation of a
20 p38-2 polypeptide by bradykinin may be evaluated using immunoprecipitation assays
and/or measurements of ICa v Measurerments of ICa v may be performed using a standard
whole-cell patch-clamp technique. Briefly, at least two neurotransmitters, bradykinin
and leu-enkephalin, modulate the amplitude of lCaV in NGl08-15 cells. The effect of
bradykinin, but not that of leu-enkephalin, is sensitive to the block of p38-2 activity.
25 These transmitters may be applied via a capillary tubing positioned near the NG108-15
cells. The ICa v can be recorded on magnetic tape or on computer disk for subsequent
analysis.
Substitutions and/or modifications may also render the polypeptide
constitutively active or inactive. Constitutively active polypeptides display the ability
30 to stimulate substrate phosphorylation in the absence of stimulation, as described
below. Such polypeptides may be identified using the representative assays for p38-2
.. . . .
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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.
DNA sequences encoding a native p38-2 polypeptide may be prepared
by arnplification from a suitable 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. A 1.6 kb cDNA identified using a human cDNA library hasthe sequence provided in SEQ ID NO: I and Figure 5. The encoded p38-2 polypeptide,
shown in SEQ ID NO:2 and Figure l, has a predicted size of 364 arnino 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 Figure l, as well as SEQ ID NO:3 and SEQ ID NO:4.
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 m~mm~ n 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~es~ion vector may, but need not, include DNA encoding an epitope, such that therecombinant 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 oldinary skill in the art.
The DNA sequences expressed in this manner may encode a native p38-
2 polypeptide, or may encode alternate splice variants, portions or other variants of
p38-2. DNA molecules encoding variants of p38-2 may generally be prepared using
-
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standard mutagenesis techniques, such as oligonucleotide-directed site-specific
mutagenesis, and sections of the DNA sequence may be removed to permit prepa~dlion
of truncated polypeptides. For variants of p38-2, any such changes should not flimini~h
the ability of the variant to stimulate phosphorylation of substrates such as ATF2 (see,
5 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, Mc~ aughlin 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 s~lbst~nti~lly change the properties of p38-2.
10 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.
Modifications may also be made in critical regions of p38-2, provided
that the resulting variant substantially retains the ability to stimulate substrate
15 phosphorylation. The effect of any modification on the ability of the variant to
stimul~te substrate phosphorylation may generally be evaluated using any assay for
p38-2 kinase activity, such as the representative assays described herein.
Expressed polypeptides of this invention are generally isolated in
substantially pure form. Preferably, the polypeptides are isolated to a purity of at least
20 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
25 recombinant.
In one aspect of the present invention, p38-2 polypeptides may be used
to identify agents, which may be antibodies or drugs, that modulate (preferably inhibit)
signal transduction via the p38-2 cascade. Modulation includes the suppression of
expression of p38-2 when it is ove[~ essed, as well as suppression of phosphorylation
3~ of p38-2 or the inhibition of the ability of activated (i.e., phosphorylated) p38-2 to
phosphorylate a substrate. For example, a mo~ ting agent may modulate the kinase
.. . . . . ..
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activity of one or more MAPKKs, such as MEK6, thereby inhibiting p38-2 activation.
Known activators of MAPKKs include, but are not limited to, stress-inducing signals
(e.g, UV, osmotic shock, DNA-dslm:~ging agents), anisomycin, LPS, and cytokines.Similarly, compositions that inhibit p38-2 activity by inhibiting p38-2 phosphorylation
5 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 ~nti.~n.~e
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
10 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.
In general, mod~ ting agents may be identified by combining a test
compound with an activated p38-2 polypeptide, or a polynucleotide encoding such a
polypeptide, in ~itro or in vivo, and evaluating the effect of the test compound on the
15 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 [~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
20 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 ofp38-2 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
25 such an assay ranges from about 0.1 ~uM to about 10 ~LM. The effect of the agent on
p38-2 kinase activity may then be evaluated by quan~ g the incorporation of
[3~P]phosphate into ATF2, and comparing the level of incorporation with that achieved
using activated p38-2 without the addition of a candidate agent. Alternativelyl the
incorporation of phosphate into ATF2 may be measured using an antibody specific for
30 phosphorylated substrate, using well known techniques. Within another alternative, a
polynucleotide encoding the kinase may be inserted into an expression vector and the
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11
effect of a composition on transcription of the kinase measured, for example, byNorthern blot analysis
In another aspect of the present invention, a p38-2 polypeptide may be
used for phosphorylating and activating a substrate of p38-2. In one embodiment, a
5 substrate may be phosphorylated in vi~ro 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~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 ~g to about 10 ,ug of
10 p38-2 polypeptide, from about 0.1 ~g to about 10 ~lg 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 [~-32P]ATP to a test aliquot~ and evaluating the level
of substrate phosphorylation as described below.
One or more p38-2 polypeptides, mod~ tin~ agents as described above
and/or polynucleotides encoding such polypeptides and/or mod~ tin~ agents may also
be used to modulate p38-2 activity in a patient. As used herein, a "patient" may be any
m~mm~l, including a human, and may be afflicted with a condition associated with the
p38-2 cascade or may be free of detectable disease. Accordingly, the treatment may be
20 of an existing disease or may be prophylactic. Conditions associated with the p38-2
cascade include any disorder which is etiologically linked to MAP kinase activity,
including cardiovascular disease, immune-related (li.se~ees (e.g., infl~mm~tory diseases,
autoimmune tliee~ee~s7 m~ n~nt cytokine production or endotoxic shock), cell growth-
related diseases (e.g., cancer, metabolic tli.se~ses, abnormal cell growth and
25 proliferation or cell cycle abnormalities) and cell regeneration-related diseases (e.g
cancer, degenerative ~lise~ees, 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 apl,ropliate for treatment with p38-2
30 polypeptides include osteoarthritis, ischemia, reperfusion injury, trauma, certain cancers
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12
and viral disorders, and autoimmune ~iee~es such as rheumatoid arthritis, multiple
sclerosis, psoriasis, infl~mm~tory bowel disease, and other acute phase responses.
It has been found, within the context of the present invention, that pain is
also a condition associated with the p38-2 c~ec~de, and that pain may be treated by
5 inhibiting p38-2 kinase activity. Bradykinin is a major mediator of pain, acting on the
peripheral endings of dorsal root ganglion neurons. One of bradykinin's actions in
these cells is the inhibition of ICav~ which allows a faster rate of action potential
generation (the electrophysical substrate of the pain sensation). Pain within a patient
may be reduced by inhibiting p38-2 kinase activity, without the vascular side effects of
10 bradykinin antagonists and without the addictive risks of opioids.
Treatment may include ~.lmini.ctration of a p38-2 polypeptide and/or a
compound which modulates p38-2 activity. For ~mini.ctration to a patient, one ormore polypeptides (and/or mod~ ting agents) are generally formulated as a
pharrnaceutical composition. A pharmaceutical composition may be a sterile aqueous
lS 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
ph~rm~ceutical composition may additionally contain preservatives and/or other
additives such as, for example, antimicrobial agents, anti-oxidants, chelatin~ agents
and/or inert gases, and/or other active ingredients.
2~ Alternatively, a pharmaceutical composition may comprise a
polynucleotide encoding a p38-2 polypeptide, and/or mod~ ting agent, such that the
polypeptide and/or mod~ ting agent is generated in situ, in combination with a
physiologically acceptable carrier. In such ph~rm~reutical compositions, the
polynucleotide may be present within any of a variety of delivery systems known to
30 those of ordinary skill in the art, including nucleic acid, bacterial and viral expression
systems, as well as colloidal dispersion systems, including liposomes. Appropriate
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nucleic acid expression systems contain the necessary polynucleotide sequences for
expression in the patient (such as a suitable promoter and terrnin~ting signal). DNA
may also be "naked," as described, for example, in Ulmer et al., Science 259:1745-1749
(1993).
5Various 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
10limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus
(HaMuSV), murine m~mm~ry 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
15specific). 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
Viral vectors are typically non-pathogenic (defective), replication
competent viruses, which require ~ t~nce in order to produce infectious vector
20particles. 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 mech~ni~m to recognize an RNA transcript for
encapsulation. Such helper cell lines include (but are not limited to) ~2, PA317 and
25PA12. 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
vlrlons.
Another targeted delivery system for p38-2 polynucleotides is a colloidal
30dispersion system. Colloidal dispersion systems include macromolecule complexes,
nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water
. . .
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14
emulsions, micelles, mixed micelles, and liposomes. A prefe.led 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 l-nil~mell~r vesicles (LUV), which range in size
from 0.2-4.0 !lm can encapsulate a substantial percentage of an aqueous buffer
5 cont~ining 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 m~nnm~ n 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
10 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
1 5 6:882, 1 988).
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.
20 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 ch~nging the composition or size of the liposome in order to achieve
25 targeting to organs and cell types other than the naturally occurring sites of localization.
Routes and frequency of ~(lmini.~tration, as well doses, will vary from
patient to patient. In general, the pharmaceutical compositions may be ~imini.~tered
intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity or
tr~n~lerm~lly. Between 1 and 6 doses may be ~mini.~tered daily. A suitable dose is
30 an amount of polypeptide or polynucleotide that is sufficient to show improvement in
the symptoms of a patient afflicted with a condition associated with the p38-2 cascade.
CA 022~79 1998-11-18
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~ 15
Such improvement may be detected based on a determination of relevant cytokine
levels (e.g., IL-l, IL-6 and/or IL-8), by monitoring infl~mm~tory responses (e.g,
~ edema, transplant rejection, hypersensitivity) or through an improvement in clinical
symptoms associated with the condition (e.g., pain). In general, the amount of
5 polypeptide present in a dose, or produced in si~u by DNA present in a dose, ranges
from about 1 ,ug to about 250 llg per kg of host, typically from about 1 ~lg to about 60
,ug. 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.
The present invention also provides methods for ~letectin~ the level of
10 mitogen activated protein kinase kinase (such as MEK6) activity in a sarnple. 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
15 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 [~-32P]ATP in asuitable buffer (such as 20 mM HEPES (pH 7.6), 5 mM MnCI2, 10 mM MgCl2, 1 mM
dithiothreitol) for 30 minutes at 30~C. In general, approximately l ~g recombinant
p38-2, with 50 nM [7/-32P]ATP, is sufficient. Proteins may then be separated by SDS-
20 PAGE on 10% gels and subjected to autoradiography. Incorporation of [32P]phosphateinto p38-2 may be quantitated using techniques well known to those of ordinary skill in
the art, such as with a phosphorimager. lt will be apparent to those of ordinary skill in
the art, that this assay may also be performed with unlabeled phosphate, using an
antibody specific for phosphorylated substrate (i. e., the antibody binds to
25 phosphorylated substrate, and does not bind significantly to unphosphorylatedsubstrate, such that the antibody can be used to distinguish between the two forms) and
standard methods.
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
30 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
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16
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 MgCl2, the p3~-2
(0.1-10 ~g) may be incubated with ATF2 (0.1-10,ug) and ~y-32P]ATP (10-500 nM) in a
buffer cont~ining 20 mM HEPES (pH 7.6), 20 mM MgC12. It should be noted that
5 alternative buffers may be used and that buffer composition can vary without
significant effect 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.
To evaluate the effect of an antibody or other candidate mo(l~ ting
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 moc~ ting agent is added to the
incubation mixture. The candidate agent may be preincubated with MAPKK before
15 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.
All these assays can further be modified by removing the candidate agent after the
20 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 ~LM to about 10 ~M. The effect of
the agent on phosphorylation of p38-2 may then be evaluated by quantitating the
incorporation of ~32P]phosphate into p38, as described above, and compa~ g the level
of incorporation with that achieved using MAPKK without the addition of the
25 candidate agent.
p38-2 activity may also be measured in whole cells transfected with a
reporter gene whose expression is dependent upon the activation of an ap~ ,liatesubstrate, such as ATF2. For example, ap~)ro~l;ate cells (i.e., cells that express p38-2)
may be transfected with an ATF2-dependent promoter linked to a reporter gene such as
30 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
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17
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 aconstitutively active variant of MAPKK, such as MEK6. Candidate mocl~ tin~ agents
may be added to the system? as described above, to evaluate their effect on the p38-2
5 cascade.
Altematively, a whole cell system may employ only the transactivation
domain of ATF2 fused to a suitable DNA binding domain, such as GHF-l or GAL~.
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
10 on ATF2-specific gene activation.
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
15 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:l. To detect p38-2 protein, thereagent 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
20 antibody to detect a polypeptide in a sample. See, e.g., Harlow and I,ane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For exarnple? 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 ~lçtect~hle
25 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
30 sample. Suitable reporter groups for use in these methods include, but are not limited
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18
to, enzymes (e.g., horseradish peroxidase), substrates, cofactors, inhibitors, dyes,
radionuclides, l-lmin~ sc~nt groups, fluorescent groups and biotin.
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.
5 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 ~mini~tered to a patient
(either prophylactically or for treatment of an existing disease) to modulate the
activation of p38-2 in vivo.
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, Anfibodies. A Lahoratory
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
15 incorporating one or more booster immunizations, and the ~nim~ are bled
periodically. Polyclonal antibodies specific for the polypeptide may then be purified
from such antisera by, for exarnple, affinity chromatography using the polypeptide
coupled to a suitable solid support.
Monoclonal antibodies specific for p38-2 or a variant thereof may be
20 prepared, for example, using the technique of Kohler and Milstein, ~ur. J. ~mmunol
6:511-519, 1976, and improvements thereto. Briefly, these methods involve the
aldtion 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
25 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 immunizedanimal. 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
30 selection techni~ue uses HAT (hypox~nthine, aminopterin, thymidine) selection. After
a sufficient time~ usually about 1 to 2 weeks, colonies of hybrids are observed. Single
CA 022~79 1998-11-18
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19
colonies are selected and tested for binding activity against the polypeptide.
Hybridomas having high reactivity and specificity are preferred.
Monoclonal antibodies may be isolated from the supernatants of
growing hybridoma colonies. In addition, various techniques may be employed to
5 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. ContAnnin~nt~ may be removed from the
antibodies by conventional techniques, such as chromatography, gel filtration,
precipitation, and extraction.
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. MAPKE~ 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
org~ni.cmc and fluids found within living org~ni.~m~. 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.
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.
CA 022~79 1998-11-18
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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
5 may be a substrate that is phosphorylated on}y 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
10 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 kinaseactivity (e.g., antibody specific for phosphorylated substrate, which may be used to
distinguish phosphorylated substrate from unphosphorylated substrate) may also be
15 provided. Such kits may be employed in direct or coupled kinase assays, which may be
performed as described above.
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
20 proteins that interact with p38-2. Alternatively, an expression library may be
sequenced for cDNAs that phosphorylate p38-2.
The following Examples are offered by way of illustration and not by
way of limitation.
.
CA 022.,.,.,79 1998 - 11 - 18
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21
EXAMPLES
Example 1
Clonin~ and Sequencin~ cDNA Encoding p38-2
This Example illustrates the cloning of a cDNA molecule encoding the
human MAPK p38-2.
The Expressed Sequence Tags (EST) subdivision of the National Center
for Biotechnology Information (NCBI) Genbank ~ b~nk was searched with the
tblastn prograrn and the human p38 amino acid sequence (Han et al., Science 265:808-
811, 1994; Lee etal., Nature 372:739-746, 1994) as query using the BLAST e-mail
server. The EST sequence R72598 from a breast cDNA library displayed the highestsimilarity score. A clone corresponding to the EST sequence R72598 was obtained
from Research Genetics Inc., (Huntsville, AL), and the insert size was deterrnined 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 mi~.cing. 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, CA), and verified by miniprep analysis.
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, CA), and the sequence of
the full length clone without intron is shown in SEQ ID NO: 1 and Figure 5.
Example 2
In vivo Expression of p38-2
This Example illustrates the expression of p38-2, as compared to p38, in
various human tissues.
Northern blots were performed using 2 llg of polyA~ RNA isolated from
16 different adult human tissues~ fractionated by denaturing formaldehyde 1.2%
CA 022~79 1998-11-18
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22
agarose gel electrophoresis, and transferred onto a charge-modified nylon membrane
(Clontech Laboratories, Palo Alto, CA). 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, CA) according to the manufacturer's instructions.
5 Both probes were prepared by labeling the cDNA with [a-3~P]dCTP (NEN, Boston,
MA) by random priming (Stratagene, La Jolla, CA). For control purposes, the blots
were also hybridized to a radiolabeled ~-actin probe.
The results, shown in Figures 2A and 2B, demonstrate that p38-2 is
widely expressed in many adult human tissues, with highest levels in heart and skeletal
10 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 ,B-actin mRNA (not shown).
Example 3
Preparation of p38-2
This Example illustrates the in vitro translation of HA-tagged p38-2.
HA tagged p38-2 was in vitro transcribed and tr~n.~l~ted using the
Promega TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI) in the
20 presence of 35S-methionine using SP6 polymerase and the template DNA 3xHA-p38-
2-SRa3). Radioactive, in vitro-translated proteins were separated by SDS-PAGE and
vi~ li7Pd by autoradiography (Figure 3).
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-inducin~ Agents
This Example illustrates the activation of p38-2 by a variety of
stimulators of the MAPK pathway.
An ex,ulession vector encoding epitope-tagged p38-2 (3xHA-p38-2-
SRa3) was constructed by adding sequence encoding three copies of a 10 amino acid
CA 022~79 1998-11-18
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23
hemagglutinin (HA) epitope to the N-terminus of p38-2 and ligating the resultingcDNA into the expression vector SRoc3. To investigate the pattern of regulation of p38-
2, COS cells were transiently transfected with HA-p38-2-SRa3 (as described above) by
the DEAE-Dextran method (Kawai and Nishizawa, Mol. Cell. Biol. 4:1172-1174,
5 1984). These cells were then treated with various stimulators of the MAPK pathway
(i.e., treated for 45 minllte~ with UV (250 nm~ 120 J/m2), anisomycin (50 ng/ml) or
NaCI (200 ~lM) or cotransfected with 1000 ng of the empty expression vector Sra3, the
expression vector for the constitutively active mutant MEK6(DD) or the MAPK
TAKl~N).
Following treatment, cell Iysates were prepared by solubilization in Iysis
buffer as described (Derijard et al., Cell 76:1025-1037, 1994), and protein
concentration of Iysates was deterrnined by Bradford assay (Bradford, Ann. Biochem
72:248-254, 1976). Cell Iysates were used in an immune complex kinase assay withGST-ATF2 substrate, prepared as previously described (Gupta et al., Science 267:389
15 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 [~-~2P]ATP was 50 nM, and 30 ~g cell Iysate was
immunoprecipitated for 2 hours with the anti-HA antibody 12CA5 (Boehringer-
Mannheim Corp.. Indi~n:~polis7 IN) and then incubated with I ,ug of recombinant
20 substrate. Reactions were separated by SDS-PAGE, and the results are presented in
Figure 4.
These experiments showed a strong induction of p38-2 by UV (254 nm;
120 J/m2 for 45 minutes), anisomycin (50 ng/mL for 45 minutes) and MEK6(DD),
constitutively active variant of MEK6 (Fig. 4). No increase in p38-2 activity was
25 observed when the cells were treated with NaCI, expression vector alone or the MAP
kinase TAKl~N (see Yamaguchi et al., Science 270:2008-2011, 1995). This clearly
indicates that the inducible phosphorylation of ATF2 depends on a kinase cascadecomprised of MEK6 and p38-2.
CA 022~79 1998-11-18
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24
Example 5
Selective Activation of BRK by Bradykinin
This Exarnple illustrates the ability of bradykinin to potently and
selectively activate BRK.
An initial experiment was performed to identify the MAPK pathways
represented in NG108-lS cells (ATCC Accession No. HB-12317). The cells were
grown according to published methods (see Hamprecht et al., Methods in Enzymol.
109:316-347, 1985). Immunoblot analyses were perforrned on extracts using
polyclonal antibodies raised against p38 and a small peptide uni~ue to BRK. As shown
in Figure 6, these kinases could be clearly detected.
Immunoprecipitation assays were then performed to investigate
stimulation by bradykinin. The results, presented in Figure 7, show that bradykinin
stimulates BRK with kinetics similar to the effect of bradykinin on lCaV. Thus, in
NG108-15 cells, bradykinin strongly stimulates BRK, only modestly stimulates JNKl S and ERK, and does not measurably activate type 1 p38 kinases.
Example 6
Effect of Inhibitors of ERK and BRK Kinase Activity on
Inhibition of Ir~ v bv Bradykinin
This Example illustrates the use of an inhibitor of BRK to block the
inhibition of ICa v by bradykinin.
Activation of ERK and p38 kinases can be selectively blocked by the
compounds PD98059 (see Pang et al., J. Biol. Chem. 270:13585-13588, 1995) and
SB203580 (see Lee et al., Nature 372:739-746, 1994), respectively. PD98059 (Parke-
Davis) and SB203580 (SmithKline Beecham) were used to investigate which MAPK
pathway mediates inhibition of ICav by bradykinin (Figures 8A-F). After intracellular
dialysis of each compound, the inhibition of lCa v by bradykinin was examined first and
the inhibition by Leu-Enkphalin (Leu-Enk) was measured thereafter. The inhibition of
ICaV by bradykinin was blocked after application of SB203580 (20 ~IM), whereas that
produced by Leu-Enk was retained (Figures 8A-B). Application of an inactive analog
SKF106978 (20 ~lM (SmithKline Beecham); see Lee et al., Nature 372:739-746, 1994)
CA 022~79 1998-11-18
WO 97144467 PCT/US97/08738
did not block the inhibition of ICav by bradykinin or Leu-Enk (Figures 8C-D). After
application of PD98059 (20 ~lM), the inhibition ~f ICDV by either transmitter was not
~ttenl~ted (Figures 8E-F). Similarly, application of the dimethylsulfoxide-cont~ining
vehicle was without effect. A sum~nary of the results of the experiments with PD98059
5 and SB203580 is presented in Figures 9E-F.
Bradykinin and Leu-Enk inhibit the same component of ICav (see Wilk-
Bl~7r7~k et al., Neuron 13:1215-1224, 1994) but only the bradykinin response is
blocked by the p38 kinase inhibitor SB203580. Therefore, it is unlikely that the effect
of this compound is due to direct suppression of ICa v This conclusion was confirmed
10 by studying the direct effect on peak ICa v ~f SB203580 over a broad range of membrane
potentials (Figure 9B). As a control, current-voltage curves were obtained afterdialyzing the cells with SKF106978 (Figure 9A). In all cells treated with SB203580 (n
= 2), the current-voltage relation was not altered significantly compared to control cells
(n = 1), while the inhibition by BK was suppressed over the entire range of membrane
15 potentials examined. These fin-ling~ were consistent in all cells studied. The lack of
any direct action of SB203580 on ICav was further conf1rmed by monitoring the peak
lCa v for the entire duration of the experiment (Figure I OC; n = 11).
The specificity of SB203580 for the G,3 pathway was further tested by
e~c~mining whether this compound blocks a second response to BK, activation of a20 voltage-independent K+ current (IK BK). This response is mediated by a distinct
heterotrimeric G protein Gq/, l (see Wilk-Bl~c7~7slk et al., Neuron 12: 1 09, 1 994). IK BK
was measured as described in Wilk-BI~7~7~k et al., Neuron 12:109, 1994 in cells
dialyzed either with the p38 inhibitor SB203580 or with the inactive analog
SKF106978 (see Figure 10A). In all cells tested, dialysis with SB203580 did not
25 reduce activation ~f IKBK by BK, compared to dialysis with the inactive SKF106978
(Figure 9C).
The above results demonstrate that activation of BRK is required for the
inhibitory effect of BK on lCaV. Involvement of this kinase in the pathway of BK is
strongly supported by the observation that BK potently activates this isoform of the
30 enzyme only, following a time course similar to that of the BK response, and that the
p38 inhibitor SB203580 blocks the effects of BK both on the kinase and on the lca
.
CA 022~79 1998-11-18
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26
The effect of SB203580 is selective, because it does not extend to the activation Of IKB~
by BK, or to the inhibition of ICa v by Leu-Enk. Involvement of JNK in the inhibition of
ICaV is unlikely, because this kinase is only weakly stimulated by BK, and is blocked
only by high concentrations of SB203580. The ERK kinase is also not involved,
5 because the inhibition of ICa v by BK is not sensitive to the MEK inhibitor PD98059.
The above data define a novel role for MAP kinase pathways in the
modulation of ion channels by neurotransmitters. In contrast to the growth factor-
initiated effects of MAP kinases on cellular proliferation and differentiation, the action
of BRK kinase on ICav is triggered by a G protein-coupled serpentine receptor and
10 unfolds over a short time scale. Thus, MAP kinase pathways play roles similar to those
of the classical second messenger pathways.
From the foregoing, it will be appreciated that, although specific
embodiments of the invention have been described herein for the purpose of
15 illustration, various modifications may be made without deviating from the spirit and
scope of the invention.
~_ .
CA 02255579 l998-ll-l8
W 0 97/44467 27 PCT~US97/08738
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Signal Pharmaceuticals, Inc.
(ii) TITLE OF INVENTION: MITOGEN-ACTIVATED PROTEIN KINASE p38-2
AND METHODS OF USE THEREFOR
(iii) NUMBER OF SEQUENCES: 4
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SEED and BERRY LLP
(B) STREET: 6300 Columbia Center, 701 Fifth Avenue
(C) CITY: Seattle
(D) STATE: Washington
(E) COUNTRY: USA
(F) ZIP: 98104-7092
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC ~ompatible
(C) OPERATING SYSTEM: PC-DOS/MS-DO.5
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 20-MAY-1997
(C) CLASSIFICATION:
(viii) ATTORNEY/AGEN~ INFORMATION:
(A) NAME: Maki, David J.
(B) REGISTRATION NUMBER: 31,392
(C) REFERENCE/DOCKET NUMBER: 860098.412PC
.
CA 022~79 1998-11-18
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28
~ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (206) 622-4900
(B) TELEFAX: (206) 682-6031
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQ~ENCE CHARACTERISTICS:
(A) LENGTH: 1502 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQ~ENCE DESCRIPTION: SEQ ID NO:l:
GGACATGTCG GGCCCTCGCG CCGGCTTCTA CCGGCAGGAG CTGAACAAGA CCGTGTGGGA 60
GGTGCCCCAC CCCCTCCAGG GGCTGCGCCC GGTCGGCTCC GGCGCCTACG GCTCCGTCTG 120
TTCGGCCTAC GACGCCCGGC TGCGCCAGAA GGTGGCGGTG AAGAAGCTGT CGCGCCCCTT 180
CCAGTCGCTG ATCCACGCGC GCAGAACGTA CCGGGAGCTG CGGCTGCTCA AGCACCTGAA 240
GCACGAGAAC GTCATCGGGC TTCTGGACGT CTTCACGCCG GCCACGTCCA TCGAGGACTT 300
CAGCGAAGTG TACTTGGTGA CCACCCTGAT GGGCGCCGAC CTGAACAACA TCGTCAAGTG 360
CCAGGCGCTG AGCGACGAGC ACGTTCAATT CCTGGTTTAC CAGCTGCTGC GCGGGCTGAA 420
GTACATCCAC TCGGCCGGGA TCATCCACCG GGACCTGAAG CCCAGCAACG TGGCTGTGAA 480
CGAGGACTGT GAGCTCAGGA TCCTGGATTT CGGGCTGGCG CGCCAGGCGG ACGAGGAGAT 540
CA 022~79 l998-ll-l8
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GACCGGCTAT GTGGCCACGC GCTGGTACCG GGCACCTGAG ATCATGCTCA ACTGGATGCA 600
TTACAACCAA ACAGTGGATA TCTGGTCCGT GGGCTGCATC ATGGCTGAGC TGCTCCAGGG 660
CAAGGCCCTC TTCCCGGGAA GCGACTACAT TGACCAGCTG AAGCGCATCA TGGAAGTGGT 720
GGGCACACCC AGCCCTGAGG TTCTGGCAAA AATCTCCTCG GAACACGCCC GGACATATAT 780
CCAGTCCCTG CCCCCCATGC CCCAGAAGGA CCTGAGCAGC ATCTTCCGTG GAGCCAACCC 840
CCTGGCCATA GACCTCCTTG GAAGGATGCT GGTGCTGGAC AGTGACCAGA GGGTCAGTGC 900
AGCTGAGGCA CTGGCCCACG CCTACTTCAG CCAGTACCAC GACCCCGAGG ATGAGCCAGA 960
GGCCGAGCCA TATGATGAGG GCGTTGAGGC CAAGGAGCGC ACGCTGGAGG AGTGGAAGGA 1020
GCTCACTTAC CAGGAAGTCC TCAGCTTCAA GCCCCCAGAG CCACCGAAGC CACCTGGCAG 1080
.
CCTGGAGATT GAGCAGTGAG GTGCTGCCCA GCAGCCCCTG AGAGCCTGTG GAGGGGCTTG 1140
GGCCTGCACC CTTCCACAGC TGGCCTGGTT TCCTCGAGAG GCACCTCCCA CACTCCTATG 1200
GTCACAGACT TCTGGCCTAG GACCCCTCGC CTTCAGGAGA ATCTACACGC ATGATGGAGC 1260
TGATCCAGTA ACCTCGGAGA CGGGACCCTG CCCAGAGCCG AGTTGGGGGT GTGGCTCTCC 1320
CCTGGAAAGG GGGTGACCTC TTGCCTCGAG GGGCCCAGGG AAGCCTGGGT GTCAAGTGCC 1380
TGCACCAGGG GTGCACAATA AAGGGGGTTC TCTCTAAAAA AAAAAAAAAA AAAAAAAAAA 1440
AAAAAAAA~G CGGCCGCTGA ATTCTACCTG CCCGGGCGGC CGCTCGAGCC CTATAGTGAG 1500
TA 1502
(2) INFORMATION FOR SEQ ID NO:2:
.. . . . .
CA 022~79 l998-ll-l8
W O 97/44467 PCT~US97/08738
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 364 amino acids
(B) TYPE: amlno acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQ~ENCE DESCRIPTION: SEQ ID NO:2:
Met Ser Gly Pro Arg Ala Gly Phe Tyr Arg Gln Glu Leu Asn Lys Thr
1 5 10 15
Val Trp Glu Val Pro Gln Arg Leu Gln Gly Leu Arg Pro Val Gly Ser
Gly Ala Tyr Gly Ser Val Cys Ser Ala Tyr Asp Ala Arg Leu Arg Gln
Lys Val Ala Val Lys Lys Leu Ser Arg Pro Phe Gln Ser Leu Ile His
Ala Arg Arg Thr Tyr Arg Glu Leu Arg Leu Leu Lys His Leu Lys His
Glu Asn Val Ile Gly Leu Leu Asp Val Phe Thr Pro Ala Thr Ser Leu
Glu Asp Phe Ser Glu Val Tyr Leu Val Thr Thr Leu Met Gly Ala Asp
100 105 110
Leu Asn Asn Ile Val Lys Cys Gln Ala Leu Ser Asp Glu His Val Gln
115 120 125
CA 022~79 l998-ll-l8
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31
Phe Leu Val Tyr Gln Leu Leu Arg Gly Leu Lys Tyr Ile His Ser Ala
130 135 140
Gly Ile Ile His Arg Asp Leu Lys Pro Ser Asn Val Ala Val Asn Glu
145 150 155 160
~sp Cys Glu Leu Arg Ile Leu Asp Phe Gly Leu Ala Arg Gln Ala Asp
165 170 175
~lu Glu Met Thr G~ y Tyr Val Ala Thr Arg Trp Tyr Arg Ala Pro Glu
180 185 190
Ile Met Leu Asn Trp Met His Tyr Asn Gln Thr Val Asp Ile Trp Ser
195 200 205
Val Gly Cys Ile Met Ala Glu Leu Leu Gln Gly Lys Ala Leu Phe Pro
210 215 220
Gly Ser Asp Tyr Ile Asp Gln Leu Lys Arg Ile Met Glu Val Val Gly
225 230 235 240
~hr Pro Ser Pro Glu Val Leu Ala Lys Ile Ser Ser Glu His Ala Arg
245 250 255
~hr Tyr Ile Gln Ser Leu Pro Pro Met Pro Gln Lys Asp Leu Ser Ser
260 265 270
Ile Phe Arg Gly Ala Asn Pro Leu Ala Ile Asp Leu Leu Gly Arg Met
275 280 285
Leu Val Leu Asp Ser Asp Gln Arg Val Ser Ala Ala Glu Ala Leu Ala
290 295 300
His Ala Tyr Phe Ser Gln Tyr His Asp Pro Glu Asp Glu Pro Glu Ala
305 310 315 320
CA 022~79 1998-11-18
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Glu Pro Tyr Asp Glu Gly Val Glu Ala Lys Glu Arg Thr Leu Glu Glu
325 330 335
Trp Lys Glu Leu Thr Tyr Gln Glu Val Leu Ser Phe Lys Pro Pro Glu
340 3~5 350
Pro Pro Lys Pro Pro Gly Ser Leu Glu Ile Glu Gln
355 360
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 amino acids
(B) TYPE: amlno acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Ser Gly Pro Arg Ala Gly Phe Tyr Arg Gln Glu Leu Asn Lys Thr
1 5 10 15
Val Trp Glu Val Pro Gln Arg Leu Gln Gly Leu Arg Pro Val Gly Ser
Gly Ala Tyr Gly Ser Val Cys Ser Ala Tyr Asp Ala Arg Leu Arg Gln
Lys Val Ala Val Lys Lys Leu Ser Arg Pro Phe Gln Ser Leu Ile His
CA 022~79 1998-11-18
W 0 97/44467 PCTtUS97tO8738
33
Ala Arg Arg Thr Tyr Arg Glu Leu Arg Leu Leu Lys His Leu Lys His
Glu Asn Val Ile Gly Leu Leu Asp Val Phe Thr Pro Ala Thr Ser Ile
Glu Asp Phe Ser Glu Val
100
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEOUENCE CHARACTERISTICS:
(A) LENGTH: 155 amino acids
(B) TYPE: amino acid
~C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Me~ Ser Gly Pro Arg Ala Gly Phe Tyr Arq Gln Glu Leu Asn Lys Thr
1 5 10 15
Val Trp Glu Val Pro Gln Arg Leu Gln Gly Leu Arg Pro Val Gly Ser
Gly Ala Tyr Gly Ser Val Cys Ser Ala Tyr Asp Ala Arg Leu Arg Gln
Lys Val Ala Val Lys Lys Leu Ser Arg Pro Phe Gln Ser Leu Ile His
Ala Arg Arg Thr Tyr Arg Glu Leu Arg Leu Leu Lys His Leu Lys His
.,
CA 02255579 l998-ll-l8
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34
Glu Asn Val Ile Gly Leu Leu Asp Val Phe Thr Pro Ala Thr Ser Ile
~lu Asp Phe Ser Glu Val Tyr Leu Val Thr Thr Leu Met Gly Ala Asp
100 105 110
Leu Asn Asn Ile Val Lys Cys Gln Ala Leu Ser Asp Glu Hls Val Gln
115 120 125
Phe Leu Val Tyr Gln Leu Leu Arg Gly Leu Lys Tyr Ile Hls Ser Ala
130 135 140
Gly Ile Ile Hls Arg Val Gly Ala Thr Ala Gly
145 150 155
