Activation of Protein Kinase Cascades by Osmotic Shock

Osmotic shock induces a variety of biochemical and physiological responses in vertebrate cells. By analyzing extracts obtained from rat 3Y1 fibroblastic cells exposed to hyper-osmolar media, we have found that mitogen-activated protein kinases (MAPKs) and stress-activated protein kinases (SAPKs, also known as JNKs) are both activated in response to osmotic shock. MAPKK1 (MEK1) was also activated markedly. Furthermore, Raf-1 and MEKK were activated strikingly by the osmotic shock. Activation of Raf-1 and MEKK in response to osmotic shock was detected also in PC12 cells, in which MEKK activation by the osmotic shock was much stronger than that by epidermal growth factor. Activation of SAPKs in PC12 cells by the osmotic shock was also more marked than that by epidermal growth factor. The activated MEKK phosphorylated not only MAPKKs but also XMEK2, which is distantly related to MAPKK. Recombinant wild-type XMEK2, but not kinase-negative XMEK2, was able to phosphorylate and activate recombinant SAPKα in vitro. In addition, this activity of XMEK2 was activated by the activated MEKK. These results suggest that the MAPK cascade consisting of Raf-1, MAPKK, and MAPK and the SAPK cascade consisting of MEKK, XMEK2, and SAPK are both activated in response to osmotic shock. Finally, it was found that XMEK2 is a good substrate for SAPK. Osmotic shock induces a variety of biochemical and physiological responses in vertebrate cells. By analyzing extracts obtained from rat 3Y1 fibroblastic cells exposed to hyper-osmolar media, we have found that mitogen-activated protein kinases (MAPKs) and stress-activated protein kinases (SAPKs, also known as JNKs) are both activated in response to osmotic shock. MAPKK1 (MEK1) was also activated markedly. Furthermore, Raf-1 and MEKK were activated strikingly by the osmotic shock. Activation of Raf-1 and MEKK in response to osmotic shock was detected also in PC12 cells, in which MEKK activation by the osmotic shock was much stronger than that by epidermal growth factor. Activation of SAPKs in PC12 cells by the osmotic shock was also more marked than that by epidermal growth factor. The activated MEKK phosphorylated not only MAPKKs but also XMEK2, which is distantly related to MAPKK. Recombinant wild-type XMEK2, but not kinase-negative XMEK2, was able to phosphorylate and activate recombinant SAPKα in vitro. In addition, this activity of XMEK2 was activated by the activated MEKK. These results suggest that the MAPK cascade consisting of Raf-1, MAPKK, and MAPK and the SAPK cascade consisting of MEKK, XMEK2, and SAPK are both activated in response to osmotic shock. Finally, it was found that XMEK2 is a good substrate for SAPK. INTRODUCTIONMitogen-activated protein kinase (MAPK)1( 1The abbreviation used are: MAPKmitogen-activated protein kinaseMAPKKMAPK kinaseMAPKK-KMAPKK kinaseSAPKstress-activated protein kinaseJNKjun N-terminal kinaseEGFepidermal growth factorMBPmyelin basic proteinMAP2microtubule-associated protein 2PAGEpolyacrylamide gel electrophoresis.) and its direct activator, MAPK kinase (MAPKK), are activated in a large number of signal transduction pathways in vertebrate cells (for review, see Refs. 1Cobb M.H. Boulton T.G. Robbins D.J. Cell Regul. 1991; 2: 965-978Crossref PubMed Scopus (426) Google Scholar, 2Sturgill T.W. Wu J. Biochim. Biophys. Acta. 1991; 1092: 350-357Crossref PubMed Scopus (328) Google Scholar, 3Pelech S.L. Sanghera J.S. Trends Biochem. Sci. 1992; 17: 233-238Abstract Full Text PDF PubMed Scopus (339) Google Scholar, 4Leevers S.J. Marshall C.J. Trends Cell Biol. 1992; 2: 283-286Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 5Nishida E. Gotoh Y. Trends Biochem. Sci. 1993; 18: 128-131Abstract Full Text PDF PubMed Scopus (958) Google Scholar, 6Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar). MAPKK and MAPK are thought to form a linear pathway called the MAPKK/MAPK cascade, which has been shown to play a pivotal role in diverse biological processes including fibroblastic cell proliferation and transformation (7Pages G. Lenormand P. L'Allemain G. Chabard J.-C. Meloche S. Pouyssegur J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8319-8323Crossref PubMed Scopus (923) Google Scholar, 8Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar, 9Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1254) Google Scholar), PC12 cell differentiation (8Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar), Xenopus oocyte maturation (10Kosako H. Gotoh Y. Nishida E. EMBO J. 1994; 13: 2131-2138Crossref PubMed Scopus (189) Google Scholar), and metaphase II arrest of unfertilized eggs (11Haccard O. Sarcevic B. Lewellyn A. Hartley R. Roy L. Izumi T. Erikson E. Maller J.L. Science. 1993; 267: 1262-1265Crossref Scopus (236) Google Scholar, 12Kosako H. Gotoh Y. Nishida E. J. Biol. Chem. 1994; 269: 28354-28358Abstract Full Text PDF PubMed Google Scholar). Several serine/threonine kinases such as Raf-1 (13Dent P. Haster W. Haystead T.A.J. Vincent L.A. Roberts T.M. Sturgill T.W. Science. 1992; 257: 1404-1407Crossref PubMed Scopus (496) Google Scholar, 14Kyriakis J.M. App H. Zhang Z.-F. Banerjee P. Brautigan D.L. Rapp U. Avruch J. Nature. 1992; 358: 417-421Crossref PubMed Scopus (966) Google Scholar, 15Howe L.R. Leevers S.J. Gomez N. Nakielny S. Cohen P. Marshall C.J. Cell. 1992; 71: 335-342Abstract Full Text PDF PubMed Scopus (627) Google Scholar), Mos (16Nebreda A.R. Hunt T. EMBO J. 1993; 12: 1979-1986Crossref PubMed Scopus (251) Google Scholar, 17Posada J. Yew N. Ahn N.G. Vande Woude G.F. Cooper J.A. Mol. Cell. Biol. 1993; 13: 2546-2553Crossref PubMed Scopus (335) Google Scholar), and MEKK (18Lange-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) have been identified as a direct activator for MAPKK. Raf-1 is a proto-oncogene product and is one of targets of p21ras (for review, see Ref. 19Avruch J. Zhang X-f. Kyriakis J.M. Trends Biochem. Sci. 1994; 19: 279-283Abstract Full Text PDF PubMed Scopus (540) Google Scholar and 20Daum G. Eisenmann-Tappe I. Fries H-W. Troppmair J. Rapp U.R. Trends Biochem. Sci. 1994; 19: 474-480Abstract Full Text PDF PubMed Scopus (483) Google Scholar). Mos is also a proto-oncogene product and is shown to function during oocyte maturation (21Sagata N. Oskersson M. Copeland T. Brumbaugh J. Vande Woude G.F. Nature. 1988; 335: 519-526Crossref PubMed Scopus (462) Google Scholar). MEKK is found to be activated by epidermal growth factor (EGF) and nerve growth factor through a p21ras -dependent pathway in PC12 cells (22Lange-Carter C.A. Johnson G.L. Science. 1994; 265: 1458-1461Crossref PubMed Scopus (294) Google Scholar), but the activation in this system is not so strong, suggesting that MEKK may be activated also in other signaling pathways.In yeast a number of MAPKK/MAPK cascades have been found and each kinase cascade is functioning in its specific cellular pathway (5Nishida E. Gotoh Y. Trends Biochem. Sci. 1993; 18: 128-131Abstract Full Text PDF PubMed Scopus (958) Google Scholar, 23Errede B. Levin D.E. Curr. Opin. Cell Biol. 1993; 5: 254-260Crossref PubMed Scopus (186) Google Scholar, 24Ammerer G. Curr. Opin. Cell Biol. 1994; 4: 90-95Google Scholar). In contrast, in vertebrate cells the same MAPKK and the same MAPK function in diverse biological processes as other of the MAPK in vertebrate stress-activated protein kinases (SAPKs, also known as JNKs) J.M. Avruch J. J. Biol. Chem. 265: Full Text PDF PubMed Google Scholar, J.M. Brautigan Avruch J. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar, M. A. T. A. M. 1993; PubMed Scopus Google Scholar, B. M. Wu T. B. T. M. R.J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, J.M. Banerjee P. E. T. Avruch J. Nature. 1994; PubMed Scopus Google Scholar, A. A. T. B. M.H. R.J. M. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar) and J. L. R.J. Science. 1994; 265: PubMed Scopus Google Scholar, J. Cohen P. S. M. A. Hunt T. A.R. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, L. E. S. J. J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, S. J.L. Nature. 1994; PubMed Scopus Google Scholar). These kinases are distantly related to and may have see of of the MAPK of that activation of kinases and of signaling pathways are a to we that to activation of both and of the MAPK and found that osmotic shock induces marked activation of both and SAPKs in fibroblastic cells. both Raf-1 and MEKK have been found to be activated also by osmotic shock. In addition, we have found that XMEK2 K. B. Mol. Cell. Biol. 1993; 13: PubMed Scopus Google Scholar), which is distantly related to MAPKK, function as a SAPK kinase and be activated by both and of was from used was M. Gotoh Y. H. S. Nishida E. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). MAPKK we H. Gotoh Y. S. M. Nishida E. EMBO J. 1992; PubMed Scopus Google Scholar) was shown to MAPKK1 Y. and E. in and used for of rat were to the Y. Nishida E. T. M. M. H. J. Biochem. PubMed Scopus Google Scholar) was by the gel and in a the gel was for of a consisting of and basic protein protein was used as a substrate in These substrate were to the gel to of was from was from E. M. Y. H. T. M. S. H. T. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and was as a protein and by a In recombinant XMEK2 were of Recombinant XMEK2 and XMEK2 K. B. Mol. Cell. Biol. 1993; 13: PubMed Scopus Google Scholar) was by and of the and to protein and protein The were by (for by (for protein by The were a consisting of and and was both of recombinant XMEK2 in of the The kinase-negative XMEK2 was obtained by a in the as K. B. Mol. Cell. Biol. 1993; 13: PubMed Scopus Google Scholar), and and protein were as The SAPKα J.M. Banerjee P. E. T. Avruch J. Nature. 1994; PubMed Scopus Google Scholar) was by and the of the The protein was from by a and of Cell 3Y1 cells were in PC12 cells were in and of rat 3Y1 cells were exposed to of of rat PC12 cells were exposed to of the cells were in a consisting of and and for The cellular was to kinase kinase and of the was to of of cellular and for of protein was to the and for a The were the for the to of protein was in of a consisting of and to as kinase of Xenopus recombinant MAPK Y. K. S. E. T. H. K. I. H. Nishida E. EMBO J. 1991; PubMed Scopus Google Scholar, S. H. K. K. H. T. Gotoh Y. Nishida E. EMBO J. 1992; PubMed Scopus Google Scholar), and for of of and the was for a The was by of was by and by for MAPKK activity the to of protein was in of the kinase of Xenopus recombinant MAPKK H. Nishida E. Gotoh Y. EMBO J. 1993; 12: PubMed Scopus Google Scholar), of recombinant kinase-negative MAPK see Ref. and The was for The was by of kinase-negative MAPK was by and by for XMEK2 the MEKK was in of the kinase of recombinant kinase-negative XMEK2 and The was by of XMEK2 was by and for XMEK2 the MEKK was in of the kinase of recombinant wild-type XMEK2 and of recombinant and for The was to kinase to activity of recombinant of in the activity of XMEK2 to activate of recombinant SAPKα was of recombinant XMEK2 in a of of kinase of and The was for the The was by of was by and by for SAPKα of kinase-negative XMEK2 wild-type XMEK2 was of recombinant SAPKα in a of of the kinase The was for The was by of for MAPK of recombinant MAPK was of recombinant MAPKK of recombinant XMEK2 in a of of kinase The was for of and were to the and for a The was by of was by and by that activation of both and of the MAPK in rat 3Y1 fibroblastic cells by kinase substrate kinase as the are and to activation of both of MAPK and MAPK and have been to rat PC12 cells and 3Y1 cells Y. Nishida E. T. M. M. H. J. Biochem. PubMed Scopus Google Scholar, E. Gotoh Y. 1992; PubMed Scopus Google Scholar). 3Y1 cells were exposed to such as growth and osmotic and extracts from cells were by kinase protein Y. and E. hyper-osmolar were found to activation of both and SAPKs is a good substrate for Y. Nishida E. T. M. M. H. J. Biochem. PubMed Scopus Google Scholar, E. Gotoh Y. 1992; PubMed Scopus Google Scholar, Y. Nishida E. S. N. H. K. T. K. H. Nature. 1991; PubMed Scopus Google Scholar, M. Nishida E. H. J. Biochem. PubMed Scopus Google Scholar, E. A. T. R.J. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar), but not for SAPKs J.M. Avruch J. J. Biol. Chem. 265: Full Text PDF PubMed Google Scholar, B. M. Wu T. B. T. M. R.J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar), and is a good substrate for SAPKs J.M. Avruch J. J. Biol. Chem. 265: Full Text PDF PubMed Google Scholar, B. M. Wu T. B. T. M. R.J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar), but not for as a good substrate for both SAPKs and J.M. Avruch J. J. Biol. Chem. 265: Full Text PDF PubMed Google Scholar, Y. Nishida E. T. M. M. H. J. Biochem. PubMed Scopus Google Scholar, E. Gotoh Y. 1992; PubMed Scopus Google Scholar, Y. Nishida E. S. N. H. K. T. K. H. Nature. 1991; PubMed Scopus Google Scholar, M. Nishida E. H. J. Biochem. PubMed Scopus Google Scholar, E. A. T. R.J. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar), but the used activation of SAPKs was more than that of in and are for both and SAPKs J.M. Avruch J. J. Biol. Chem. 265: Full Text PDF PubMed Google Scholar, Y. Nishida E. T. M. M. H. J. Biochem. PubMed Scopus Google Scholar, E. Gotoh Y. 1992; PubMed Scopus Google Scholar, Y. Nishida E. S. N. H. K. T. K. H. Nature. 1991; PubMed Scopus Google Scholar, M. Nishida E. H. J. Biochem. PubMed Scopus Google Scholar, E. A. T. R.J. J. Biol. Chem. 1991; Full Text PDF PubMed Google and were activated in a 3Y1 cells were exposed to hyper-osmolar The activation was detected of of the cells to and the the activation as to the was in the not the activation of and were activated by the osmotic the of activation was to that of MAPK activation and of as SAPK was shown by substrate in kinase to phosphorylate and and to phosphorylate and not and to that been by recombinant rat SAPKα not Activation of of the was the as by the in not The activation was the of the activation was The activation was the cells were exposed to for and to of SAPKs suggesting that SAPKs may be Activation of and SAPKs in 3Y1 cells be by hyper-osmolar as as not of MAPKK activity also cells were exposed to hyper-osmolar the of the cells to MAPKK1 was from the cell extracts and for MAPKK activity MAPKK1 was activated in a was to that of MAPK activation of MAPKK1 in response to osmotic shock. 3Y1 cells were exposed to and cell were the of cell was to and the was for MAPKK1 activity as and have shown that Raf-1 and MEKK as a MAPKK kinase that and MAPKK, we activity in rat 3Y1 cells osmotic shock. were obtained from the cells that been exposed to for and and were to and each was for the MAPKK Raf-1 and MEKK were found to be activated by the of the cells to hyper-osmolar These were and of MEKK and Raf-1 in response to osmotic shock. 3Y1 cells were exposed to and cell were and the of cell was to protein and for MAPKK activity as and activation of both Raf-1 and MEKK in response to was also in rat PC12 cells was that MEKK activation by osmotic shock was much stronger than that by Raf-1 activation by both was and were activated by the osmotic shock in PC12 cells were activated by SAPK activation MEKK activation and MEKK function of SAPK. it is to that MEKK may phosphorylate and activate a kinase which be distantly related to MAPKK, MAPKK is shown to be of SAPK in not we that XMEK2 K. B. Mol. Cell. Biol. 1993; 13: PubMed Scopus Google Scholar), which a of the MAPKK and is distantly related to MAPKK in be a SAPK kinase and as a substrate for MEKK. this XMEK2 was phosphorylated by the obtained from rat 3Y1 cells In addition, the of MEKK to phosphorylate XMEK2 was activated by the osmotic shock and the MAPKK activity not that MEKK of MEKK, Raf-1, and SAPK in PC12 cells. cell were from PC12 cells for for of cell was to protein and the was for the activity as in PC12 cells were exposed to for for was to kinase The of and are and activation of XMEK2 by MEKK. 3Y1 cells were exposed to Cell were and of cell was to protein The was for XMEK2 activity as and The of phosphorylated XMEK2 is shown The same was for XMEK2 activity XMEK2 activity was by the SAPKα and the SAPKα activity was by that recombinant wild-type XMEK2, but not kinase-negative XMEK2, activate SAPKα in and that XMEK2 phosphorylated SAPKα in XMEK2 not activate recombinant MAPK These results suggest that XMEK2 as a SAPK and of SAPKα by of recombinant SAPKα recombinant wild-type XMEK2 kinase-negative XMEK2 for the the SAPKα was for the activity to phosphorylate as and The activity of SAPKα was in each was recombinant SAPKα in the of for The phosphorylated were by and The of recombinant XMEK2 and recombinant SAPKα are as and recombinant MAPK was recombinant wild-type MAPKK recombinant wild-type XMEK2 for the MAPK activity was as and the activity of XMEK2, the SAPK was activated by the XMEK2 activity of MEKK was activated by the of the cells to hyper-osmolar The activation of XMEK2 of XMEK2 and results are the that XMEK2 is a SAPK kinase which is activated by this we that XMEK2 be phosphorylated by as shown in in which of kinase-negative XMEK2 was in the of SAPK. we the kinase from 3Y1 cells exposed to of were by this kinases of and were found to be activated by hyper-osmolar and the of activation are to of and kinases may be and XMEK2, activator for is a in for of XMEK2 by SAPK. 3Y1 cells were exposed to and for Cell were and to kinase recombinant wild-type The is are the The to and are by and the shown in were by for and kinases as activity to the activity The same cell were to kinase and the of and were as that hyper-osmolar a marked in activity in cells B. Wu R.J. Science. 1994; 265: PubMed Scopus Google Scholar) and a in MAPK activity in cells Y. K. H. T. T. Y. S. J. Biol. Chem. 1994; Full Text PDF Google Scholar), The results in this have shown that osmotic shock hyper-osmolar induces activation of both MAPK and and SAPK and in fibroblastic 3Y1 cells. The are and for more than and are that both MAPK and SAPK function in the osmotic signal transduction the to pathways in the activation of and SAPKs in this we have found that osmotic shock induces marked activation of MEKK as as and Johnson (22Lange-Carter C.A. Johnson G.L. Science. 1994; 265: 1458-1461Crossref PubMed Scopus (294) Google Scholar) nerve growth p21ras -dependent activation of MEKK in PC12 cells. was the that identified physiological activation of MEKK. results that MEKK is activated more by osmotic shock than by in PC12 cells. Furthermore, in PC12 cells SAPKs were found to be activated more by osmotic shock than by activation of MEKK activation of SAPKs than of A. A. M. B. R.J. Johnson G.L. M. Science. 1994; PubMed Scopus Google Scholar) has shown that of MEKK MEKK activation of than in cells. In have identified one of the that activation of MEKK in cells. osmotic pathway be for of activation and function of MEKK in that XMEK2 K. B. Mol. Cell. Biol. 1993; 13: PubMed Scopus Google Scholar), which has been as a of the MAPKK but is from MAPKK, be a SAPK this recombinant wild-type XMEK2, but not kinase-negative XMEK2, activate recombinant SAPKα in vitro. Furthermore, the SAPK activity of XMEK2 was activated by MEKK. In addition, the XMEK2 activity of MEKK was found to be activated by osmotic shock in 3Y1 cells. of that a of XMEK2 as a SAPK kinase I. K. Avruch J. Kyriakis J.M. Nature. 1994; PubMed Scopus Google Scholar), and that MEKK activate Y. T. Kyriakis J.M. D.J. Nature. 1994; PubMed Scopus Google Scholar). results are and suggest that MEKK as activator for the cascade in cells in signal transduction In XMEK2 protein in 3Y1 cells was activated in response to hyper-osmolar H. S. Y. and E. it is to not that osmotic shock a kinase cascade consisting of MEKK, XMEK2 and SAPK. as it is that Raf-1 as a MAPKK kinase (13Dent P. Haster W. Haystead T.A.J. Vincent L.A. Roberts T.M. Sturgill T.W. Science. 1992; 257: 1404-1407Crossref PubMed Scopus (496) Google Scholar, 14Kyriakis J.M. App H. Zhang Z.-F. Banerjee P. Brautigan D.L. Rapp U. Avruch J. Nature. 1992; 358: 417-421Crossref PubMed Scopus (966) Google Scholar, 15Howe L.R. Leevers S.J. Gomez N. Nakielny S. Cohen P. Marshall C.J. Cell. 1992; 71: 335-342Abstract Full Text PDF PubMed Scopus (627) Google Scholar) and activation of Raf-1 and MAPKK in to activation of MAPK was to in cells exposed to it is that osmotic shock induces activation of the kinase cascade consisting of Raf-1, MAPKK, and are to the Raf-1 a MAPKK kinase in this MEKK also as a MAPKK kinase in to a are Raf-1 and MEKK a SAPK kinase in this biochemical this osmotic pathway and other signaling pathways are in this has shown that XMEK2 is one of the in for SAPK. In this it be that MAPKK is phosphorylated by MAPK S. Gotoh Y. Nishida E. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). of INTRODUCTIONMitogen-activated protein kinase (MAPK)1( 1The abbreviation used are: MAPKmitogen-activated protein kinaseMAPKKMAPK kinaseMAPKK-KMAPKK kinaseSAPKstress-activated protein kinaseJNKjun N-terminal kinaseEGFepidermal growth factorMBPmyelin basic proteinMAP2microtubule-associated protein 2PAGEpolyacrylamide gel electrophoresis.) and its direct activator, MAPK kinase (MAPKK), are activated in a large number of signal transduction pathways in vertebrate cells (for review, see Refs. 1Cobb M.H. Boulton T.G. Robbins D.J. Cell Regul. 1991; 2: 965-978Crossref PubMed Scopus (426) Google Scholar, 2Sturgill T.W. Wu J. Biochim. Biophys. Acta. 1991; 1092: 350-357Crossref PubMed Scopus (328) Google Scholar, 3Pelech S.L. Sanghera J.S. Trends Biochem. Sci. 1992; 17: 233-238Abstract Full Text PDF PubMed Scopus (339) Google Scholar, 4Leevers S.J. Marshall C.J. Trends Cell Biol. 1992; 2: 283-286Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 5Nishida E. Gotoh Y. Trends Biochem. Sci. 1993; 18: 128-131Abstract Full Text PDF PubMed Scopus (958) Google Scholar, 6Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar). MAPKK and MAPK are thought to form a linear pathway called the MAPKK/MAPK cascade, which has been shown to play a pivotal role in diverse biological processes including fibroblastic cell proliferation and transformation (7Pages G. Lenormand P. L'Allemain G. Chabard J.-C. Meloche S. Pouyssegur J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8319-8323Crossref PubMed Scopus (923) Google Scholar, 8Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar, 9Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1254) Google Scholar), PC12 cell differentiation (8Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar), Xenopus oocyte maturation (10Kosako H. Gotoh Y. Nishida E. EMBO J. 1994; 13: 2131-2138Crossref PubMed Scopus (189) Google Scholar), and metaphase II arrest of unfertilized eggs (11Haccard O. Sarcevic B. Lewellyn A. Hartley R. Roy L. Izumi T. Erikson E. Maller J.L. Science. 1993; 267: 1262-1265Crossref Scopus (236) Google Scholar, 12Kosako H. Gotoh Y. Nishida E. J. Biol. Chem. 1994; 269: 28354-28358Abstract Full Text PDF PubMed Google Scholar). Several serine/threonine kinases such as Raf-1 (13Dent P. Haster W. Haystead T.A.J. Vincent L.A. Roberts T.M. Sturgill T.W. Science. 1992; 257: 1404-1407Crossref PubMed Scopus (496) Google Scholar, 14Kyriakis J.M. App H. Zhang Z.-F. Banerjee P. Brautigan D.L. Rapp U. Avruch J. Nature. 1992; 358: 417-421Crossref PubMed Scopus (966) Google Scholar, 15Howe L.R. Leevers S.J. Gomez N. Nakielny S. Cohen P. Marshall C.J. Cell. 1992; 71: 335-342Abstract Full Text PDF PubMed Scopus (627) Google Scholar), Mos (16Nebreda A.R. Hunt T. EMBO J. 1993; 12: 1979-1986Crossref PubMed Scopus (251) Google Scholar, 17Posada J. Yew N. Ahn N.G. Vande Woude G.F. Cooper J.A. Mol. Cell. Biol. 1993; 13: 2546-2553Crossref PubMed Scopus (335) Google Scholar), and MEKK (18Lange-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) have been identified as a direct activator for MAPKK. Raf-1 is a proto-oncogene product and is one of targets of p21ras (for review, see Ref. 19Avruch J. Zhang X-f. Kyriakis J.M. Trends Biochem. Sci. 1994; 19: 279-283Abstract Full Text PDF PubMed Scopus (540) Google Scholar and 20Daum G. Eisenmann-Tappe I. Fries H-W. Troppmair J. Rapp U.R. Trends Biochem. Sci. 1994; 19: 474-480Abstract Full Text PDF PubMed Scopus (483) Google Scholar). Mos is also a proto-oncogene product and is shown to function during oocyte maturation (21Sagata N. Oskersson M. Copeland T. Brumbaugh J. Vande Woude G.F. Nature. 1988; 335: 519-526Crossref PubMed Scopus (462) Google Scholar). MEKK is found to be activated by epidermal growth factor (EGF) and nerve growth factor through a p21ras -dependent pathway in PC12 cells (22Lange-Carter C.A. Johnson G.L. Science. 1994; 265: 1458-1461Crossref PubMed Scopus (294) Google Scholar), but the activation in this system is not so strong, suggesting that MEKK may be activated also in other signaling pathways.In yeast a number of MAPKK/MAPK cascades have been found and each kinase cascade is functioning in its specific cellular pathway (5Nishida E. Gotoh Y. Trends Biochem. Sci. 1993; 18: 128-131Abstract Full Text PDF PubMed Scopus (958) Google Scholar, 23Errede B. Levin D.E. Curr. Opin. Cell Biol. 1993; 5: 254-260Crossref PubMed Scopus (186) Google Scholar, 24Ammerer G. Curr. Opin. Cell Biol. 1994; 4: 90-95Google Scholar). In contrast, in vertebrate cells the same MAPKK and the same MAPK function in diverse biological processes as other of the MAPK in vertebrate stress-activated protein kinases (SAPKs, also known as JNKs) J.M. Avruch J. J. Biol. Chem. 265: Full Text PDF PubMed Google Scholar, J.M. Brautigan Avruch J. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar, M. A. T. A. M. 1993; PubMed Scopus Google Scholar, B. M. Wu T. B. T. M. R.J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, J.M. Banerjee P. E. T. Avruch J. Nature. 1994; PubMed Scopus Google Scholar, A. A. T. B. M.H. R.J. M. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar) and J. L. R.J. Science. 1994; 265: PubMed Scopus Google Scholar, J. Cohen P. S. M. A. Hunt T. A.R. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, L. E. S. J. J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, S. J.L. Nature. 1994; PubMed Scopus Google Scholar). These kinases are distantly related to and may have see of of the MAPK of that activation of kinases and of signaling pathways are a to we that to activation of both and of the MAPK and found that osmotic shock induces marked activation of both and SAPKs in fibroblastic cells. both Raf-1 and MEKK have been found to be activated also by osmotic shock. In addition, we have found that XMEK2 K. B. Mol. Cell. Biol. 1993; 13: PubMed Scopus Google Scholar), which is distantly related to MAPKK, function as a SAPK kinase and be activated by MEKK.