CA2148898A1 - P54 stress-activated protein kinases - Google Patents

Abstract

The p54 Stress-Activated Protein Kinases (SAPKs) are members of a large gene family and are structurally related to the previously described mitogen-activated protein kinases (MAPKs). They are distinguished by their strong activation in response to heat shock, TNF-.alpha., IL-1-.beta., sphingomyelinases, chemical protein synthesis inhibitors, and ischemia. These kinases have potential utility in the modulation of the inflammatory response and the up-regulation of repair or protective cellular proteins following injury or chemical insult.

Description

PATENT
A.~O~N~Y DOCKET NO: 00786/217CAl p54 STRESS-ACTIVATED PROTEIN KINASES
Statement as to Federally Sponsored Research This invention was funded at least in part by the following grants from the United States Government:
GM46577 to JMK, DK17776 and DK41513 to JA (DK41513 transferred to JMK as of 1/94), and the U.S. Government has certain rights in the invention.
Background of the Invention The field of the invention is second messenger protein kinases which regulate activation of transcription factors, which in turn modulate cellular responses to extracellular stimuli.
The extracellular signal-regulated kinases (ERKs) are a family of proline-directed kinases that are activated via concomitant Tyr and Thr phosphorylation and share sequence homology in the Ser/Thr protein kinase catalytic domain. They include the mitogen activated 20 protein kinases, or MAPKs. Stimuli for the activation of these kinases are diverse, and induce discrete second messenger pathways to effect specific cellular responses.
ERKs participate in the regulation of other protein kinases and several transcription factors including c-25 Jun, c-Myc, c-Fos, ATF-2, and p62TCF/Elk-l. These functions indicate that the ERKs mediate the expression of genes in response to extracellular agonists.
The first well characterized members of this kinase family were p42 and p44 MAPKs, which are 30 stimulated by insulin and require both Tyr and Thr phosphorylation for activity (Sturgill et al, Nature 334:715-718, 1988; Anderson et al., Nature 343:651-653, 1990). They are also stimulated by a variety of mitogens, phorbol esters, and activated ras.

2148~9~

The ERKs share sequence homology in the Ser/Thr protein kinase catalytic domain, as mentioned above, and this functions to phosphorylate c-Jun on serine and threonine residues that have been localized to c-Jun 5 tryptic peptides termed X and Y. X and Y are located near the N-terminal trans-activation domain (Pulverer et al., Nature 353:670-673, 1992). Phosphorylation of these peptides regulates transactivating binding activity, and thus their function as promoters of gene expression.
Summary of the Invention The invention features a molecule consisting of either DNA or amino acids encoding a p54 stress-activated protein kinase (SAPK), or a biologically active fragment thereof, characterized by its ability to modulate 15 transcription pathways in response to extracellular stress stimuli. By "biologically active fragment" it is meant that the fragment can exert a physiological effect (e.g., binding to its biological substrate, phosphorylation, causing an antigenic response, etc.) in 20 vivo or in vitro.
In preferred embodiments, the molecule of the invention is a polypeptide at least 95% identical to p54aI, p54aII, p54~I, p54~II, or p54y, preferably from a mammalian source (e.g., SEQ ID N0s: 1, 2, 3, 4, or 5).
25 Any polypeptide sequence containing an "X" is intended to denote a position that could be any amino acid. By "polypeptide", it is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
Other preferred embodiments include polypeptides that are substantially identical to SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, and 8. By "substantially identical", it is meant an amino acid sequence which differs only by conservative amino acid substitutions, for example, 35 substitution of one amino acid for another of the same 21~8~8 class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy 5 the biological function of the polypeptide.
In one preferred embodiment, the molecule of the invention is a polypeptide fragment that contains a biologically active portion of a p54 polypeptide, at least 10 amino acids in length. Examples include, but 10 are not limited to, the ATP binding site, which includes Y 55 Gly33, 35, and 38; the site of regulatory phosphorylation by upstream activators (Thr183-Pro-Tyrl85); and the SAPK catalytic domain (between amino acids 22 and 321; see Fig. 1).
In another preferred embodiment the polypeptide or a fragment thereof is useful for producing antibodies which specifically bind to a p54 stress-activated protein kinase (e.g., SEQ ID NOs 6, 7, and 8). In this context, fragment means at least the smallest antigenic epitope, 20 generally at least 10 contiguous amino acids.
The invention also features a DNA molecule encoding a p54 stress-activated protein kinase polypeptide, its degenerate variants, or a fragment thereof including at least 30 contiguous nucleotides.
25 The DNA sequence may be 90% identical to p54~I (e.g., SEQ
ID N0: 9), p54~II (e.g., SEQ ID N0: 10), p54~I (e.g., SEQ
ID N0: 11), p54~II (e.g., SEQ ID N0: 12), or p54y (e.g., SEQ ID N0: 13), or be a fragment of any of the above nucleotide sequences containing at least 30 contiguous 30 nucleic acids, or a degenerate variant thereof. By "degenerate variant" it is meant any nucleotide sequence that encodes the same amino acid sequence as the polypeptide translated from the DNA, or a substantially identical polypeptide.

21~8~98 The molecules of the invention are preferably derived from a mammal, more preferably from a rat or human.
The invention also includes DNA molecules which 5 hybridize under stringent conditions to one or more of the DNAs encoding p54 SAPKs (SEQ ID NOs 9, 10, 11, 12, and 13). By "stringent conditions" it is meant conditions under which molecules without significant homology (e.g., at least 90%) to the DNAs of the 10 invention could not hybridize (protocols to determine stringency and melting point of DNA sequences are well known to those skilled in the art and may be found in Sambrook et al. (eds), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989; hereby 15 incorporated by reference). Included in the term "DNA(s)" are the sequences of both strands of double stranded DNA.
The invention also features methods of screening potentially therapeutic compounds involving applying test 20 compounds in physiologically relevant concentrations in a suitable excipient to cultured cells, with or without previous, prior, or concurrent stress-activated protein kinase-activating stimuli (e.g., TNF-~, IL-l-~, sphingomyelinase, heat shock, etc.), preparing cellular 25 extracts, and assaying the isolated recombinant stress-activated protein kinase for c-Jun kinase activity. By "physiologically relevant" it is meant a concentration that is achievable during non-toxic administration to a human patient. By suitable excipient it is meant a non-30 toxic or non-injurious solvent or carrier for the molecules of the invention or test compounds used in the screening assay.
In another embodiment, the cells treated with the test compound and with or without previous, prior, or 35 concurrent SAPK activating stimuli are extracted, and ` 2148898 combined with inactive recombinant stress-activated protein kinase. The recombinant SAPK is then isolated and assayed for c-Jun kinase activating ability.
The advantages and uses of the invention are in 5 the treatment and prevention of inflammation and the deleterious effects of hypoxia, heat stress, reperfusion injury, and other tissue insults which are currently difficult to manage clinically. The molecules of the invention may be useful for reducing inflammation in such 10 chronic disorders as autoimmune diseases or allergies, and in acute conditions such as anaphylactic shock, or soft tissue injury where swelling may aggravate the condition (e.g, around broken bones). other uses include prophylactic treatment of patients about to undergo 15 surgeries where there is a high likelihood of ischemia-reperfusion injury (e.g., vascular surgery, organ transplants, etc.), or treatment of sepsis and fever.
Additionally, these molecules could be used to up-regulate AP-l expression via c-Jun phosphorylation, and 20 thus enhance levels of IL-2 expression as a cancer therapeutic.
The molecules of the invention may also be useful for drug design based on an underst~n~;ng of the enzymes' structure and function (e.g., the polypeptide's binding 25 site on c-Jun), or as indicators in an assay to screen large numbers of drugs for beneficial effects on the conditions listed above.
Other features and advantages of the invention will be apparent from the following description of the 30 preferred embodiments, and from the claims.

Brief Description of the Drawings Figure 1: Deduced amino acid sequences (SEQ ID
NOs: 1, 2, 3, 4, 5, 6, 7, and 8) alignment of p54 stress-activated protein kinase (SAPK) cDNAs (single 21~8898 -letter code). Sequences determined by protein microsequencing are underlined, and the black diamonds flank the catalytic domains of the SAPKs.
Figures 2 a) and b): Specificity of anti p54 SAPK
5 antisera for p54 SAPKs. a) In vitro translation of ERKs.
RNAs (2~g) transcribed from plasmids containing the cDNAs of rat p54~I (lane 1), p54~ (lane 2), and HA-epitope-tagged p44 MAPK (lane 3) were translated using rabbit reticulocyte lysates. Lane 4 shows lysate programmed 10 with water; a 43-kDa background band is indicated by an open arrowhead. b) Aliquots of the translated proteins were immunoprecipitated in RIPA buffer with antisera raised against bacterially expressed p54~. Lanes are the same as in the left panel (lanes 1, 2, and 3).
Figures 3 a) and b): HepG2 cells were treated with TNF-~ (lOOng/ml, 15 min). Cell extracts were prepared and depleted of SAPKs by exhaustive immunoprecipitation for the times indicated (a) or for 4 hrs (b), at which time extracts were subjected to GST-c-20 Jun chromatography as a means of determining the c-Jun kinase activity remaining in the extracts. As a control, immunodepletions were done with preimmune serum or without serum (b). Activity in the immunoprecipitates and on the c-Jun columns was measure (a). The results 25 indicate that around 70% of the c-Jun kinase activity present in the extracts can be removed with the anti-SAPK
serum.
Figures 4 a) and b): Comparative activation of p42/44 MAPKs (open bars) and p54 SAPKs (filled bars) 30 showing that NIH3T3 cell (a) and HT-29 cell (b) p54 SAPKs are poorly activated by mitogens and strongly activated by heat stress and cycloheximide.
Figure 5 a) and b): Activation of c-Jun phosphorylation in situ by various stimuli. a) c-Jun 35 phosphorylated in vivo in response to cycloheximide (lane 1) and heat shock (lane 2); retardation of the Jun polypeptide upon SDS-PAGE (compare lanes 1 and 2 with non-phosphorylated c-Jun in lane 3). The c-Jun polypeptide is indicated with an arrowhead. b) Two 5 dimensional tryptic phosphopeptide mapping of c-Jun polypeptides immunoprecipitated from control (left), or heat shock-treated (right) HepG2 cells indicates enhanced phosphorylation of peptides X and Y in response to heat shock. The origin is marked with an arrowhead. The 10 dotted circle indicates the position of the xylene cyanol marker.
Figures 6 a) and b): Detection and isolation of c-Jun kinases activated by heat shock and other stimuli on GST-c-Jun immobilized on glutathione agarose (b);
15 comparison with activation of p54 SAPKs (a). The black and white bars each represent results from one experiment.
Figures 7 a) and b): Activation of SAPKs and c-Jun kinases by tunicamycin. p54 SAPK activity is shown 20 in a, closed circles (mean + SD for triplicate determinations), total c-Jun kinases binding to immobilized GST-c-Jun, a, open circles; b shows the fold activation of the p42/p44 MAPKs by tunicamycin.
Figure 8 a) and b): Comparison of activation of 25 p54 SAPKs and p42/44 MAPKs by various stimuli in human CCD-18Co cells. a) Activation of p54 SAPKs by TNF-~ and other stimuli. Mean + SD for triplicate determinations is shown. b) Parallel assays for relative activation of p42/44 MAPKs by the same stimuli shown in a).
Figure 9 a) and b): Comparison of activation of p54 SAPKs and p42/44 MAPKs by various stimuli in primary porcine hepatocytes. a) Activation of p54 SAPKs by TNF-~ and other stimuli. Mean + SD for triplicate determinations is shown. b) Comparison of fold activation by various stimuli of p54 SAPKs (filled bars) and p42/44 MAPKs (open bars).
Figure 10: Activation of HepG2 cell p54 SAPKs by TNF-~ and S. aureus sphingomyelinase. SMase =
5 sphingomyelinase.
Figures 11 a) and b): Comparison of activation of p54 SAPKs and p42/44 MAPKs by IL-l-~. EL-4 murine thyoma cells were treated with 20 ng/ml recombinant human IL-l-~. Cells were extracted as described in the legend of 10 Table 2. SAPKs (a and filled bars, b) and p42/44 MAPKs (b) were assayed in standard experimental paradigms. For a) data are mean + SD for triplicate determinations. For b) data are presented as percent of control for comparative purposes.
Figures 12 a) and b): Effect of ischemia/
reperfusion on MAPK and SAPK activation in vivo.
Activation of p54 SAPK (a) and p42/44 MAPKs (b) was measured at times from 0-90 min after initiating reperfusion of rat kidneys made ischemic for 45 min. as 20 described in the Methods section. MAPKs experienced rapid activation and inactivation (b), while SAPKs were rapidly activated and remained at elevated levels for periods well in excess of 90 min.
Figure 13: Bacterially expressed rat SAPK-~ was 25 expressed as a GST fusion protein and purified on glutathione agarose. The SAPK was treated with buffer alone, or with an extract from HepG2 cells treated with TNF and prepared as described in the Table 2 legend. The SAPK was recovered with glutathione beads and assayed for 30 c-Jun kinase activity. As an additional control, blank beads were exposed to cell extracts (extract alone bar).

214~8 g Description of the Preferred Embodiments Methods Cloning p54 SAP kinase was purified to homogeneity from 5 livers of cycloheximide injected rats (Kyriakis and Avruch, J. Biol. Chem. 265:17355-17363) and the sequences of several tryptic peptides were determined following RP-HPLC. Two of these peptides (HRDLKPSN and MLVIDPDKRISVDEAL) were homologous to protein kinase 10 subdomains VIb and XI, respectively (Hanks et al ., Science, 241:42-52, 1988) and were used to design degenerate sense and antisense primers, respectively. A
467 bp fragment was amplified by PCR from rat brain cDNA
and used as a probe to screen 250,000 plaques of a rat 15 brain cDNA library in AZAP (Stratagene). Twenty seven positive plaques were purified and representatives sequenced on both strands using nested deletion series.

Antisera/Antibodies To generate antisera, the p54 SAPK-~ isoform was 20 subcloned into pGEX-KG and expressed as a glutathione S-transferase (GST) fusion protein. Expression p54 SAPK-~polypeptide was induced with IPTG (50~g/ml) and the fusion purified to homogeneity by glutathione-agarose chromatography, followed by thrombin cleavage to release 25 the kinase moiety from the fusion protein. This material was then used as an immunogen and antisera collected and tested. The antisera tested have been shown to cross-react with human, mouse, rat, and pig tissues. The SAPK
sera react with several human cell lines, including human 30 hepatocellular carcinoma cells (HepG2), human colon fibroblasts (CCD-18Co), human monocytic lymphoma cells (U937), and human colon carcinoma cells (HT-29).
Monoclonal antibodies may be made by fusing immune B cells from the spleen with tumor cells to produce hybridomas specifically secreting each antibody, using methods well known in the art (see, for instance, Coligan et al., eds. Current Protocols in Immunology, John Wiley and Sons, 1992; Kohler et al., Nature 256: 495, 1975;
5 Hammerling et al., in Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981) Peptide antisera were generated using standard methods to the p54~, p54~, and p54y classes using the least conserved region of the molecules (SEQ ID NOs: 6, 10 7, and 8) to enhance the probability of class-specificity.

Cell Cul ture Treatments and Assay Confluent cultures of NIH3T3 cells were treated with H22 (5 mM, 15 min) phorbol-12-myristate-13-acetate (PMA, 500 nM, 20 min) FGF (10 ng/ml, 20 min), A23187 (100 nM 20 min), heat (42C, 30 min) or cycloheximide.
HT-29 cells were treated with EGF (50 ng/ml, 20 min) or heat shock (42C, 30 min). Cells were washed three times in PBS and lysed in ice-cold lysis buffer (20 mM Na-20 Hepes, pH 7.5, 2 mM EGTA, 1 mM DTT, 1 mM Na3V04. 50 mM ~-glycerophosphate, 1% (w/v) Triton X-100, 10% (v/v) glycerol, 2 ~M leupeptin, 10 kallikrein-inhibiting units/ml aprotinin, 200 ~m PMSF). Extracts were normalized to identical protein concentration (1 mg/ml).
25 A portion (1 ml) was immunoprecipitated with 5/13-99, and assayed for GST-Jun kinase activity as follows. To 40 ~l of a 1:1 suspension of p54 SAPK beads were added 20 ~l 0.2 mg/ml GST-c-Jun-1-135 (Adler et al., (1992), Proc.
Natl. Acad. Sci. U.S.A., 89:5341-5345) or 0.01 mg/ml 30 holo-c-Jun (Pulverer et al., supra; Pulverer et al., (1993), Oncogene, 8:407-415). ATP (100 ~M) and MgCl2 (10 mM) were added to start the reaction. The reaction was allowed to proceed for 20 min at 30C at which time the reaction was stopped with SDS sample buffer and the - 21 188~

mixtures resolved by SDS-PAGE. The 40-kDa GST-Jun band was excised and counted by liquid scintillation spectroscopy (mean + SD for triplicate determinations are shown in the figures). Another portion (1 ml) was 5 assayed for p42/p44 MAPK activity by Mono-Q
chromatography, and by a myelin basic protein (MBP) kinase activity assay (Ahn et al., (1991), J . Biol .
Chem ., 266:4220-4227). A peak of stimulated MBP kinase activity was always detected eluting between 200 and 350 10 nM NaCl. Total p42/p44 MAPK activity was taken as that contained in those fractions and is shown as percent of control for comparative purposes.
U937 cells were labeled with 32p orthophosphate (1 mCi/ml) and treated with cycloheximide heat stress or 15 vehicle. c-Jun was immunoprecipitated using a polyclonal antibody specific for the C-terminal 15 amino acids of c-Jun and subjected to SDS-PAGE (Pulverer et al.). HepG2 cells were labeled with 32p as above and subjected to heat shock. c-Jun was immunoprecipitated and, after SDS-PAGE, 20 was subjected to two dimensional tryptic phosphopeptide mapping. In other experiments, U937 cells were treated with actinomycin-D (10 ~g/ml, 30 min), anisomycin (10 ~g/ml, 30 min) emetine (10 ~g/ml, 30 min), puromycin (10 ~g/ml, 30 min) or heat stress (42C, 30 min). Lysates 25 were prepared and were passed over columns of immobilized GST-Jun. After washing away unbound proteins, Mg/y32P-ATP
was added and phosphorylation of the immobilized c-Jun was assayed as described above. Parallel aliquots were subjected to immunoprecipitation and assayed for p54 30 SAPK. In both assays, 1 U of kinase activity transferred 1 pmol P04/min from ATP to GST-Jun. HT29 cells were treated with various concentrations of tunicamycin for 5 h. Lysis, assay of p42/p44 MAPK activity, and p54 SAPK
immune complex kinase assay were done by standard 35 methods. Detection of total tunicamycin-stimulated c-Jun 21~88g8 kinase activity in HT-29 cells isolated on columns of immobilized c-Jun was performed as described above.
CCD-18Co cells, HepG2 cells, or primary porcine hepatocytes were cultured to 80% confluence, serum 5 starved (0.5% serum, 16 hours) and treated with the following agonists as shown in Figures 16 and 17: heat shock (42C, 30 min), PMA (200 nM, 20 min), EGF (100 ng/ml, 20 min) or TNF-~ (50 ng/ml, 10 or 20 min).
Extracts were prepared and p54 SAPKs were 10 immunoprecipitated and assayed. Parallel assays of p42/p44 MAPKs were performed. For treatment with sphingomyelinase, 100 mU/ml S. aureus sphingomyelinase were added to HepG2 cells as known in the art (Dressler et al., (1992), Science, 255:1715-1718) for 15 min. lU
15 S. aureus sphingomyelinase hydrolyzed 1 ~mole TNP-sphingomyelin/min at pH 7.4, 37C.

Ischemia/Reperfusion Male Sprague-Dawley rats, 120-150 g each, were anesthetized with sodium pentobarbital (65 mg/kg). In 20 order to induce ischemia, the renal artery of one kidney was clamped for 45 min. The contralateral kidney served as a control. Reperfusion was accomplished by releasing the clamp and allowing blood flow for 0 to 90 min.
Control and ischemia/reperfusion kidneys were harvested 25 and homogenized in lysis buffer (20 mM Hepes, pH 7.4, 2 mM EGTA, 50 mM ~-glycerophosphate, 1 mM DTT, 250 mM
sucrose, 400 ~M PMSF, 2 ~M leupeptin, 2 ~M aprotinin).
After centrifugation for 1 hr at 100,000 x g, the supernatants were collected and Na3VO4 added to 0.1%
(w/v). Immunoprecipitation and SAPK assay were as described in the Table 2 legend.

Isolated clones p54 protein kinase was isolated from rat liver that had been stimulated with cycloheximide, using the above methods (also described in Kyriakis and Avruch, 5 supra; hereby incorporated by reference) and amino acid sequences were derived from peptides generated by tryptic digests. These sequences aligned with the consensus Ser/Thr protein kinase catalytic domain of known MAPKs (Hanks et al . ) . Two of these peptide sequences were used 10 to design overlapping degenerate oligonucleotide probes for use in a nested PCR reaction from which a 467 bp cDNA
was generated using rat brain cDNA as the template. This probe was used to screen a rat brain cDNA library from which 27 clones were isolated and sequenced. The cDNAs 15 encoded five separate polypeptides (Fig. 1, SEQ ID NOs 1-5). This was quite unexpected, since it was assumed that only one peptide had been purified to homogeneity from the rat liver in this and previous work (Kyriakis and Avruch). One set of clones (p54~I, SEQ ID NO. 9) encoded 20 a protein that contained all of the tryptic peptides derived from rat liver p54 kinase. Two additional sets (p54~I/II, SEQ ID NOs 11 and 12; and p54~, SEQ ID NO. 13) encoded closely related polypeptides (88-90% identity, respectively to ~I, Fig. 1). A fourth group of cDNAs (p54~II, SEQ ID NO. 10) was identical to ~I kinase except for a region of 71 base pairs which results in the substitution of 15 amino acids in subdomains IX and X
(see Fig. 1). The ~I and ~II RNAs are most likely derived from the same gene by alternate splicing. The 30 predicted proteins encoded by the full length clones are:
~I, 48,076 Da; ~II, 47,986 Da; and ~I, 48,095 Da.
Northern blotting analysis of several tissues revealed ubiquitous, low expression of all three gene classes.

214~898 ProPerties of p54 SAPKs The distinct characteristics of the isolated p54 clones and expressed and native polypeptides have led to the nomenclature of Stress-_ctivated Protein _inases (SAPKs), which will be used throughout to distinguish them from the MAPKs.
Sequence alignment of the catalytic domains of the p54 SAPK clones with those of the known mammalian MAPKs, and with the yeast MAPK homologs RSS1, HOG-l FUS3, SLT-2, 10 spk-1 and erk-l (Courchesne et al., (1989), Cell, 58:1107-1119; Brewster et al., (1993), Science, 259:1760-1763; Levin et al ., ( 1993), J. NIH Res., 5:49-52) shows that the p54 polypeptides exhibit nearly equal identity to the mammalian 44 kDa MAPK (43-44% sequence identity) 15 and the kinases from lower eukaryotes (41-44% identity).
By contrast, p44 MAPK is closer in sequence to the yeast kin~ces (between 49-52% for the S. cerevisiae enzymes and 56% identity for Spk-l) than it is to the p54 SAPKs.
From these and other comparative data, we conclude that 20 none of the MAP kinase-like genes identified thus far in lower eukaryotes is likely to be a functional homologue of the p54 SAPKs. All of the p54 isoforms contain the sequence Thr183-Pro-Tyrl85 in an analogous position to the Thr and Tyr residues of p42/p44 MAPKs (Payne et al., (1991) EMBO J. 10:885-892) that must be phosphorylated for activity. These residues in the p42/p44 MAPKs are phosphorylated by the _APK or ERK _inases (MEKs), a family of dual specificity protein Tyr/Thr kinases (Ahn et al.; G6mez et al., (1991) Nature, 253:170-173;
30 Nakielny et al., (1992), EMBO J., 11:2123-2129).
However, in vitro, neither purified dephosphorylated liver p54 SAPK nor bacterially expressed p54 SAPK
isoforms are phosphorylated or reactivated by p42/p44 MAPK-specific MEKs, suggesting the existence of a 35 specific p54 SAPK kinase.

In vivo administration of cycloheximide activates p54 SAPK, and we have also characterized the stimuli and signal transduction pathways that activate p54 SAPKs in cultured cells. A polyclonal antiserum (5/13-99), raised 5 against the prokaryotic recombinant p54 SAPK ~-isoform, immunoblots p54 SAPK and immunoprecipitates in vitro translated p54~ and p54~ polypeptides but not p44 MAP
kinase (Fig. 2). Purified rat liver p54 SAPK
phosphorylates c-Jun exclusively at Ser73 and Ser63 in the 10 c-Jun trans activation domain, two sites located on c-Jun tryptic phosphopeptides designated X and Y, respectively (Pulverer et al.; Smeal et al., (1991), Nature, 354:494-496). The anti-p54 SAPK antiserum (5/13-99) immunoprecipitates from HT-29 human colon carcinoma cells 15 a protein kinase activity that phosphorylates recombinant GST-c-Jun, as well as full-length c-Jun, selectively at tryptic peptides X and Y. Pretreatment of NIH3T3 cells or HT-29 cells with cycloheximide substantially augments this c-Jun (X/Y) kinase activity. Thus, the p54 20 antiserum is reactive with p54 SAPKs but not p42 MAPK or p44 MAPK. Moreover, p54 SAPK activity, measured as a c-Jun "X/Y" kinase, is stimulated by cycloheximide in tissue culture as well as in vivo.
SAPKs are the major c-Jun kinase activated by TNF-25 ~. SAPK immunodepletion experiments removed 60-70% of the recombinant c-Jun kinase activity induced in TNF-~treated cells (Fig. 3). This indicates that the SAPKs account for around 70% of the c-Jun-associated c-Jun kinase activity.

30 Requlation of p54 SAPKs Effects of NAPR Activators We examined the regulation of the p54 SAPK, in NIH3T3 and human HT-29 cells, by agents known to regulate p42/p44 MAPKs. The activity of p54 SAPKs was measured in 214~98 -an immune complex kinase assay using GST-c-Jun as a substrate, whereas the p42/p44 MAPKs in the same extracts were assayed, after Mono-Q anion exchange chromatography, using MBP as a substrate. Figure 4 compares the 5 activation of each set of kinases in response to the various treatments to the baseline values. As expected, NIH3T3 cell p42/44 MAPKs are strongly activated by mitogen (10-fold activation by FGF) and phorbol esters (6-fold), and are activated to a lesser extent by Ca2+
10 influx stimulated by the ionophore A23187 (2-fold). In addition, the p42/p44 MAPKs are activated by H2O2 and cycloheximide (4- and 6-fold, respectively). In striking contrast, the NIH3T3 cell p54 SAPKs are not activated by FGF, phorbol esters, H22 or by Ca2+ ionophore (top panel, 15 filled bars). A similar result is seen for HT-29 cells (bottom panel, filled bars). In these cells, EGF
substantially stimulates the p42/p44 MAPKs (6 fold, bottom panel, open bars) while only slightly stimulating p54 SAPKs (bottom panel, filled bars).
These results suggest that signals generated by activation of receptor tyrosine kinases and phospholipase C are not likely to represent the primary regulatory input to the p54 SAPKs. Therefore, stimuli other than mitogens were investigated to see if they could activate 25 p54 SAPKs more vigorously than they activated the p42/p44 MAPKs. Cycloheximide, although capable of strongly activating the NIH3T3 cell p54 SAPK (10-fold), also gives substantial (5-fold) activation of endogenous p42/p44 MAPKs. By contrast, heat shock (42C, 30 min) gives a 30 large induction of NIH3T3 cell p54 SAPK activity (7-fold), while p42/p44 MAPK activity is only slightly (1.5-fold) stimulated (Fig. 4, top panel). In HT-29 cells as well, heat shock dramatically stimulates HT-29 cell p54 SAPKs (8.4 fold, Fig. 4, bottom panel, filled bars) while 35 only modestly stimulating p42/p44 MAPKs (2.9 fold, Fig.

21~89~
-4, bottom, open bars). Activation of p54 SAPK by heat shock (42C) is evident by 10 min, maximal at 30 min, and declines slightly after 1 hr.

Effect of p54 SAP~ Activation on c-~un Since the p54 SAPKs are potent c-Jun kinases in vitro, we inquired whether stimuli such as heat shock and cycloheximide, that activate p54 SAPKs preferentially, also increase the phosphorylation of c-Jun in situ. 32p_ labeled U937 and HepG2 cells were exposed to heat shock 10 or cycloheximide, and the phosphorylation of endogenous c-Jun was examined. Both treatments induce a robust phosphorylation of c-Jun, accompanied by a dramatic retardation of c-Jun mobility on SDS-PAGE (Fig. 5a).
This retardation is characteristic of phosphorylation on 15 tryptic peptides X and Y (corresponding to Ser 73 and 63, respectively) within the c-Jun N-terminal trans activation domain (Pulverer et al.); the induction of X/Y
phosphorylation was verified directly by tryptic peptide maps of 32P-c-Jun isolated from heat-shocked 32P-labeled 20 HepG2 cells (Fig. 5b). Activation of c-Jun N-terminal phosphorylation by heat shock represents a previously unrecognized mode of c-Jun regulation. The likelihood that p54 SAPKs contribute substantially to the c-Jun phosphorylation elicited by heat shock and cycloheximide 25 in situ is supported by the ability of the immunoprecipitated p54 SAPK activated by cycloheximide to phosphorylate recombinant c-Jun in vitro exclusively on tryptic 32p peptides that co-migrate with tryptic peptides X and Y (see Fig. 3).

30 Effects of Protein Synthesis Inhibitors on p54 SAPRs Maneuvers that increase c-Jun X/Y phosphorylation in situ, such as W light or PMA, have been shown to activate c-Jun kinases that bind tightly to immobilized 21~8898 GST-Jun (Adler et al .; Hibi et al ., Genes & Dev., 7: 2135-2148, 1993). Extracts from heat-shocked or cycloheximide-treated cells also contain activated c-Jun kinase(s) that bind to immobilized GST-c-Jun (Fig. 6, 5 right panel). Moreover, two other inhibitors of polypeptide chain elongation, emetine and especially anisomycin, also activate both p54 SAPK and GST-Jun-bound c-Jun kinase (Fig. 6), whereas puromycin and the RNA
synthesis inhibitor, actinomycin D, are each unable to 10 activate either c-Jun kinase (Fig. 6, right panel) or p54 SAPK activity (Fig. 6, left panel). A summary of these results is presented in Table 1, below. Thus, heat shock, a variety of protein synthesis inhibitors as well as the glycosylation inhibitor, tunicamycin (see below), 15 alter p54 SAPK, and the GST-Jun-bound c-Jun kinase in parallel, suggesting that p54 SAPK is likely to be one of the physiologic Jun kinases activated by this class of perturbations.

Activation of p54s and c-Jun-associated k;n~s~c by protein and RNA synthesis inhibitors.

c-Jun-associated Treatmentp54 MAPK (mU) kinase (mU) Control 31.0 14.5 25 Anisomycin 370.0 385.0 Cycloheximide 166.0 243.0 Heat-shock 168.0 180.0 Emetine 75.0 85.0 Puromycin 19.3 21.5 30 Actinomycin-D 22.0 24.0 Activation of p54s and c-Jun-associated kinases by protein and RNA synthesis inhibitors. U937 cells were treated with anisomycin (lO~g/ml, 30 min), cycloheximide 21~8~98 , (200~M, 60 min), heat shock (42C, 30 min), or actinomycin D (lO~g/ml, 30 min). Extracts were prepared as described in the methods, and parallel aliquots were subjected to GST-c-Jun chromatography/assay or 5 immunoprecipitation and assay for p54. In both assays, 1 unit of kinase activity transferred 1 pmol PO4/min from ATP to GST-c-Jun. Shown are mean results for two experiments.

Polypeptide Misfolding Stimulates p54 SAPRs Although heat shock and protein synthesis inhibitors could activate p54 SAPK through entirely unrelated mechanisms, a shared property of heat shock and translational inhibitors (but not the RNA synthesis inhibitor, actinomycin D), is their ability to promote 15 polypeptide misfolding and denaturation. Consequently, we investigated whether the accumulation of misfolded polypeptides might be an initial stimulus common to protein synthesis inhibitors and heat shock that leads to the preferential activation of p54 SAPK. Tunicamycin 20 inhibits the synthesis and proper folding of proteins destined for membrane insertion or secretion through inhibition of N-linked glycosylation in the Golgi.
Increasing doses of tunicamycin added to HT29 cells promotes a striking activation (up to 12-fold) of p54 25 SAPK activity (Fig. 7, top, filled circles), whereas the p42/p44 MAPKs, assayed after separation by Mono-Q
chromatography, are only modestly activated (Fig. 7, bottom). In addition, tunicamycin stimulates total HT-29 Jun kinases which bind to GST-c-Jun with a similar dose 30 response to p54 SAPK activation (Fig. 7, top, open circles). The activation of the p54 SAPKs by tunicamycin, together with the data in Figures 4 and 6 support the idea that cellular stresses which result in the accumulation of misfolded polypeptides, can, to a 35 degree greater than mitogens, generate a signal to activate the p54 SAPKs. By contrast, the p42/p44 MAPKs 21~8898 -are more strongly activated by mitogenic signaling, relative to stress signaling.

i nf~c l'NF--~ and IL~
In mammals, integration of the multicellular and 5 inter-organ response to a variety of "stressful" noxious stimuli is mediated by a diverse array of inflammatory cytokines, such as TNF-~ and IL-1-~. TNF-~ was initially detected by its ability to induce the hemorrhagic necrosis of some transplantable tumors in inbred mice (Buetler et al., Ann. Rev. Biochem., 57:505-517, 1988;
Goeddel et al., Cold Spring Harbor Symp. Quant. Biol., 51:597-609, 1986). The cellular responses to inflammatory cytokines are quite diverse and cell-specific, and are directed at optimizing overall host 15 defense against infection. For example, TNF-a acts on adipose tissue to inhibit insulin action and energy storage; on liver to yield the protein secretory pattern known as the acute phase response; on neutrophils to enhance superoxide radical production and cytotoxic 20 efficacy; and on T cells to promote the secretion of additional cytokines, e.g. IL-2 and IL-6 (see Buetler et al. and Goeddel et al. for review). The multifarious actions of TNF-~ are due in part to TNF-~-directed programs of gene expression. Notably, TNF-~ has been 25 shown to be a potent activator of the trans activation function and autoinduction of c-Jun (Brenner et al., Nature, 337:661-663, 1989), as well as an activator of NF-KB (Osborn et al., Proc. Natl. Acad. Sci. U.S.A., 86:2336-2340, 1989), both of which are needed by 30 lymphocytes for trans activation of the IL-2 gene.
The profile of physiologic and cellular responses to TNF-~ and IL-l-~ led us to inquire whether these agents could activate p54 SAPK. Human CCD-18Co colon fibroblasts are acutely responsive to TNF-~ (Goeddel et 21~8~98 -al . ); in these cells, TNF-~ elicited a striking activation of p54 SAPKs, whereas EGF and PMA were without noteworthy effect (Fig. 8a). The 10-fold stimulation of p54 SAPK by TNF-~ was slightly greater than that provoked 5 by heat stress. Comparing the responses of p42/p44 MAPKs in CCD-18Co cells, it is clear that heat stress and TNF-a are far more potent activators of p54 SAPK than are EGF
and PMA, while the converse is true for the p42/44 MAPKs (Fig. 8b).
Liver is also a target tissue for TNF-~ (Buetler et al ., and Goeddel et al . ); addition of TNF-~ to primary cultures of freshly isolated porcine hepatocytes stimulates p54 SAPK activity by ~ 6-fold (Fig. 9a), whereas EGF increases p54 SAPK activity 3-fold. In these 15 cells, EGF and PMA activate the p42/p44 MAPKs and the p54 SAPKs to a comparable degree. However, as is seen in CCD-18Co cells, heat shock and TNF-a are much more potent activators of p54 SAPK than they are p42/p44 MAPK
activators (Fig. 9b). The present results are thus 20 consistent with recent reports showing that TNF-~activates p42/p44 MAPKs (Van Lint et al., J. Biol. Chem., 267:25916-25921, 1992); however, it is clear that the p42/p44 MAP kinases are much more potently activated by ligands like EGF and FGF, that operate through receptor 25 tyrosine kinase, whereas the ability of TNF-~ to activate the p54 SAPKs greatly exceeds the ability of FGF and EGF
to activate the p54 SAPK, at least in most cell types.
Little is known of the molecular mechanisms of TNF-~ signaling. TNF-~ binds to one of two receptors, 30 55-kDa and 70-kDa, whose intracellular extensions show no homology with receptors whose signal transduction mechanisms are better understood (Loetscher et al., Cell, 61:351-359, 1990; Heller et al ., Proc. Natl . Acad . sci .
U.S .A., 87:6151-6155, 1990). TNF-~ has been shown to 35 stimulate rapid sphingomyelin hydrolysis and the accumulation of ceramide, through the activation of a neutral sphingomyelinase (Dressler et al.; Yang et al., J. Biol . Chem., 268:20520-20523, 1993; Dobrowsky et al., J. Biol. Chem., 267:5048-5051, 1992; Dbaibo et al., J.
5 Biol . Chem., 268:17762-17766, 1993; Schutze et al., Cell, 71:42-52, 1992). Ceramide has been proposed to serve as a second messenger for TNF-~, analogous to the role envisioned for diacylglycerol in the action of hormones that activate phosphatidylinositol-specific phospholipase 10 C enzymes (Dressler, et al.; Yang, Z., et al.;
Dobrowsky, et al.; Dbaibo, et al.; Schutze et al.; and Sch~tze, et al . ) . Many of the responses to TNF-~, including growth inhibition, apoptosis, activation of heterotrimeric forms of protein phosphatase-2A, 15 activation of a membrane bound Ser/Thr kinase and, possibly, activation of NF-~B can be elicited by the addition of various ceramide derivatives or bacterial sphingomyelinase to intact or permeabilized cells (Dressler et al.; Yang, et al.; Dobrowsky, et al.;
20 Dbaibo, et al .; and Schutze et al . ) . Based on these considerations, we compared the ability of TNF-~, IL-1-~and S. aureus sphingomyelinase treatment of HepG2 or EL-4 cells to alter p54 SAPK activity. TNF-~ stimulates a robust activation of the p54 SAPKs (15-fold) in these 25 cells, and a substantial activation of p54 SAPKs (5.3-fold) is also evident in response to sphingomyelinase (Fig. 10). These results are consistent with the possibility that sphingomyelin hydrolysis, as is known to occur in response to inflammatory cytokines, may be an 30 early step in the p54 SAPK signal transduction pathway.
When EL-4 murine thyoma cells were treated with 20 ng/ml recombinant human IL-1-~, p54 SAPK activity increased over 4-fold, while p42/44 MAPK activity remained unchanged by the treatment (Fig. 11 a and b).
35 This strong induction of SAPK activity without concomitant activation of MAPKs mirrors the results with cytokine TNF-~, and indicates a similar inflammation-mediated pathway.

Ischemia/Reperfusion Induces p54 SAP~s An additional distinction for the p54 SAPKs from the p42/44 MAPKs is the time course of activation in response to reperfusion of ischemic tissue. Rat kidney was made ischemic in vivo by the methods described above.
The results of this experiment (Fig. 12) showed that the 10 p54 SAPKs and the p42/44 MAPKs both were activated immediately upon reperfusion of the kidney, and that the activation of p54 SAPKs peaked after about 20 min (Fig.
12a), while the p42/44 MAPKs peaked at about 5 min after initiation of reperfusion (Fig. 12b). The duration of 15 p54 SAPK activation was also considerably longer, lasting considerably longer than 90 min before returning to control levels, whereas p42/44 MAPK activation returned to control levels within 20 min.
These findings have important implications in the 20 pathogenesis of chronic problems such as acute renal failure, infarction arising from ischemic heart disease, and surgical induction of ischemia/reperfusion injury.
The p54 SAPKs clearly play a more significant role in tissue stresses such as ischemia than do the p42/44 25 MAPKs, and their effects are more sustained. These results are the first to delineate a signal transduction cascade specifically activated during reperfusion of ischemic tissue. It is known that c-fos and some heat shock protein genes are transcribed during reperfusion, 30 and that ischemia results in the generation of oxygen radicals and can lead to tissue damage. The data presented here indicate that SAPKs are activated during reperfusion, and SAPKs target c-Jun. Tissue repair genes, such as the collagenase gene, are regulated by AP-~.

1, and thus, SAPK activation may represent the initiation of repair processes at the molecular level.

Therapeutic Drug Screening with pS4 SAPKs The experimental approaches described above can be S used to screen compounds to identify those with therapeutic potential. Two examples of possible screening protocols follow. These examples are not intended to be limiting.
Exampl e Human (or other mammalian) cell lines such as HepG2, CCD-18Co, U937, and HT-29 can be treated with any test compound, and treated with SAPK activators (e.g., TNF-~, IL~ , ATP-depletion/refeeding analogous to ischemia/reperfusion, etc.). The order of treatment can 15 be test compound, then SAPK-activator; SAPK activator then test compound, or both treatments simultaneously.
Treatment times can vary from 1 min to several hours, depending on the time necessary to induce changes in the SAPK pathway.
The cells are then extracted, and the endogenous SAPKs are immunoprecipitated as described above. The SAPKs are then tested in a st~n~rd c-Jun kinase assay for activity. This method of assaying for SAPK
activation can also be employed with tissue samples 25 obtained from patient biopsy, and has been used successfully to detect the activity of SAPKs from samples of rat kidney. In this way, therapeutics can be evaluated for their ability to activate or inhibit the SAPK pathway.
30 Example 2 Any of the human or rat SAPK clones may be used in an expression vector for recombinant bacterial (or viral, etc.) expression of inactive forms of the SAPK. Because of the high homology between rat and human SAPKs, either 21~8898 -may be used in the assay for therapeutic compounds.
Human cell lines such as HepG2, CCD-18Co, U937, and HT-29 can be treated with any compound to evaluate its toxicity and efficacy as a therapeutic in modulating the SAPK/c-5 Jun kinase pathway.
Cells are treated with the test compound either alone or with a SAPK activating compound (such as TNF-~, etc.; the activator may be applied before or concurrently with the application of the test compound), and then 10 extracted to produce a cytoplasmic lysate. This extract is then combined with the recombinantly expressed, inactive SAPK. The SAPK can be expressed as a GST fusion protein so that it is easily purified on glutathione agarose. After a suitable length of time (minutes to 15 hours) for interaction between the cell lysate and the recombinant inactive SAPK, the recombinant SAPK is isolated by binding to glutathione beads (leaving behind endogenous SAPKs) or by immunoprecipitation with an anti-glutathione antibody (commercially available; any other 20 isolation technique may be used also), and assayed for c-Jun kinase activity. If there is no c-Jun kinase activity, and the test compound was applied to the cells alone, then it may be concluded that this compound does not activate SAPKs. If the test compound was applied 25 with a SAPK activator, then it may be concluded that the test compound inhibits the activation of the SAPK
pathway. If c-Jun kinase activity is detected, and the test compound was applied to the cells alone, then the compound activates the SAPK pathway. If the test 30 compound was applied with a SAPK activator, and c-Jun kinase activity is detected, then it may be concluded that a) the compound does not inhibit the SAPK pathway, b) the compound may enhance SAPK activity (which can be quantitatively evaluated), or c) the drug has no effect.

214~898 Both of these screening systems allow rapid and extensive testing of many compounds, regardless of their preconceived potential therapeutic use, and may facilitate identification of therapeutics which 5 accelerate tissue repair (increased SAPK pathway activation), or prevent or alleviate allergy, excess swelling, anaphylaxis, etc. (decreased SAPK pathway activation). An example of this assay is shown in Figure 13, using TNF-~ as the test compound, and showing 10 controls with the expressed SAPK alone, and the HepG2 cell extract alone to demonstrate that the recombinant form is inactive as expressed, and that the cell extract has no intrinsic activity in the assay.

Summary of Results This invention identifies the molecular structures of a new subfamily of proline-directed protein kinases whose upstream activators include heat stress, protein synthesis inhibitors, ischemia/reperfusion injury, and the inflammatory cytokines, TNF-~ and IL-1-~. This array 20 of regulatory inputs suggests that this kinase subfamily may serve to monitor and form part of the cellular response to a variety of intracellular and extracellular stress signals. The p42/p44 MAPKs, although capable of being activated by these stress treatments, are to a much 25 greater degree activated by mitogens (see Table 2 for summary of results). The mitogenic agents such as EGF, FGF and PMA, which have been shown to activate the p42/p44 MAPKs through a Ras and c-Raf-l-dependent pathway, activate the SAPKs weakly if at all. This 30 suggests that the SAPKs, although reguiring both Ser/Thr and Tyr phosphorylation for activity (Kyriakis et al., (1991), J. Biol . Chem., 266:10043-10046) as do the p42/p44 MAPKs (Anderson et al . ), lie on an entirely distinct signal transduction pathway. Consistent with 21~8898 -this conclusion is our inability, using highly purified MEK, to reactivate phosphatase-2A inactivated rat liver SAPK, or to activate prokaryotic recombinant SAPK-~ under conditions that provide near complete MEK activation of 5 comparable preparations of purified or recombinant p42/p44 MAPK. The SAPKs are thus the proline-directed kinase elements in what is likely to be a new protein kinase cascade, the second such pathway uncovered in mammalian cells. Three independently regulated protein 10 kinase cascades upstream of distinct proline-directed kinases (FUS/RSS; HOG-1; MPR-l) have been uncovered thus far in S. cerevisiae (Levin et al.). We propose that the SAPK cascade is an important, perhaps central, component of the cellular response system to TNF-~, analogous to 15 the role of the MAPK cascade in the response to activation of receptor tyrosine kinases. Lipid regulators derived from sphingomyelin hydrolysis may participate at one of the earlier intracellular steps upstream of the SAPKs; a similar role for phospholipid-20 derived mediators upstream of the MAPKs has recently beenproposed (Cai et al., (1993), Mol. Cell. Biol., 13:7645-7651).

2148~98 Activation of p54 and MAPKs P42/44 In various cell lines by various treatments. ND = not done.
p54 Activity p42/44 5 Cells Treatment (mU) MAPK
Activity NIH3T3 Control 5.8 + 0.6 71.4 PMA 5.8 + 0.9 409.7 FGF 8.5 + 0.6 795.5 A23187 8.9 + 0.7 172.0 H2O2 5.9 + 0.1 272.2 Heat shock38.8 + 1.7 169.7 Cycloheximide57.1 + 4.1 419.9 HT-29 Control 20.7 + 1.7 95.0 EGF 35.7 + 1.4 576.0 Heat shock172.0 + 28.5 281.0 HT-29 Control 69.4 + 7.5 195.5 Tunicamycin803.9 + 77.1 480.9 CCD-18Co Control 7.0 + 1.0 12.2 PMA 8.8 + 0.7 601.0 EGF 13.0 + 2.1 394.0 Heat shock48.0 + 1.3 83.7 TNF-a 60.0 + 5.0 117.6 25 Primary Control 179 + 13 1294 Porcine PMA 479 + 58 3529 Hepatocytes EGF 665 + 58 4882 Heat shock697 + 49 2427 TNF-~, 10 min 1008 + 31 3064 TNF-~, 20 min 924 + 38 ND
HepG2 Control 29.8 + 4.3 ND
TNF-~ 434.8 + 36.4 ND
Sphingomyelinase 159.0 + 17.3 ND
Confluent cultures of NIH3T3 cells were 35 treated with H22 (5 mM, 15 min) phorbol-12-myristate-13-acetate (PMA, 500 nM, 20 min) FGF (10 ng ml~1, 20 min), A23187 (100 nM 20 min), heat (42C, 30 min) or cycloheximide (200 ~M, 60 min). HT-29 cells were treated with EGF (50 ng ml~ , 20 min), heat shock (42C, 30 min) 40 or tunicamycin (50 ~ ml~l). CCD-18Co Cells (100%
confluent) were treated with PMA 200 nM, 15 min), EGF (50 ng ml~1, 15 min), heat shock (42C, 40 min) or TNF-~ (50 ng ml~l, 20 min). Primary porcine hepatocytes were treated the same as the CCD-18Co cells. HepG2 cells (80%
45 confluent) were treated with TNF-~ (50 ng ml~1, 15 min) or S. aureus sphingomyelinase (100 U ml~l). lU 5. aureus sphingomyelinase will hydrolyze 1 ~mole TNP-sphingomyelin min~1 at pH 7.4, 37C. Cell lysis and immunoprecipitation were performed as described in Kyriakis et al, Nature 5 358:417-421, 1992 (hereby incorporated by reference).
Precipitates were assayed for GST-Jun kinase activity as follows. To 40 ~l of a 1:1 suspension of protein G
Sepharose beads containing immunocomplexed p54 were added 20 ~1 0.2 mg ml~l GST-C-Jun-1-135 (26,37) or 0.01 mg ml~
10 holo-c-Jun. [y-32P]ATP (100 ~M) and MgC12 (10 mM) were added to start the reaction. The reaction was allowed to proceed for 20 min at 30C at which time the reaction was stopped with SDS sample buffer and the mixtures resolved by SDS-PAGE. The 40-kDa GST-Jun band was excised and 15 counted by liquid scintillation spectrometry. p54 kinase assays were performed in triplicate. Mean + SD are shown. Another portion of extracts (1 ml) was subjected to Mono-Q chromatography; fractions were assayed for MAPK
p42/44 activity as MBP kinase activity. A peak of 20 stimulated MBP kinase activity was always detected eluting between 200 and 350 mM NaCl; total MAPK p42/44 activity was taken as MBP kinase activity in those fractions combined. 1 U p54 activity will transfer 1 pmol min~1 PO4 from ATP to c-Jun. 1 U MAPK p42/44 will 25 transfer 1 pmol min~1 P04 from ATP to MBP.
A central conundrum in the field of signal transduction is how agonists such as EGF and TNF-~ can exert such different effects while apparently only activating one MAPK (the p42/p44 MAPK) pathway. The data 30 in Figures 16 and 17, taken in combination with the known differences in SAPK and p42/p44 MAPK substrate specificity, suggest that signaling specificity may arise in part from the differential recruitment of signaling pathways such as the SAPK and p42/p44 MAPK pathways.
Whatever the identity of the elements that couple heat shock, protein synthesis inhibition, the cytokine receptor(s), and other cellular stresses to the SAPKs, the results presented here support the contention that the c-Jun polypeptide is a downstream target of the 40 SAPKs. The SAPKs are a group of c-Jun N-terminal kinases. These kinases and members of the related superfamily focus a broad variety of regulatory signals, including phorbol esters, Ha-Ras, ultraviolet radiation 21~8898 -(Pulverer et al.; Smeal et al.; Adler et al.; Hibi et al .; Binétruy et al .; Devary et al ., ( 1992 ), Cel l, 71:1081-1091), heat shock, protein synthesis inhibitors and cytokines into the phosphorylation of the c-Jun trans 5 activation domain. The multiplicity of these c-Jun kinases and their activating inputs attest to the diversity of c-Jun function, and the complexity of its regulation. The role of c-Jun phosphorylation in the heat shock response and in the actions of TNF-~ remain to 10 be clarified. The consequences of c-Jun mediated transcriptional trans activation exhibit a great deal of cell specificity, and whether the outcome of MAPK- or SAPK-mediated c-Jun transactivation is mitogenesis, growth inhibition or a stable new phenotype resulting lS from altered gene expression is likely to depend on the array of signaling components acting in concert with AP-1.

Uses of the Invention The extracellular signal regulated family of 20 kinases are activated in response to different extracellular stimuli, and this specificity allows cells to diverge in their function in order to target a response to a given stimulus. The molecules of the invention can intervene in or stimulate a basic pathway 25 modulating transcription of proteins that mediate an inflammatory or cell-stress response. Their use in the treatment and prevention of inflammation and the deleterious effects of hypoxia, heat stress, reperfusion injury, and other tissue insults may resolve many 30 difficulties that arise with current therapies that rely on amelioration of the aftermath of damage instead of being able to redirect the course of the syndrome.
An additional use for these molecules may be in the upregulation of IL-2, which has proven useful in the 21~88~8 treatment of cancer. AP-1 activation is known to induce IL-2, and since c-Jun is a component of the AP-1 dimer, p54 phosphorylation of c-Jun would be expected to modulate AP-l levels. p54 immunodepletion experiments 5 have been shown to have a major effect on the phosphorylation of c-Jun.
A use for the p54 kinases is as a template for drug design, or a functional component for therapeutic drug assays. Many functional motifs of the molecules are 10 already known (e.g., the ATP-binding site, sites of regulatory phosphorylation, c-Jun binding site, etc.) which, if an effective antagonist or agonist could be derived, could play an important role in therapies for conditions such as are listed above. Assays to screen 15 for such drugs exist, and are described above.
Monitoring the enzymatic activity following incubation with potentially therapeutic compounds such as was done with the drugs described herein would allow a simple, fast method to determine efficacy and dose-response for 20 up or down regulation of the SAPKs. Large numbers of candidate compounds can be screened easily and evaluated for specificity, efficacy, and toxicity. Such information would allow rational evaluation of many drugs for their ability to up- or down-regulate cellular 25 responses to physiological stresses, and be useful in clinical management of inflammation, ischemia, and many other stimuli that activate the SAPKs.
The molecules of the invention may be useful for reducing inflammation in such chronic disorders as 30 autoimmune diseases or allergies, and in acute conditions such as anaphylactic shock, or soft tissue injury where swelling may aggravate the condition (e.g, around broken bones). Other uses include prophylactic treatment of patients about to undergo surgeries where there is a high 35 likelihood of ischemia-reperfusion injury (e.g., vascular -surgery, organ transplants, etc.), or treatment of sepsis and fever.
For such conditions as mentioned above, a systemic application of the molecules of the invention, 5 or antibodies derived from them, is generally desirable, although local application may be more appropriate in certain cases. Transport of the molecules to their site of action may be effected by, for example, liposome delivery systems, antisense technology, plasmid or 10 retroviral vectors, or any of a number of other methods known in the art. The vehicle for application may be any excipient compatible with the molecules and the health of the patient.

214889~

SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: KYRIAKIS, JOHN M.
AVRUCH, JOSEPH
BANERJEE, PAPIA
WOODGETT, JAMES R.
(ii) TITLE OF INVENTION: p54 STRESS-ACTIVATED PROTEIN
KINASES
(iii) NUMBER OF SEQUENCES: 16 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: FISH & RICHARDSON
(B) STREET: 225 FRANKLIN STREET
(C) CITY: BOSTON
(D) STATE: MA
(E) COUNTRY: US
(F) ZIP: 02110 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COM~ K: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US UNASSIGNED
(B) FILING DATE: 04-MAY-1994 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: CLARK, PAUL C.
(B) REGISTRATION NUMBER: 30,162 (C) REFERENCE/DOCKET NUMBER: 00786/217001 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 617/542-5070 (B) TELEFAX: 617/542-8906 (C) TELEX: 200154 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 423 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single -(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Met Ser Asp Ser Lys Ser Asp Gly Gln Phe Tyr Ser Val Gln Val Ala Asp Ser Thr Phe Thr Val Leu Lys Arg Tyr Gln Gln Leu Lys Pro Ile Gly Ser Gly Ala Gln Gly Ile Val Cys Ala Ala Phe Asp Thr Val Leu Gly Ile Asn Val Ala Val Lys Lys Leu Ser Arg Pro Phe Gln Asn Gln Thr His Ala Lys Arg Ala Tyr Arg Glu Leu Val Leu Leu Lys Cys Val Asn His Lys Asn Ile Ile Ser Leu Leu Asn Val Phe Thr Pro Gln Lys Thr Leu Glu Glu Phe Gln Asp Val Tyr Leu Val Met Glu Leu Met Asp Ala Asn Leu Cys Gln Val Ile His Met Glu Leu Asp His Glu Arg Met Ser Tyr Leu Leu Tyr Gln Met Leu Cys Gly Ile Lys His Leu His Ser Ala Gly Ile Ile His Arg Asp Leu Lys Pro Ser Asn Ile Val Val Lys Ser Asp Cys Thr Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg Thr Ala Cys Thr Asn Phe Met Met Thr Pro Tyr Val Val Thr Arg Tyr Tyr Arg Ala Pro Glu Val Ile Leu Gly Met Gly Tyr Lys Glu Asn Val Asp Ile Trp Ser Val Gly Cys Ile Met Ala Glu Met Val Leu His Lys Ser Cys Ser Pro Gly Arg Asp Tyr Ile Asp Gln Trp Asn Lys Val Ile Glu Gln Leu Gly Thr Pro Ser Ala Glu Phe Met Lys Lys Leu Gln Pro Thr Val Arg Asn Tyr Val Glu Asn Arg Pro Lys Tyr Pro Gly Ile Lys Phe Glu Glu Leu Phe Pro Asp Trp Ile Phe Pro Ser Glu Ser Glu Arg Asp Lys Ile Lys Thr Ser Gln Ala Arg Asp Leu Leu Ser Lys Met Leu Val Ile Asp Pro Asp Lys Arg Ile Ser Val ABP Glu Ala Leu Arg His Pro Tyr le Thr Val Trp Tyr Asp Pro Ala Glu Ala Glu Ala Pro Pro Pro Gln le Tyr Asp Ala Gln Leu Glu Glu Arg Glu His Ala Ile Glu Glu Trp Lys Glu Leu Ile Tyr Lys Glu Val Met Asp Trp Glu Glu Arg Ser Lys Asn Gly Val Lys Asp Gln Pro Ser Asp Ala Ala Val Ser Ser Lys Ala Thr Pro Ser Gln Ser Ser Ser Ile Asn Asp Ile Ser Ser Met Ser Thr Glu His Thr Leu Ala Ser Asp Thr Asp Ser Ser Leu Asp Ala Ser Thr Gly Pro Leu Glu Gly Cys Arg (2) INFORMATION FOR SEQ ID NO:2:
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A LENGTH: 423 amino acids ~B TYPE: amino acid C STRANDEDNESS: single D, TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ser Asp Ser Lys Ser Asp Gly Gln Phe Tyr Ser Val Gln Val Ala Asp Ser Thr Phe Thr Val Leu Lys Arg Tyr Gln Gln Leu Lys Pro Ile Gly Ser Gly Ala Gln Gly Ile Val Cys Ala Ala Phe Asp Thr Val Leu Gly Ile Asn Val Ala Val Lys Lys Leu Ser Arg Pro Phe Gln Asn Gln Thr His Ala Lys Arg Ala Tyr Arg Glu Leu Val Leu Leu Lys Cys Val sn Hi~ Lys Asn Ile Ile Ser Leu Leu Asn Val Phe Thr Pro Gln Lys hr Leu Glu Glu Phe Gln Asp Val Tyr Leu Val Met Glu Leu Met Asp Ala Asn Leu Cys Gln Val Ile His Met Glu Leu Asp His Glu Arg Met Ser Tyr Leu Leu Tyr Gln Met Leu Cys Gly Ile Lys His Leu His Ser Ala Gly Ile Ile His Arg Asp Leu Lys Pro Ser Asn Ile Val Val Lys 21~8898 er Asp Cys Thr Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg Thr Ala ys Thr Asn Phe Met Met Thr Pro Tyr Val Val Thr Arg Tyr Tyr Arg Ala Pro Glu Val Ile Leu Gly Met Gly Tyr Lys Glu Asn Val Asp Ile Trp Ser Val Gly Cys Ile Met Gly Glu Leu Val Lys Gly Cys Val Ile Phe Gln Gly Thr Asp His Ile Asp Gln Trp Asn Lys Val Ile Glu Gln eu Gly Thr Pro Ser Ala Glu Phe Met Lys Lys Leu Gln Pro Thr Val rg Asn Tyr Val Glu Asn Arg Pro Lys Tyr Pro Gly Ile Lys Phe Glu Glu Leu Phe Pro Asp Trp Ile Phe Pro Ser Glu Ser Glu Arg Asp Lys Ile Lys Thr Ser Gln Ala Arg Asp Leu Leu Ser Lys Met Leu Val Ile Asp Pro Asp Lys Arg Ile Ser Val Asp Glu Ala Leu Arg His Pro Tyr le Thr Val Trp Tyr Asp Pro Ala Glu Ala Glu Ala Pro Pro Pro Gln le Tyr Asp Ala Gln Leu Glu Glu Arg Glu His Ala Ile Glu Glu Trp Lys Glu Leu Ile Tyr Lys Glu Val Met Asp Trp Glu Glu Arg Ser Lys Asn Gly Val Lys Asp Gln Pro Ser Asp Ala Ala Val Ser Ser Lys Ala Thr Pro Ser Gln Ser Ser Ser Ile Asn Asp Ile Ser Ser Met Ser Thr Glu His Thr Leu Ala Ser Asp Thr Asp Ser Ser Leu Asp Ala Ser Thr Gly Pro Leu Glu Gly Cys Arg (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
~A' LENGTH: 426 amino acids B TYPE: amino acid ,C STRANDEDNESS: single l,DI TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Ser Lys Ser Lys Val Asp Asn Gln Phe Tyr Ser Val Glu Val Gly sp Ser Thr Phe Thr Val Leu Lys Arg Tyr Gln Asn Leu Lys Pro Ile Gly Ser Gly Ala Gln Gly Ile Val Cys Ala Ala Tyr Asp Ala Val Leu Asp Arg Asn Val Ala Ile Lys Lys Leu Ser Arg Pro Phe Gln Asn Gln Thr His Ala Lys Arg Ala Tyr Arg Glu Leu Val Leu Met Lys Cys Val sn His Lys Asn Ile Ile Ser Leu Leu Asn Val Phe Thr Pro Gln Lys hr Leu Glu Glu Phe Gln Asp Val Tyr Leu Val Met Glu Leu Met Asp Ala Asn Leu Cys Gln Val Ile Gln Met Glu Leu Asp His Glu Arg Met Ser Tyr Leu Leu Tyr Gln Met Leu Ser Ala Ile Lys His Leu His Ser Ala Gly Ile Ile His Arg Asp Leu Lys Pro Ser Asn Ile Val Val Lys er Asp Cys Thr Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg Thr Ala ly Thr Ser Phe Met Met Thr Pro Tyr Val Val Thr Arg Tyr Tyr Arg Ala Pro Glu Val Ile Leu Gly Met Gly Tyr Lys Glu Asn Val Asp Ile Trp Ser Val Gly Cys Ile Met Gly Glu Met Val Arg His Lys Ile Leu Phe Pro Gly Arg Asp Tyr Ile Asp Gln Trp Asn Lys Val Ile Glu Gln eu Gly Thr Pro Cys Pro Glu Phe Met Lys Lys Leu Gln Pro Thr Val rg Asn Tyr Val Glu Asn Arg Pro Lys Tyr Ala Gly Leu Thr Phe Pro Lys Leu Phe Pro Asp Ser Leu Phe Pro Ala Asp Ser Glu His Asn Lys Leu Lys Ala Ser Gln Ala Arg Asp Leu Leu Ser Lys Met Leu Val Ile Asp Pro Ala Lys Arg Ile Ser Val Asp Asp Ala Leu Gln His Pro Tyr le Asn Val Trp Tyr Asp Pro Ala Glu Val Glu Ala Pro Pro Pro Gln le Tyr Asp Lys Gln Leu Asp Glu Arg Glu His Thr Ile Glu Glu Trp ys Glu Leu Ile Tyr Lys Glu Val Met Asn Ser Glu Glu Lys Thr Lys 21~8898 Asn Gly Val Val Lys Gly Gln Pro Ser Pro Ser Gly Ala Ala Val Asn Ser Ser Glu Ser Leu Pro Pro Ser Ser Ser Val Asn Asp Ile Ser Ser Met Ser Thr Asp Gln Thr Leu Ala Ser Asp Thr Asp Ser Ser Leu Glu la Ser Ala Gly Pro Leu Gly Cys Cys Arg 2) INFORMATION FOR SEQ ID NO:4:
(i) ~U~N~: CHARACTERISTICS:
'A' LENGTH: 385 amino acids B TYPE: amino acid C STRANDEDNESS: single D, TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Ser Lys Ser Lys Val Asp Asn Gln Phe Tyr Ser Val Glu Val Gly sp Ser Thr Phe Thr Val Leu Lys Arg Tyr Gln Asn Leu Lys Pro Ile Gly Ser Gly Ala Gln Gly Ile Val Cys Ala Ala Tyr Asp Ala Val Leu Asp Arg Asn Val Ala Ile Lys Lys Leu Ser Arg Pro Phe Gln Asn Gln Thr His Ala Lys Arg Ala Tyr Arg Glu Leu Val Leu Met Lys Cys Val sn His Lys Asn Ile Ile Ser Leu Leu Asn Val Phe Thr Pro Gln Lys hr Leu Glu Glu Phe Gln Asp Val Tyr Leu Val Met Glu Leu Met Asp Ala Asn Leu Cys Gln Val Ile Gln Met Glu Leu Asp His Glu Arg Met Ser Tyr Leu Leu Tyr Gln Met Leu Ser Ala Ile Lys His Leu His Ser Ala Gly Ile Ile His Arg Asp Leu Lys Pro Ser Asn Ile Val Val Lys er Asp Cys Thr Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg Thr Ala ly Thr Ser Phe Met Met Thr Pro Tyr Val Val Thr Arg Tyr Tyr Arg Ala Pro Glu Val Ile Leu Gly Met Gly Tyr Lys Glu Asn Val Asp Ile Trp Ser Val Gly Cys Ile Met Gly Glu Met Val Arg His Lys Ile Leu Phe Pro Gly Arg Asp Tyr Ile Asp Gln Trp Asn Lys Val Ile Glu Gln eu Gly Thr Pro Cys Pro Glu Phe Met Lys Lys Leu Gln Pro Thr Val rg Asn Tyr Val Glu Asn Arg Pro Lys Tyr Ala Gly Leu Thr Phe Pro Lys Leu Phe Pro Asp Ser Leu Phe Pro Ala Asp Ser Glu His Asn Lys Leu Lys Ala Ser Gln Ala Arg Asp Leu Leu Ser Lys Met Leu Val Ile Asp Pro Ala Lys Arg Ile Ser Val Asp Asp Ala Leu Gln His Pro Tyr le Asn Val Trp Tyr Asp Pro Ala Glu Val Glu Ala Pro Pro Pro Gln le Tyr Asp Lys Gln Leu Asp Glu Arg Glu His Thr Ile Glu Glu Trp ys Glu Leu Ile Tyr Lys Glu Val Met Asn Ser Glu Glu Lys Thr Lys sn Gly Val Val Lys Gly Gln Pro Ser Pro Ser Xaa Xaa Gly Ala Ala Val (2) INFORM~TION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
A' LENGTH: 411 amino acids B TYPE: amino acid ,C STRANDEDNESS: single ,DI TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Ser Arg Ser Ly~ Arg Asp Asn Asn Phe Tyr Ser Val Glu Ile Ala Asp Ser Thr Phe Thr Val Leu Lys Arg Tyr Gln Asn Leu Lys Pro Ile Gly Ser Gly Ala Gln Gly Ile Val Cys Ala Ala Tyr Asp Ala Ile Leu Glu Arg Asn Val Ala Ile Lys Lys Leu Ser Arg Pro Phe Gln Asn Gln Thr His Ala Lys Arg Ala Tyr Arg Glu Leu Val Leu Met Lys Cys Val Asn His Lys Asn Ile Ile Gly Leu Leu Asn Val Phe Thr Pro Gln Lys Ser Leu Glu Glu Phe Gln Asp Val Tyr Ile Val Met Glu Leu Met Asp Ala Asn Leu Cys Gln Val Ile Gln Met Glu Leu Asp His Glu Arg Met Ser Tyr Leu Leu Tyr Gln Met Leu Cys Gly Ile Lys His Leu His Ser Ala Gly Ile Ile His Arg Asp Leu Lys Pro Ser Asn Ile Val Val Lys Ser Asp Cys Thr Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg Thr Ala Gly Thr Ser Phe Met Met Thr Pro Tyr Val Val Thr Arg Tyr Tyr Arg Ala Pro Glu Val Ile Leu Gly Met Gly Tyr Lys Glu Asn Val Asp Leu Trp Ser Val Gly Cys Ile Met Gly Glu Met Val Cys Leu Lys Ile Leu Phe Pro Gly Arg Asp Tyr Ile ABP Gln Trp Asn Lys Val Ile Glu Gln Leu Gly Thr Pro Cys Pro Glu Phe Met Lys Lys Leu Gln Pro Thr Val Arg Thr Tyr Val Glu Asn Arg Pro Lys Tyr Ala Gly Tyr Ser Phe Glu Lys Leu Phe Pro Asp Val Leu Phe Pro Ala Asp Ser Glu His A~n Lys Leu Lys Ala Ser Gln Ala Arg Asp Leu Leu Ser Lys Met Leu Val Ile Asp Ala Ser Lys Arg Ile Ser Val Asp Glu Ala Leu Gln His Pro Tyr Ile Asn Val Trp Tyr Asp Pro Ser Glu Ala Glu Ala Pro Pro Pro Lys Ile Pro Asp Lys Gln Leu Asp Glu Arg Glu His Thr Ile Glu Glu Trp Lys Glu Leu Ile Tyr Lys Glu Val Met Asp Leu Glu Glu Arg Thr Lys Asn Gly Val Ile Arg Gly Gln Pro Ser Pro Leu Gly Ala Ala Val Ile Asn Gly Ser Gln His Pro Val Ser Ser Pro Ser Val Asn Asp Met Ser Ser Met Ser Thr Asp Pro Thr Leu Ala Ser Asp (2) lNrOR~ATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 17 amino acids B TYPE: amino acid C, STRANDEDNESS: single ,D TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Ser Asp Ala Ala Val Ser Ser Lys Ala Thr Pro Ser Gln Ser Ser Ser Ile (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 21 amino acids B TYPE: amino acid ,C STRANDEDNESS: single l,D, TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Gly Ala Ala Val Asn Ser Ser Glu Ser Leu Pro Pro Ser Ser Ser Val Gln Pro Ser Pro Ser (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 20 amino acids B TYPE: amino acid C STRANDEDNESS: single ,D, TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Ser Pro Leu Gly Ala Ala Val Ile Asn Gln Ser Gln His Pro Val Ser Ser Pro Ser Val (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 2629 base pairs B TYPE: nucleic acid C, STRANDEDNESS: single ~D~ TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GGAl~CC~ ATGACACTAC ATCATGAGTG ACAGTA~AAG CGATGGCCAG TTTTACAGTG 60 21~88~8 GCTCTGGAGC CCAAGGAATT G~..~lGCTG CTTTTGATAC A~.~-.GGA ATAAATGTTG 180 CTGTCAAGAA GTTAAGTCGT C~....CAGA ACrAAACGCA TGrAAAr-Ar-A GCCTACCGTG 240 AA~ C~. CCTAAAGTGT GTCAATCATA AAAATATAAT TAG~..~..A AA~ ~A 300 rACrArAAAA AACGCTAGAA GAATTCCAAG ATGTGTACTT GGTTATGGAG TTAATGGACG 360 CTAACTTATG TCAGGTTATT CATATGGAGC TGGACCATGA AAGAATGTCA TAC~.C~ ~. 420 AcrAr,ATGCT TTGTGGCATT AAGCACCTGC ATTCAGCTGG CATAATTCAT AGGGATTTGA 480 CACGGACAGC CTGTACCAAC TTTATGATGA ~.CC~.ATGT GGTAACTCGC TACTATCGGG 600 CTCr-Ar-AAr,T CATCCTGGGC ATGGGCTACA AGGAGAATGT GGACATCTGG ~ ,.CGGC,~ 660 GCATCATGGC AGAAATGGTC CTCrATAAAT C~.~..CCCC AGr-AAr-Ar-AC TATATTGATC 720 AGCCAACTGT AAGGAATTAT GTGGAAAAÇA GACrAAAr,TA CCCTGGAATC AAATTTGAAG 840 AG~.~...CC AGATTGGATA TTTCCGTCAG AATCCGAACG Ar7ArAAAATA AAAArAAr,Tc goo AAGCrAGAr,A .~.~..ATCG AAAATGTTAG TGATTGATCC GGACAAGCGG A.~.~.~,.GG 960 rACrACCTCA AATTTATGAT GCCCAGTTGG AArAAAr,AÇA GCATGCGATT GAAGAGTGGA 1080 AAr-AAcTAAT TTAr~AAAr7AA GTGATGGACT GGr7AAr-AAAr7 AAGçAAGAAT GGGGTGAAAG 1140 ATGACATCTC ATCCATGTCC ACTGAGCACA CCCTGGCCTC AGAC'-ACAGAC AGCAGTCTCG 1260 ATGCCTCAAC CGGACCCCTG GAAGGCTGCC GATGAAACCT CGCAGATGGC GCA~..~.~. 1320 GCTCCATGTT CTGCATGTAA rAAArACrAr GCCTTGCCCC CACTCAGTTC CAGTAGGATT 1440 GCCTGCGTAG ACTGTAACAT GAGGCAGACG A.~.~.GGAG AAAAAGTACA AACrArACTG 1500 TTAr,AAATTT TGTTCAAGAT CATTCAGGTG AGCAATTAGA ATAGCCGAGT .~.l..CAAG 1560 .C~.~.GGTG TCCTTGGTGA CAGATCATGT GTAACTGTGG GGACTCGTAT GCATGTGACC 1620 TAA.~..C~A GGTAGTTCTG CTTCTAGAAT AA.~.~ .AA .C~.C...AG TAATTTGGTG 1740 .~.~.C~ACA AAAAAATArA TTA.~.~.~,. ATGAATTGGC CACTATCATA TTATCATATT 1800 TTACCCACTT TTATGGTATG ATTTATTCTG ~.-..~-AT TTCAGAAGGA ~TATAATTAA 1860 ATTTATTTAA TAAATAAAAC TACAGCTTTT CTTAAATTTG TGA.~-....A GGCTGAGAAT 1920 TACCACTGCT TTATATCGAC A~.~l~.~,.C CTTTAAACTG CCCACTATGG GAAACTTTAC 1980 CACCTTGAAT CCTr-ACr-ArA CA...C~... TTCTTGGTCC TCTGAACTTG GATCTAGAAT 2100 21~8898 AGATTCCTGA GCCCCGCTGC CTAATGTAGA GCTGACAGGG TGG~,,CCCC AGAACGGTGG 2220 GTGGGTGCAT C~1~CC~GA GCCCACCCAT CCTTTGCTCC C~ A TTTAAGGTGA 2280 AAGGTGATTG GGTCTCATAG C~C~1~ TGTAGCATTG CCTAACTTGT ~ ACT 2340 r~rArAAGCC ACCACGTCCA GCCAGAGCAC ATG~ ,, AGGAGACCGG GCTTACTTAC 2400 CATGCATGTT TGCTGCTGTC ~ C~ATT TTGTGGAGGC A~1~C~ TCTAAGGGAA 2460 ~ C~CAGAT GTTCTAGAAA CATTCAGAAG AACGCAGAAG AAATATTCTA GAGAATTGGG 2520 CCTGATCTTG TAAATTACTC GAGATTTGGT AAGATGCTGA ~ C~V~ 2629 (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
~A LENGTH: 2629 ba~e pair~
~B TYPE: nucleic acid C STRANDEDNESS: ~ingle ~D TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GGA, C~,~, ATGACACTAC ATCATGAGTG ACAGTAAAAG CGATGGCCAG TTTTACAGTG 60 TGCAAGTGGC AGACTCAACT TTCACTGTTC TAAAACGTTA CCAGCAGTTG AAAcrAATTG 120 GCTCTGGAGC CCAAGGAATT ~,,,~,GCTG CTTTTGATAC AG1~ GGA ATAAATGTTG 180 CTGTCAAGAA GTTAAGTCGT C~,,,.~AGA ACrAAACGCA TGrAAA~Ar,A GCCTACCGTG 240 AA~,,~,C~, CCTAAAGTGT GTCAATCATA AAAATATAAT TAG~,,~A AA~ A 300 rArrAr~AAA AACGCTAGAA GAATTCCAAG ATGTGTACTT GGTTATGGAG TTAATGGACG 360 CTAACTTATG TCAGGTTATT CATATGGAGC TGGACCATGA AAGAATGTCA TAC~,C~,~-, 420 CTCrAr~A~T CATCCTGGGC ATGGGCTACA AGGAGAATGT TGATATCTGG TCAGTGGGTT 660 AATGGAATAA AGTTATTGAA CAGCTAGGAA CACCATCCGC AGAGTTCATG AAr-AAACTTC 780 AG~ CC AGATTGGATA ,,-CC~,CAG AATCCGAACG A~rAAAATA AAAArAA~TC 900 AAGCCAGAGA 1~ ATCG AAAATGTTAG TGATTGATCC GGACAAGCGG A.~.~.~.GG 960 ACGAAGCCTT GCGCr~CCCG TATATTACTG TTTGGTATGA CCCCGCTGAA GCAGAAGCGC 1020 r~rrACCTCA AATTTATGAT GCCCAGTTGG AA~AAA~A~A GCATGCGATT GAAGAGTGGA 1080 AAGAACTAAT TTArAAA~AA GTGATGGACT GG~AA~AAA~ AAGrAA~AT GGGGTGAAAG 1140 21488~8 ATGACATCTC ATCCATGTCC ACTGAGCACA CCCTGGCCTC Ar,ACAÇAr-AC AGCAGTCTCG 1260 ATGCCTCAAC CGGACCCCTG GAAGGCTGCC GATGAAACCT CGCAGATGGC GCA~ll~l`l 1320 GCTCCATGTT CTGCATGTAA r-AAA~ACGAC GCCTTGCCCC CACTCAGTTC CAGTAGGATT 1440 GCCTGCGTAG ACTGTAACAT GAGGCAGACG ATGTCTGGAG AAAAAGTACA AACCAÇACTG 1500 TTAr-AAATTT TGTTCAAGAT CATTCAGGTG AGCAATTAGA ATAGCCGAGT l~ AAG 1560 l~l~lGGTG TCCTTGGTGA CAGATCATGT GTAACTGTGG GGACTCGTAT GCATGTGACC 1620 TAA.`llC~A GGTAGTTCTG CTTCTAGAAT AA.~l~llAA ~C~l~lllAG TAATTTGGTG 1740 .~l~lC~ACA AAAAAATpr.A TTA~l~l~l ATGAATTGGC CACTATCATA TTATCATATT 1800 TTACCr-Ar,TT TTATGGTATG ATTTATTCTG l~ ~lAT TTCAGAAGGA ATATAATTAA 1860 ATTTATTTAA TAAATAAAAc TACAGCTTTT CTTAAATTTG TGAl~llllA GGCTGAGAAT 1920 TACCACTGCT TTATATCGAC A~l~l~l~lC CTTTA~ACTG CCCACTATGG GAAACTTTAC 1980 CACCTTGAAT CCTr-ACr-AÇA CA.~lC~lll TTCTTGGTCC TCTGAACTTG GATCTAGAAT 2100 CCCTCACAGA ACTTCACCTT CTTTATCACA AAGr-AcccrA TCTCAGTAGA ATGAATCGGC 2160 AGATTCCTGA GCCCCGCTGC CTAATGTAGA GCTGACAGGG TGG~lCCCC AGAACGGTGG 2220 GTGGGTGCAT C~llCC~lGA GCCCACCCAT CCTTTGCTCC C~l`l`ll lA TTTAAGGTGA 2280 AAGGTGATTG GGTCTCATAG C~lllC~.l' TGTAGCATTG CCTAACTTGT ~ lCACT 2340 ~-A~AGAAGCC ACCACGTCCA GCCAGAGCAC ATG~l~l~ll AGr-Ar-ACCGG GCTTACTTAC 2400 CATGCATGTT TGCTGCTGTC ~l lllC~ATT TTGTGGAGGC AlllC~llll TCTAAGGGAA 2460 llC~l~AGAT GTTCTAGAAA CATTCAGAAG AACGCAGAAG AAATATTCTA GAGAATTGGG 2520 CCTGATCTTG TAAATTACTC GAGATTTGGT AAGATGCTGA Gll`l~l~ 2629 ~2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
'A LENGTH: 1975 base pairq B TYPE: nucleic acid C STRANDEDNESS: ~ingle D, TOPOLOGY: 1inear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CCClC~llAT TCCGGTTTGG AATGTGGCTA ATGAAAGCCC AGTAGGAGGA TTTCTGGGGC 60 AAACAGGTGG ACCAGGATCC TG~l~l~AG GCACGGAATG GCTATTGTGA GAGCGCCACC 120 21~8898 CC~...CCCG TAGATGAGAA ATÇAÇAC~AG CAGTGGTATT TATGAGCCTC CA... AT 240 ACTACTGCAG TGAAC~AACC TTGGATGTGA AAATTGCCTT TTGTCAGGTG .~7.~..C~.. 300 ACAGGTAAAA CAAAGGGATT CGAÇAAAÇA~ GTGGATGTGT ~..~ ~7..~. ÇAAAÇATTAC 360 AACATGAGCA AAAGCAAGGT Ar-ATAACCAG TTCTACAGTG TGGAAGTGGG AGACTCAACC 420 TTCACAGTTC TAAAGCGCTA C~AGAACCTG AAGCCGATCG GCTCTGGGGC TCAGGGAATA 480 ~...~.GCTG CGTATGACGC l~.C~lCGAC AGAAATGTGG CCATTAAGAA GCTCAGCAGA 540 CC~..CCAGA ACCAAACTCA TGC~AA~-AGG GCTTACCGGG AGCTGGTCCT CATGAAGTGT 600 GT~-~ACcATA AAAA~-ATTAT TAGCTTATTA AA.~ A ~-Accc~A~-~A AACACTGGAG 660 CAGATGGAGC TG~-AC~AC~-A GCGGATGTCG TACTTGCTGT ACCAGATGCT GTCGGCGATC 780 TTCATGATGA ~lCCG.ATGT GGTG~C~-A~-~ TATTAcA~-AG CCCCCGAGGT CATCCTGGGC 960 ATGGGCTACA AG~-A~-AACGT G~Ac~TATGG TCTGTGGGCT GCATCATGGG AGAAATGGTT 1020 CGTCAC~AAA .C~l..CC CGGAAGGGAC TATATTGACC AGTGGAAÇAA AGT~ATAGAG 1080 CAGCTAGGAA ~lCC~l~.CC AGAATTCATG AA~AAATTGC AGCCCACCGT ~A~-AAACTAC 1140 GTG~-A~-AACC GGCCCAAGTA TGCAGGCCTC ACCTTCCCCA AG~.~l.lCC AGAl.CC~.C 1200 TTCCCAGCGG ATTCCGAGCA ~AATAAACTT AAAGCCAGCC AAGCCAGGGA ~ 7.CA 1260 AAGATGTTAG TGATTGACCC AGCGAAr-AGG ATATCGGTGG ATGACGCATT GCAGCATCCG 1320 TACATCAACG TTTGGTACGA CCCTGCTGAA GTGGAGGCGC CTCCGCCTCA ~-ATATATGAC 1380 AAGCAATTGG ATGAAAGGGA GcAÇAC~ATC ~AA~AATGGA AAGAACTCAT CTACAAGGAA 1440 GTAATGAACT ~7AGAAGAGAA GACTAA~-AAC GGCGTAGTCA AAGGCCAGCC CTCACCTTCA 1500 GGTGCAGCAG TGAACAGCAG TGAGAGTCTC CCTCCATCCT CAl~-~l~AA CGACATCTCC 1560 TCCATGTCCA CC~-AC~A~-AC CCTCGCATCC GACACTGACA GCAGCCTGGA AGCCTCGGCG 1620 GGACCGCTGG Gll~7llGCAG GTGACTAGCC GCCTGCCTGC ~AAAcc~A~c ~ll~l ~AGG 1680 AGATGACGCC AT~-ATAGAAC ACAGCGCACA TGcAcAçAr-~ CAGAGCTTGT AcAcAcAcAC 1740 A~ArA~ACAC A~AC~CGCAC GCACGCACGC ACGCAAGCAC GCACGCACGC ACAAATGCAC 1800 TCACGCAATG T~-AA~-AA~AA A~AAAGTAGC GA~-A~-AG~GC GAGA~-AGCCA ACGTAAAACT 1860 AAGTTAAATC TTTCTGCGTG ~ll lC~AGA Gll~l~lATC GCAGCTGAGC TGAAATGTAT 1920 ACTTAACTTC TAGTCGCGCT CGCTCGACTT TGGl~lCC~l CCGGCAGTGC TTACT 1975 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1986 base pairs `- 21~8898 -(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

TGGGGCAAAC AGGTGGACCA GGA.C~.G~ TCTCAGGCAC GGAATGGCTA TTGTGAGAGC 120 GC~A~-AGCA G~'AC~-ATCGC AGATCTTGGT TATGGCTGCT CACGCAAGAG GCTGTTGATG 180 TA~ACCCCCT ,CCC~7.AGA T~-A~-AAATCA CACGAGCAGT GGTATTTATG AGCCTCCATT 240 TCTTATACTA CTGCAGTGAA CCAACCTTGG ATGTGAAAAT TGC~.~ 7~ CAGGl~7 ~7~G 300 l~C~.ACAG GTAAAA~AAA GGGATTCGAC AAP~PCGTGG A~ 7~ ~C ~71~71 AAA 360 CATTACAA~A TGAGCAAAAG CAAGGTAGAT AACCAGTTCT ACAGTGTGGA AGTGGGAGAC 420 GGAATAGTTT GTGCTGCGTA TGACGCTGTC CTC~-ACA~-AA ATGTGGCCAT TAA~-AAGCTC 540 AG~A~ACCCT TCCA~-AAC~A AACTCATGCC AAGAGGGCTT ACCGGGAGCT GGlCC-~ATG 600 AA~7~ 7LGA AC~ATPAAAA CATTATTAGC TTATTAAATG TCTTTACACC C~A~-AAAA~A 660 CTG~-A~,AGT TC~-AA~-ATGT TTACTTAGTG ATGGAACTGA TGGACGCCAA ~7~7L AG 720 GTGATTCAGA TGGAGCTGGA CCACGAGCGG Ai~lC~7lACT TGCTGTACCA GATGCTGTCG 780 CTGGGCATGG GCTA~-AAr~r-A GAACGTGGAC ATATGGTCTG TGGGCTGCAT CATGGGAGAA 1020 ATG~7..C~7~C A~AAAATCCT ~.~CCCGGA AGGGACTATA TTGACCAGTG ~AA~AAAGTC 1080 AACTACGTGG A~AACCGGCC CAAGTATGCA GGCCTCACCT TCCC~AAGCT ~l..C~AGAT 1200 .CC~.~..CC CAGCGGATTC CGAGCACAAT AAACTTAAAG CCAGCCAAGC CAGGGACTTG 1260 CATCCGTACA TCAACGTTTG GTAC~-ACCCT GCTGAAGTGG AGGCGCCTCC GCCTCAGATA 1380 CCTTCAGCAC AGGTGCAGCA GTGAACAGCA GTGAGAGTCT CCCTCCATCC TCA.~.~7.~A 1560 AC~A~ATCTC CTCCATGTCC ACCGACCAGA CCCTCGCATC CGACACTGAC AGCAGCCTGG 1620 AAGCCTCGGC GGGACCGCTG G~7..~7..GCA GGTGACTAGC CGCCTGCCTG C~-AAACCCAG 1680 CG.. ~ AG GAGATGACGC CAT~-ATAGAA CACAGCGCAC ATGCACACAC ACAGAGCTTG 1740 21488~8 TArArAçAcA cAçArlAr-AçA r-~rArAcGcA CGCACGCACG CACGCAAGCA CGCACGCACG 1800 rlArAAATGCA CTCACGCAAT GTrAAGAAA~A- AAAAAAGTAG cr-Ar-ArArAr, Cr-AGAGAGCC 1860 AACGTAAAAC TAAGTTAAAT ~,, GCGT G~ C~AG AG~ ~ AT CGCAGCTGAG 1920 CTGAAATGTA TACTTAACTT CTAGTCGCGC TCGCTCGACT TTGG~CCC TCCGGCAGTG 1980 (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
A) LENGTH: 1408 base pairs B) TYPE: nucleic acid C) STRANDEDNESS: single D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

CAGAAGTAAA cGTr~Ar-AAr-~ ATTTTTATAG TGTArAr-ATC GCAGATTCTA CATTCACAGT 240 CcTAAAAcr-A TACCAGAACT TAAAGCCTAT AGGCTCAGGA GCTCAAGGAA TAG.~GC 300 AGCTTATGAT GCTATTCTTG AAAr-AAATGT TGCAATCAAG AAGCTCAGCC GGCCATTTCA 360 rAAAAATAT~ ATTGGCCTTT TGAATGTTTT CACACCACAG AAATCCCTAG AAGAATTTCA 480 GTTAGATCAT rAAAr-AATGT CCTACCTTCT CTATCAAATG ~ GGAA TCAAGCACCT 600 TCACTCTGCT GGAATTATTC ATCGGGACTT AAAGCCTAGT AATATAr,TAG TCAAATCAGA 660 GACGCCTTAC GTGGTAACTC GTTACTACAG AGrAcr-AGAG GTCATTCTCG GCATGGGCTA 780 CAAGGAGAAC GTGGATTTAT GG~ GGG GTGCATTATG Gr-Ar-AAATGG TTTGCCTCAA 840 AA.C~ CCAGGAAGGG ACTATATTGA TCAGTGGAAT AAAGTTATTG AACAGCTCGG 900 AACACCTTGT CCTGAATTCA Tr-AAGAAACT ArAACÇAAr-~ GTAAGGACTT ACGTTGAAAA 960 CAGACCTAAG TACGCTGGCT ATAGCTTTGA GAAACTGTTT CCTGATGTGC ~CC~AGC 1020 TGACTCAGAA çATAAçAAAC TTAAAGCCAG TCAGGCGAGA GA~ AT CTAAAATGCT 1080 GGTr-ATAr-AT GCGTCCAAAA GGAl~CCG~ Ar-ACGAAGCT CTCCAGCACC CGTACATCAA 1140 C~ AT GATCCTTCAG AAGCAGAGGC CCÇArrArrA AAGATCCCTG ACAAGCAGTT 1200 TTTGGAGGAG CGAACTAAGA ATGGCGTCAT AAGAGGGCAG CC~,~,C~,, TAGGTGCAGC 1320 21~88~8 AGTGATCAAT GGCTCTCAGC ATCCGGTCTC TTCGCCGTCT GTCAATGACA ~.~.-~AAT 1380 GTC~-ACAr-AT CCGACTCTGG CCTCGGAT 1408 (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 51 base pairs B TYPE: nucleic acid ,C, STRANDEDNESS: single ,D, TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
TCAGATGCAG CAGTAAGCAG CAAGGCTACT C~--~.~AGT CGTCATCCAT C 51 (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
~'A'~ LENGTH: 63 base pairs B, TYPE: nucleic acid C STRANDEDNESS: single ,D,I TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
CAGCCCTCAC CTTCAGGTGC AGCAGTGAAC AGCAGTGAGA ~.CC~.CC ATCCTCATCT 60 (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
~A' LENGTH: 60 base pairs ~B, TYPE: nucleic acid C STRANDEDNESS: single ,D, TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
.C~.l.AG GTGCAGCAGT GATCAATGGC TCTCAGCATC CGG.~ C GCCG.`:.~.C 60 What is claimed is:

Claims (32)

1. A recombinant p54 stress-activated protein kinase polypeptide or biologically active fragment thereof at least 10 amino acids in length.

2. The polypeptide of claim 1, wherein said polypeptide or fragment thereof is derived from an amino acid sequence substantially identical to SEQ ID NO: 1.

3. The polypeptide of claim 1, wherein said polypeptide or fragment thereof is derived from an amino acid sequence at least 95% identical to SEQ ID NO: 1.

4. The polypeptide of claim 1, wherein said polypeptide or fragment thereof is derived from an amino acid sequence substantially identical to SEQ ID NO: 2.

5. The polypeptide of claim 1, wherein said polypeptide or fragment thereof is derived from an amino acid sequence at least 95% identical to SEQ ID NO: 2.

6. The polypeptide of claim 1, wherein said polypeptide or fragment thereof is derived from an amino acid sequence substantially identical to SEQ ID NO: 3.

7. The polypeptide of claim 1, wherein said polypeptide or fragment thereof is derived from an amino acid sequence at least 95% identical to SEQ ID NO: 3.

8. The polypeptide of claim 1, wherein said polypeptide or fragment thereof is derived from an amino acid sequence substantially identical to SEQ ID NO: 4.

9. The polypeptide of claim 1, wherein said polypeptide or fragment thereof is derived from an amino acid sequence at least 95% identical to SEQ ID NO: 4.

10. The polypeptide of claim 1, wherein said polypeptide or fragment thereof is derived from an amino acid sequence substantially identical to SEQ ID NO: 5.

11. The polypeptide of claim 1, wherein said polypeptide or fragment thereof is derived from an amino acid sequence at least 95% identical to SEQ ID NO: 5.

12. The polypeptide of claim 1 wherein said polypeptide is derived from a mammal.

13. The polypeptide of claim 12 wherein said mammal is a rat.

14. The polypeptide of claim 12 wherein said mammal is a human.

15. The polypeptide of claim 1 wherein said polypeptide or fragment thereof is useful for producing antibodies which specifically bind to a p54 stress-activated protein kinase.

16. The antibody of claim 15 wherein said polypeptide fragment is chosen from the group consisting of SEQ ID NOs: 6, 7, and 8.

17. A DNA and its degenerate variants which encode a p54 stress-activated protein kinase polypeptide, or a biologically active fragment thereof at least 30 nucleotides in length.

18. The DNA of claim 17 comprising a nucleotide sequence encoding a p54.alpha.I polypeptide at least 90%
identical to SEQ ID NO 9.

19. The DNA of claim 17 comprising a nucleotide sequence encoding a p54.alpha.II polypeptide at least 90%
identical to SEQ. ID NO.: 10.

20. The DNA of claim 17 comprising a nucleotide sequence encoding a p54.beta.I polypeptide at least 90%
identical to SEQ. ID NO.: 11.

21. The DNA of claim 17 comprising a nucleotide sequence encoding a p54.beta.II polypeptide at least 90%
identical to SEQ. ID NO.: 12.

22. The DNA of claim 17 comprising a nucleotide sequence encoding a p54.gamma. polypeptide at least 90%
identical to SEQ. ID NO.: 13.

23. The DNA of claim 17 wherein said fragment is chosen from the group consisting of SEQ ID NOs: 14, 15, and 16.

24. The DNA of claim 17 wherein said DNA is derived from a mammal.

25. The DNA of claim 24 wherein said mammal is a rat.

26. The DNA of claim 24 wherein said mammal is a human.

27. A DNA which hybridizes under stringent conditions to one or more of the DNAs chosen from the group SEQ ID NO. 9, 10, 11, 12, and 13.

28. A method of screening potentially therapeutic compounds comprising the steps of a) treating cultured cells by applying said compounds and stress-activated protein kinase-activating stimuli, b) preparing cytoplasmic extracts of said treated cells, then c) assaying the isolated recombinant stress-activated protein kinases for c-Jun kinase activity.

29. The method of claim 28 wherein said cultured cells are selected from the group consisting of human cell lines HepG2, CCD-18Co, U937, and HT-29.

30. A method of screening potentially therapeutic compounds comprising the steps of a) treating cultured cells by applying said compounds, b) preparing cytoplasmic extracts of said treated cells, c) combining said extracts with an inactive recombinant stress-activated protein kinase, and then d) assaying the isolated recombinant stress-activated protein kinases for c-Jun kinase activity.

31. The method of claim 30 wherein said cultured cells are selected from the group consisting of human cell lines HepG2, CCD-18Co, U937, and HT-29.

32. The method of claim 30 wherein said cultured cells have been treated previously or concurrently with stress-activated protein kinase-activating stimuli.