Cloning and Characterization of a Human STE20-like Protein Kinase with Unusual Cofactor Requirements

Abstract

We cloned and characterized a novel human member of the STE20 serine/threonine protein kinase family named mst-3. Based on its domain structure, mst-3 belongs to the SPS1 subgroup of STE20-like proteins, which includes germinal center (GC) kinase, hematopoietic progenitor kinase (HPK), kinase homologous to STE20/SPS-1 (KHS), kinases responsive to stress (KRS1/2), the mammalian STE20-like kinases (mst1/2), and the recently published STE20/oxidant stress response kinase SOK-1. mst-3 is most closely related to SOK-1, with 88% amino acid similarity in the kinase domain. The similarity of the mst-3 kinase domain to STE20 is 42%. The mst-3 transcript is ubiquitously expressed, and the protein was found in all human, mouse, and monkey cell lines tested. An in vitro kinase assay showed that mst-3 can phosphorylate basic exogenous substrates as well as itself. Interestingly, mst-3 prefers Mn2+ to Mg2+ as a divalent cation and can use both GTP and ATP as phosphate donors. Like SOK-1, mst-3 is activated by autophosphorylation. However, a physiological stimulus of mst-3 activity was not identified. mst-3 activity does not change upon exposure to several mitogenic and stress stimuli. Overexpression of mst-3 wild-type or kinase dead protein affects neither the extracellular signal-regulated kinases (ERK1/2 or ERK6), c-Jun N-terminal kinase (JNK), p38, nor pp70S6 kinase, suggesting that mst-3 is part of a novel signaling pathway. We cloned and characterized a novel human member of the STE20 serine/threonine protein kinase family named mst-3. Based on its domain structure, mst-3 belongs to the SPS1 subgroup of STE20-like proteins, which includes germinal center (GC) kinase, hematopoietic progenitor kinase (HPK), kinase homologous to STE20/SPS-1 (KHS), kinases responsive to stress (KRS1/2), the mammalian STE20-like kinases (mst1/2), and the recently published STE20/oxidant stress response kinase SOK-1. mst-3 is most closely related to SOK-1, with 88% amino acid similarity in the kinase domain. The similarity of the mst-3 kinase domain to STE20 is 42%. The mst-3 transcript is ubiquitously expressed, and the protein was found in all human, mouse, and monkey cell lines tested. An in vitro kinase assay showed that mst-3 can phosphorylate basic exogenous substrates as well as itself. Interestingly, mst-3 prefers Mn2+ to Mg2+ as a divalent cation and can use both GTP and ATP as phosphate donors. Like SOK-1, mst-3 is activated by autophosphorylation. However, a physiological stimulus of mst-3 activity was not identified. mst-3 activity does not change upon exposure to several mitogenic and stress stimuli. Overexpression of mst-3 wild-type or kinase dead protein affects neither the extracellular signal-regulated kinases (ERK1/2 or ERK6), c-Jun N-terminal kinase (JNK), p38, nor pp70S6 kinase, suggesting that mst-3 is part of a novel signaling pathway. Eukaryotic cells are able to couple extracellular signals to specific biological processes such as cell growth, differentiation, and stress responses through the activation of distinct evolutionarily conserved intracellular signaling cascades, collectively known as mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; MBP, myelin basic protein. cascades. In both mammals and lower eukaryotes, the core of MAP kinase cascades is a three-component module consisting of a generic MAPK kinase kinase, which phosphorylates and activates a dual-specificity MAPK kinase, which in turn activates the MAPK. In many cases the activated MAP kinases translocate to the nucleus where they phosphorylate transcription factors, thus eliciting the biological response (1Chen R.-H. Sarnecki C. Blenis J. Mol. Cell. Biol. 1992; 12: 915-927Crossref PubMed Google Scholar). In the budding yeast Saccharomyces cerevisiae, at least six MAPK pathways have been identified. They regulate diverse biological processes such as mating and invasive growth, cell wall integrity, and the response to high osmolarity as well as pseudohyphal development and spore formation in diploid cells (2Ammerer G. Curr. Opin. Cell Biol. 1994; 5: 254-260Google Scholar, 3Blumer K.J. Johnson G.L. Trends Biochem. 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Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 21Raingeaud J. Whitmarsh A.J. Barret T. Derijard B. Davis R.J. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1146) Google Scholar, 22Stein 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). Based on epistasis experiments, the S. cerevisiaeserine-threonine kinase STE20 was placed upstream of the MAPK module consisting of the MAPK kinase kinase STE11, the MAPK kinase STE7, and the MAPKs FUS3/KSS1 in the mating pathway (23Leberer E. Dignard D. Harcus D. Thomas D.Y. Whiteway M. EMBO J. 1992; 11: 4815-4824Crossref PubMed Scopus (347) Google Scholar, 24Ramer S.W. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 452-456Crossref PubMed Scopus (169) Google Scholar, 25Leberer E. Dignard D. Hougan L. Thomas D.Y. Whiteway M. EMBO J. 1992; 11: 4805-4813Crossref PubMed Scopus (76) Google Scholar). Other S. cerevisiae STE20-like kinases are Cla4 (26Cvrckova F. De Virgilio C. Manser E. Pringle J.R. Nasmyth K. Genes Dev. 1995; 9: 1817-1830Crossref PubMed Scopus (310) Google Scholar), which is involved in budding and cytokinesis and SPS1 (27Friesen H. Lunz R. Doyle S. Segall J. Genes Dev. 1994; 8: 2162-2175Crossref PubMed Scopus (108) Google Scholar), a kinase required for the late events of sporulation. The mammalian STE20-related kinases represent a rapidly growing kinase family. Recently identified mammalian members of this family are the rat and mouse p21-activated protein kinases (p65PAK and mPAK-3) (28Manser E. Leung T. Salhuddin H. Zhao Z.S. Lim L. Nature. 1994; 367: 40-46Crossref PubMed Scopus (1300) Google Scholar, 29Bagrodia S. Taylor J.S. Creasy C.L. Chernoff J. Cerione R.A. J. Biol. Chem. 1995; 270: 22731-22737Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar) and its human counterparts, hPAK65 (hPAK2) (30Martin G.A. Bollag G. McCormick F. Abo A. EMBO J. 1995; 14: 1970-1978Crossref PubMed Scopus (303) Google Scholar, 31Knaus U.G. Morris S. Dong H. Chernoff J. Bokoch G.M. Science. 1995; 269: 221-223Crossref PubMed Scopus (357) Google Scholar) and hPAK1 (32Brown J.L. Stowers L. Baer M. Trejo J. Coughlin S. Chant J. Curr. Biol. 1996; 6: 598-605Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 31Knaus U.G. Morris S. Dong H. Chernoff J. Bokoch G.M. Science. 1995; 269: 221-223Crossref PubMed Scopus (357) Google Scholar), thegerminal center kinase, GCK (33Katz P. Whalen G. Kehrl J.H. J. Biol. Chem. 1994; 269: 16802-16809Abstract Full Text PDF PubMed Google Scholar), and its murine equivalent, the rab8-interactingprotein (rabip) (34Ren M. Zeng J. de Lemos-Chiarandini C. Rosenfeld M. Adesnik M. Sabatini D.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5151-5155Crossref PubMed Scopus (88) Google Scholar), a hematopoieticprogenitor kinase (HPK) (35Hu M.C.-T. Qiu W.R. Wang X. Meyer C.F. Tan T.H. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (194) Google Scholar), akinase homologous to STE20/SPS1 (KHS) (36Tung R.M. Blenis J. Oncogene. 1997; 14: 653-659Crossref PubMed Scopus (66) Google Scholar), the mammalian STE20-like kinases mst-1 (37Creasy C.L. Chernoff J. J. Biol. Chem. 1995; 270: 21695-21700Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) and mst-2 (37Creasy C.L. Chernoff J. J. Biol. Chem. 1995; 270: 21695-21700Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), kinases responsive to stress (KRS1/2) (48Taylor L.K. Wang H.C. Erikson R.L. Proc. Natl. Acad. Sci U. S. A. 1996; 93: 10099-10104Crossref PubMed Scopus (141) Google Scholar), and theSTE20/oxidant stress responsekinase-1 (SOK-1) (38Pombo C.M. Bonventre J.V. Molnar A. Kyriakis J. Force T. EMBO J. 1996; 15: 4537-4546Crossref PubMed Scopus (133) Google Scholar). Based on their and members of the STE20 family can be like STE20 and the mammalian of a kinase domain and N-terminal domain and have a to and not (28Manser E. Leung T. Salhuddin H. Zhao Z.S. Lim L. Nature. 1994; 367: 40-46Crossref PubMed Scopus (1300) Google Scholar, 29Bagrodia S. Taylor J.S. Creasy C.L. Chernoff J. Cerione R.A. J. Biol. Chem. 1995; 270: 22731-22737Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, G.A. Bollag G. McCormick F. Abo A. EMBO J. 1995; 14: 1970-1978Crossref PubMed Scopus (303) Google Scholar, 31Knaus U.G. Morris S. Dong H. Chernoff J. Bokoch G.M. Science. 1995; 269: 221-223Crossref PubMed Scopus (357) Google Scholar). they and are activated (28Manser E. Leung T. Salhuddin H. Zhao Z.S. Lim L. Nature. 1994; 367: 40-46Crossref PubMed Scopus (1300) Google Scholar, 29Bagrodia S. Taylor J.S. Creasy C.L. Chernoff J. Cerione R.A. J. Biol. Chem. 1995; 270: 22731-22737Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, G.A. Bollag G. McCormick F. Abo A. EMBO J. 1995; 14: 1970-1978Crossref PubMed Scopus (303) Google Scholar, 31Knaus U.G. Morris S. Dong H. Chernoff J. Bokoch G.M. Science. 1995; 269: 221-223Crossref PubMed Scopus (357) Google Scholar). all STE20-like kinases identified thus SPS1 in that they N-terminal kinase domain and a domain and not a domain. have that mammalian STE20 are able to activate mammalian MAPK can specifically activate the JNK and p38 pathways not the pathway. and can also activate JNK not p38 or the ERKs (35Hu M.C.-T. Qiu W.R. Wang X. Meyer C.F. Tan T.H. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (194) Google Scholar, R.M. Blenis J. Oncogene. 1997; 14: 653-659Crossref PubMed Scopus (66) Google Scholar, C.M. Kehrl J.H. I. P. Avruch J. Zon L.I. Woodgett J.R. Force T. Kyriakis J.M. Nature. 1995; PubMed Scopus Google Scholar). kinases not the and JNK activation is thus to be In this the and of a human STE20 with kinase activity mst-3 belongs to the of STE20-like kinases with N-terminal kinase domain and a domain. mst-3 and protein is ubiquitously mst-3 kinase prefers to as a and can use both ATP and GTP as phosphate donors. mst-3 high activity and does not activate of the known mammalian MAPK In mst-3 kinase activity is by autophosphorylation. that mst-3 is in the on conserved of and in the kinase domain STE20 and and used to a human cell and the with S. was kinase domain of novel kinases related to the yeast STE20 protein. STE20-like was used to of a human cell of F. and mst-3 upstream of the and both was and used to of a human cell of R. with and in all upstream of the cloned was used as a for to for of J. was used to mst-3 N-terminal of amino The mst-3 was and cloned for mammalian was used to a to mst-3 for mst-3 was by to and in the mst-3 protein of was used for and was used for of of protein. human of L. was with and The was used to the to Cell as (1Chen R.-H. Sarnecki C. Blenis J. Mol. Cell. Biol. 1992; 12: 915-927Crossref PubMed Google Scholar). on or to for at least 1 in and with or a for 1 to was with or mst-3 was as (1Chen R.-H. Sarnecki C. Blenis J. Mol. Cell. Biol. 1992; 12: 915-927Crossref PubMed Google Scholar) with a with for 1 with protein with The with 1 with with high and with 1 with 1 of 1 and 1 for mst-3 kinase activity as (1Chen R.-H. Sarnecki C. Blenis J. Mol. Cell. Biol. 1992; 12: 915-927Crossref PubMed Google Scholar). However, kinase in the of or for at to assay in a with 4 of as a The was by the of of and by The gel was and was a with cells with in 4 of in of as and with of or with of of for 1 at 4 by with protein for 1 at 4 in and in of and and as as The on the gel was and the by cell used in with cells in with and cells at a of 4 cells 1 and for 4 the J. T. Scholar). of of was used cells at cells and the with to myelin basic protein and and as (2Ammerer G. Curr. Opin. Cell Biol. 1994; 5: 254-260Google Scholar). cells on placed in and as in PBS, for with in for in in for and with for 1 in PBS, The was used at a for with PBS, with and a to conserved in the kinase of yeast STE20 and used to a a cell was used as a to a cell of F. to be a was and a upstream of the a mst-3 was and used to a cell was that a upstream of the and in However, was identified The of amino with a of mst-3 all of serine/threonine kinases A.M. T. Science. PubMed Scopus Google Scholar). the to to kinases, mst-3 was identified as a member of the STE20 family of protein kinases. mst-3 of N-terminal kinase domain and a acid domain. The of mst-3 closely the of the STE20 family of protein kinases mst-3 is related to the recently published kinase (38Pombo C.M. Bonventre J.V. Molnar A. Kyriakis J. Force T. EMBO J. 1996; 15: 4537-4546Crossref PubMed Scopus (133) Google Scholar), with 88% in the kinase domain and amino acid The mst-3 kinase domain is related to the kinase of (37Creasy C.L. Chernoff J. J. Biol. Chem. 1995; 270: 21695-21700Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, L.K. Wang H.C. Erikson R.L. Proc. Natl. Acad. Sci U. S. A. 1996; 93: 10099-10104Crossref PubMed Scopus (141) Google Scholar), C.L. Chernoff J. 1995; PubMed Scopus Google Scholar, L.K. Wang H.C. Erikson R.L. Proc. Natl. Acad. Sci U. S. A. 1996; 93: 10099-10104Crossref PubMed Scopus (141) Google Scholar), GCK (33Katz P. Whalen G. Kehrl J.H. J. Biol. Chem. 1994; 269: 16802-16809Abstract Full Text PDF PubMed Google Scholar), (35Hu M.C.-T. Qiu W.R. Wang X. Meyer C.F. Tan T.H. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (194) Google Scholar), SPS1 (27Friesen H. Lunz R. Doyle S. Segall J. Genes Dev. 1994; 8: 2162-2175Crossref PubMed Scopus (108) Google Scholar), hPAK65 (30Martin G.A. Bollag G. McCormick F. Abo A. EMBO J. 1995; 14: 1970-1978Crossref PubMed Scopus (303) Google Scholar), and STE20 (23Leberer E. Dignard D. Harcus D. Thomas D.Y. Whiteway M. EMBO J. 1992; 11: 4815-4824Crossref PubMed Scopus (347) Google Scholar, 24Ramer S.W. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 452-456Crossref PubMed Scopus (169) Google Scholar) with amino acid The mst-3 domain does not a and does not have several human that the mst-3 transcript is ubiquitously high a upstream of the kinase domain as a a was that is at in and of the was a human not The of mst-3 protein was a of the mst-3 protein a protein of in all human mouse and monkey cells The be to or with the the of the not An mst-3 that a acid at the be as a on cells 4 also a protein of the cell 4 that was with 4 or 4 for the was used to mst-3 to vitro kinase activity the was and as substrates not However, mst-3 and at at the of mst-3 was also suggesting that mst-3 and the of a protein in the that phosphorylates mst-3 be is the was in and in mst-3 cell and was was with the the mst-3 mst-3 activity was Mn2+ or Mg2+ in vitro kinase wild-type or was and and or a cell with or with to vitro kinase in the of or and 1 ATP as a Interestingly, mst-3 activity was found to be in the of Mn2+ with the activity in the of was with activity can be suggesting that the activity is not to and The intracellular of Mn2+ is in the whereas the Mg2+ is in the M.F. Scholar). able to activate mst-3 at physiological mst-3 activity was a of Mg2+ and Mn2+ mst-3 was and in vitro kinase the Mn2+ and Mg2+ Mn2+ at activate mst-3 to Mg2+ at mst-3 activity at to The Mn2+ at which a in mst-3 activity was was activation was at 1 Mn2+ was and mst-3 activity Mg2+ not activate mst-3 at mst-3 activity was with and was not that Mn2+ a in mst-3 physiological acid of in vitro and that mst-3 is a serine/threonine protein on and and on also on of protein kinases GTP as well as ATP as a phosphate the mst-3 phosphate of GTP used to with ATP in the GTP with ATP the of GTP as a phosphate in vitro kinase in the of or and mst-3 was with cells with or with vitro kinase in the of of or at specific mst-3 use GTP and ATP as phosphate ATP was and As and a of the of mst-3 in and was to be mst-3. mst-3 lead to mst-3 activity was a with ATP and Mn2+ or In the of Mn2+ not this in a in kinase activity with that not and a in not as a not suggesting that mst-3 high was not activity was also by cells with a of and serine/threonine not by with a suggesting that mst-3 is by the of mst-3 in signaling and with several MAP kinases JNK, p38, and In neither nor activated or JNK, p38, and of for their to activate or mst-3 activity in cell In of the high activity of all in with to kinase assay or of stress and which have been to activate the closely related kinase (38Pombo C.M. Bonventre J.V. Molnar A. Kyriakis J. Force T. EMBO J. 1996; 15: 4537-4546Crossref PubMed Scopus (133) Google Scholar), not activate of mst-3 activity in cell growth growth growth in a the of with and the protein was with and was in the was in the cells or cells with We have cloned and characterized a novel human member of the growing family of STE20-related protein kinases. the of a physiological this kinase was named mst-3. Based on its structure, mst-3 belongs to the of STE20-like kinases with N-terminal and a domain. mst-3 is most closely related to the recently identified kinase (38Pombo C.M. Bonventre J.V. Molnar A. Kyriakis J. Force T. EMBO J. 1996; 15: 4537-4546Crossref PubMed Scopus (133) Google Scholar) with amino acid of and in the kinase domain and protein However, mst-3 is not in the as and distinct kinase activity of mst-3 that is ubiquitously in all cell lines human monkey and mouse An in vitro kinase assay as well as mst-3 was and that mst-3 The to phosphorylate and exogenous substrates is in the of Mn2+ Mg2+ and is with the kinase activity of kinases. the ATP used in in vitro kinase Mn2+ is also the most divalent cation of for the kinases of the M. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and growth factor G.L. L. S. Nature. PubMed Scopus Google Scholar) Interestingly, a protein kinase with also a for Mn2+ C. 1992; PubMed Scopus Google Scholar). the of Mn2+ the of Mg2+ on mst-3 Mn2+ the of mst-3 activity whereas a of Mn2+ and of the ATP is as and the Mn2+ does not lead to a M. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of mst-3 activity in response to Mn2+ and thus that formation of the of Mn2+ to a distinct on mst-3 be required for kinase The of Mn2+ be to physiological the of in the of the intracellular Mg2+ like the ATP in the The that the Mn2+ required for mst-3 activation was of lower the Mg2+ at the ATP used in with the that Mn2+ activate mst-3 to Mg2+ that Mn2+ is a physiological of mst-3. However, the physiological of Mn2+ in mst-3 activation is and of protein kinases that are conserved among members of the serine/threonine kinases or the kinases A.M. T. Science. PubMed Scopus Google Scholar). The most of amino acid in kinases whereas serine/threonine kinases In this the kinases whereas the in as a serine/threonine kinase A.M. T. Science. PubMed Scopus Google Scholar). acid that mst-3 is a serine/threonine kinase mst-3 of the signaling pathways or a novel pathway, to or its activity several mitogenic and stress to the high activity of and kinase kinase assay not be in the and thus all kinase assay However, stimulus of mst-3 activity was identified. activity does not change upon growth to or growth agents such as growth growth and The stress and UV exposure not mst-3 activity in the cell lines mst-3 not be activated by stress to or which have been to activate the closely related kinase (38Pombo C.M. Bonventre J.V. Molnar A. Kyriakis J. Force T. EMBO J. 1996; 15: 4537-4546Crossref PubMed Scopus (133) Google Scholar). for this is that mst-3 and have distinct that Mn2+ is not a for SOK-1, and that their closely related mst-3 and are and are part of signaling mst-3 activity does not change upon the intracellular or We also serine/threonine and on mst-3 activation of mst-3 activity with suggesting that mst-3 activation also be through and that mst-3 can not In this mst-3 which been to C.L. Chernoff J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). mst-3 wild-type or protein not activate or of the MAPK cascades JNK, p38, nor kinase in not suggesting that mst-3 is part of a novel signaling pathway. mst-3 activity be not for the activation of the pathways tested. mst-3 is not a kinase, mst-3 like and is activated by autophosphorylation. on cell and the a in the of ATP by with in a in mst-3 mst-3 high is not a this the physiological of mst-3 is as is the of its similarity to of have to the of physiological substrates and We members of the Blenis for and R. for with We also F. and R. for the cell and the cell