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• W2022202654 abstract "Mitogen-activated protein (MAP) kinase pathways are three-kinase modules that mediate diverse cellular processes and have been highly conserved among eukaryotes. By using a functional complementation screen in yeast, we have identified a human MAP kinase kinase kinase (MAPKKK) that shares homology with members of the mixed lineage kinase (MLK) family and therefore was called MRK (MLK-related kinase). We report the structure of the MRK gene, from which are generated two splice forms of MRK, MRK-α and MRK-β, encoding for proteins of 800 and 456 amino acids, respectively. By using a combination of solid phase protein kinase assays, transient transfections in cells, and analysis of endogenous proteins in stably transfected Madin-Darby canine kidney cells, we found that MRK-β preferentially activates ERK6/p38γ via MKK3/MKK6 and JNK through MKK4/MKK7. We also show that expression of wild type MRK increases the cell population in the G2/M phase of the cell cycle, whereas dominant negative MRK attenuates the G2 arrest caused by γ-radiation. In addition, exposure of cells to γ-radiation induces MRK activity. These data suggest that MRK may mediate γ-radiation signaling leading to cell cycle arrest and that MRK activity is necessary for the cell cycle checkpoint regulation in cells. Mitogen-activated protein (MAP) kinase pathways are three-kinase modules that mediate diverse cellular processes and have been highly conserved among eukaryotes. By using a functional complementation screen in yeast, we have identified a human MAP kinase kinase kinase (MAPKKK) that shares homology with members of the mixed lineage kinase (MLK) family and therefore was called MRK (MLK-related kinase). We report the structure of the MRK gene, from which are generated two splice forms of MRK, MRK-α and MRK-β, encoding for proteins of 800 and 456 amino acids, respectively. By using a combination of solid phase protein kinase assays, transient transfections in cells, and analysis of endogenous proteins in stably transfected Madin-Darby canine kidney cells, we found that MRK-β preferentially activates ERK6/p38γ via MKK3/MKK6 and JNK through MKK4/MKK7. We also show that expression of wild type MRK increases the cell population in the G2/M phase of the cell cycle, whereas dominant negative MRK attenuates the G2 arrest caused by γ-radiation. In addition, exposure of cells to γ-radiation induces MRK activity. These data suggest that MRK may mediate γ-radiation signaling leading to cell cycle arrest and that MRK activity is necessary for the cell cycle checkpoint regulation in cells. In a wide range of organisms, from yeast to mammals, mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPKmitogen-activated protein kinaseMAPKKMAPK kinaseMAPKKKMAPK kinase kinaseMAPmitogen-activated proteinERKextracellular signal-regulated protein kinaseMEKMAPK/ERK kinaseMEKKMEK kinaseMLKmixed lineage kinaseMDCKMadin-Darby canine kidneyJNKc-Jun NH2-terminal kinaseRACErapid amplification of cDNA endsRTreverse transcriptaseFBSfetal bovine serumPBSphosphate-buffered salineFACSfluorescence-activated cell sortingGSTglutathione S-transferaseUTRuntranslated regionGygrayMBPmyelin basic protein1The abbreviations used are: MAPKmitogen-activated protein kinaseMAPKKMAPK kinaseMAPKKKMAPK kinase kinaseMAPmitogen-activated proteinERKextracellular signal-regulated protein kinaseMEKMAPK/ERK kinaseMEKKMEK kinaseMLKmixed lineage kinaseMDCKMadin-Darby canine kidneyJNKc-Jun NH2-terminal kinaseRACErapid amplification of cDNA endsRTreverse transcriptaseFBSfetal bovine serumPBSphosphate-buffered salineFACSfluorescence-activated cell sortingGSTglutathione S-transferaseUTRuntranslated regionGygrayMBPmyelin basic proteinpathways mediate a variety of signals that regulate multiple physiological processes, including cell proliferation, cell differentiation, and cell death as well as stress-induced responses (1.Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 2.Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2249) Google Scholar, 3.Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1397) Google Scholar). These MAPK modules consist of distinct cascades of kinases, beginning with a serine/threonine kinase, MAPKKK, which phosphorylates and activates a dual specificity kinase, MAPKK or MEK, that in turn transfers phosphates onto threonine and tyrosine residues of a third enzyme, MAP kinase. The MAP kinase subsequently phosphorylates and activates various transcription factors, among other substrates. In mammals, the best characterized MAPK pathways are defined by the four main classes of MAPK they activate: extracellular signal-regulated protein kinases (ERK-1 and -2), Jun amino-terminal kinases (JNK-1, -2, and -3), p38 proteins (p38α, -β, -γ, and -δ), and ERK5 (4.Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4334) Google Scholar). The MAPKKK family consists of at least 14 members that include the MEKK group (MEKK1–4), the mixed lineage kinase group (MLK1–3, DLK, and LZK), the ASK proteins (ASK1 and -2), TAK1, TAO, and Tpl2/Cot. Although members within each group are highly homologous, with identity ranging between 50 and more than 90%, the homology between groups is significantly reduced and is restricted to the kinase domain. The large number of structurally diverse MAPKKKs may reflect tissue specificity or stimulus-specific signaling. Although substantial progress has been made in linking each of the known MAPKKK proteins to specific MAP kinase pathways, their precise contribution has not been clearly defined. For instance, MEKK1–3, DLK, MLK, and Tpl2 have been reported to activate preferentially JNK or ERK, rather than the p38 MAPK (5.Blank 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 (207) Google Scholar, 6.Deacon K. Blank J.L. J. Biol. Chem. 1999; 274: 16604-16610Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 7.Hirai S. Izawa M. Osada S. Spyrou G. Ohno S. Oncogene. 1996; 12: 641-650PubMed Google Scholar, 8.Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (706) Google Scholar, 9.Rana A. Gallo K. Godowski P. Hirai S. Ohno S. Zon L. Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 19025-19028Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 10.Salmeron 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). Conversely, TAK1, MEKK4, TAO, and ASK1 more effectively activate the p38 pathway (11.Hutchison M. Berman K.S. Cobb M.H. J. Biol. Chem. 1998; 273: 28625-28632Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 12.Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (1999) Google Scholar, 13.Takekawa M. Posas F. Saito H. EMBO J. 1997; 16: 4973-4982Crossref PubMed Scopus (157) Google Scholar, 14.Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1169) Google Scholar). The link between individual MAPKKKs and specific upstream control molecules has only been identified for some family members and remains to be firmly established for most. Despite our growing knowledge of the signaling elements involved in each cascade, no upstream MAPKKKs have yet been described for some MAP kinases, such as ERK3 (15.Zhu A.X. Zhao Y. Moller D.E. Flier J.S. Mol. Cell. Biol. 1994; 14: 8202-8211Crossref PubMed Google Scholar) and ERK6/p38γ (16.Wang X.S. Diener K. Manthey C.L. Wang S. Rosenzweig B. Bray J. Delaney J. Cole C.N. Chan-Hui P.Y. Mantlo N. Lichenstein H.S. Zukowski M. Yao Z. J. Biol. Chem. 1997; 272: 23668-23674Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar).In Saccharomyces cerevisiae, there are five MAPK modules, one of which is the well characterized mating pheromones pathway (17.Herskowitz I. Cell. 1995; 80: 187-197Abstract Full Text PDF PubMed Scopus (863) Google Scholar). In this system, Ste11 is the MAPKKK that activates Ste7, the MEK counterpart, which in turn activates the MAPK, Fus3 (18.Elion E.A. Grisafi P.L. Fink G.R. Cell. 1990; 60: 649-664Abstract Full Text PDF PubMed Scopus (308) Google Scholar). We and others (19.Freed E. Symons M. Macdonald S.G. McCormick F. Ruggieri R. Science. 1994; 265: 1713-1716Crossref PubMed Scopus (352) Google Scholar, 20.Irie K. Gotoh Y. Yashar B.M. Errede B. Nishida E. Matsumoto K. Science. 1994; 265: 1716-1719Crossref PubMed Scopus (255) Google Scholar) have shown that loss of Ste11 by gene knock out can be functionally complemented in this system by an active mammalian Raf protein and its substrate MEK. In the present study we conducted a functional screen in this system to identify novel components of MAPK pathways, and we discovered a gene that encodes a serine/threonine kinase, designated MRK forMLK-related kinase. Here we describe the characterization of the structure of the MRKgene and the effect of the MRK protein on the known mammalian MAP kinase cascades. We found that MRK expression preferentially activated the ERK6/p38γ and JNK pathways, both in transiently transfected and stable cell lines, whereas it had a marginal effect on p38α and no significant effect on ERK. The activation of these pathways is accompanied by stimulation of their respective MKKs, in particular MKK3/MKK6 and MKK4. We also report that expression of wild type MRK induces an increase in the G2/M cell population. Conversely, γ-radiation-mediated G1 and G2arrest are decreased in cells expressing the dominant negative allele of MRK. The effect of γ-radiation is accompanied by activation of endogenous MRK. These findings suggest a role for MRK in the regulation of cell cycle checkpoints.DISCUSSIONIn this study, we describe the identification of a human serine/threonine kinase, MRK, discovered as an activator of S. cerevisiae Ste7, the yeast MEK homolog that mediates the mating pheromone response. We also characterize MRK-β as a member of the MAPKKK family and show that MRK-β preferentially activates the ERK6/p38γ and the JNK MAP kinase pathways. In addition, we provide evidence that the MRK-β-mediated pathway is activated by γ-radiation and is necessary for the G1 and G2 arrest induced by DNA damage.We identified two splice variants of the MRK gene, as supported by the characterization of the genomic structure of the MRK locus. The gene is spread over more than 200 kb, a rather long stretch of genomic sequence. Interestingly, the MRK-β mRNA has an unusually long 5.6-kb 3′-UTR that could be involved in post-transcriptional regulation. Although rare, a long 3′-UTR has been reported for other mRNAs, such as one of the FGF-2 mRNA species (40.Prats H. Kaghad M. Prats A.C. Klagsbrun M. Lelias J.M. Liauzun P. Chalon P. Tauber J.P. Amalric F. Smith J.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1836-1840Crossref PubMed Scopus (399) Google Scholar) where it has been implicated in modulating translation (41.Touriol C. Roussigne M. Gensac M.C. Prats H. Prats A.C. J. Biol. Chem. 2000; 275: 19361-19367Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The role of this region in MRK mRNA stability or in protein expression remains to be investigated. It is also possible that the transcript length may control splicing, yielding a much less abundant mRNA encoding the alternative splice form, MRK-α. This form is, in fact, expressed at much lower levels than MRK-β in all tissues with the exception of liver, where it appears to be the major species.The MRK proteins share significant homology in the kinase domain with proteins of the MAPKKK family. The most closely related members are those in the MLK subfamily. However, the homology is restricted to the kinase domain and remains in the 50% similarity range. There is a single leucine zipper domain in the MRK proteins, whereas this is found as a double domain in the MLK family members.The functional identification and the primary sequence suggest that MRK-β is a member of the MAPKKK family. This was confirmed by its activation of specific MAP kinase pathways. Although activation of the three major MAP kinase pathways was observed when MRK was greatly overexpressed with the respective MAP kinases in cells, we found that the effects of relatively low levels of MRK on endogenous MAP kinases were more specific. Of the pathways tested, the ERK6/p38γ and JNK cascades were predominantly activated, whereas the ERK and the p38α pathways were marginally affected. In vitro phosphorylation studies demonstrated that the effect on ERK is indirect, as shown by the inability of MRK-β to phosphorylate MEK directly. Therefore, the activation of MEK, when co-transfected with MRK in cells, is likely to be secondary to new gene expression of autocrine factors. In line with this interpretation, we did not observe any effect on the activation of endogenous ERK1 and -2 in MRK-expressing MDCK cells (Fig. 6). In contrast, the MKK proteins upstream of JNK and p38, MKK4 and MKK3/MKK6, respectively, were found to be good substrates in vitro as well as in cells. Remarkably, the activation of endogenous MKK3/MKK6 and ERK6/p38γ proteins was already obvious at a time when recombinant MRK is expressed at relatively low levels, 8 h after induction, underlining the preferential stimulation of this pathway. The activation of these stress-activated kinases did not appear to be the result of cellular stress caused by overexpression of proteins in cells, because expression of similar levels of the catalytically inactive MRK kinase did not elicit any of the responses observed with the expression of the wild type protein. This observation, therefore, supports the specificity of the MRK-induced effects.While this work was in progress, Gotoh et al. (42.Gotoh I. Adachi M. Nishida E. J. Biol. Chem. 2001; 276: 4276-4286Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) reported the isolation of two mouse clones orthologous to the MRKgenes, called MLTK. However, they reported indiscriminate activation of the ERK, JNK, p38, and ERK5 pathways. It is possible that the experimental approach used in their study, namely co-expression of the kinases with each of the potential substrates in cells, could explain the lack of discrimination observed among these signaling pathways. As discussed above, autocrine factors induced by the recombinant proteins may account for the observed effects on some of the pathways tested.Members of the p38 pathway, such as MKK3, MKK6, and ERK6/p38γ, are preferentially expressed in heart or skeletal muscle (16.Wang X.S. Diener K. Manthey C.L. Wang S. Rosenzweig B. Bray J. Delaney J. Cole C.N. Chan-Hui P.Y. Mantlo N. Lichenstein H.S. Zukowski M. Yao Z. J. Biol. Chem. 1997; 272: 23668-23674Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 43.Stein B. Brady H. Yang M.X. Young D.B. Barbosa M.S. J. Biol. Chem. 1996; 271: 11427-11433Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 44.Lechner C. Zahalka M.A. Giot J.F. Moller N.P. Ullrich A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4355-4359Crossref PubMed Scopus (274) Google Scholar, 45.Han 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). Interestingly, the levels of MRK-β are particularly elevated in these tissues. It will be of interest to explore the possibility that MRK-β, via the ERK6/p38γ pathway, plays an important role in the physiology of these tissues.This work also identifies a role for MRK in the cell cycle checkpoint regulation in response to DNA damage-inducing radiation. In the MDCK cell system, the effect on the cell cycle caused by wild type MRK suggests that this kinase mediates signals leading to G2arrest. This hypothesis is supported by the finding that dominant negative MRK reduces the effects of γ-radiation on the G1and G2 phases of the cell cycle. This observation is consistent with a physiological function of MRK in the regulation of cell cycle checkpoints. These data were supported by the finding that endogenous MRK is activated by γ-irradiation shortly after treatment. How does MRK affect checkpoint regulation? The ERK6/p38γ cascade has been implicated in γ-radiation-induced cell cycle arrest (35.Wang X. McGowan C.H. Zhao M. He L. Downey J.S. Fearns C. Wang Y. Huang S. Han J. Mol. Cell Biol. 2000; 20: 4543-4552Crossref PubMed Scopus (235) Google Scholar). Given the prominent activation of ERK6/p38γ by MRK, this member of the p38 family was expected to be a good candidate. Surprisingly, no activation of ERK6/p38γ was detected in response to γ-irradiation (data not shown). Therefore, at this time we cannot implicate ERK6/p38γ in the observed cell cycle effects of γ-radiation in this system. It is possible that cell type differences are responsible for this discrepancy.In conclusion, we have shown that MRK, a member of the MAPKKK family, preferentially activates the ERK6/p38γ and JNK pathways and plays a role in the regulation of DNA damage-induced checkpoints. Future studies will address the identification of the elements that relay the signals initiated by γ-radiation to MRK and those that act downstream of MRK in response to DNA damage. In a wide range of organisms, from yeast to mammals, mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPKmitogen-activated protein kinaseMAPKKMAPK kinaseMAPKKKMAPK kinase kinaseMAPmitogen-activated proteinERKextracellular signal-regulated protein kinaseMEKMAPK/ERK kinaseMEKKMEK kinaseMLKmixed lineage kinaseMDCKMadin-Darby canine kidneyJNKc-Jun NH2-terminal kinaseRACErapid amplification of cDNA endsRTreverse transcriptaseFBSfetal bovine serumPBSphosphate-buffered salineFACSfluorescence-activated cell sortingGSTglutathione S-transferaseUTRuntranslated regionGygrayMBPmyelin basic protein1The abbreviations used are: MAPKmitogen-activated protein kinaseMAPKKMAPK kinaseMAPKKKMAPK kinase kinaseMAPmitogen-activated proteinERKextracellular signal-regulated protein kinaseMEKMAPK/ERK kinaseMEKKMEK kinaseMLKmixed lineage kinaseMDCKMadin-Darby canine kidneyJNKc-Jun NH2-terminal kinaseRACErapid amplification of cDNA endsRTreverse transcriptaseFBSfetal bovine serumPBSphosphate-buffered salineFACSfluorescence-activated cell sortingGSTglutathione S-transferaseUTRuntranslated regionGygrayMBPmyelin basic proteinpathways mediate a variety of signals that regulate multiple physiological processes, including cell proliferation, cell differentiation, and cell death as well as stress-induced responses (1.Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 2.Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2249) Google Scholar, 3.Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1397) Google Scholar). These MAPK modules consist of distinct cascades of kinases, beginning with a serine/threonine kinase, MAPKKK, which phosphorylates and activates a dual specificity kinase, MAPKK or MEK, that in turn transfers phosphates onto threonine and tyrosine residues of a third enzyme, MAP kinase. The MAP kinase subsequently phosphorylates and activates various transcription factors, among other substrates. In mammals, the best characterized MAPK pathways are defined by the four main classes of MAPK they activate: extracellular signal-regulated protein kinases (ERK-1 and -2), Jun amino-terminal kinases (JNK-1, -2, and -3), p38 proteins (p38α, -β, -γ, and -δ), and ERK5 (4.Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4334) Google Scholar). The MAPKKK family consists of at least 14 members that include the MEKK group (MEKK1–4), the mixed lineage kinase group (MLK1–3, DLK, and LZK), the ASK proteins (ASK1 and -2), TAK1, TAO, and Tpl2/Cot. Although members within each group are highly homologous, with identity ranging between 50 and more than 90%, the homology between groups is significantly reduced and is restricted to the kinase domain. The large number of structurally diverse MAPKKKs may reflect tissue specificity or stimulus-specific signaling. Although substantial progress has been made in linking each of the known MAPKKK proteins to specific MAP kinase pathways, their precise contribution has not been clearly defined. For instance, MEKK1–3, DLK, MLK, and Tpl2 have been reported to activate preferentially JNK or ERK, rather than the p38 MAPK (5.Blank 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 (207) Google Scholar, 6.Deacon K. Blank J.L. J. Biol. Chem. 1999; 274: 16604-16610Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 7.Hirai S. Izawa M. Osada S. Spyrou G. Ohno S. Oncogene. 1996; 12: 641-650PubMed Google Scholar, 8.Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (706) Google Scholar, 9.Rana A. Gallo K. Godowski P. Hirai S. Ohno S. Zon L. Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 19025-19028Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 10.Salmeron 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). Conversely, TAK1, MEKK4, TAO, and ASK1 more effectively activate the p38 pathway (11.Hutchison M. Berman K.S. Cobb M.H. J. Biol. Chem. 1998; 273: 28625-28632Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 12.Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (1999) Google Scholar, 13.Takekawa M. Posas F. Saito H. EMBO J. 1997; 16: 4973-4982Crossref PubMed Scopus (157) Google Scholar, 14.Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1169) Google Scholar). The link between individual MAPKKKs and specific upstream control molecules has only been identified for some family members and remains to be firmly established for most. Despite our growing knowledge of the signaling elements involved in each cascade, no upstream MAPKKKs have yet been described for some MAP kinases, such as ERK3 (15.Zhu A.X. Zhao Y. Moller D.E. Flier J.S. Mol. Cell. Biol. 1994; 14: 8202-8211Crossref PubMed Google Scholar) and ERK6/p38γ (16.Wang X.S. Diener K. Manthey C.L. Wang S. Rosenzweig B. Bray J. Delaney J. Cole C.N. Chan-Hui P.Y. Mantlo N. Lichenstein H.S. Zukowski M. Yao Z. J. Biol. Chem. 1997; 272: 23668-23674Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). mitogen-activated protein kinase MAPK kinase MAPK kinase kinase mitogen-activated protein extracellular signal-regulated protein kinase MAPK/ERK kinase MEK kinase mixed lineage kinase Madin-Darby canine kidney c-Jun NH2-terminal kinase rapid amplification of cDNA ends reverse transcriptase fetal bovine serum phosphate-buffered saline fluorescence-activated cell sorting glutathione S-transferase untranslated region gray myelin basic protein mitogen-activated protein kinase MAPK kinase MAPK kinase kinase mitogen-activated protein extracellular signal-regulated protein kinase MAPK/ERK kinase MEK kinase mixed lineage kinase Madin-Darby canine kidney c-Jun NH2-terminal kinase rapid amplification of cDNA ends reverse transcriptase fetal bovine serum phosphate-buffered saline fluorescence-activated cell sorting glutathione S-transferase untranslated region gray myelin basic protein In Saccharomyces cerevisiae, there are five MAPK modules, one of which is the well characterized mating pheromones pathway (17.Herskowitz I. Cell. 1995; 80: 187-197Abstract Full Text PDF PubMed Scopus (863) Google Scholar). In this system, Ste11 is the MAPKKK that activates Ste7, the MEK counterpart, which in turn activates the MAPK, Fus3 (18.Elion E.A. Grisafi P.L. Fink G.R. Cell. 1990; 60: 649-664Abstract Full Text PDF PubMed Scopus (308) Google Scholar). We and others (19.Freed E. Symons M. Macdonald S.G. McCormick F. Ruggieri R. Science. 1994; 265: 1713-1716Crossref PubMed Scopus (352) Google Scholar, 20.Irie K. Gotoh Y. Yashar B.M. Errede B. Nishida E. Matsumoto K. Science. 1994; 265: 1716-1719Crossref PubMed Scopus (255) Google Scholar) have shown that loss of Ste11 by gene knock out can be functionally complemented in this system by an active mammalian Raf protein and its substrate MEK. In the present study we conducted a functional screen in this system to identify novel components of MAPK pathways, and we discovered a gene that encodes a serine/threonine kinase, designated MRK forMLK-related kinase. Here we describe the characterization of the structure of the MRKgene and the effect of the MRK protein on the known mammalian MAP kinase cascades. We found that MRK expression preferentially activated the ERK6/p38γ and JNK pathways, both in transiently transfected and stable cell lines, whereas it had a marginal effect on p38α and no significant effect on ERK. The activation of these pathways is accompanied by stimulation of their respective MKKs, in particular MKK3/MKK6 and MKK4. We also report that expression of wild type MRK induces an increase in the G2/M cell population. Conversely, γ-radiation-mediated G1 and G2arrest are decreased in cells expressing the dominant negative allele of MRK. The effect of γ-radiation is accompanied by activation of endogenous MRK. These findings suggest a role for MRK in the regulation of cell cycle checkpoints. DISCUSSIONIn this study, we describe the identification of a human serine/threonine kinase, MRK, discovered as an activator of S. cerevisiae Ste7, the yeast MEK homolog that mediates the mating pheromone response. We also characterize MRK-β as a member of the MAPKKK family and show that MRK-β preferentially activates the ERK6/p38γ and the JNK MAP kinase pathways. In addition, we provide evidence that the MRK-β-mediated pathway is activated by γ-radiation and is necessary for the G1 and G2 arrest induced by DNA damage.We identified two splice variants of the MRK gene, as supported by the characterization of the genomic structure of the MRK locus. The gene is spread over more than 200 kb, a rather long stretch of genomic sequence. Interestingly, the MRK-β mRNA has an unusually long 5.6-kb 3′-UTR that could be involved in post-transcriptional regulation. Although rare, a long 3′-UTR has been reported for other mRNAs, such as one of the FGF-2 mRNA species (40.Prats H. Kaghad M. Prats A.C. Klagsbrun M. Lelias J.M. Liauzun P. Chalon P. Tauber J.P. Amalric F. Smith J.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1836-1840Crossref PubMed Scopus (399) Google Scholar) where it has been implicated in modulating translation (41.Touriol C. Roussigne M. Gensac M.C. Prats H. Prats A.C. J. Biol. Chem. 2000; 275: 19361-19367Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The role of this region in MRK mRNA stability or in protein expression remains to be investigated. It is also possible that the transcript length may control splicing, yielding a much less abundant mRNA encoding the alternative splice form, MRK-α. This form is, in fact, expressed at much lower levels than MRK-β in all tissues with the exception of liver, where it appears to be the major species.The MRK proteins share significant homology in the kinase domain with proteins of the MAPKKK family. The most closely related members are those in the MLK subfamily. However, the homology is restricted to the kinase domain and remains in the 50% similarity range. There is a single leucine zipper domain in the MRK proteins, whereas this is found as a double domain in the MLK family members.The functional identification and the primary sequence suggest that MRK-β is a member of the MAPKKK family. This was confirmed by its activation of specific MAP kinase pathways. Although activation of the three major MAP kinase pathways was observed when MRK was greatly overexpressed with the respective MAP kinases in cells, we found that the effects of relatively low levels of MRK on endogenous MAP kinases were more specific. Of the pathways tested, the ERK6/p38γ and JNK cascades were predominantly activated, whereas the ERK and the p38α pathways were marginally affected. In vitro phosphorylation studies demonstrated that the effect on ERK is indirect, as shown by the inability of MRK-β to phosphorylate MEK directly. Therefore, the activation of MEK, when co-transfected with MRK in cells, is likely to be secondary to new gene expression of autocrine factors. In line with this interpretation, we did not observe any effect on the activation of endogenous ERK1 and -2 in MRK-expressing MDCK cells (Fig. 6). In contrast, the MKK proteins upstream of JNK and p38, MKK4 and MKK3/MKK6, respectively, were found to be good substrates in vitro as well as in cells. Remarkably, the activation of endogenous MKK3/MKK6 and ERK6/p38γ proteins was already obvious at a time when recombinant MRK is expressed at relatively low levels, 8 h after induction, underlining the preferential stimulation of this pathway. The activation of these stress-activated kinases did not appear to be the result of cellular stress caused by overexpression of proteins in cells, because expression of similar levels of the catalytically inactive MRK kinase did not elicit any of the responses observed with the expression of the wild type protein. This observation, therefore, supports the specificity of the MRK-induced effects.While this work was in progress, Gotoh et al. (42.Gotoh I. Adachi M. Nishida E. J. Biol. Chem. 2001; 276: 4276-4286Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) reported the isolation of two mouse clones orthologous to the MRKgenes, called MLTK. However, they reported indiscriminate activation of the ERK, JNK, p38, and ERK5 pathways. It is possible that the experimental approach used in their study, namely co-expression of the kinases with each of the potential substrates in cells, could explain the lack of discrimination observed among these signaling pathways. As discussed above, autocrine factors induced by the recombinant proteins may account for the observed effects on some of the pathways tested.Members of the p38 pathway, such as MKK3, MKK6, and ERK6/p38γ, are preferentially expressed in heart or skeletal muscle (16.Wang X.S. Diener K. Manthey C.L. Wang S. Rosenzweig B. Bray J. Delaney J. Cole C.N. Chan-Hui P.Y. Mantlo N. Lichenstein H.S. Zukowski M. Yao Z. J. Biol. Chem. 1997; 272: 23668-23674Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 43.Stein B. Brady H. Yang M.X. Young D.B. Barbosa M.S. J. Biol. Chem. 1996; 271: 11427-11433Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 44.Lechner C. Zahalka M.A. Giot J.F. Moller N.P. Ullrich A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4355-4359Crossref PubMed Scopus (274) Google Scholar, 45.Han 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). Interestingly, the levels of MRK-β are particularly elevated in these tissues. It will be of interest to explore the possibility that MRK-β, via the ERK6/p38γ pathway, plays an important role in the physiology of these tissues.This work also identifies a role for MRK in the cell cycle checkpoint regulation in response to DNA damage-inducing radiation. In the MDCK cell system, the effect on the cell cycle caused by wild type MRK suggests that this kinase mediates signals leading to G2arrest. This hypothesis is supported by the finding that dominant negative MRK reduces the effects of γ-radiation on the G1and G2 phases of the cell cycle. This observation is consistent with a physiological function of MRK in the regulation of cell cycle checkpoints. These data were supported by the finding that endogenous MRK is activated by γ-irradiation shortly after treatment. How does MRK affect checkpoint regulation? The ERK6/p38γ cascade has been implicated in γ-radiation-induced cell cycle arrest (35.Wang X. McGowan C.H. Zhao M. He L. Downey J.S. Fearns C. Wang Y. Huang S. Han J. Mol. Cell Biol. 2000; 20: 4543-4552Crossref PubMed Scopus (235) Google Scholar). Given the prominent activation of ERK6/p38γ by MRK, this member of the p38 family was expected to be a good candidate. Surprisingly, no activation of ERK6/p38γ was detected in response to γ-irradiation (data not shown). Therefore, at this time we cannot implicate ERK6/p38γ in the observed cell cycle effects of γ-radiation in this system. It is possible that cell type differences are responsible for this discrepancy.In conclusion, we have shown that MRK, a member of the MAPKKK family, preferentially activates the ERK6/p38γ and JNK pathways and plays a role in the regulation of DNA damage-induced checkpoints. Future studies will address the identification of the elements that relay the signals initiated by γ-radiation to MRK and those that act downstream of MRK in response to DNA damage. In this study, we describe the identification of a human serine/threonine kinase, MRK, discovered as an activator of S. cerevisiae Ste7, the yeast MEK homolog that mediates the mating pheromone response. We also characterize MRK-β as a member of the MAPKKK family and show that MRK-β preferentially activates the ERK6/p38γ and the JNK MAP kinase pathways. In addition, we provide evidence that the MRK-β-mediated pathway is activated by γ-radiation and is necessary for the G1 and G2 arrest induced by DNA damage. We identified two splice variants of the MRK gene, as supported by the characterization of the genomic structure of the MRK locus. The gene is spread over more than 200 kb, a rather long stretch of genomic sequence. Interestingly, the MRK-β mRNA has an unusually long 5.6-kb 3′-UTR that could be involved in post-transcriptional regulation. Although rare, a long 3′-UTR has been reported for other mRNAs, such as one of the FGF-2 mRNA species (40.Prats H. Kaghad M. Prats A.C. Klagsbrun M. Lelias J.M. Liauzun P. Chalon P. Tauber J.P. Amalric F. Smith J.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1836-1840Crossref PubMed Scopus (399) Google Scholar) where it has been implicated in modulating translation (41.Touriol C. Roussigne M. Gensac M.C. Prats H. Prats A.C. J. Biol. Chem. 2000; 275: 19361-19367Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The role of this region in MRK mRNA stability or in protein expression remains to be investigated. It is also possible that the transcript length may control splicing, yielding a much less abundant mRNA encoding the alternative splice form, MRK-α. This form is, in fact, expressed at much lower levels than MRK-β in all tissues with the exception of liver, where it appears to be the major species. The MRK proteins share significant homology in the kinase domain with proteins of the MAPKKK family. The most closely related members are those in the MLK subfamily. However, the homology is restricted to the kinase domain and remains in the 50% similarity range. There is a single leucine zipper domain in the MRK proteins, whereas this is found as a double domain in the MLK family members. The functional identification and the primary sequence suggest that MRK-β is a member of the MAPKKK family. This was confirmed by its activation of specific MAP kinase pathways. Although activation of the three major MAP kinase pathways was observed when MRK was greatly overexpressed with the respective MAP kinases in cells, we found that the effects of relatively low levels of MRK on endogenous MAP kinases were more specific. Of the pathways tested, the ERK6/p38γ and JNK cascades were predominantly activated, whereas the ERK and the p38α pathways were marginally affected. In vitro phosphorylation studies demonstrated that the effect on ERK is indirect, as shown by the inability of MRK-β to phosphorylate MEK directly. Therefore, the activation of MEK, when co-transfected with MRK in cells, is likely to be secondary to new gene expression of autocrine factors. In line with this interpretation, we did not observe any effect on the activation of endogenous ERK1 and -2 in MRK-expressing MDCK cells (Fig. 6). In contrast, the MKK proteins upstream of JNK and p38, MKK4 and MKK3/MKK6, respectively, were found to be good substrates in vitro as well as in cells. Remarkably, the activation of endogenous MKK3/MKK6 and ERK6/p38γ proteins was already obvious at a time when recombinant MRK is expressed at relatively low levels, 8 h after induction, underlining the preferential stimulation of this pathway. The activation of these stress-activated kinases did not appear to be the result of cellular stress caused by overexpression of proteins in cells, because expression of similar levels of the catalytically inactive MRK kinase did not elicit any of the responses observed with the expression of the wild type protein. This observation, therefore, supports the specificity of the MRK-induced effects. While this work was in progress, Gotoh et al. (42.Gotoh I. Adachi M. Nishida E. J. Biol. Chem. 2001; 276: 4276-4286Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) reported the isolation of two mouse clones orthologous to the MRKgenes, called MLTK. However, they reported indiscriminate activation of the ERK, JNK, p38, and ERK5 pathways. It is possible that the experimental approach used in their study, namely co-expression of the kinases with each of the potential substrates in cells, could explain the lack of discrimination observed among these signaling pathways. As discussed above, autocrine factors induced by the recombinant proteins may account for the observed effects on some of the pathways tested. Members of the p38 pathway, such as MKK3, MKK6, and ERK6/p38γ, are preferentially expressed in heart or skeletal muscle (16.Wang X.S. Diener K. Manthey C.L. Wang S. Rosenzweig B. Bray J. Delaney J. Cole C.N. Chan-Hui P.Y. Mantlo N. Lichenstein H.S. Zukowski M. Yao Z. J. Biol. Chem. 1997; 272: 23668-23674Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 43.Stein B. Brady H. Yang M.X. Young D.B. Barbosa M.S. J. Biol. Chem. 1996; 271: 11427-11433Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 44.Lechner C. Zahalka M.A. Giot J.F. Moller N.P. Ullrich A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4355-4359Crossref PubMed Scopus (274) Google Scholar, 45.Han 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). Interestingly, the levels of MRK-β are particularly elevated in these tissues. It will be of interest to explore the possibility that MRK-β, via the ERK6/p38γ pathway, plays an important role in the physiology of these tissues. This work also identifies a role for MRK in the cell cycle checkpoint regulation in response to DNA damage-inducing radiation. In the MDCK cell system, the effect on the cell cycle caused by wild type MRK suggests that this kinase mediates signals leading to G2arrest. This hypothesis is supported by the finding that dominant negative MRK reduces the effects of γ-radiation on the G1and G2 phases of the cell cycle. This observation is consistent with a physiological function of MRK in the regulation of cell cycle checkpoints. These data were supported by the finding that endogenous MRK is activated by γ-irradiation shortly after treatment. How does MRK affect checkpoint regulation? The ERK6/p38γ cascade has been implicated in γ-radiation-induced cell cycle arrest (35.Wang X. McGowan C.H. Zhao M. He L. Downey J.S. Fearns C. Wang Y. Huang S. Han J. Mol. Cell Biol. 2000; 20: 4543-4552Crossref PubMed Scopus (235) Google Scholar). Given the prominent activation of ERK6/p38γ by MRK, this member of the p38 family was expected to be a good candidate. Surprisingly, no activation of ERK6/p38γ was detected in response to γ-irradiation (data not shown). Therefore, at this time we cannot implicate ERK6/p38γ in the observed cell cycle effects of γ-radiation in this system. It is possible that cell type differences are responsible for this discrepancy. In conclusion, we have shown that MRK, a member of the MAPKKK family, preferentially activates the ERK6/p38γ and JNK pathways and plays a role in the regulation of DNA damage-induced checkpoints. Future studies will address the identification of the elements that relay the signals initiated by γ-radiation to MRK and those that act downstream of MRK in response to DNA damage. We thank Silvio Gutkind (National Institutes of Health) for the generous gift of reagents and for inspiring discussions. We also thank John Lyons, George Martin, and Jerry Beltman (Onyx Pharmaceuticals, CA), Roger Davis (University of Massachusetts Medical School), and Amy Yee (Tufts University) for the gifts of plasmids. We thank Kunihiro Matsumoto (Nagoya University, Japan) for providing some of the yeast strains and Keith Mostov (University of California, San Francisco) for the parental MDCK T23 clone. We are very thankful to Marc Symons and Kirk Manogue for critical reading of the manuscript and helpful discussions. As this project was initiated while some of the authors were at Onyx Pharmaceuticals, we thank Onyx and Bayer, Inc., for interactive support. AF480461" @default.
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