AU736316B2 - Mitogen-activated protein kinase p38-2 and methods of use therefor - Google Patents

Description

WO 97/44467 PCT/US97/08738 Description MITOGEN-ACTIVATED PROTEIN KINASE p38-2 AND METHODS OF USE THEREFOR Technical Field The present invention relates generally to compositions and methods useful for the study of mitogen-activated protein kinase cascades and for treating conditions associated with such cascades. The invention is more particularly related to a mitogen-activated protein kinase p 3 8-2, 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 p 3 8-2 kinase cascade. Such agents may be used, for example, 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 other target molecules in response to a variety of extracellular signals. MAPKs are activated by phosphorylation at a dual phosphorylation motif with the sequence Thr-X- Tyr by mitogen-activated protein kinase kinases (MAPKKs). In higher eukaryotes, the physiological role of MAPK signaling has been correlated with cellular events such as proliferationoncogenesis,development-anddifferentiation.-Accordingly,-theability-toregulate signal transduction via these pathways could lead to the development of treatments and preventive therapies for human diseases associated with MAPK signaling, such as inflammatory diseases, autoimmune diseases and cancer.

In mammalian cells, three parallel MAPK pathways have been described. The best characterized pathway leads to the activation of the extracellularsignal-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 3U- b-01:16:42 ;DAVIES COLLISON CAVE Pat.&Trad ;61 7 3368 2262 15/ 27 2 Mahadevan, Trends Biochem. Sci 20:117-122, 1995). The identification and characterization of members of these cascades is critical for understanding the signal transduction pathways involved and for developing methods for activating or inactivating MAPKs in vivo.

Three MAPKKs capable of activating p38 in vitro have been identified.

MKK3 appears to be specific for p38 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 Serial Number 5,948,885, issued September 7, 1999). These proteins appear to have utility in therapeutic methods for treating conditions associated with the p38 signal transduction pathway. However, in "0 order to precisely tailor such therapeutic methods, and to gain an understanding of the 0 pathways involved, it would be advantageous to identify and characterize other proteins that participate in this cascade and related MAP kinase cascades.

15 Accordingly, there is a need in the art for improved methods for modulating the activity of proteins involved in the MAP kinase cascades, and for treating conditions associated with such cascades. 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-ID- 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 amino RA acid residues, such that said polypeptide is rendered constitutively inactive.

r- In related aspects, the present invention provides isolated DNA o r molecules encoding polypeptides as described above, as well as recombinant WO 97/44467 PCT/US97/08738 3 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 above with a substrate of p38-2, thereby phosphorylating the substrate of p38-2.

In a related aspect, methods are provided for activating a substrate of p38-2 in a patient, comprising administering to a patient a polypeptide as described above in combination with a pharmaceutically acceptable carrier, thereby activating a substrate of p 3 8-2.

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: 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: 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 subsequently measuring the ability of the candidate agent to modulate p 3 8 -2 activity.

In-yet-another-aspect,thepresentinvention providesantibodies that bind to a polypeptide as described above.

In further aspects, methods are provided for treating a condition associated with the p 3 8-2 cascade, comprising administering 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.

WO 97/44467 PCT/US97/08738 4 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 apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

Brief Description of the Drawings 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 Northern blot 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 translated HA-tagged p38-2, as determined 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/m 2 lane anisomycin ng/ml;jane orNaCl_(200_jM;_lane orcotransfected_with_10Q0_0ngof the empty expression vector Sra3 (lane the expression vector for the constitutively active mutant MEK6(DD) (lane 6) or the MAPK TAK1AN (lane 7).

Figure 5 presents the nucleotide and amino acid sequence of a native p38-2 polypeptide.

Figure 6 is an immunoblot 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 identified as lysate. In the left panel, antibodies were raised against the full length p38 protein, and recombinant p38 was used as a standard to WO 97/44467 PCT/US97/08738 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 "S-methionine migrates as a band of about 40 kD.

Figure 7 is an autoradiogram showing 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, and 30 minutes, respectively. Lanes 6-10 show the levels in cells treated with 1 JM 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 p.M SB203580 (Figures 8A and B) or SKF106978 (Figures 8C and D) are shown. The Ica.v was activated by a 100 ms test command to 0 mV, applied 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 4M). The continuous line marks the zero current.

Figure 8E and F present a summary of the responses to bradykinin (Figure 8E) and Leu-Enk (Figure 8F). Mean and standard deviations are displayed. The numbers on the left of each bar indicate the number of cells studied.

Figures 9A and 9B show peak Ica,v-voltage relations in two NG108-15 cells before (open circles) and during (closed circles) application of bradykinin (0.1 after intracellular dialysisofSKFl0697_8_(Eigure 9-A;-20-M)-orSB203580 (Figure 9B; 20 piM). Figure 9C presents the time course of the peak Ica.v during perfusion with SB203580 (20 pM) and application of bradykinin and Leu-Enk (both at 0.1 gM). Between and 20 minutes, Icv was activated every 30 s. In the remaining portion of the time course, Ica,v was activated every 10 s. Data were acquired at kHz.

Figure 10A shows the activation of l BK by BK (0.1 utM) obtained from two NG108-15 cells, after intrapipette dialysis with SB203580 (20 tM, panel or SKF106978 (20 jiM, panel A 2 Data were acquired at 100 Hz. Figure 10B is a graph WO 97/44467 PCT/US97/08738 6 showing a summary of the IK.K responses to BK (0.1 tM). The graph displays means and standard deviations. Figure 10C presents a proposed pathway for the inhibition of Ica.v by BK.

Detailed Description of the Invention As noted above, the present invention is generally directed to compositions and methods for modulating stimulating or inhibiting) signal transduction via MAP kinase cascades, and for treating conditions associated with such cascades. In particular, the present invention is directed to compositions comprising a 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 by bradykinin. When active, a p38-2 polypeptide is generally capable of activating at least one substrate of p38-2 ATF-2, MAPKAP kinase 2, MAPKAP kinase 3, MNK1, PHAS-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 diminished.

The present invention also encompasses compositions and methods for modulating p38-2 activity. In general, compositions that inhibit p38-2 activity may inhibit phosphorylation of p 3 8 or may inhibit the ability of p38-2 to phosphorylate a substrate. As used herein,the term "p38-2cascade" refers to any signal transduction pathway that involves p38-2, and such a cascade may include any compound that modulates p38-2 activity or acts as a substrate for p38-2.

p38-2 polypeptide variants within the scope of the 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 diminished. In certain preferred embodiments, a variant contains substitutions and/or modifications at no more than 25% of the amino acid residues, more preferably at no more than 20% of the amino acid residues and most WO 97/44467 PCT/US97/08738 7 preferably at no more than 10% of residues. Such substitutions, which are preferably conservative, may be made in non-critical and/or critical regions of the native protein.

Variants may also, or alternatively, contain other modifications, including the deletion or addition of amino acids that have minimal influence on the activity of the polypeptide. In particular, variants may contain additional amino acid sequences at the amino and/or carboxy termini. Such sequences may be used, for example, to facilitate purification or detection of the polypeptide.

As noted above, p38-2 polypeptides, including native p38-2 sequences and variants thereof, may be selectively activated by bradykinin. Bradykinin (BK) 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 term "selectively activated," as used herein refers to a strong stimulation at least three fold) of kinase activity of a p38-2 polypeptide under conditions that produce at most only a modest stimulation between about 1.5 and 3 fold) of JNK and/or ERK activity and little or no measurable activation less than fold) of type 1 and/or type 3 p 3 8 kinases. In general, the selective activation of a p38-2 polypeptide by bradykinin may be evaluated using immunoprecipitation assays and/or measurements of ca,v. Measurements of Icav may be performed using a standard whole-cell patch-clamp technique. Briefly, at least two neurotransmitters, bradykinin _andleu=enkephalin, modulate the amplitude of Ic.vin NGl108.15_cells. The_effectof bradykinin, but not that of leu-enkephalin, is sensitive to the block of p38-2 activity.

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 to stimulate substrate phosphorylation in the absence of stimulation, as described below. Such polypeptides may be identified using the representative assays for p38-2 WO 97/44467 PCTIUS97/08738 8 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 amplification 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 adapterligated 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 has the sequence provided in SEQ ID NO:1 and Figure 5. The encoded p38-2 polypeptide, shown in SEQ ID NO:2 and Figure 1, has a predicted size of 364 amino acids, with a molecular weight of about 42 kD as determined by calculation and SDSpolyacrylamide gel electrophoresis. p38-2 is 73% identical to its closest homolog p38 (see, 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 p 3 8 -2 have also been identified, with the sequences provided in Figure 1, 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 mammalian cells. The recombinant DNA_maybeclonedinto_anyexpressionvector-suitable-for-use-within the host cell, using techniques well known to those of ordinary skill in the art. An expression vector may, but need not, include DNA encoding an epitope, such that the recombinant protein contains the epitope at the N- or C-terminus. Epitopes such as glutathione-S transferase protein (GST), HA (hemagglutinin)-tag, FLAG and Histidinetag may be added using techniques well known to those of ordinary skill in the art.

The DNA sequences expressed in this manner may encode a native p 3 8 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 WO 97/44467 PCT/US97/08738 9 standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis, and sections of the DNA sequence may be removed to permit preparation of truncated polypeptides. For variants of p38-2, any such changes should not diminish the ability of the variant to stimulate phosphorylation of substrates such as ATF2 (see, Gupta et al., Science 267:389-393, 1995), MAPKAP kinase 2 (see, Rouse et al., Cell 78:1027-1037, 1994 Ben Levy et al., EMBO J.:14:5920-6930, 1995) or MAPKAP kinase 3 (see McLaughlin et al., J. Biol. Chem. 271:8488-8492, 1996).

In general, modifications may be more readily made in non-critical regions, which are regions of the native sequence that do not substantially change the properties of p38-2.

Non-critical regions may be identified by modifying the p38-2 sequence in a particular region and assaying the ability of the resulting variant in a kinase assay, using a suitable substrate, as described herein.

Modifications may also be made in critical regions of p38-2, provided that the resulting variant substantially retains the ability to stimulate substrate phosphorylation. The effect of any modification on the ability of the variant to stimulate substrate phosphorylation may generally be evaluated using any assay for p 3 8-2 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 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-PAGEelectrophoresis, andaffinity_chromatography._ p38-2 polypeptides for use in the methods of the present invention may be native, purified or 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 overexpressed, as well as suppression of phosphorylation of p38-2 or the inhibition of the ability of activated phosphorylated) p38-2 to phosphorylate a substrate. For example, a modulating agent may modulate the kinase WO 97/44467 PCT/US97/08738 activity of one or more MAPKKs, such as MEK6, thereby inhibiting p 3 8 -2 activation.

Known activators of MAPKKs include, but are not limited to, stress-inducing signals UV, osmotic shock, DNA-damaging agents), anisomycin, LPS, and cytokines.

Similarly, compositions that inhibit p38-2 activity by inhibiting p38-2 phosphorylation may include one or more agents that inhibit or block MAPKK activity, such as an antibody that neutralizes a MAPKK, a competing peptide that represents the substrate binding domain of a MAPKK or the dual phosphorylation motif of p38-2, an antisense polynucleotide or ribozyme that interferes with transcription and/or translation of a MAPKK, a molecule that inactivates a MAPKK by binding to the kinase, a molecule that binds to p 3 8-2 and prevents phosphorylation by a MAPKK or a molecule that prevents transfer of phosphate groups from the kinase to the substrate.

In general, modulating agents may be identified by combining a test compound with an activated p38-2 polypeptide, or a polynucleotide encoding such a polypeptide, in vitro or in vivo, and evaluating the effect of the test compound on the p38-2 kinase activity using, for example, a representative assay described herein. An increase or decrease in kinase activity can be measured by adding a radioactive compound, such as [y 32 P]-ATP, to the mixture of components, and observing radioactive incorporation into a suitable substrate for p 3 8-2, to determine whether the compound inhibits or stimulates kinase activity. Briefly, a candidate agent may be included in a mixture of active p 3 8-2 polypeptide and substrate (such as ATF2), with or without pre-incubation with one or more components of the mixture. Activation of p38-2 may be achieved by any of a variety of means. Typically, activation involves the addition of aMAP_kinase kinase,_which-may-in-tum-be-activated-via-stimulation-as described above. In general, a suitable amount of antibody or other agent for use in such an assay ranges from about 0.1 gM to about 10 gM. The effect of the agent on p38-2 kinase activity may then be evaluated by quantitating the incorporation of 3 2 P]phosphate into ATF2, and comparing the level of incorporation with that achieved using activated p38-2 without the addition of a candidate agent. Alternatively, the incorporation of phosphate into ATF2 may be measured using an antibody specific for phosphorylated substrate, using well known techniques. Within another alternative, a polynucleotide encoding the kinase may be inserted into an expression vector and the WO 97/44467 PCT/US97/08738 11 effect of a composition on transcription of the kinase measured, for example, by Northern blot analysis.

In another aspect of the present invention, a p38-2 polypeptide may be used for phosphorylating and activating a substrate of p 3 8-2. In one embodiment, a substrate may be phosphorylated in vitro by incubating a p38-2 polypeptide with a substrate and ATP in a suitable buffer (described in more detail below) for 30 minutes at 30 0 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 gg to about 10 upg of p 3 8 2 polypeptide, from about 0.1 tg to about 10 gg of substrate, and from about 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 [y- 32 P]ATP to a test aliquot, and evaluating the level of substrate phosphorylation as described below.

One or more p38-2 polypeptides, modulating agents as described above and/or polynucleotides encoding such polypeptides and/or modulating agents may also be used to modulate p38-2 activity in a patient. As used herein, a "patient" may be any mammal, including a human, and may be afflicted with a condition associated with the p38-2 cascade or may be free of detectable disease. Accordingly, the treatment may be of an existing disease or may be prophylactic. Conditions associated with the p38-2 cascade include any disorder which is etiologically linked to MAP kinase activity, including cardiovascular disease, immune-related diseases inflammatory diseases, autoimmunediseases,-malignantcytokineproduction-or-endotoxic-shock),cell-growthrelated diseases cancer, metabolic diseases, abnormal cell growth and proliferation or cell cycle abnormalities) and cell regeneration-related diseases cancer, degenerative diseases, trauma, environmental stress by heat, UV or chemicals or abnormalities in development and differentiation). In particular, the high expression of p38-2 in heart tissue suggests an important role for p38-2 in cardiovascular diseases.

Immunological-related cell proliferative diseases appropriate for treatment with p38-2 polypeptides include osteoarthritis, ischemia, reperfusion injury, trauma, certain cancers WO 97/44467 PCT/US97/08738 12 and viral disorders, and autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory 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 cascade, and that pain may be treated by 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 bradykinin antagonists and without the addictive risks of opioids.

Treatment may include administration of a p 3 8-2 polypeptide and/or a compound which modulates p38-2 activity. For administration to a patient, one or more polypeptides (and/or modulating agents) are generally formulated as a pharmaceutical composition. A pharmaceutical composition may be a sterile aqueous or non-aqueous solution, suspension or emulsion, which additionally comprises a physiologically acceptable carrier a non-toxic material that does not interfere with the activity of the active ingredient). Any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of the present invention. Representative carriers include physiological saline solutions, gelatin, water, alcohols, natural or synthetic oils, saccharide solutions, glycols, injectable organic esters such as ethyl oleate or a combination of such materials. Optionally, a pharmaceutical composition may additionally contain preservatives and/or other additives such as, for example, antimicrobialagents,_anti-oxidants,-chelating-agentsand/or inert gases, and/or other active ingredients.

Alternatively, a pharmaceutical composition may comprise a polynucleotide encoding a p38-2 polypeptide, and/or modulating agent, such that the polypeptide and/or modulating agent is generated in situ, in combination with a physiologically acceptable carrier. In such pharmaceutical compositions, the polynucleotide may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid, bacterial and viral expression systems, as well as colloidal dispersion systems, including liposomes. Appropriate WO 97/44467 PCT/US97/08738 13 nucleic acid expression systems contain the necessary polynucleotide sequences for expression in the patient (such as a suitable promoter and terminating signal). DNA may also be "naked," as described, for example, in Ulmer et al., Science 259:1745-1749 (1993).

Various viral vectors that can be used to introduce a nucleic acid sequence into the targeted patient's cells include, but are not limited to, vaccinia or other pox virus, herpes virus, retrovirus, or adenovirus. Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus including, but not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).

A retroviral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a gene that encodes the ligand for a receptor on a specific target cell (to render the vector target specific). For example, retroviral vectors can be made target specific by inserting a nucleotide sequence encoding a sugar, a glycolipid, or a protein. Targeting may also be accomplished using an antibody, by methods known to those of ordinary skill in the art.

Viral vectors are typically non-pathogenic (defective), replication competent viruses, which require assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids that encode all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR, but that are missing a nucleotide sequence which enables the packaging mechanism_to recognizean RNA transcript_for_ encapsulation. Such helper cell lines include (but are not limited to) T2, PA317 and PA12. A retroviral vector introduced into such cells can be packaged and vector virion produced. The vector virions produced by this method can then be used to infect a tissue cell line, such as NIH 3T3 cells, to produce large quantities of chimeric retroviral virions.

Another targeted delivery system for p38-2 polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water WO 97/44467 PCT/US97/08738 14 emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome an artificial membrane vesicle). It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 jim can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci. 6:77, 1981). In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells. In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: encapsulation of the genes of interest at high efficiency while not compromising their biological activity; preferential and substantial binding to a target cell in comparison to non-target cells; delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques 6:882, 1988).

The targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity and may be, for example, organ-specific, cell-specific and/or organelle-specific.

Mechanistic targeting can be distinguished based upon whether it is passive or active.

Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticuloendothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.

Routes and frequency of administration, as well doses, will vary from patient to patient. In general, the pharmaceutical compositions may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity or transdermally. Between 1 and 6 doses may be administered daily. A suitable dose is an amount of polypeptide or polynucleotide that is sufficient to show improvement in the symptoms of a patient afflicted with a condition associated with the p38-2 cascade.

WO 97/44467 PCT/US97/08738 Such improvement may be detected based on a determination of relevant cytokine levels IL-1, IL-6 and/or IL-8), by monitoring inflammatory responses edema, transplant rejection, hypersensitivity) or through an improvement in clinical symptoms associated with the condition pain). In general, the amount of polypeptide present in a dose, or produced in situ by DNA present in a dose, ranges from about 1 ptg to about 250 pg per kg of host, typically from about 1 gg to about 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 detecting the level of mitogen activated protein kinase kinase (such as MEK6) activity in a sample. The level of MAPKK activity may generally be determined by evaluating the ability of the sample to phosphorylate a p38-2 polypeptide, thereby rendering the p38-2 polypeptide active capable of phosphorylating in vivo substrates such as ATF2). In one embodiment, a kinase assay may be performed substantially as described in Derijard et al., Cell 76:1025-1037, 1994 and Lin et al., Science 268:286-290, 1995, with minor modifications. Briefly, a sample may be incubated with p38-2 and [y- 32 P]ATP in a suitable buffer (such as 20 mM HEPES (pH 5 mM MnCl 2 10 mM MgCl 2 1 mM dithiothreitol) for 30 minutes at 30 0 C. In general, approximately 1 ug recombinant p 3 8-2, with 50 nM [y- 32 P]ATP, is sufficient. Proteins may then be separated by SDS- PAGE on 10% gels and subjected to autoradiography. Incorporation of 32 P]phosphate into p38-2 may be quantitated using techniques well known to those of ordinary skill in the art, such as with a phosphorimager. It will be apparent to those of ordinary skill in theart,_that_tha isassaymay alsobeperformed-with unlabeled-phosphate,-using-an antibody specific for phosphorylated substrate the antibody binds to phosphorylated substrate, and does not bind significantly to unphosphorylated substrate, such that the antibody can be used to distinguish between the two forms) and standard methods.

To determine whether p 3 8-2 phosphorylation results in activation, a coupled in vitro kinase assay may be performed using a substrate for p38-2, such as ATF2, with or without an epitope tag. ATF2 for use in such an assay may be prepared as described in Gupta et al., Science 267:389-393, 1995. Briefly, following WO 97/44467 PCT/US97/08738 16 phosphorylation of p38-2 as described above, isolation of the protein by binding to GSH-sepharose and washing with 20 mM HEPES (pH 20 mM MgCl 2 the p38-2 (0.1-10 pg) may be incubated with ATF2 (0.1-10 ptg) and [y-"P]ATP (10-500 nM) in a buffer containing 20 mM HEPES (pH 20 mM MgC1 2 It should be noted that 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 modulating agent on the level of signal transduction via the p38-2 cascade, a kinase assay may be performed as described above, except that a MAPKK, such as MEK6 (rather than a sample) is generally employed and the candidate modulating agent is added to the incubation mixture. The candidate agent may be preincubated with MAPKK before addition of ATP and p38-2 polypeptide. Alternatively, the p38-2 may be preincubated with the candidate agent before the addition of MAPKK. Further variations include adding the candidate agent to a mixture of MAPKK and ATP before the addition of p38-2, or to a mixture of p38-2 and ATP before the addition of MAPKK, respectively.

All these assays can further be modified by removing the candidate agent after the initial preincubation step. In general, a suitable amount of antibody or other candidate agent for use in such an assay ranges from about 0.1 pM to about 10 jM. The effect of the agent on phosphorylation of p38-2 may then be evaluated by quantitating the incorporationof [3P]phosphateintop38,_asdescribed above, and_comparing the_level of incorporation with that achieved using MAPKK without the addition of the 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 appropriate substrate, such as ATF2. For example, appropriate cells cells that express p38-2) may be transfected with an ATF2-dependent promoter linked to a reporter gene such as luciferase. In such a system, expression of the luciferase gene (which may be readily detected using methods well known to those of ordinary skill in the art) depends upon WO 97/44467 PCT/US97/08738 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 a constitutively active variant of MAPKK, such as MEK6. Candidate modulating agents may be added to the system, as described above, to evaluate their effect on the p 3 8-2 cascade.

Alternatively, a whole cell system may employ only the transactivation domain of ATF2 fused to a suitable DNA binding domain, such as GHF-1 or GAL4.

The reporter system may then comprise the GH-luciferase or GAL4-luciferase plasmid.

Candidate modulating agents may then be added to the system to evaluate their effect on ATF2-specific gene activation.

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 p 3 8 2 may generally be determined using a reagent that binds to p38-2, or to DNA or RNA encoding p38-2. To detect nucleic acid encoding p38-2, standard hybridization and/or PCR techniques may be employed using a nucleic acid probe or a PCR primer.

Suitable probes and primers may be designed by those of ordinary skill in the art based on the p38-2 cDNA sequence provided in SEQ ID NO:1. To detect p38-2 protein, the reagent is typically an antibody, which may be prepared as described below. There are a variety of assay formats known to those of ordinary skill in the art for using an antibody to detect a polypeptide in a sample. See, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, the antibody may be immobilized on a solid support such that it can bind to and remove the polypeptidefromthe sample._Theboundpolypeptidemaythenbe detected_using a second antibody that binds to the antibody/peptide complex and contains a detectable reporter group. Alternatively, a competitive assay may be utilized, in which polypeptide that binds to the immobilized antibody is labeled with a reporter group and allowed to bind to the immobilized antibody after incubation of the antibody with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the antibody is indicative of the level of polypeptide within the sample. Suitable reporter groups for use in these methods include, but are not limited WO 97/44467 PCT/US97/08738 18 to, enzymes horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent 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.

Preferred antibodies are those antibodies that inhibit or block p38-2 activity in vivo and within an in vitro assay, as described above. As noted above, antibodies and other agents having a desired effect on p38-2 activity, may be administered to a patient (either prophylactically or for treatment of an existing disease) to modulate the activation of p38-2 in vivo.

Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art (see, Harlow and Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In one such technique, an immunogen comprising the polypeptide is initially injected into a suitable animal mice, rats, rabbits, sheep and goats), preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for p38-2 or a variant thereof may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol.

6:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity-(i. e.,-reactivity-with-the-polypeptide-of-interest).-Such-celllinesmaybe produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single WO 97/44467 PCT/US97/08738 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 enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction.

In a related aspect of the present invention, kits for detecting p38-2 and p38-2 kinase activity, as well as MAPKK kinase activity, are provided. Such kits may be designed for detecting the level of p38-2 or nucleic acid encoding p38-2, or may detect kinase activity of p38-2 or MAPKK in a direct kinase assay or a coupled kinase assay, in which both the level of phosphorylation and the kinase activity of p38-2 may be determined. MAPKK or p38-2 kinase activity may be detected in any of a variety of samples, such as eukaryotic cells, bacteria, viruses, extracts prepared from such organisms and fluids found within living organisms. In general, the kits of the present invention comprise one or more containers enclosing elements, such as reagents or buffers, to be used in the assay.

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-p3 8 2 -protein-the-reagent-is-typically-an-antibody-Such-kits-also-contain-areporter group suitable for direct or indirect detection of the reagent 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 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.

WO 97/44467 PCT/US97/08738 Kits for detecting p38-2 activity typically comprise a p38-2 substrate in combination with a suitable buffer, p38-2 activity may be specifically detected by performing an immunoprecipitation step with a p38-2-specific antibody prior to performing a kinase assay as described above. Alternatively, the substrate provided may be a substrate that is phosphorylated only by p38-2 is not phosphorylated by p38). A kit for detecting MAPKK kinase activity based on measuring the phosphorylation of p38-2 generally comprises a p38-2 polypeptide in combination with a suitable buffer. A kit for detecting MAPKK kinase activity based on detecting p38-2 activity generally comprises a p38-2 polypeptide in combination with a suitable p38-2 substrate, such as ATF2. Optionally, a kit may additionally comprise a suitable buffer and/or material for purification of p38 after activation and before combination with ATF2. Other reagents for use in detecting phosphorylation of p38-2 and/or kinase activity antibody specific for phosphorylated substrate, which may be used to distinguish phosphorylated substrate from unphosphorylated substrate) may also be 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 kinases that phosphorylate and activate p38-2 in vivo). A p38-2 polypeptide may be used in a yeast two-hybrid system to identify proteins that interact with p38-2. Alternatively, an expression library may be sequenced for cDNAs that phosphorylate p38-2.

The following Examples are offered byway ofillustrationandnot by way of limitation.

WO 97/44467 PCT/US97/08738 21

EXAMPLES

Example 1 Cloning and Sequencing cDNA Encoding p38-2 This Example illustrates the cloning of a cDNA molecule encoding the human MAPK p 3 8 -2.

The Expressed Sequence Tags (EST) subdivision of the National Center for Biotechnology Information (NCBI) Genbank databank was searched with the tblastn program and the human p38 amino acid sequence (Han et al., Science 265:808- 811, 1994; Lee et al., Nature 372:739-746, 1994) as query using the BLAST e-mail server. The EST sequence R72598 from a breast cDNA library displayed the highest similarity score. A clone corresponding to the EST sequence R72598 was obtained from Research Genetics Inc., (Huntsville, AL), and the insert size was determined to be about 0.9 kb. Sequencing revealed that this clone encodes the 5' portion of a previously unknown gene and that the 3' end with the polyA tail was missing. The 3' portion was obtained from a skeletal muscle cDNA library by RACE PCR using a gene specific forward primer and an adapter-based reverse primer. The complete cDNA was obtained by fusion ligation of the 5' portion and the 3' portion using a common KpnI site into pBluescript (Stratagene, La Jolla, 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 _AutomatedSequencer_(AppliedBiosystems, Inc.,_EosterCity,CA), andthesequenceof the full length clone without intron is shown in SEQ ID NO:1 and Figure 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 of polyA' RNA isolated from 16 different adult human tissues, fractionated by denaturing formaldehyde 1.2% WO 97/44467 PCT/US97/08738 22 agarose gel electrophoresis, and transferred onto a charge-modified nylon membrane (Clontech Laboratories, Palo Alto, CA). The blots were hybridized to a p 3 8 -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.

Both probes were prepared by labeling the cDNA with [a-3 2 P]dCTP (NEN, Boston, MA) by random priming (Stratagene, La Jolla, CA). For control purposes, the blots were also hybridized to a radiolabeled p-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 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 p-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 translated using the Promega TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI) in the 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 visualized by autoradiography (Figure 3).

Theseresults_demonstratethat themolecular weight_ofthe epitope tagged p38-2 is approximately 42 kDa.

Example 4 Activation of p38-2 by Stress-inducing Agents This Example illustrates the activation of p38-2 by a variety of stimulators of the MAPK pathway.

An expression vector encoding epitope-tagged p38-2 (3xHA-p38-2- SRa3) was constructed by adding sequence encoding three copies of a 10 amino acid WO 97/44467 PCT/US97/08738 23 hemagglutinin (HA) epitope to the N-terminus of p38-2 and ligating the resulting cDNA into the expression vector SRa3. 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, 1984). These cells were then treated with various stimulators of the MAPK pathway treated for 45 minutes with UV (250 nm, 120 J/m 2 anisomycin (50 ng/ml) or NaCI (200 tM) or cotransfected with 1000 ng of the empty expression vector Sra3, the expression vector for the constitutively active mutant MEK6(DD) or the MAPK TAK1AN).

Following treatment, cell lysates were prepared by solubilization in lysis buffer as described (Derijard et al., Cell 76:1025-1037, 1994), and protein concentration of lysates was determined by Bradford assay (Bradford, Ann. Biochem.

72:248-254, 1976). Cell lysates were used in an immune complex kinase assay with GST-ATF2 substrate, prepared as previously described (Gupta et al., Science 267:389- 39, 1995). The assay was generally performed as described previously (Derijard et al., Cell 76:1025-1037, 1994; Lin et al., Science 268:286-290, 1995) with minor modifications. The concentration of [y- 32 P]ATP was 50 nM, and 30 p.g cell lysate was immunoprecipitated for 2 hours with the anti-HA antibody 12CA5 (Boehringer- Mannheim Corp., Indianapolis, IN) and then incubated with 1 ug of recombinant 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/m 2 for 45_minutes),_anisomycin (50 ng/mL for 45_minutes) and MEK6(DD), a constitutively active variant of MEK6 (Fig. No increase in p38-2 activity was observed when the cells were treated with NaC1, expression vector alone or the MAP kinase TAK1AN (see Yamaguchi et al., Science 270:2008-2011, 1995). This clearly indicates that the inducible phosphorylation of ATF2 depends on a kinase cascade comprised of MEK6 and p38-2.

WO 97/44467 PCT/US97/08738 24 Example Selective Activation of BRK by Bradvkinin This Example illustrates the ability of bradykinin to potently and selectively activate BRK.

An initial experiment was performed to identify the MAPK pathways represented in NG108-15 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 performed on extracts using polyclonal antibodies raised against p38 and a small peptide unique 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 Icav. Thus, in NG108-15 cells, bradykinin strongly stimulates BRK, only modestly stimulates JNK and ERK, and does not measurably activate type 1 p 3 8 kinases.

Example 6 Effect of Inhibitors of ERK and BRK Kinase Activity on Inhibition of by Bradykinin This Example illustrates the use of an inhibitor of BRK to block the inhibition of Ic,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 Ica,V by bradykinin (Figures 8A-F). After intracellular dialysis of each compound, the inhibition of Ica,v by bradykinin was examined first and the inhibition by Leu-Enkphalin (Leu-Enk) was measured thereafter. The inhibition of Ica,v by bradykinin was blocked after application of SB203580 (20 uM), whereas that produced by Leu-Enk was retained (Figures 8A-B). Application of an inactive analog SKF106978 (20 PM (SmithKline Beecham); see Lee et al., Nature 372:739-746, 1994) WO 97/44467 PCT/US97/08738 did not block the inhibition of Icav by bradykinin or Leu-Enk (Figures 8C-D). After application of PD98059 (20 uM), the inhibition of Ic,,v by either transmitter was not attenuated (Figures 8E-F). Similarly, application of the dimethylsulfoxide-containing vehicle was without effect. A summary of the results of the experiments with PD98059 and SB203580 is presented in Figures 9E-F.

Bradykinin and Leu-Enk inhibit the same component of Ica,v (see Wilk- Blaszczak 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 Ic,,v. This conclusion was confirmed by studying the direct effect on peak Ic,.v of SB203580 over a broad range of membrane potentials (Figure 9B). As a control, current-voltage curves were obtained after dialyzing the cells with SKF106978 (Figure 9A). In all cells treated with SB203580 (n the current-voltage relation was not altered significantly compared to control cells (n while the inhibition by BK was suppressed over the entire range of membrane potentials examined. These findings were consistent in all cells studied. The lack of any direct action of SB203580 on Ic,,v was further confirmed by monitoring the peak Ica,v for the entire duration of the experiment (Figure 10C; n 11).

The specificity of SB203580 for the G,3 pathway was further tested by examining whether this compound blocks a second response to BK, activation of a voltage-independent

K

4 current (IK,BK). This response is mediated by a distinct heterotrimeric G protein Gq,, (see Wilk-Blaszczak et al., Neuron 12:109, 1994). IK,BK was measured as described in Wilk-Blaszczak et al., Neuron 12:109, 1994 in cells dialyzed either with the p38_inhibitor_SB203580_or withthe inactive_analog SKF106978 (see Figure 10A). In all cells tested, dialysis with SB203580 did not reduce activation of 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 Ica.v. Involvement of this kinase in the pathway of BK is strongly supported by the observation that BK potently activates this isoform of the 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 Ic.v.

5-01;16:42 ;DAVIES COLLISON CAVE Pat. &Trad ;61 7 3368 2262 16/ 27 26 The effect of SB203580 is selective, because it does not extend to the activation of I"K by BK, or to the inhibition of Ilv by Leu-Enk. Involvement of JNK in the inhibition of Icv 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, because the inhibition of Ic,.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 factorinitiated effects of MAP kinases on cellular proliferation and differentiation, the action of BRK kinase on Iv, is triggered by a G protein-coupled serpen tne receptor and unfolds over a short time scale. Thus, MAP kinase pathways play roles similar to those t* '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.

.i Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood 20 to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

*oo The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

SRA4 0 WO 97/44467 27 PCT/US97/08738 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Signal Pharmaceuticals, Inc.

(ii) TITLE OF INVENTION: MITOGEN-ACTIVATED PROTEIN KINASE p 3 8-2 AND METHODS OF USE THEREFOR (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: SEED and BERRY LLP STREET: 6300 Columbia Center, 701 Fifth Avenue CITY: Seattle STATE: Washington COUNTRY: USA ZIP: 98104-7092 COMPUTER READABLE

FORM:

MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION

DATA:

APPLICATION

NUMBER:

FILING DATE: 20-MAY-1997

CLASSIFICATION:

(viii) ATTORNEY/AGENT

INFORMATION:

NAME: Maki, David J.

REGISTRATION NUMBER: 31,392 REFERENCE/DOCKET NUMBER: 860098.412PC WO 97/44467 PCTIUS97/08738 28 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: (206) 622-4900 TELEFAx: (206) 682-6031 INFORMATION FOR SEQ ID NO:1: SEQUENCE

CHARACTERISTICS:

LENGTH: 1502 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GGACATGTCG GGCCCTCGCG CCGGCTTCTA

CCGGCAGGAG

GGTGCCCCAC

TTCGGCCTAC

CCAGTCGCTG

GCACGAGAAC

CAGCGAAGTG

CCAGGCGCTG

GTACATCCAC

CGAGGACTGT

CCCCTCCAGG

GACGCCCGGC

ATCCACGCGC

GTCATCGGGC

TACTTGGTGA

AGCGACGAGC

TCGGCCGGGA

GAGCTCAGGA

GGCTGCGCCC

TGCGCCAGAA

GCAGAACGTA

TTCTGGACGT

CCACCCTGAT

ACGTTCAATT

TCATCCACCG

TCCTGGATTT

GGTCGGCTCC

GGTGGCGGTG

CCGGGAGCTG

CTTCACGCCG

GGGCGCCGAC

CCTGGTTTAC

GGACCTGAAG

CGGGCTGGCG

CTGAACAAGA

GGCGCCTACG

AAGAAGCTGT

CGGCTGCTCA

GCCACGTCCA

CTGAACAACA

CAGCTGCTGC

CCCAGCAACG

CGCCAGGCGG

CCGTGTGGGA

GCTCCGTCTG

CGCGCCCCTT

AGCACCTGAA

TCGAGGACTT

TCGTCAAGTG

GCGGGCTGAA

TGGCTGTGAA

ACGAGGAGAT

WO 97/44467 WO 9744467PCTIUS97/08738 GACCGGCTAT GTGGCCACGC GCTGGTACCG TTACAACCAA ACAGTGGATA TCTGGTCCGT CAAGGCCCTC TTCCCGGGAA GCGACTACAT GGGCACACCC AGCCCTGAGG TTCTGGCAAA CCAGTCCCTG CCCCCCATGC CCCAGAAGGA CCTGGCCATA GACCTCCTTG GAAGGATGCT AGCTGAGGCA CTGGCCCACG CCTACTTCAG GGCCGAGCCA TATGATGAGG GCGTTGAGGC GCTCACTTAC CAGGAAGTCC TCAGCTTCAA CCTGGAGATT GAGCAGTGAG GTGCTGCCCA GGCCTGCACC CTTCCACAGc TGGCCTGGTT GTCACAGACT TCTGGCCTAG GACCCCTCGC TGATCCAGTA ACCTCGGAGA CGGGACCCTG CCTGGAAAGG GGGTGACCTC TTGCCTCGAG TGCACCAGGG GTGCACAATA AAGGGGGTTC AAAAAAAAAG CGGCCGCTGA ATTCTACCTG

TA

GGCACCTGAG

GGGCTGCATC

TGACCAGCTG

AATCTCCTCG

CCTGAGCAGC

GGTGCTGGAC

CCAGTACCAC

CAAGGAGCGC

GCCCCCAGAG

GCAGCCCCTG

TCCTCGAGAG

CTTCAGGAGA

CCCAGAGCCG

GGGCCCAGGG

TCTCTAAAAA

CCCGGGCGGC

ATCATGCTCA

ATGGCTGAGC

AAGCGCATCA

GAACACGCCC

ATCTTCCGTG

AGTGACCAGA

GACCCCGAGG

ACGCTGGAGG

CCACCGAAGC

AGAGCCTGTG

GCACCTCCCA

ATCTACACGC

AGTTGGGGGT

AAGCCTGGGT

CGCTCGAGCC

ACTGGATGCA

TGCTCCAGGG

TGGAAGTGGT

GGACATATAT

GAGCCAACCC

GGGTCAGTGC

ATGAGCCAGA

AGTGGAAGGA

CACCTGGCAG

GAGGGGCTTG

CACTCCTATG

ATGATGGAGC

GTGGCTCTCC

GTCAAGTGCC

AAAAAAA

CTATAGTGAG

600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1502 INFORMATION FOR SEQ ID NO:2: WO 97/44467 WO 9744467PCTIUS97/08738 SEQUENCE CHARACTERISTICS: LENGTH: 364 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Ser Gly Pro Arq Ala Gly Phe Tyr Gln Glu Leu Asn Lys Thr Val Trp Glu Pro Gln Arg Leu Gly Leu Arg Pro Val Gly Ser Gly Ala Tyr Gly Ser Val Cys Ser 40 Ala Tyr Asp Ala Arg Leu Arg Gln Lys Val Ala Val Lys Lys Ser Arg Pro Phe Gln Ser Leu Ile His Al a Arg Arg Thr Tyr Glu Leu Arg Leu Lys His Leu Lys His Glu Asn Val Ile Gly Leu Leu Asp Val Phe 90 Thr Pro Ala Thr Ser Leu Glu Asp Phe Leu Asn Asn 115 Glu Val Tyr Leu Val Thr Thr 105 Ala Leu Ser Leu Met Gly Ala Asp 110 Asp Glu His Val Gln 125 Ile Val Lys Cys Gln 120 WO 97/44467 PCT/US97/08738 Phe Leu Val Tyr Gin Leu Leu 130 135 Arg Gly Leu Lys Tyr 140 Ile His Ser Ala Gly 145 Ile Ile His Arg Leu Lys Pro Ser Val Ala Val Asn Asp Cys Glu Leu Arg 165 Ile Leu Asp Phe Gly 170 Leu Ala Arg Gin Ala Asp 175 Glu Glu Met Ile Met Leu 195 Gly Tyr Val Ala Arg Trp Tyr Arg Ala Pro Glu 190 Ile Trp Ser Asn Trp Met His Tyr 200 Asn Gin Thr Val Val Gly 210 Cys Ile Met Ala Leu Leu Gin Gly Lys Ala Leu Phe Pro 220 Met Glu Val Val Glv Ser Asp Tyr Ile Gin Leu Lys Arg Ile 235 Thr Pro Ser Pro Val Leu Ala Lys Ser Ser Glu His Ala Arg 255 Thr Tyr Ile Gin 260 Ser Leu Pro Pro Met 265 Pro Gin Lys Asp Leu Ser Ser 270 Ile Phe Arg 275 Gly Ala Asn Pro Leu 280 Ala Ile Asp Leu Leu Gly Arg Met 285 Leu Val 290 Leu Asp Ser Asp Gin Arg Val Ser Ala Ala Glu Ala Leu Ala 300 His 305 Ala Tyr Phe Ser Gin Tyr 310 His Asp Pro Glu Asp 315 Glu Pro Glu Ala 320 WO 97/44467 PCT/US97/08738 Glu Pro Tyr Asp Gly Val Glu Ala Glu Arg Thr Leu Glu Glu 335 Trp Lys Glu Leu 340 Thr Tyr Gin Glu Val 345 Leu Ser Phe Lys Pro Pro Glu 350 Pro Pro Lys 355 Pro Pro Gly Ser Glu Ile Glu Gin INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 102 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Ser Gly Pro Arg Ala Gly Phe Tyr Arg Gin Glu Leu Asn Lys Thr 1 5 10 Val Trp Glu Val Pro Gin Arg Leu.Gln Gly Leu Arg Pro Val Gly Ser 25 Gly Ala Tyr Gly Ser Val Cys Ser Ala Tyr Asp Ala Arg Leu Arg Gin 40 Lys Val Ala Val Lys Lys Leu Ser Arg Pro Phe Gin Ser Leu Ile His 55 WO 97/44467 WO 9744467PCTIUS97/08738 Al a Arg.Arg Thr Tyr Glu Leu Arg Leu Leu Lys His Leu Lys His 75 Phe Thr Pro Ala Thr Ser Ile 90 Glu Asn Val Ile Gly Leu Leu Asp Val Glu Asp Phe Ser Glu Val 100 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 155 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Ser Gly Pro Ala Gly Phe Tyr Arg Gin Glu Leu Asn Lys Thr Val Trp Giu Val Pro Gin Arg Leu Gin Gly Leu Arq Pro 25 Val GlySer Leu Arg Gin Gly Ala Tyr Giy Ser Val Cys Ser 40 Ala Tyr Asp Ala Arg Lys Val Ala Val Lys Lys Ser Arg Pro Phe Ser Leu Ile His Ala Arg Arg Thr Tyr Arg Glu Leu Arg Leu Leu Lys His Leu Lys His WO 97/44467 WO 9744467PCTIUS97/08738 Glu Asn Val Ile Gly Leu Len Asp Val Phe Thr Pro Ala Thr 90 Ser Ile Gin Asp Phe Leu Asn Asn 115 Gin Val Tyr Leu Thr Thr Len Met Ile Val Lys Cys Gin 120 Ala Leu Ser Asp Gin 125 Gly Ala Asp 110 His Vai Gin His Ser Ala Phe Len 130 Val Tyr Gin Len Len Arg Gly 135 Gly Ala Thr Leu Lys Tyr Ile 140 Gly Ile Ile His Arg Val 150 Ala Gly 155

Claims (23)

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

2. A constitutively active variant as hereinbefore defined of a Spolypeptide according to claim 1. 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 phosphorylating a substrate of p38-2, wherein said polypeptide is selectively activated by bradykinin. A polypeptide according to claim 4, wherein said polypeptide comprises a variant as hereinbefore defined of SEQ ID NO:2 that differs in substitutions and/or modifications at no more than 25% of the amino acid residues.

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

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. 01/06 '01 FRI 15:31 [TX/RX NO 9912] 5-01;16:42 ;DAVIES COLLISON CAVE Pat.&Trad ;61 7 3368 2262 18/ 27 36

9. A host cell transformed or transfected with an expression vector according to claim 8. 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 p 3 8-2, comprising contacting the polypeptide of claim 1 with a substrate of p38-2, thereby phosphorylating the substrate ofp38-2.

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 I or 4 in combination with a pharmaceutically acceptable carrier, for use in the manufacture of a medicament o: 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. A method for screening for an agentthat modulates-signal transduction via the p38-2 cascade, comprising: 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 5-01:16:42 ;DAVIES COLLISON CAVE Pat.&Trad ;61 7 3368 2262 19/ 27 37 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: 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 specifically binds to a polypeptide according to either of claims 1 or 4. iii

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

19. An agent that modulates signil transduction via the p38-2 cascade, for use in the manufacture of a medicament for treating a condition associated with the p38-2 cascade. 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. 5-01;16:42 ;DAVIES COLLISON CAVE Pat. &Trad ;*61 7 3368 2262 20/ 27 38

22. An agent that modulates phosphorylation ofp38-2, 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. o 25. The agent of any one of claims 19, 20 or 22, wherein said agent comprises a polynucleotide. *e.

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