CA2255579A1 - Mitogen-activated protein kinase p38-2 and methods of use therefor - Google Patents

Abstract

Compositions and methods are provided for the treatment of conditions associated with mitogen-activated protein kinase cascades. In particular, the mitogen-activated protein kinase p38-2, and polypeptide variants thereof that stimulate phosphorylation and activation of substrates such as ATF2, are provided. The polypeptides may be used, for example, to identify antibodies and other agents that inhibit signal transduction via the p38-2 kinase cascade. The polypeptides and agents may be used in a variety of methods, such as in the reduction of pain sensations.

Description

CA 022~5~79 1998-11-18 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 .. . ..

CA 022~79 1998-11-18 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 CA 022~79 1998-11-18 W O 97/44467 PCT~US97tO8738 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.

.. . ..

CA 022~79 1998-11-18 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 CA 022~79 1998-11-18 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 .. , . , , , . , _ .

CA 022~79 1998-11-18 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 .

CA 022~79 1998-11-18 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 .. . . .

CA 022~79 1998-11-18 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 -CA 022~79 1998-11-18 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 .. . . . . ..

CA 022~79 1998-11-18 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 CA 022~79 1998-11-18 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 CA 0225~579 1998-11-18 W O 97/44467 PCT~US97/08738 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 CA 022~79 l998-ll-l8 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 . . .

CA 022~79 1998-11-18 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 ~ 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 CA 022~79 1998-ll-18 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 CA 022.?.?.?79 1 998 - I I - I 8 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 CA 022~79 1998-11-18 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 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 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 WO 97/44467 PCT~US97/08738 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 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 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 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 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 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
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CA 022~79 1998-11-18 ~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:

CA 022~79 l998-ll-l8 W O 97/44467 PCTrUS97/08738 .

AAAAAAAA~G CGGCCGCTGA ATTCTACCTG CCCGGGCGGC CGCTCGAGCC CTATAGTGAG 1500 (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 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 Leu Asn Asn Ile Val Lys Cys Gln Ala Leu Ser Asp Glu His Val Gln CA 022~79 l998-ll-l8 WO 97t44467 PCT/US97/08738 Phe Leu Val Tyr Gln Leu Leu Arg Gly Leu Lys Tyr Ile His Ser Ala Gly Ile Ile His Arg Asp Leu Lys Pro Ser Asn Val Ala Val Asn Glu ~sp Cys Glu Leu Arg Ile Leu Asp Phe Gly Leu Ala Arg Gln Ala Asp ~lu Glu Met Thr G~ y Tyr Val Ala Thr Arg Trp Tyr Arg Ala Pro Glu Ile Met Leu Asn Trp Met His Tyr Asn Gln Thr Val Asp Ile Trp Ser Val Gly Cys Ile Met Ala Glu Leu Leu Gln Gly Lys Ala Leu Phe Pro Gly Ser Asp Tyr Ile Asp Gln Leu Lys Arg Ile Met Glu Val Val Gly ~hr Pro Ser Pro Glu Val Leu Ala Lys Ile Ser Ser Glu His Ala Arg ~hr Tyr Ile Gln Ser Leu Pro Pro Met Pro Gln Lys Asp Leu Ser Ser Ile Phe Arg Gly Ala Asn Pro Leu Ala Ile Asp Leu Leu Gly Arg Met Leu Val Leu Asp Ser Asp Gln Arg Val Ser Ala Ala Glu Ala Leu Ala His Ala Tyr Phe Ser Gln Tyr His Asp Pro Glu Asp Glu Pro Glu Ala CA 022~79 1998-11-18 W O 97/44467 PCTrUS97/08738 Glu Pro Tyr Asp Glu Gly Val Glu Ala Lys Glu Arg Thr Leu Glu Glu 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 (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 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 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 (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 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 W O 97/44467 PCTruS97/08738 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 Leu Asn Asn Ile Val Lys Cys Gln Ala Leu Ser Asp Glu Hls Val Gln Phe Leu Val Tyr Gln Leu Leu Arg Gly Leu Lys Tyr Ile Hls Ser Ala Gly Ile Ile Hls Arg Val Gly Ala Thr Ala Gly

Claims (30)

Claims

1. A polypeptide comprising an amino acid sequence as recited in SEQ ID NO:2, or a variant thereof that differs only in substitutions and/or modifications at no more than 25% of the amino acid residues.

2. A constitutively active variant of a polypeptide according to claim 1.

3. A polypeptide comprising the amino acid sequence recited in SEQ ID NO:2, modified at no more than 25% of the amino acid residues, such that said polypeptide is rendered constitutively inactive.

4. A polypeptide capable of activating a substrate of p38-2, wherein said polypeptide is selectively activated by bradykinin.

5. A polypeptide according to claim 4, wherein said polypeptide comprises a variant of SEQ ID NO:2 that differs in substitutions and/or modifications at more than 25% of the amino acid residues.

6. An isolated DNA molecule encoding a polypeptide according to any of claims 1-5.

7. An isolated DNA molecule according to claim 6, wherein the DNA molecule comprises the nucleotide sequence provided in SEQ ID NO: 1.

8. A recombinant expression vector comprising a DNA molecule according to claim 6.

9. A host cell transformed or transfected with an expression vector according to claim 8.

10. A host cell according to claim 9, wherein the host cell is selected from the group consisting of bacteria, yeast, baculovirus infected insect cells and mammalian cells.

11. A method for phosphorylating a substrate of p38-2, comprising contacting a polypeptide according to either of claims 1 or 4 with a substrate of p38-2, thereby phosphorylating the substrate of p38-2.

12. The method of claim 11, wherein the substrate of p38-2 is selected from the group consisting of ATF2, MAPKAP kinase 2 and MAPKAP kinase 3.

13. A polypeptide according to either of claims 1 or 4 in combination with a pharmaceutically acceptable carrier, for use in the manufacture of a medicament for activating a substrate of p38-2 in a patient.

14. The polypepdite of claim 13, wherein the substrate of p38-2 is selected form the group consisting of ATF2, MAPKAP kinase 2 and MAPKAP kinase 3.

15. A method for screening for an agent that modulates signal transduction via the p38-2 cascade, comprising:
(a) contacting a candidate agent with a polypeptide according to either of claims 1 or 4, wherein the step of contacting is carried out under conditions and for a time sufficient to allow the candidate agent and the polypeptide to interact;
and (b) subsequently measuring the ability of said candidate agent to modulate p38-2 activity, and thereby evaluating the ability of the candidate agent to modulate signal transduction via the p38 cascade.

16. A method for screening for an agent that modulates signal transduction via the p38-2 cascade, comprising:
(a) contacting a candidate agent with a polynucleotide encoding a polypeptide according to either of claims 1 or 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 said candidate agent to modulate p38-2 activity, and thereby evaluating the ability of the candidate agent to modulate signal transduction via the p38-2 cascade.

17. An antibody that binds to a polypeptide according to either of claims 1 or 4.

18. An antibody according to claim 17, wherein said antibody inhibits the phosphorylation of substrate by said polypeptide.

19. An agent that modulates signal transduction via the p38-2 cascade, for use in the manufacture of a medicament for treating a condition associated with the p38-2 cascade.

20. An agent that modulates p38-2 kinase activity, for use in the manufacture of a medicament for treating a patient afflicted with a condition associated with the p38-2 cascade.

21. An agent according to claim 20, wherein said agent inhibits p38-2 kinase activity.

22. An agent that modulates phosphorylation of p38-2, for use in the manufacture of a medicament for treating a patient afflicted with a condition associated with the p38-2 cascade.

23. An agent according to claim 22, wherein said agent inhibits phosphorylation of p38-2.

24. The agent of any one of claims 19, 20 or 22, wherein said agent comprises a monoclonal antibody.

25. The agent of any one of claims 19, 20 or 22, wherein said agent comprises a polynucleotide.

26. The agent of any one of claims 19, 21 or 23, wherein the condition associated with the p38-2 cascade is pain.

27. A method for detecting mitogen activated protein kinase kinase activity in a sample, comprising evaluating the ability of the sample to phosphorylate a polypeptide according to either of claims 1 or 4, thereby detecting mitogen activated protein kinase kinase activity in the sample.

28. The method of claim 27, wherein the mitogen activated protein kinase kinase is MEK6.

29. The method of claim 28, wherein the ability of the sample to phosphorylate a polypeptide is evaluated using a coupled kinase assay.

30. A kit for detecting mitogen activated protein kinase kinase activity in a sample, comprising p38-2 in combination with a suitable buffer.