+

WO1999009181A1 - Proteine kinase humaine skk4 activee par le stress - Google Patents

Proteine kinase humaine skk4 activee par le stress Download PDF

Info

Publication number
WO1999009181A1
WO1999009181A1 PCT/GB1998/002475 GB9802475W WO9909181A1 WO 1999009181 A1 WO1999009181 A1 WO 1999009181A1 GB 9802475 W GB9802475 W GB 9802475W WO 9909181 A1 WO9909181 A1 WO 9909181A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
variant
fragment
fusion
derivative
Prior art date
Application number
PCT/GB1998/002475
Other languages
English (en)
Inventor
Sean Edward Lawler
Ana Isabel Cuenda-Mendez
Philip Cohen
Original Assignee
Medical Research Council
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Medical Research Council filed Critical Medical Research Council
Priority to AU87432/98A priority Critical patent/AU8743298A/en
Priority to EP98938835A priority patent/EP1005555A1/fr
Publication of WO1999009181A1 publication Critical patent/WO1999009181A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to polypeptides, polynucleotides and uses thereof, in particular to members of the stress-activated protein kinase kinase (SKK) family.
  • SKK stress-activated protein kinase kinase
  • MAP mitogen-activated protein
  • LPS bacterial lipopolysaccharide
  • ILl interleukin-1
  • TNF tumour necrosis factor
  • SAPK1 also called c-Jun N-terminal kinases (JNKs)] phosphorylate Ser- 63 and Ser-73 in the activation domain of c-Jun (Pulverer et al, 1991), thereby increasing its transcriptional activity.
  • JNKs c-Jun N-terminal kinases
  • the same sites in c-Jun also become phosphorylated when cells are exposed to the stresses and cytokines that activate SAPK1 (Pulverer et al, 1991; Hibi et al, 1993; Derijard et al, 1994; Kyriakis et al, 1994), suggesting that c-Jun is a physiological substrate for SAPK1.
  • SAPK2a [also termed ⁇ 38 (Han et al, 1994), p40 (Freshney et al, 1994), RK (Rouse et al, 1994), CSBP (Lee et al, 1994) and Mxi2 (Zervos et al, 1995)] is inhibited very specifically by the pyridinyl imidazoles SB 203580 and SB 202190 (Lee et al, 1994; Cuenda et al, 1995; reviewed in Cohen, 1997) which have been exploited to identify several physiological substrates.
  • MAP kinase-activated protein kinase-2 MAPKAP-K2
  • MAPKAP-K3 MAPKAP-K3
  • Mnkl and Mnk2 MAP kinase interacting protein kinases- 1 and -2
  • Physiological substrates of MAPKAP-K2/K3 include heat shock protein (HSP) 27 (Cuenda et al, 1995; Huot et al, 1995) and the transcription factor CREB (Tan et al, 1996), whereas transcription factor eIF4E is a physiological substrate of Mnkl/2 (Waskiewicz et al, 1997).
  • SAPK2a also mediates the stress-induced phosphorylation and activation of the CEBP ⁇ - related transcription factor CHOP (Wang and Ron, 1996) and the ternary complex factor Elk-1 (Price et al, 1996).
  • the activation of SAPK2a is rate- limiting in the LPS-induced production of ILl and TNF in monocytes (Lee et al, 1994), in the TNF-stimulated transcription of IL6 and GM-CSF in fibroblasts (Beyaert et al, 1996), in the ILl-induced stimulation of glucose uptake in epithelial cells (Gould et al, 1995), in collagen-induced platelet aggregation (Saklatvala et al, 1996) and in the stress-induced transcription of c-Jun and c-Fos in fibroblasts (Hazzalin et al, 1996; Price et al, 1996).
  • SAPK2a catalysed phosphorylation of Elk-1 (Price et al, 1996) and the MAPKAP-K2 catalysed phosphorylation of CREB (Tan et al, 1996) are both likely to contribute to the stress-induced transcription of c-fos (Ginty et al, 1994).
  • SAPK2b [or p38 ⁇ (Jiang et al, 1996)] and SAPK3 (Mertens et al, 1996) [also called ERK6 (Lechner et al, 1996) and p38 ⁇ (Li et al, 1996)].
  • SAPK2b The amino acid sequence of S APK2b is 73 % identical to S APK2a and it is inhibited by SB 202190 and SB 203580 at similar concentrations to SAPK2a.
  • the amino acid sequence of SAPK3 is only 60% identical to SAPK2a and SAPK3 is not inhibited by SB 203580 (Cuenda et al, 1997).
  • SAPK2b and SAPK3 have been introduced into mammalian cells by transient transfection and shown to be activated in response to pro-inflammatory cytokines and stressful stimuli in a manner similar to SAPKl and SAPK2a.
  • the physiological roles of SAPK2b and SAPK3 are unknown.
  • the mRNAs encoding these enzymes are present in all mammalian tissues examined (Jiang et al, 1996; Mertens et al, 1996; Goedert et al., 1997), with the mRNA encoding SAPK3 being highest in skeletal muscle. Expression of wild-type SAPK3 and an inactive mutant in the muscle cell line C2C12 enhanced and inhibited differentiation into myotubes, respectively (Lechner et al, 1996).
  • SAPK2b and SAPK3 phosphorylated several proteins that are also substrates for SAPK2a.
  • SAPK2b was reported to phosphorylate die transcription factor ATF2 more efficiently than SAPK2a (Jiang et al., 1996) but, since the stress- and cytokine- induced phosphorylation of ATF2 in fibroblasts is unaffected by SB 203580 (Hazzalin et al, 1996; Beyaert et al., 1996), neither SAPK2a nor SAPK2b appears to be rate-limiting for ATF2 phosphorylation in vivo, in contrast to earlier studies using transfection- based approaches (Gupta et al, 1995).
  • SAPK4 is the seventh MAP kinase family member. This enzyme, which also contains a TGY sequence in the activation domain, shows about 60% identity to SAPK2a, SAPK2b and SAPK3 and its mRNA is widely expressed in human tissues. SAPK4 is activated by me same stimuli that activate other SAP kinases, has an in vitro substrate specificity similar to that of SAPK3 and, like SAPK3, is not inhibited by SB 203480 or SB 202190.
  • SAPKKs Five chromatographically distinct SAP kinase kinases (SAPKKs or SKKs) have been identified in mammalian cells (Meier et al, 1996; Cuenda et al, 1996).
  • SKK1 also termed MKK4 (Derijard et al, 1995), SEK1 (Sanchez et al, 1994) and XMEK2 (Yashar et al, 1993)] activates all four SAPKs (Sanchez et al, 1994; Derijard et al, 1995; Doza et al, 1995; Jiang et al, 1996; Cuenda et al, 1997), although SAPK2b and SAPK3 are phosphorylated less efficiently.
  • SKK2 [also termed MKK3 (Derijard et al, 1995)] and SKK3 (Cuenda et al, 1996) [also called MKK6 (Han et al, 1996; Moriguchi et al, 1996; Raingeaud et al, 1996) and MEK6 (Stein et al, 1996)] activate SAPK2a but not SAPKl, while SKK3 was the only detectable activator of SAPK3 induced by pro-inflammatory cytokines and stressful stimuli in human epithelial KB cells or human embryonic kidney 293 cells (Cuenda et al, 1997).
  • SKK3 was also the most efficient activator of SAPK2b in co-transfection experiments (Jiang et al, 1996), and was the only detectable activator of SAPK4 induced by pro- inflammatory cytokines and stressful stimuli in KB cells [Goedert et al (1997) EMBO J 16, 3563-3571].
  • SKK4 and SKK5 activate SAPKl but not SAPK2a (Meier et al, 1996) or SAPK3 (Cuenda et al, 1997).
  • MKK7 mouse MAP kinase kinase family member termed MKK7 has recendy been reported in Tournier et al (1997) PNAS 94, 7337-7342.
  • SKK1/MKK4 is the only activator of SAPK1/JNK that can be detected biochemically in extracts prepared from PC 12 cells that have been exposed to adverse stimuli (Meier et al (1996)), and in 293 cells the activation of transfected SAPKl /JNK by anisomycin can be prevented by overexpressing a catalytically inactive form of SKK1/MKK4 (Sanchez et al (1994) Nature 372, 794-798). In addition, heat shock or anisomycin failed to activate SAPKl /JNK in murine embryonic stem cells lacking
  • SAPKl /JNK is activated strongly by cotransfection with MEK kinase (MEKK) (Yan et al (1994) Nature 372, 798-800).
  • MEK Kinase is an upstream activator of SKK1 mat is activated by osmotic shock in rat 3Y1 fibroblasts and PC 12 cells (Yan et al (1994)
  • Altiiough SKK1/MKK4 is clearly essential for the activation of SAPK1/JNK in some cells, the dominant activator of SAPK1/JNK detected biochemically in human epithelial KB cells (Meier et al (1996) or in Rat 3Y1 fibroblasts (Moriguchi et al (1995) is a chromatographically distinct enzyme(s).
  • SKK4 was partially resolved from SKK3 by elution from Mono S using a sodium chloride gradient, but purification of SKK4 was not achieved.
  • Moriguchi et al, (1995) identified multiple SAP kinase kinases using a method employing Q-sepharose and heparin-sepharose chromatography. This enabled the presence of a kinase that may correspond to SKK4 as identified in Meier et al, (1996) to be detected, but purification of the kinase was not reported.
  • SKK4 activates SAPKl/JNK in vitro, but not SAPK2a/p38, SAPK3/ERK6 or SAPK4.
  • SKK4 and SKK1/MKK4 another activator of SAPKl/JNK
  • SKK4 and SKK1/MKK4 are both activated by stressful stimuli, but only SKK4 is activated strongly by promflammatory cytokines.
  • Recombinant SKK4 can be activated by incubation with MgATP and MEKK.
  • the identification of SKK4 explains why the major SAPKl/JNK activator detected in many mammalian cell extracts is chromatographically separable from SKK1/MKK4.
  • SKK4 may be a more attractive target for an anti-inflammatory drug than SAPKl/JNK because inhibitors of SKK4 may prevent the activation of SAPKl/JNK by proinflammatory cytokines, without affecting the activation of SAPKl/JNK by other agents.
  • a first aspect of the invention provides a substantially pure polypeptide comprising the amino acid sequence
  • This polypeptide is considered to be a stress-activated protein kinase kinase.
  • polypeptide with the amino acid sequence as shown above is herein referred to as SKK4 (stress-activated protein kinase kinase 4).
  • SKK4 stress-activated protein kinase kinase 4
  • substantially pure we mean that the said polypeptide is substantially free of other proteins.
  • any composition that includes at least 30% of the protein content by weight as the said polypeptide, preferably at least 50%, more preferably at least 70%, still more preferably at least 90% and most preferably at least 95% of the protein content is the said polypeptide.
  • the invention also includes compositions comprising the said polypeptide and a contaminant wherein the contanrinant comprises less than 70% of the composition by weight, preferably less than 50% of the composition, more preferably less than 30% of the composition, still more preferably less than 10% of the composition and most preferably less than 5 % of the composition by weight.
  • the invention also includes the substantially pure said polypeptide when combined with other components ex vivo, said other components not being all of the components found in the cell in which said polypeptide is found.
  • Variants may be made using the methods of protein engineering and site-directed mutagenesis well known in the art using the recombinant polynucleotides described below.
  • fragment of said polypeptide we include any fragment which retains activity or which is useful in some other way, for example, for use in raising antibodies or in a binding assay.
  • fusion of said polypeptide we include said polypeptide fused to any other polypeptide.
  • the said polypeptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said polypeptide.
  • GST glutathione-S-transferase
  • the said polypeptide may be fused to an oligo-histidine tag such as His 6 or to an epitope recognised by an antibody such as the well known Myc tag epitope. Fusions to any variant, fragment or derivative of said polypeptide are also included in the scope of the invention.
  • variants of the polypeptide we include insertions, deletions and substitutions, either conservative or non-conservative. In particular we include variants of the polypeptide where such changes do not substantially alter the activity of the said polypeptide.
  • variants of SKK4 do not include polypeptides which have d e amino acid sequence of human SKK1 , SKK2, SKK3 or D. melanogaster HEP.
  • substitutions is intended combinations such as Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • the polypeptide variant has an amino acid sequence which has at least 98.8% identity with the amino acid sequence given above, more preferably at least 99.1 % , still more preferably at least 99.4% , yet more preferably at least 99.7% , and most preferably at least 99.9% identity with the amino acid sequence given above.
  • the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequences have been aligned optimally.
  • a particular embodiment of the invention provides a substantially pure human SKK4 polypeptide which consists of the amino acid sequence
  • amino acid sequence is also shown as me translation of a polynucleotide sequence in Figure 1.
  • variant or fragment or derivative or fusion of the said polypeptide, or the fusion of the variant or fragment or derivative has at least 30% of the enzyme activity of SKK4 with respect to the phosphorylation of SAPKl. It is more preferred if the variant or fragment or derivative or fusion of die said polypeptide, or the fusion of the variant or fragment or derivative has at least 50%, preferably at least 70% and more preferably at least 90% of the enzyme activity of SKK4 with respect to the phosphorylation of SAPKl.
  • variants or fusions or derivatives or fragments which are devoid of enzymatic activity may nevertheless be useful, for example by interacting with another polypeptide, or as antigens in raising antibodies.
  • a second aspect of the invention provides a recombinant polynucleotide encoding a polypeptide as defined in the first aspect of tihe invention or encoding a variant or fragment or derivative of fusion of said polypeptide or a fusion of a said variant or fragment or derivative.
  • Preferences and exclusions for the said polynucleotide variant are the same as in the first aspect of the invention, except that the following Expressed Sequence Tags (ESTs) are also excluded:
  • nucleotide sequence is shown in Figure 1 together wim the translation of the relevant open reading frame.
  • the invention also includes a polynucleotide comprising a fragment of the recombinant polynucleotide of me second aspect of me invention.
  • the polynucleotide comprises a fragment which is at least 10 nucleotides in lengm, more preferably at least 14 nucleotides in length and still more preferably at least 18 nucleotides in length.
  • Such polynucleotides are useful as PCR primers.
  • the polynucleotide or recombinant polynucleotide may be DNA or RNA, preferably DNA.
  • the polynucleotide may or may not contain nitrons in the coding sequence; preferably the polynucleotide is a cDNA.
  • a “variation" of the polynucleotide includes one which is (i) usable to produce a protein or a fragment thereof which is in turn usable to prepare antibodies which specifically bind to me protein encoded by the said polynucleotide or (ii) an antisense sequence corresponding to me gene or to a variation of type (i) as just defined.
  • different codons can be substituted which code for the same amino acid(s) as the original codons.
  • the substitute codons may code for a different amino acid that will not affect the activity or i munogenicity of the protein or which may improve or otherwise modulate its activity or immunogenicity.
  • site-directed mutagenesis or other techniques can be employed to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, as described in Botstein and Shortle, "Strategies and Applications of In Vitro Mutagenesis," Science, 229: 193-210 (1985), which is incorporated herein by reference. Since such modified polynucleotides can be obtained by the application of known techniques to the teachings contained herein, such modified polynucleotides are witiiin me scope of the claimed invention.
  • polynucleotide sequence (or fragments tiiereof) of me invention can be used to obtain o er polynucleotide sequences tiiat hybridise with it under conditions of high stringency.
  • polynucleotides includes any genomic DNA.
  • me polynucleotide of me invention includes polynucleotide mat shows at least 89.5% , preferably 92%, and more preferably at least 95% and most preferably at least 99% homology with the polynucleotide identified in d e method of me invention, provided that such homologous polynucleotide encodes a polypeptide which is usable in at least some of the methods described below or is otherwise useful.
  • Per cent homology can be determined by, for example, me GAP program of the University of Wisconsin Genetic Computer Group.
  • DNA-DNA, DNA-RNA and RNA-RNA hybridisation may be performed in aqueous solution containing between 0.1XSSC and 6XSSC and at temperatures of between 55 °C and 70 °C. It is well known in me art that the higher the temperature or the lower the SSC concentration the more stringent the hybridisation conditions. By “high stringency” we mean 2XSSC and 65°C. 1XSSC is 0.15M NaCl/0.015M sodium citrate. Polynucleotides which hybridise at high stringency are included within the scope of the claimed invention.
  • "Variations" of d e polynucleotide also include polynucleotide in which relatively short stretches (for example 20 to 50 nucleotides) have a high degree of homology (at least 95% and preferably at least 99 or 99.9%) witii equivalent stretches of die polynucleotide of me invention even tiiough me overall homology between me two polynucleotides may be much less. This is because important active or binding sites may be shared even when me general architecture of the protein is different.
  • a furtiier aspect of the invention provides a replicable vector comprising a recombinant polynucleotide encoding a said polypeptide or a variant, fragment, derivative or fusion of said polypeptide or a fusion of said variant, fragment or derivative.
  • a variety of methods have been developed to operably link polynucleotides, especially DNA, to vectors for example via complementary cohesive termini.
  • complementary homopolymer tracts can be added to me DNA segment to be inserted to me vector DNA.
  • the vector and DNA segment are men joined by hydrogen bonding between me complementary homopolymeric tails to form recombinant DNA molecules.
  • Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors.
  • the DNA segment generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single-stranded termini with their 3'-5'-exonucleolytic activities, and fill in recessed 3' -ends with their polymerizing activities.
  • the combination of these activities tiierefore generates blunt-ended DNA segments.
  • the blunt-ended segments are men incubated wim a large molar excess of linker molecules in me presence of an enzyme mat is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • me products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved wim e appropriate restriction enzyme and ligated to an expression vector mat has been cleaved wim an enzyme that produces termini compatible witii those of the DNA segment.
  • Synmetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CN, USA.
  • a desirable way to modify me DNA encoding a polypeptide of die invention is to use me polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491.
  • This method may be used for introducing me DNA into a suitable vector, for example by engineering in suitable restriction sites, or it may be used to modify the DNA in other useful ways as is known in me art.
  • me DNA to be enzymatically amplified is flanked by two specific primers which themselves become incorporated into me amplified DNA.
  • the said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in me art.
  • RNA (or in the case of retroviral vectors, RNA) is then expressed in a suitable host to produce a polypeptide comprising me compound of me invention.
  • me DNA encoding me polypeptide consti ting me compound of me invention may be used in accordance wim known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is men used to transform an appropriate host cell for the expression and production of the polypeptide of me invention.
  • Such techniques include tiiose disclosed in US Patent Nos.
  • the DNA (or in me case of retroviral vectors, RNA) encoding me polypeptide constituting me compound of me invention may be joined to a wide variety of omer DNA sequences for introduction into an appropriate host.
  • the companion DNA will depend upon me nature of me host, me manner of the introduction of me DNA into die host, and whetiier episomal maintenance or integration is desired.
  • me DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression.
  • an expression vector such as a plasmid
  • the DNA may be linked to me appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, altiiough such controls are generally available in the expression vector.
  • the vector is then introduced into the host through standard techniques. Generally, not all of die hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells.
  • One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance. Alternatively, the gene for such selectable trait can be on another vector, which is used to co- transform the desired host cell.
  • Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to tiiose skilled in die art in view of die teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.
  • the vectors include a prokaryotic replicon, such as the ColEl ori, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic, cell types.
  • the vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli, transformed tiierewith.
  • a promoter is an expression control element formed by a DNA sequence tiiat permits binding of RNA polymerase and transcription to occur.
  • Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
  • Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA, USA) and p7/r99A and pKK223-3 available from Pharmacia, Piscataway, NJ, USA.
  • a typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, NJ, USA. This vector uses the SV40 late promoter to drive expression of cloned genes, d e highest level of expression being found in T antigen-producing cells, such as COS-1 cells.
  • an inducible mammalian expression vector is pMSG, also available from Pharmacia.
  • This vector uses tiie glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of d e cloned gene.
  • Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
  • Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3.
  • Plasmids pRS413-416 are Yeast Centromere plasmids (YCps).
  • the present invention also relates to a host cell transformed with a polynucleotide vector construct of the present invention.
  • the host cell can be either prokaryotic or eukaryotic.
  • Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (No ATCC 31343).
  • Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line.
  • Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
  • Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, and monkey kidney- derived COS-1 cells available from the ATCC as CRL 1650.
  • Preferred insect cells are Sf9 cells which can be transfected with baculovirus expression vectors.
  • Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275, 104-109 is also useful.
  • reagents useful in transfecting such cells for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA.
  • Electroporation is also useful for transforming and/or transfecting cells and is well known in the art for transforming yeast cell, bacterial cells, insect cells and vertebrate cells.
  • Successfully transformed cells ie cells mat contain a DNA construct of the present invention
  • cells resulting from die introduction of an expression construct of the present invention can be grown to produce the polypeptide of the invention.
  • Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208.
  • the presence of the protein in the supernatant can be detected using antibodies as described below.
  • the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.
  • a further aspect of the invention provides a method of making the polypeptide of the invention or a variant, derivative, fragment or fusion thereof or a fusion of a variant, fragment or derivative me method comprising culturing a host cell comprising a recombinant polynucleotide or a replicable vector which encodes said polypeptide, and isolating said polypeptide or a variant, derivative, fragment or fusion thereof or a fusion of a variant, fragment or derivative from said host cell.
  • Methods of cultivating host cells and isolating recombinant proteins are well known in the art.
  • the invention also includes a polypeptide, or a variant, fragment, derivative or fusion tiiereof, or fusion of a said variant or fragment or derivative obtainable by the above method of the invention.
  • a still further aspect of the invention provides an antibody reactive towards a polypeptide of the invention. Examples of such antibodies are given in Example 1.
  • Antibodies reactive towards the said polypeptide of the invention may be made by methods well known in the art.
  • the antibodies may be polyclonal or monoclonal.
  • Suitable monoclonal antibodies which are reactive towards die said polypeptide may be prepared by known techniques, for example those disclosed in “Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and Applications", SGR Hurrell (CRC Press, 1982).
  • die antibody is raised using any suitable peptide sequence obtainable from the given amino acid sequence of SKK4. It is preferred if polyclonal antipeptide antibodies are made.
  • the antibody does not react substantially with another stress-activated protein kinase kinase such as SKK1, SKK2 or SKK3. Accordingly, it may be preferred if peptides based on die SKK4 sequence are used which vary significantly from any peptides found in any other stress-activated protein kinases such as SKK1, SKK2 or SKK3.
  • Peptides in which one or more of the amino acid residues are chemically modified, before or after the peptide is synthesised may be used providing that the function of the peptide, namely the production of specific antibodies in vivo, remains substantially unchanged.
  • modifications include forming salts with acids or bases, especially physiologically acceptable organic or inorganic acids and bases, forming an ester or amide of a terminal carboxyl group, and attaching amino acid protecting groups such as N-t-butoxycarbonyl. Such modifications may protect the peptide from in vivo metabolism.
  • the peptides may be present as single copies or as multiples, for example tandem repeats. Such tandem or multiple repeats may be sufficiently antigenic themselves to obviate the use of a carrier.
  • the peptide may be formed as a loop, witii the N-terrninal and C-terminal ends joined togetiier, or to add one or more Cys residues to an end to increase antigenicity and/or to allow disulphide bonds to be formed.
  • a carrier preferably a polypeptide
  • the arrangement is preferably such that the peptide of the invention forms a loop.
  • a carrier function should be present in any immunogenic formulation in order to stimulate, or enhance stimulation of, the immune system. It is thought that the best carriers embody (or, together with the antigen, create) a T-cell epitope.
  • the peptides may be associated, for example by cross-linking, with a separate carrier, such as serum albumins, myoglobins, bacterial toxoids and keyhole limpet haemocyanin.
  • More recently developed carriers which induce T-cell help in the immune response include the hepatitis-B core antigen (also called the nucleocapsid protein), presumed T-cell epitopes such as Thr-Ala-Ser-Gly-Val-Ala-Glu-Thr-Thr-Asn-Cys, beta- galactosidase and the 163-171 peptide of interleukin-1.
  • the latter compound may variously be regarded as a carrier or as an adjuvant or as both.
  • several copies of the same or different peptides of the invention may be cross-linked to one another; in this situation there is no separate carrier as such, but a carrier function may be provided by such cross-linking.
  • Suitable cross-linking agents include those listed as such in the Sigma and Pierce catalogues, for example glutaraldehyde, carbodiimide and succinimidyl 4-(N-maleimidomethyl)cyclohexane-l- carboxylate, the latter agent exploiting the -SH group on the C-terminal cysteine residue (if present).
  • the peptide is prepared by expression of a suitable nucleotide sequence in a suitable host, then it may be advantageous to express die peptide as a fusion product with a peptide sequence which acts as a carrier. Kabigen's "Ecosec" system is an example of such an arrangement.
  • the peptide of the invention may be linked to other antigens to provide a dual effect.
  • Peptides may be synthesised by the Fmoc-polyamide mode of solid-phase peptide syntiiesis as disclosed by Lu et al (1981) J. Org. Chem. 46, 3433 and references therein. Temporary N-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is effected using 20% piperidine in N,N-dimethylformamide.
  • Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case of arginine).
  • glutamine or asparagine are C-terminal residues, use is made of die 4,4'-dimethoxybenzhydryl group for protection of the side chain amido functionalities.
  • the solid-phase support is based on a polydimethyl-acrylamide polymer constituted from the three monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalising agent).
  • the peptide-to-resin cleavable linked agent used is die acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives with the exception of asparagine and glutamine, which are added using a reversed N,N-dicyclohexyl-carbodiimide/l- hydroxybenzotriazole mediated coupling procedure.
  • Any scavengers present are removed by a simple extraction procedure which on lyophilisation of die aqueous phase affords the crude peptide free of scavengers.
  • Reagents for peptide synthesis are generally available from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK. Purification may be effected by any one, or a combination of, techniques such as size exclusion chromatography, ion-exchange chromatography and (principally) reverse-phase high performance liquid chromatography. Analysis of peptides may be carried out using thin layer chromatography, reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis.
  • FAB fast atom bombardment
  • a further aspect of the invention provides a method of identifying a compound that modulates the activity of a polypeptide as defined in the first aspect of the invention, the method comprising contacting a compound witii the polypeptide or a suitable variant, fragment, derivative or fusion tiiereof or a fusion of a variant, fragment or derivative thereof and determining whether the protein kinase activity of the said polypeptide is changed compared to the activity of the said polypeptide or said variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof in the absence of said compound.
  • the compound decreases the activity of said polypeptide.
  • the compound may bind substantially reversibly or substantially irreversibly to die active site of said polypeptide.
  • the compound may bind to a portion of said polypeptide that is not the active site so as to interfere with the binding of the said polypeptide to its substrate.
  • the compound may bind to a portion of said polypeptide so as to decrease said polypeptide' s activity by an allosteric effect.
  • This allosteric effect may be an allosteric effect that is involved in the natural regulation of the said polypeptide 's activity.
  • the compound increases the activity of said polypeptide.
  • the compound may bind to a portion of said polypeptide that is not ti e active site so as to aid die binding of die said polypeptide to its substrate.
  • me compound may bind to a portion of said polypeptide so as to increase said polypeptide 's activity by an allosteric effect.
  • This allosteric effect may be an allosteric effect that is involved in the natural regulation of the said polypeptide' s activity.
  • the method makes use of the fact that SKK4 phosphorylates SAPKl as described in Example 1 or Example 2, but any suitable substrate may be used.
  • the method makes use of an assay which may be substantially the same as that described in Example 1.
  • an assay which may be substantially the same as that described in Example 1.
  • phosphorylation of ATF2 by SAPKl is measured.
  • phosphorylation of GST-c-Jun( 1-194) by SAPKl may be measured. It is preferred mat the SKK4 is recombinant SKK4.
  • a still further aspect of the invention provides a method of identifying a compound which binds to SAPKl (or other substrate of the polypeptide as defined in the first aspect of the invention) and enhances or prevents its activation by the polypeptide as defined in the first aspect of the invention, the method comprising dete ⁇ nining whether a compound enhances or prevents the interaction of SAPKl (or other substrate of the polypeptide as defined in the first aspect of the invention) or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative with the polypeptide as defined in die first aspect of the invention or deternrining whether the compound substantially blocks activation of SAPKl (or otiier substrate of the polypeptide as defined in the first aspect of the invention) or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative by the polypeptide as defined in the first aspect of the invention.
  • a still further aspect of the invention provides a method of identifying a compound which modulates the activation of the polypeptide as defined in the first aspect of the invention by an "upstream activator” , for example MEKK.
  • upstream activator is meant a molecule that interacts with the polypeptide of the invention with the result that the protein kinase activity of the polypeptide of the invention is increased. It may be a polypeptide. Preferably, it is a physiological activator of native SKK4.
  • the method comprises deterrnining whether a compound enhances or disrupts the interaction between (a) a polypeptide as defined in the first aspect of the invention or a suitable fragment, variant, derivative or fusion tiiereof or a suitable fusion of a fragment, variant or derivative and (b) an "upstream activator" , for example MEKK, or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a variant, derivative or fragment, or detei ⁇ nining whether the compound substantially blocks activation of the said polypeptide or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion by an "upstream activator” or a suitable variant, derivative, fragment or fusion thereof.
  • an upstream activator for example MEKK
  • MEKK is a known activator of SKK1 that is shown here to also be an activator of the polypeptide of die invention, known as SKK4. Prior to the present invention, it was not known how SKK4 could be activated. By “activation of SKK4" it is meant ti at the abtiity of SKK4 to phosphorylate SAPKl is increased following the treatment of SKK4, for example by MgATP and MEKK.
  • MEKK The sequence of MEKK is given in Lange-Carter et al (1993) Science 260, 315-319, and its expression as a histidine-tagged fusion protein and purification using nickel-nitrilotriacetate-agarose is described in Meier et al (1996) and Example 1.
  • a further aspect of the invention is the use of MEKK for the activation of the polypeptide of die invention.
  • a still further aspect of the invention provides a method of identifying a polypeptide tiiat interacts with the protein kinase (polypeptide) of die invention, die method comprising 1) contacting a) the said protein kinase as defined in the first aspect of the invention or a suitable variant fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof with b) a composition that may contain a polypeptide that interacts with the said protein kinase, 2) detecting the presence of a complex containing the said protein kinase and a polypeptide, and optionally 3) identifying any polypeptide bound to d e said protein kinase.
  • the composition may comprise material from cells.
  • the cells may be selected from the following types: 1) cells which do not have SKK4 activity even when stimulated, 2) cells which have SKK4 activity after exposure to a stimulus, but which have not been so exposed and 3) cells of type 2 after exposure to the stimulus.
  • Polypeptides tiiat are found in a subset only of types 1-3 are of particular interest and may be characterised further. Such a peptide may be an activator of SKK4. Alternatively, it may be an inactivator of SKK4.
  • tiiat the method may be performed within a cell, for example using the yeast two hybrid system as is well known in the art.
  • cDNAs copied from mRNA from the three cell types described above would be used.
  • a still further aspect of the invention provides a method of identifying a compound which blocks die activation of the polypeptide as defined in the first aspect of the invention by an interacting polypeptide, for example MEKK, die metiiod comprising dete ⁇ riining whether a compound enhances or disrupts the interaction between (a) a protein kinase as defined in the first aspect of the invention or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative and (b) said interacting polypeptide or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a variant, derivative or fragment, or deterrnining whether the compound substantially blocks activation of the polypeptide according to the first aspect of the invention or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion by said interacting polypeptide or a suitable variant, derivative, fragment or fusion thereof.
  • an interacting polypeptide for example MEKK
  • the said polypeptide according to the first aspect of the invention or fragment, derivative, variant or fusion thereof used in the method is one which is produced by recombinant DNA technology.
  • die SAPKl or fragment, derivative, variant or fusion tiiereof used in the method of identifying compounds tiiat modulate activity of the said protein kinase is one which is produced by recombinant DNA technology.
  • MEKK or other "upstream activator" or fragment, derivative, variant or fusion thereof used in the method is one which is produced by recombinant DNA technology.
  • polypeptide of the invention (SKK4) is activated in vitro by treating the polypeptide witii MEKK and MgATP, as described in Example 1. It is particularly preferred if die SKK4 is the recombinant polypeptide produced according to die methods of die invention. It will be appreciated tiiat by "suitable” we mean that the said components in the method are those that have interactions or activities which are substantially the same as those of SKK4 or SAPKl or other substrates, or the upstream activator such as MEKK as the case may be but which may be more convenient to use in an assay. For example, fusions of SKK4 or SAPKl are particularly useful since said fusion may contain a moiety which may allow the fusion to be purified readily.
  • reporter gene constructs may be prepared by methods known to those skilled in the art, using the teaching herein.
  • a reporter gene construct may be made with a c-jun-dependent promoter sequence. This construct may be introduced togedier with an SKK4 construct into a cell line, in the parent cell line of which SAPKl is activated in response to known stimuli, and in which die endogenous SKK4 gene has been inactivated.
  • die reporter gene construct could be introduced into the cell line in which SAPKl is activated in response to known stimuli. The expression of the reporter gene will be dependent on the activity of SKK4 and tiius the effect of compounds can be measured.
  • the reporter gene may be fatal to the cells, or alternatively may allow cells to survive under otherwise fatal conditions.
  • Cell survival can tiien be measured, for example using colorimetric assays for mitochondrial activity, such as reduction of WST-1 (Boehringer).
  • WST-1 is a formosan dye that undergoes a change in absorbance on receiving electrons via succinate dehydrogenase.
  • the yeast two-hybrid system is used.
  • the enhancement or disruption of the interaction between the said polypeptide of die invention and SAPKl or an interacting polypeptide as defined above, or suitable derivatives, fragments, fusions or variants can be measured in vitro using methods well known in the art of biochemistry and include any methods which can be used to assess protein-protein interactions.
  • the said interaction can also be measured within a cell, for example using the yeast two hybrid system as is well known in the art.
  • the invention provides screening assays for drugs which may be useful in modulating the activity of SKK4 or its interactions with upstream activators.
  • the compounds identified in the methods may themselves be useful as a drug or tiiey may represent lead compounds for the design and synthesis of more efficacious compounds.
  • screening assays which are capable of high throughput operation will be particularly preferred.
  • Examples may include the cell based assays described and protein-protein binding assays.
  • a further example is an SPA-based (Scintillation Proximity Assay) system as described in Example 2.
  • a further aspect of the invention provides a compound identifiable by die screening methods of d e invention.
  • a still further aspect provides such a compound for use in medicine.
  • SKK4 becomes activated in response to proinflammatory cytokines (interleukin-1, tumour necrosis factor) in human epithelial cells, whereas the other known activator of SAPKl (SKK1/MKK4) is not. Since overproduction and/or inappropriate production of interleukin-1 and tumour necrosis factor is known to be a major cause of inflammatory diseases, such as rheumatoid arthritis, drugs tiiat inhibit SKK4 are likely to suppress a number of the intracellular effects of these cytokines and may therefore be useful for treating inflammation.
  • a further aspect of the invention is the use of any of the screening methods of the invention in the identification of a molecule that may be useful in treating inflammatory disease.
  • Inflammatory diseases include rheumatoid artiiritis, psoriasis, septic shock, asthma and inflammatory bowel disease.
  • a further aspect of the invention provides a metiiod of treating a patient with an inflammatory disease the method comprising administering to the patient an effective amount of a compound identifiable by the screening methods of die invention.
  • a still further invention provides a use of a compound identifiable by die screening metiiods of the invention in the manufacture of a medicament for treating an inflammatory disease in a patient.
  • a further aspect of the invention provides a method of treating a patient with an inflammatory disease the method comprising administering to tiie patient an effective amount of a compound identifiable by die screening methods of the invention.
  • such compounds are useful in treating disease in which apoptosis is involved.
  • such compounds may suppress apoptosis, which may aid cell survival during or following cell damaging processes.
  • diseases include, but are not limited to, ischaemic disease, for example stroke and myocardial infarction, neural injury and myocardial infarction.
  • Some such compounds may aid apoptosis.
  • Conditions in which aiding apoptosis may be of benefit include resolution of inflammation.
  • a further aspect of the invention provides a method of treating a patient with an ischaemic disease die method comprising adrmnistering to d e patient an effective amount of a compound identifiable by die screening methods of die invention.
  • a still further invention provides a use of a compound identifiable by the screening methods of the invention in the manufacture of a medicament for treating an ischaemic disease in a patient.
  • a further aspect of the invention provides a method of treating a patient with an ischaemic disease the method comprising administering to the patient an effective amount of a compound identifiable by die screening methods of the invention.
  • the aforementioned compounds of the invention or a formulation thereof may be administered by any conventional method including oral and parenteral (e.g. subcutaneous or intramuscular) injection.
  • the treatment may consist of a single dose or a plurality of doses over a period of time.
  • a compound of d e invention Whilst it is possible for a compound of d e invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the compound of die invention and not deleterious to the recipients thereof.
  • the carriers will be water or saline which will be sterile and pyrogen free.
  • the invention also provides pharmaceutical compositions comprising the compound identifiable by die screening metiiods of the invention and a pharmaceutically acceptable carrier.
  • a polypeptide protein kinase
  • a further aspect of the invention provides a kit of parts tiiat are useful in carrying out the screening methods.
  • Figure 1 the nucleotide and predicted amino acid sequence of human SAP kinase kinase-4 (SKK4). Nucleotides are numbered in the 5' to 3' direction and amino acids are shown in single-letter code above d e nucleotide sequence. The in-frame termination codon is marked by an asterisk.
  • Figure 2 the sequence comparison of human SKK4, human SKK1 , human SKK2, human SKK3 and HEP from D. melanogaster . Amino acids were aligned and gaps were introduced to maximise the homology. Amino acid identities between at least tiiree of the five sequences are indicated by boxed regions.
  • Figure 3 activation of SKK4 and SKK1/MKK4 in KB cells.
  • A KB cells were incubated in the absence (control) or presence of the indicated agonist as described in Section 2.7 of Example 1 and SKK4 (open bars) and SKK1/MKK4 (filled bars) irnmunoprecipitated from die ly sates and assayed for their ability to activate SAPK1/JNK1. The results are shown as means ⁇ SEM for six dishes of ells witii each agonist (two separate experiments). The basal activity of SKK4 and SKK1/MKK4 in unstimulated cells was 5.0 ⁇ 0.4 mU/mg and 2.0 ⁇ 0.2 mU/mg, respectively.
  • SKK4 and SKK1/MKK4 were i munoprecipitated from extracts prepared from UV-irradiated KB cells. Where indicated, the antibodies were incubated witii the peptides used to raise die anti-SKK4 or anti-SKKl/MKK4 antibodies. The results are presented as means ⁇ SEM for at least three experiments.
  • FIG. 4 GST-SKK4 is activated by MEKK and activates SAPKl/JNK, but not SAPK2a/p38.
  • GST-SKK4 (0.25 ⁇ M) and GST-SKK1/MKK4 (0.1 ⁇ M) were incubated witii MgATP in the presence or absence of MEKK and then assayed for their ability to activate SAPKl/JNK (A) or SAPK2a/p38 (B) (Section 2.8 of Example 1).
  • the activation of SAPKl/JNK and SAPK2a/p38 were monitored by die phosphorylation of GST-ATF2[19-96].
  • FIG. 5 SKK4 immunoprecipitated from extracts prepared from UV- irradiated KB cells activates SAPKl/JNK, but not SAPK2/p38.
  • SKK4 and SKK1/MKK4 immunoprecipitates were incubated with MgATP and SAPK2a/p38 or SAPKl/JNK.
  • SAPK2a/p38 and SAPKl/JNK activity was tiien measured using ATF2 as substrate. The results are expressed as means ⁇ SEM for three experiments.
  • Example 1 Purification, characterisation, cloning and expression of SKK4, and generation of antibodies binding to SKK4 Summary.
  • a cDNA was cloned and expressed that encodes human stress-activated protein kinase kinase-4 (SKK4), a novel MAP kinase kinase family member whose mRNA is widely expressed in human tissues.
  • SKK4 activated SAPKl/JNK in vitro, but not SAPK2a/p38, SAPK2b/p38 ⁇ , SAPK3/ERK6 or SAPK4. It appears to be the mammalian homologue of HEP, an activator of SAPKl/JNK in Drosophila.
  • SKK4 and SKK1/MKK4 another activator of SAPKl/JNK
  • SKK4 and SKK1/MKK4 were both activated by stressful stimuli, but only SKK4 was activated strongly by proinflammatory cytokines.
  • the identification of SKK4 explains why the major SAPKl/JNK activator detected in many mammalian cell extracts is chromatographically separable from SKK1/MKK4.
  • mitogen-activated protein kinase (MAPK) family members have been identified that are activated strongly by adverse stimuli (eg chemical, heat and osmotic shock, ultraviolet radiation and die protein synthesis inhibitor anisomycin) or by signals produced/released during infection (eg lipopolysaccharide and d e proinflammatory cytokines interleukin-1 (IL-1) and tumour necrosis factor (TNF)), but only weakly (in most cell contexts) by polypeptide growth factors or phorbol esters (reviewed in [1]). For these reasons, they are termed stress-activated protein kinases (SAPKs).
  • SAPKs stress-activated protein kinases
  • the three isoforms of SAPKl/JNK bind tightly to the activation domain of c-Jun and are the only SAPKs that phosphorylate (at significant rates) the residues in this transcription factor that become phosphorylated in vivo in response to adverse stimuli and proinflammatory cytokines [204].
  • the two isoforms of SAPK2/p38 are inhibited specifically by die pyridinyl imidazoles SB 203580 and SB 202190 at submicromolar concentrations [4-6] and their physiological substrates include the transcription factors Elkl [7], SAP1 [7], CHOP [8] and MEF2C [9], as well as MAPK-activated protein kinase-2 (MAPKAP- K2) [10], MAPKAP]K3 [11,12], MAPK-interacting protein kinase-1 (Mnkl) and Mnk2 [13,14].
  • MAPKAP- K2 MAPK-activated protein kinase-2
  • Intracellular targets for MAPKAP- K2/MAPKAP-K3 include heat shock protein 27 [5,15], tyrosine hydroxylase [16] and the transcription factor CREB [17], while one of the substrates for Mnkl/Mnk2 is eukaryotic initiation factor eIF4E [13].
  • SAPK3/ERK6 [18,20] and SAPK4 [4,6] have only been identified recently and their physiological substrates are unknown.
  • SAPK3 and SAPK4 are 60% identical to each other or to SAPK2a and SAPK2b, but they are not inhibited by SB 203580 and SB 202190 [4,6,18].
  • SAPKs are activated by MAPK kinase (MKK) family members, termed here SAPK kinases (SKKs).
  • MKK MAPK kinase
  • SKKs SAPK kinases
  • the major activator of SAPK2a/p38, SAPK2b/p38 ⁇ , SAPK3 and SAPK4 that has been detected biochemically is SKK3/MKK6 [4,18,21]23], although SAPK2/p38 can also be activated by SKK2/MKK3 in vitro [24].
  • SKK2/MKK3 nor SKK3/MKK6 are capable of activating SAPKl/JNK, which is activated by a distinct enzyme termed SKK1/MKK4 [24,25].
  • SKK1/MKK4 is the only activator of SAPKl/JNK that can be detected biochemically in extracts prepared from PC 12 cells that have been exposed to adverse stimuli [21], and in 293 cells the activation of transfected SAPKl/JNK by anisomycin can be prevented by overexpressing a catalytically inactive form of SKK1/MKK4 [25] .
  • heat shock or anisomycin failed to activate SAPKl/JNK in murine embryonic stem cells lacking SKK1/MKK4 [26,27].
  • SKK1/MKK4 can activate SAPK2/p38 in vitro [24,28] but, in contrast to SAPKl/JNK which is activated strongly by cotransfection with MEK kinase (MEKK) [29] (an upstream activator of SKK1/MKK4), SAPK2/p38 is not activated [24] or activated much more weakly [30]. Moreover the activation of SAPK2/p38 by osmotic shock and anisomycin is unimpaired in murine embryonic stem cells lacking SKK1/MKK4 [26,27]. For these reasons, SKK1/MKK4 does not seem to be rate- limiting for the activation of SAPK2/p38 in vivo.
  • SKK1/MKK4 is clearly essential for the activation of SAPKl/JNK in some cells
  • the dominant activator of SAPKl/JNK detected biochemically in human epithelial KB cells [21] or in Rat 3Y1 fibroblasts [31] is a chromatographically distinct enzyme(s).
  • the activation of SAPKl/JNK by UV radiation and osmotic shock in murine embryonic stem cells lacking SKK1/MKK4 was either unimpaired [26] or only inhibited partially [27] .
  • SKK4 novel MKK family member
  • a plasmid encoding the maltose-binding protein (MalE) linked to the Xenopus homologue of SAPK2a/p38 (MalE-Mpk2) was a gift from Dr A R Nebreda (EMBL, Heidelberg, Germany), while a plasmid encoding MEKK preceded by six histidine residues (6-His-MEKK) was provided by Dr G Johnson (National Jewish Centre for Immunology and Respiratory Medicine, Denver, USA). All plasmids were transformed into E. coli strain BL21 (DE3), and expressed and purified as described [4,18,21]. 6-His SAPKl/JNKl ⁇ [32] was expressed and purified as reported previously [33].
  • TTYADFIASHGRTGRRNAIHD the specific peptide inhibitor of cAMP-dependent protein kinase
  • the purified protein was dialysed against 50 mM Tris/HCl pH 7.5, 0.1 mM EGTA, 0.03% (m/v) Brij35, 0.1 % (v/v) 2-mercaptoethanol and 50% (v/v) glycerol, and stored unfrozen at -20 °C at 2 mg/ml.
  • Peptides were synthesised corresponding to the C-terminal 12 residues of human SKK4 (GVLSQPHLPFFR) and the last 14 residues of SKK1/MKK4 (DQMPATPSSPMYVD) [24], coupled to botii bovine serum albumin and keyhole limpet haemocyanin using glutaraldehyde and injected into sheep at the Scottish Antibody Production Unit (Carluke, Ayrshire, UK).
  • the anti-SKK4 and anti-SKKl/MKK4 antibodies were purified on peptide antigen-CH-Sepharose columns [22] .
  • KB cells were cultured [18] and exposed for 15 min to osmotic stress (0.5 M sorbitol) or proinflammatory cytokines (20 ng/ml IL-l ⁇ or 100 ng/ml TNF ⁇ ), or for 30 min to anisomycin (10 ⁇ g/ml), or for 10 min to 100 ng/ml epidermal growth factor (EGF) or 300 ng/ml phorbol myristate acetate (PMA).
  • UV-C irradiation was carried out at 60 J/m2 and the cells then incubated for a further 30 min at 37 °C. Cells were lysed as described [21].
  • Immunoprecipitates ( -6.5 ⁇ l) were incubated on a shaking platform for 30 min at 30 °C with 1 :1 of 20 ⁇ M 6-His-SAPKl/JNK in 50 mM Tris/HCl (pH 7.4), 0.1 mM EGTA, 0.03% (m/v) Brij-35, 0.1 % (v/v) 2- mercaptoethanol and 5% (v/v) glycerol, and the reactions initiated witii 2.5 ⁇ l of 40 mM magnesium acetate-0.4 mM unlabelled ATP.
  • the active SAPKl/JNK generated was assayed by adding 40 ⁇ l of a solution containing 31.25 mM Tris/HCl (pH 7.4), 0.125 mM EGTA, 1.25 mM sodium orthovanadate, 3.1 ⁇ M PKI, 0.1 % (by vol) 2- mercaptoethanol and 0.25 mg/ml GST-ATF2[ 19-96], 12.5 mM magnesium acetate and 0.125 mM [( ⁇ 32 P]ATP.
  • the 32 P-labelled band corresponding to GST-ATF[ 19-96] was excised and counted.
  • One unit of SKK1/MKK4 or SKK4 activity was that amount which increased the activity of SAPKl/JNK by 1 U/min.
  • One Unit of SAPKl/JNK was that amount which incorporated 1 nmol of phosphate into GST-ATF2[19-96] in one min.
  • SKK1/MKK4 and SKK4 were also assayed for their ability to activate Xenopus SAPK2a.
  • the assay was identical except tiiat SAPK2a (2 ⁇ M) replaced SAPKl/JNK.
  • Each GST fusion protein (4 ⁇ M) was activated by incubation for 60 min at 30 °C with MEKK (1 ⁇ M) in 50 mM Tris/HCl pH 7.5, 0.1 mM EGTA, 0.1 % (v/v) 2-mercaptoethanol, 10 mM magnesium acetate and 0.1 M unlabelled ATP.
  • Activated GST-SKK4 and GST-SKK1/MKK4 were then measured by their ability to activate SAPKl/JNK or SAPK2a/p38, which were assayed by the phosphorylation of ATF2.
  • the assays (50 ⁇ l) were carried out at 30 °C and comprised 0.1 ⁇ M GST-SKK4 or GST-SKK1/MKK4, 0.2 ⁇ M 6-His SAPKl/JNK, 0.2 mg/ml GST-ATF2[ 19-96] , 50 mM Tris/HCl pH 7.5, 0.1 mM EGTA, 0.1 % (v/v) 2-mercaptoethanol, 10 mM magnesium acetate and 0.1 mM [ ⁇ 32 P]ATP.
  • the reactions were terminated by spotting 40 ⁇ l on to 2 x 2 cm squares of phosphocellulose p81 paper followed by immersion into 75 mM phosphoric acid. After washing the papers several times in phosphoric acid followed by immersion in acetone, the papers were dried and counted.
  • the open reading frame encodes a protein of 333 residues, with a predicted molecular mass of 37.9 kDa. It possesses all the conserved amino acid domains (I-XI) characteristic of protein kinases and shows 62% identity with the Drosophila MKK homologue HEP (see Discussion), 48% identity with human SKK1/MKK4, 39% identity with human SKK2/MKK3, and 41 % identity with human SKK3/MKK6 (Fig 2).
  • the sequence identity witii MKK1 and MKK2 is 36% and 35%, respectively.
  • Ser 185 and Thr 189 in kinase subdomain VIII are in an equivalent position to the Ser/Thr residues in other MKK family members, whose phosphorylation is required to generate enzymatic activity.
  • RNA transcript of ⁇ 4 kb was present in heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas (data not shown).
  • the presence of ESTs in databases revealed tiiat SKK4 is also expressed in retina and germinal centre B-cells.
  • SKK4 and mRNA is widely expressed in mammalian cells.
  • SKK4 is activated by proinflammatory cytokines and stressful stimuli.
  • An antibody was raised against the unique C-terminal sequence of SKK4 (Section 2.4) and used to immunoprecipitate SKK4 from KB cell extracts after stimulation with a variety of agonists (Fig 3).
  • SKK4 was activated rapidly in KB cells in response to the same stimuli that trigger the activation SAPKl/JNK [21], namely proinflammatory cytokines and stressful stimuli (UV radiation, osmotic shock and anisomycin).
  • SKK4 was activated weakly by EGF, but not by PMA.
  • SKK4 was also activated by stressful stimuli in COS cells (data not shown).
  • SKK4 was activated to a greater extent by interleukin-1 (IL-1) and tumour necrosis factor (TNF ⁇ ) than by UV irradiation, osmotic shock or anisomycin.
  • IL-1 interleukin-1
  • TNF ⁇ tumour necrosis factor
  • SKK1/MKK4 another activator of SAPKl/JNK (see Introduction) is also present in KB cells and, like SKK4, was activated by UV irradiation, osmotic shock and anisomycin. However, in contrast to SKK4, SKK1/MKK4 was hardly activated by interleukin-1, TNF ⁇ or EGF (Fig 3A).
  • GST-SKK4 was expressed in E. coli (Section 2.3) and 10 mg of purified enzyme could be isolated from 500 ml of bacterial culture. The preparation showed a single protein-staining band with the predicted molecular mass of 64 kDa (data not shown).
  • GST-SKK4 was inactive, but could be activated by incubation with MgATP and MEKK (Fig 4), an enzyme that also activates SKK1/MKK4 [29].
  • the activated GST-SKK4 was able to activate SAPKl/JNK in vitro, but not SAPK2a/ ⁇ 38 (Fig 4), SAPK2b/p38 ⁇ , SAPK3/ERK6 or SAPK4 (data not shown). In contrast, activated GST- SKK1/MKK4 activated SAPK2a/p38 as well as SAPKl/JNK (Fig 4).
  • SKK4 activated SAPKl/JNK but not SAPK2a/p38, while SKK1/MKK4 activated both enzymes (Fig 5).
  • SKK4 is activated in vitro by MEKK (Fig 4) one of many protein kinases [38] that have been shown to activate SKK1/MKK4 in vitro and to trigger the activation of SAPKl/JNK in cotransfection experiments.
  • SKK4 is assayed routinely by phosphorylation of SAPKl , as described in Meier et al, (1996). 10 ⁇ l of a solution containing SKK4 and inactive SAPKl (equivalent to lOOmU before inactivation with PTP IB) in 20 mM sodium glycerol 2-phosphate pH 7.4, 1.0 mM EGTA, 0.03% (by mass) Brij-35, 5 % (by vol.) glycerol, 1 mM benzamadine, 0.1 % (by vol.) 2- mercaptoethaonol, 1 mM sodium orthovanadate, 1 ⁇ M microcystin-LR was incubated for 3 min at 30 °C, and the reaction initiated with 2 ⁇ l 60 mM MgCl 2 , 0.6 mM unlabeled ATP.
  • SAPKl is assayed by phosphorylation of GST-c-Jun( 1-194), as described in Meier et al, (1996).
  • SPA Scintillation Proximity Assay
  • the sample is mixed with beads comprising scintillant and antibodies that bind GST-c-Jun(l-194). Conveniently this is done in a 96-well format.
  • the plate is then counted using a suitable scintillation counter, using known parameters for P SPA assays. Only 32 P that is in proximity to the scintillant, i.e. only that bound to GST-c- Jun(l-194) that is then bound by the antibody, is detected.
  • Example 3 Assay for compounds which modulate SKK4 activity
  • An assay is set up with SAPKl, as described in Example 1 or Example 2.
  • a yeast two hybrid assay system is set up to identify polynucleotides encoding polypeptides that are capable of associating with SKK4 in a stable enough manner to allow transcriptional activation to occur.
  • the polynucleotides are (in separate experiments) cDNAs copied from mRNA from cells that are capable of expressing SKK4, before or after stimulation capable of activating SKK4, and from cells which do not express SKK4. Interactions which are found in a subset only of these cell types are of particular interest.
  • polypeptide encoded by the polynucleotide is deteirnined by sequencing the insert by the Sanger metiiod as described in Example 1 to obtain a predicted amino acid sequence.
  • SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin- 1" FEBS Lett. 364, 229-233.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne une protéine kinase activée par le stress, sensiblement pure, qui comporte la séquence d'acides aminés (A), ou une variante, un fragment, une fusion ou un dérivé de celle-ci; ou une fusion de ladite variante, dudit fragment ou dudit dérivé. La protéine kinase activée par le stress est utile pour des techniques de recherche de médicaments.
PCT/GB1998/002475 1997-08-19 1998-08-18 Proteine kinase humaine skk4 activee par le stress WO1999009181A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU87432/98A AU8743298A (en) 1997-08-19 1998-08-18 Human stress-activated protein kinase, skk4
EP98938835A EP1005555A1 (fr) 1997-08-19 1998-08-18 Proteine kinase humaine skk4 activee par le stress

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9717416.3 1997-08-19
GBGB9717416.3A GB9717416D0 (en) 1997-08-19 1997-08-19 Polypeptides,polynucleotides and uses thereof

Publications (1)

Publication Number Publication Date
WO1999009181A1 true WO1999009181A1 (fr) 1999-02-25

Family

ID=10817610

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1998/002475 WO1999009181A1 (fr) 1997-08-19 1998-08-18 Proteine kinase humaine skk4 activee par le stress

Country Status (4)

Country Link
EP (1) EP1005555A1 (fr)
AU (1) AU8743298A (fr)
GB (1) GB9717416D0 (fr)
WO (1) WO1999009181A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999033999A1 (fr) * 1997-12-31 1999-07-08 Chiron Corporation Proteine kinase kinase 7 (mkk7) activee par mitogene
EP1008650A1 (fr) * 1997-07-03 2000-06-14 Asahi Kasei Kogyo Kabushiki Kaisha Nouvelle kinase mapk

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996036642A1 (fr) * 1995-05-19 1996-11-21 Davis Roger J Kinases de proteine kinase humaine a activation par cytokine, stress et oncoproteine

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996036642A1 (fr) * 1995-05-19 1996-11-21 Davis Roger J Kinases de proteine kinase humaine a activation par cytokine, stress et oncoproteine

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
EMBL database entry HS962239; accession number H85962; 22. Novemebr 1995 Hillier et al.:' The WashU-Merck EST project.' *
EMBL database entry HSAA14581; accession number AA194047; 24. January 1997; Hillier et al.: 'The WashU-Merck EST project.' *
EMBL database entry HSAA52650; accession number AA252650; 15. March 1997; Strausberg R.: 'National Cancer Institute, Cancer Genome Anatomy Project.' *
FOLTZ I. N. ET AL.: "Human mitogen-activated protein kinase kinase 7 (MKK7) is a highly conserved c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activated by environmental stresses and physiological stimuli.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 15, April 1998 (1998-04-01), pages 9344 - 9351, XP002085861 *
LAWLER, SEAN ET AL: "SKK4, a novel activator of stress -activated protein kinase -1 ( SAPK1 /JNK)", FEBS LETTERS, vol. 414, no. 1, 1 September 1997 (1997-09-01), pages 153 - 158, XP002085860 *
MEIER R. ET AL.: "Cellular stresses and cytokines activate multiple mitogen-activated protein kinase kinase homologues in PC12 and KB cells.", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 236, 1996, pages 796 - 805, XP002085857 *
MORIGUCHI T. ET AL.: "Evidence for multiple activators for stress-activated protein kinases/c-Jun amino-terminal kinases.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 270, no. 22, 1995, pages 12969 - 12972, XP002085858 *
TOURNIER C. ET AL.: "Mitogen-activated protein kinase kinase 7 is an activator of the c-Jun NH2-terminal kinase.", PROC. NATL. ACAD. SCI. USA, vol. 94, July 1997 (1997-07-01), pages 7337 - 7342, XP002085856 *
YAN M. ET AL.: "Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEK1.", NATURE, vol. 372, 1994, pages 798 - 800, XP002085859 *
YANG J. ET AL.: "Molecular cloning and characterization of a human protein kinase that specifically activates c-Jun N-terminal kinase.", GENE, vol. 212, 28 May 1998 (1998-05-28), pages 95 - 102, XP002085862 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1008650A1 (fr) * 1997-07-03 2000-06-14 Asahi Kasei Kogyo Kabushiki Kaisha Nouvelle kinase mapk
EP1008650A4 (fr) * 1997-07-03 2005-04-06 Asahi Kasei Pharma Corp Nouvelle kinase mapk
WO1999033999A1 (fr) * 1997-12-31 1999-07-08 Chiron Corporation Proteine kinase kinase 7 (mkk7) activee par mitogene
US6492112B1 (en) 1997-12-31 2002-12-10 Chiron Corporation Mitogen-activated protein kinase kinase 7 (MKK7)

Also Published As

Publication number Publication date
EP1005555A1 (fr) 2000-06-07
AU8743298A (en) 1999-03-08
GB9717416D0 (en) 1997-10-22

Similar Documents

Publication Publication Date Title
Li et al. The primary structure of p38γ: a new member of p38 group of MAP kinases
US5955366A (en) Polynucleotides encoding cytokine suppressive anti-inflammatory drug binding proteins
US6656716B1 (en) Polypeptide fragments of human PAK5 protein kinase
US5854003A (en) Screening method for agents that modulate human NIK activity
Lawler et al. SKK4, a novel activator of stress-activated protein kinase-1 (SAPK1/JNK)
US20100093027A1 (en) Enzyme
WO1997022704A1 (fr) Proteine kinase kinase mek6 activee par des mitogenes et ses procedes d'utilisation
EP1141003B9 (fr) Methode d'activation de la SGK par phosphorylation.
EP0932666B1 (fr) Proteine kinases activees par les mitogenes, polypeptides, polynucleotides et leur utilisation
US20030059881A1 (en) Mitogen-activated protein kinase p38-2 and methods of use therefor
EP1109918B1 (fr) Proteine kinases activees par des signaux mitogenes et le stress comprenant deux domaines kinase et leurs utilisations
EP1005555A1 (fr) Proteine kinase humaine skk4 activee par le stress
AU771307B2 (en) Identification and functional characterization of a novel ribosomal S6 protein kinase
EP0914451A1 (fr) Kinase "itak" activee par interleukine-1 ou facteur de necrose tumorale alpha et procedes correspondants de fabrication et d'utilisation
US6734001B1 (en) 3-phosphoinositide-dependent protein kinase
US7083511B1 (en) Methods
US6677130B1 (en) Mitogen-activated protein kinase p38-2 and methods of use therefor
US20030059419A1 (en) Identification of novel substrate I-TRAF of IKK-i kinase

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1998938835

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: KR

WWP Wipo information: published in national office

Ref document number: 1998938835

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 09486006

Country of ref document: US

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA

WWW Wipo information: withdrawn in national office

Ref document number: 1998938835

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载