MAP kinases: polypeptides, polynucleotides and uses thereof

Details MAP kinases: polypeptides, polynucleotides and uses thereof MAP kinases: polypeptides, polynucleotides and uses thereof

2001-03-15

2003-09-09

Prats, Francisco (Department: 1651)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S004000, C435S007910, C435S131000, C435S194000

Reexamination Certificate

active

06617117

ABSTRACT:

The present invention relates to polypeptides, polynucleotides and uses thereof, in particular to members of the stress-activated protein kinase (SAPK) family.
Four mitogen-activated protein (MAP) kinase family members are activated by cellular stresses (chemical, heat and osmotic shock, ultraviolet radiation, inhibitors of protein synthesis), bacterial lipopolysaccharide (LPS), and the cytokines interleukin-1 (IL1) and tumour necrosis factor (TNF), and have therefore been termed stress-activated protein kinases or SAPKs (reviewed in Cohen, 1997). Isoforms of 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. The transcription factors Elk1 (Cavigelli et al, 1995 and ATF2 are phosphorylated in vitro (the latter at Thr-69, Thr-71, and Ser-90 (Gupta et al, 1995; Livingstone et al, 1995), increasing the transcriptional activity of these proteins. The same sites in the transcription factor c-Jun also become phosphorylated when cells are exposed to the stresses and cytokines that activate SAPK1, or after cotransfection with protein kinases known to activate SAPK1 (Pulverer et al, 1991; Hibi et al, 1993; Dérijard et al, 1994; Kyriakis et al, 1994; Cavigelli et al, 1995; Gupta et al, 1995; Whitmarsh et al, 1995; Zinck et al, 1995), suggesting that c-Jun is a physiological substrate for SAPK1.
SAPK2a [also termed p38 (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. These include four protein kinases, namely MAP kinase-activated protein kinase-2 (MAPKAP-K2, Rouse et al, 1994) and the closely related MAPKAP-K3, which shares 75% amino acid sequence identity and has similar substrate specificity in vitro (McLaughlin et al, 1996; Ludwig et al, 1996; Clifton et al, 1996), as well as MAP kinase interacting protein kinases-1 and -2 (Mnk1 and Mnk2) (Waskiewicz et al, 1997; Fukunaga and Hunter, 1997).
Physiological substrates of MAPKAP-K2/K3 include heat shock protein (HSP) 27 (Stokoe et al, 1992a; Cuenda et al, 1995; Huot et al, 1995) and the transcription factor CREB (Tan et al, 1996), whereas transcription factor eIEF4E is a physiological substrate of Mnk1/2 (Waskiewicz et al, 1997). The phosphorylation of HSP27 appears to enhance the polymerisation of actin and is thought to help repair the actin microfilament network which becomes disrupted during cellular stress, thereby aiding cell survival (Lavoie et al, 1995). The phosphorylation of CREB (at Ser-133) is essential to allow this protein to stimulate the ascription of genes that contain cyclic AMP response elements (CREs). SAPK2a also mediates the stress-induced phosphorylation (at Ser-78 and Ser-81) and activation of the CEBP&bgr;-related transcription factor CHOP (Wang and Ron, 1996) and the ternary complex factor Elk-1 (Price et al, 1996).
Based on the effects of SB 203580, the activation of SAPK2a is rate-limiting in the LPS-induced production of IL1 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 IL1-induced stimulation of glucose uptake in epithelial cells (Gould et al, 1995), in collagen-induced platelet aggregation (Saklatvala et al, 1996), in the stress-induced transcription of c-Jun and c-Fos in fibroblasts (Hazzalin et al, 1996; Price et al, 1996) and in the LPS-induced synthesis of cyclo-oxygenase-2 (COX-2, the rate limiting enzyme in prostaglandin synthesis) in RAW264.7 macrophages (Paul et al 1996). Since the c-fos and COX2 promoters contain CREs, the MAPKAP-K2/K3 mediated phosphorylation of CREB may contribute to the stress and cytokine-induced transcription of these two genes The importance of the CRE in the induction of c-fos mRNA is well documented (Ginty et al 1994). The 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). The transcription factors ATF2 and Elk-1 are also phosphorylated by SAPK2 in vitro and after transfection of mammalian cells with the upstream activators of SAPK2 that do not activate SAPK1 (Raingeaud et al, 1996).
Recently, two additional SAP kinases were identified, called SAPK2b [or p38&bgr; (Jiang et al, 1996)] and SAPK3 (Mertens et al, 1996) [also called ERK6 (Lechner et al, 1996) and p38&ggr; (Li et al, 1996)]. The amino acid sequence of SAPK2b is 73% identical to SAPK2a and it is inhibited by SB 202190 at similar concentrations to SAPK2a. In contrast, the amino acid sequence of SAPK3 is only 60% identical to SAPK2a and SAPK2b and 47% identical to SAPK1. Like SAPK2, SAPK3 contains a TGY motif in the activation domain (which is TPY in SAPK1 and TEY in p42 and p44 MAP kinases) and subdomain VII is separated by six amino acids from the activation loop in subdomain VIII (as compared to eight residues in SAPK1 and >12 residues in any other MAP kinase family member). SAPK2b has 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 SAPK1 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). In vitro, SAPK2b and SAPK3 phosphorylated several proteins that are also substrates for SAPK2a. SAPK2b was reported to phosphorylate the 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). However, whether SAPK1 and/or SAPK3 are rate-limiting for ATF2 phosphorylation in vivo is unknown.
Five chromatographically distinct SAP kinase kinases (SKKs or SAPKKs) have been identified in mammalian cells (Meier et al, 1996; Cuenda et al, 1996). In vitro, SKK1 [also termed MKK4 (Dérijard et al, 1995), SEK1 (Sanchez et al, 1994) and XMEK2 (Yashar et al, 1993)] activates all four SAPKs 1, 2a and 2b (Sanchez et al, 1994; Dérijard et al, 1995; Doza et al, 1995; Jiang et al, 1996), although SAPK2b is phosphorylated less efficiently. SKK2 [also termed MKK3 (Dérijard 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 SAPK1. SKK3 was also the most efficient activator of SAPK2b in co-transfection experiments (Jiang et al, 1996). SKK4 and SKK5 activate SAPK1 but not SAPK2a (Meier et al, 1996). SKK4/SKK5 have not been purified or cloned and their amino acid sequences are thus still unknown. SKK1 and SKK2 are the only activators of SAPK2 generated when rat pheochromocytoma (PC12) cells are exposed to chemical stress, osmotic shock, ultraviolet irradiation or the protein synthesis inhibitor anisomycin (Meier et al, 1996). However, SKK3 is the dominant activator of SAPK2 and SKK4/SKK5 is/are the dominant activator(s) of SAPK1 when human epithelial (KB) cells are exposed to the same stresses as PC12 cells or stimulated with IL-1, or when human (THP1) monocytes are stimulated with LPS (Meier et al, 19

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