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Characterization of the Mitogen-activated Protein Kinase Kinase 4 (MKK4)/c-Jun NH2-terminal kinase 1 and MKK3/p38 Pathways Regulated by MEK Kinases 2 and 3

1997; Elsevier BV; Volume: 272; Issue: 22 Linguagem: Inglês

10.1074/jbc.272.22.14489

ISSN

1083-351X

Autores

Karl Deacon, Jonathan L. Blank,

Tópico(s)

Protein Kinase Regulation and GTPase Signaling

Resumo

We previously reported the isolation of cDNAs encoding two mammalian mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK)kinase kinases, designated MEKK2 and MEKK3 (Blank, J.L., Gerwins, P., Elliott, E.M., Sather, S. and Johnson, G.L. (1996) J. Biol. Chem. 271, 5361–5368). In the present study, cotransfection experiments were used to examine the regulation by MEKK2 and MEKK3 of the dual specificityMAP kinase kinases, MKK3 and MKK4. MKK3 specifically phosphorylates and activates p38, whereas MKK4 phosphorylates and activates both p38 and JNK. Coexpression of MEKK2 or MEKK3 with MKK4 in COS-7 cells resulted in activation of MKK4, as assessed by enhanced autophosphorylation and by its ability to phosphorylate and activate recombinant JNK1 or p38 in vitro. MKK3 autophosphorylation and activation of p38 was also observed following coexpression of MKK3 with MEKK3, but not with MEKK2. Consistent with these observations, immunoprecipitated MEKK2 directly activated recombinant MKK4 in vitro but failed to activate MKK3. The sites of activating phosphorylation in MKK3 and MKK4 were identified within kinase subdomains VII and VIII. Replacement of Ser189 or Thr193 in MKK3 with Ala abolished autophosphorylation and activation of MKK3 by MEKK3. Analogous mutations in MKK4 indicated that Ser221 and, to a lesser extent, Thr225 were necessary for MKK4 activation by MEKK2 and MEKK3.These data indicate that MKK3 is preferentially activated by MEKK3, whereas MKK4 is activated both by MEKK2 and MEKK3. Consistent with these observations, MEKK2 and MEKK3 also activated JNK1 in vivo. However, MEKK3 failed to activate p38 when coexpressed in either the absence or presence of MKK3, indicating that MEKK3 is not coupled to p38 activation in vivo. These observations suggest that regulation of p38 and JNK1 pathways by MEKK3 may involve distinct mechanisms to prevent p38 activation but to allow JNK1 activation. We previously reported the isolation of cDNAs encoding two mammalian mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK)kinase kinases, designated MEKK2 and MEKK3 (Blank, J.L., Gerwins, P., Elliott, E.M., Sather, S. and Johnson, G.L. (1996) J. Biol. Chem. 271, 5361–5368). In the present study, cotransfection experiments were used to examine the regulation by MEKK2 and MEKK3 of the dual specificityMAP kinase kinases, MKK3 and MKK4. MKK3 specifically phosphorylates and activates p38, whereas MKK4 phosphorylates and activates both p38 and JNK. Coexpression of MEKK2 or MEKK3 with MKK4 in COS-7 cells resulted in activation of MKK4, as assessed by enhanced autophosphorylation and by its ability to phosphorylate and activate recombinant JNK1 or p38 in vitro. MKK3 autophosphorylation and activation of p38 was also observed following coexpression of MKK3 with MEKK3, but not with MEKK2. Consistent with these observations, immunoprecipitated MEKK2 directly activated recombinant MKK4 in vitro but failed to activate MKK3. The sites of activating phosphorylation in MKK3 and MKK4 were identified within kinase subdomains VII and VIII. Replacement of Ser189 or Thr193 in MKK3 with Ala abolished autophosphorylation and activation of MKK3 by MEKK3. Analogous mutations in MKK4 indicated that Ser221 and, to a lesser extent, Thr225 were necessary for MKK4 activation by MEKK2 and MEKK3. These data indicate that MKK3 is preferentially activated by MEKK3, whereas MKK4 is activated both by MEKK2 and MEKK3. Consistent with these observations, MEKK2 and MEKK3 also activated JNK1 in vivo. However, MEKK3 failed to activate p38 when coexpressed in either the absence or presence of MKK3, indicating that MEKK3 is not coupled to p38 activation in vivo. These observations suggest that regulation of p38 and JNK1 pathways by MEKK3 may involve distinct mechanisms to prevent p38 activation but to allow JNK1 activation. Mammalian cells contain multiple mitogen-activated protein kinase (MAPK) 1The abbreviations used areMAPKmitogen-activated protein kinase;ERKextracellular regulated protein kinase;JNKc-Jun NH2-terminal kinase;MEKMAPK/ERK kinase;MKKMAPK kinase;SEK1stress-activated protein kinase/ERK kinase;JNKK JNKkinase;MEKK MAPK/ERKkinase kinase;TAKtransforming growth factor-β-activated kinase;p90rskp90 ribosomal protein S6 kinase;NF-κBnuclear factor-κB;ATF2activating transcription factor 2;GSTglutathioneS-transferase;HAhemagglutinin. signaling pathways that are activated by a diverse array of extracellular stimuli and regulate a variety of cellular processes (for reviews, see Refs. 1Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 2Blenis J. Proc. Natl. Acad. Sci. U. S. 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Chem. 1996; 271: 17920-17926Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar). ERKs are activated by agonists for tyrosine kinase-encoded receptors (e.g. epidermal growth factor and platelet-derived growth factor) and G protein-coupled receptors that induce mitogenesis or cellular differentiation (2Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Crossref PubMed Scopus (1151) Google Scholar, 3Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4213) Google Scholar, 4Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1653) Google Scholar). ERKs mediate the effects of these agonists by phosphorylating and regulating the activity of a number of proteins, including cytoplasmic enzymes (e.g. phospholipase A2 and p90rsk) and nuclear transcription factors (e.g. Elk-1) (1Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 2Blenis J. Proc. 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MAPKs are activated by dual phosphorylation on Thr and Tyr within the motif Thr-Xaa-Tyr, where Xaa corresponds to Glu in ERK, Pro in JNK, and Gly in p38 (4Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1653) Google Scholar, 5Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar). The dual specificity kinases involved are mostly specific for each MAPK subgroup, allowing for their independent regulation (4Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1653) Google Scholar, 5Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar). Thus, MEK1 and MEK2 selectively phosphorylate and activate the ERK subgroup (4Cobb M.H. Goldsmith E.J. J. Biol. 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Raf-1 directly phosphorylates these sites and is probably the primary MEK activator in vivo. In most cases, Raf-1 activation by receptor tyrosine kinases and G protein-coupled receptors involves Ras, which interacts directly with Raf-1, causing translocation and activation of Raf-1 at the plasma membrane (2Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Crossref PubMed Scopus (1151) Google Scholar, 3Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4213) Google Scholar, 4Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1653) Google Scholar,38Duam G. Eisenmann-Tappe I. Fries H.-W. Troppmair J. Rapp U.R. Trends Biochem. Sci. 1994; 19: 474-479Abstract Full Text PDF PubMed Scopus (483) Google Scholar). A-Raf and B-Raf have also been shown to activate the ERK pathway (42Troppmair J. Bruder J.T. Munoz H. Lloyd P.A. Kyriakis J. Banerjee P. Avruch J. Rapp U.R. J. Biol. 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Chem. 1995; 270: 26303-26310Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Whereas Raf-1 shows no MKK4-stimulating activity (37Lin A. Minden A. Marinetto H. Claret F.-X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (706) Google Scholar), members of the mammalian MEKK family of serine/threonine kinases have been demonstrated to activate MKK4 (37Lin A. Minden A. Marinetto H. Claret F.-X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (706) Google Scholar, 48Yan M. Dai T. Deak J.C. Kyriakis J.M. Zon L.I. Woodgett J.R. Templeton D.J. Nature. 1994; 372: 798-800Crossref PubMed Scopus (658) Google Scholar, 49Blank J.L. Gerwins P. Elliott E.M. Sather S. Johnson G.L. J. Biol. Chem. 1996; 271: 5361-5368Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). The cDNAs corresponding to three MEKK isoforms were isolated by virtue of their sequence homology with Ste11 and Byr2 (49Blank J.L. Gerwins P. Elliott E.M. Sather S. Johnson G.L. J. Biol. Chem. 1996; 271: 5361-5368Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 50Lange-Carter C.A. Pleiman C.M. Gardner A.M. Blumer K.J. Johnson G.L. Science. 1993; 260: 315-319Crossref PubMed Scopus (869) Google Scholar), protein kinases involved in the pheromone mating response pathway in Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The cDNA corresponding to MEKK1 encodes a protein of 161 kDa (51Xu S. Robbins D. Frost J. Dang A. Lange-Carter C. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6808-6812Crossref PubMed Scopus (148) Google Scholar), whereas those for MEKK2 and MEKK3 encode proteins of 70 and 71 kDa, respectively (49Blank J.L. Gerwins P. Elliott E.M. Sather S. Johnson G.L. J. Biol. Chem. 1996; 271: 5361-5368Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). MEKK2 and MEKK3 are also more closely related in amino acid sequence, being 94% identical to each other and ∼50% identical to MEKK1 through their respective catalytic domains (49Blank J.L. Gerwins P. Elliott E.M. Sather S. Johnson G.L. J. Biol. Chem. 1996; 271: 5361-5368Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). When transiently overexpressed, MEKKs induce constitutive activation of JNK and ERK pathways (37Lin A. Minden A. Marinetto H. Claret F.-X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (706) Google Scholar, 41Yan M. Templeton D.J. J. Biol. Chem. 1994; 269: 19067-19073Abstract Full Text PDF PubMed Google Scholar, 49Blank J.L. Gerwins P. Elliott E.M. Sather S. Johnson G.L. J. Biol. Chem. 1996; 271: 5361-5368Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 50Lange-Carter C.A. Pleiman C.M. Gardner A.M. Blumer K.J. Johnson G.L. Science. 1993; 260: 315-319Crossref PubMed Scopus (869) Google Scholar, 51Xu S. Robbins D. Frost J. Dang A. Lange-Carter C. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6808-6812Crossref PubMed Scopus (148) Google Scholar, 52Minden A. Lin A. McMahon M. Lange-Carter C. Dérijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1009) Google Scholar, 53Xu S. Robbins D.J. Christerson L.B. English J.M. Vanderbilt C.A. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5291-5295Crossref PubMed Scopus (122) Google Scholar), but not p38 (37Lin A. Minden A. Marinetto H. Claret F.-X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (706) Google Scholar, 49Blank J.L. Gerwins P. Elliott E.M. Sather S. Johnson G.L. J. Biol. 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Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). A fifth MEKK homologue, designated Tpl-2, has also recently been shown to activate the ERK and JNK pathways in vivo and to directly phosphorylate MEK1 and MKK4 in vitro (59Salmerón A. Ahmad T.B. Carlile G.W. Pappin D. Narsimhan R.P. Ley S.C. EMBO J. 1996; 15: 817-826Crossref PubMed Scopus (268) Google Scholar). The JNK activator MKK4 can also activate p38 in vivo and directly phosphorylate and activate p38 in vitro (32Dérijard B. Raingeaud J. Barrett T. Wu I.-H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1400) Google Scholar, 34Han J. Lee J.-D. Jiang Y. Li Z. Feng L. Ulevitch R.J. J. Biol. Chem. 1996; 271: 2886-2891Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar,37Lin A. Minden A. Marinetto H. Claret F.-X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (706) Google Scholar). 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