Mps1 Activation Loop Autophosphorylation Enhances Kinase Activity

The Mps1 protein kinase is required for proper assembly of the mitotic spindle, checkpoint signaling, and several other aspects of cell growth and differentiation. Mps1 regulation is mediated by cell cycle-dependent changes in transcription and protein level. There is also a strong correlation between hyperphosphorylated mitotic forms of Mps1 and increased kinase activity. We investigated the role that autophosphorylation plays in regulating human Mps1 (hMps1) protein kinase activity. Here we report that hyperphosphorylated hMps1 forms are not the only active forms of the kinase. However, autophosphorylation of hMps1 within the activation loop is required for full activity in vitro. Mass spectrometry analysis of de novo synthesized enzyme in Escherichia coli identified autophosphorylation sites at residues Thr675, Thr676, and Thr686, but phosphatase-treated and reactivated enzyme was only phosphorylated on Thr676. Mutation of Thr676 in hMps1 or the corresponding Thr591 residue within yeast Mps1 reduces kinase activity in vitro. We find that overexpression of an hMps1-T676A mutation inhibits centrosome duplication in RPE1 cells. Likewise, yeast cells harboring mps1-T591A as the sole MPS1 allele are not viable. Our data strongly support the conclusion that site-specific Mps1 autophosphorylation within the activation loop is required for full activity in vitro and function in vivo. The Mps1 protein kinase is required for proper assembly of the mitotic spindle, checkpoint signaling, and several other aspects of cell growth and differentiation. Mps1 regulation is mediated by cell cycle-dependent changes in transcription and protein level. There is also a strong correlation between hyperphosphorylated mitotic forms of Mps1 and increased kinase activity. We investigated the role that autophosphorylation plays in regulating human Mps1 (hMps1) protein kinase activity. Here we report that hyperphosphorylated hMps1 forms are not the only active forms of the kinase. However, autophosphorylation of hMps1 within the activation loop is required for full activity in vitro. Mass spectrometry analysis of de novo synthesized enzyme in Escherichia coli identified autophosphorylation sites at residues Thr675, Thr676, and Thr686, but phosphatase-treated and reactivated enzyme was only phosphorylated on Thr676. Mutation of Thr676 in hMps1 or the corresponding Thr591 residue within yeast Mps1 reduces kinase activity in vitro. We find that overexpression of an hMps1-T676A mutation inhibits centrosome duplication in RPE1 cells. Likewise, yeast cells harboring mps1-T591A as the sole MPS1 allele are not viable. Our data strongly support the conclusion that site-specific Mps1 autophosphorylation within the activation loop is required for full activity in vitro and function in vivo. The mono-polar spindle-1 (MPS1) gene was identified in Saccharomyces cerevisiae in a screen for mitotic spindle defective mutants (1Winey M. Goetsch L. Baum P. Byers B. J. Cell Biol. 1991; 114: 745-754Crossref PubMed Scopus (272) Google Scholar) and was subsequently shown to encode an essential dual specificity, autophosphorylating protein kinase (2Lauze' E. Stoelcker B. Luca F.C. Weiss E. Schutz A. Winey M. EMBO Eur. Mol. Biol. Organ. J. 1995; 14: 1655-1663Crossref PubMed Scopus (132) Google Scholar, 3Poch O. Schwob E. Fraipont F. Camasses A. Bordonne' R. Martin R. Mol. Gen. Genet. 1994; 243: 641-653Crossref PubMed Scopus (26) Google Scholar). MPS1 is conserved (4Mills G.B. Schmandt R. McGill M. Amendola A. Hill M. Jacobs K. May C. Rodricks A. Campbell S. Hogg D. J. Biol. Chem. 1992; 267: 16000-16006Abstract Full Text PDF PubMed Google Scholar, 5Fisk H.A. Winey M. Cell. 2001; 106: 95-104Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar) and is required for a variety of functions during cell growth. The mitotic checkpoint function of Mps1 is conserved among several organisms including yeast, Xenopus laevis, Zebrafish, and humans (6Abrieu A. Magnaghi-Jaulin L. Kahana J.A. Peter M. Castro A. Vigneron S. Lorca T. Cleveland D.W. Labbe J.C. Cell. 2001; 106: 83-93Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 7Liu S.T. Chan G.K. Hittle J.C. Fujii G. Lees E. Yen T.J. Mol. Biol. Cell. 2003; 14: 1638-1651Crossref PubMed Scopus (144) Google Scholar, 8He X. Jones M.H. Winey M. Sazer S. J. Cell Sci. 1998; 111: 1635-1647Crossref PubMed Google Scholar, 9Stucke V.M. Sillje H.H. Arnaud L. Nigg E.A. EMBO J. 2002; 21: 1723-1732Crossref PubMed Scopus (212) Google Scholar, 10Stucke V.M. Baumann C. Nigg E.A. Chromosoma. 2004; 113: 1-15Crossref PubMed Scopus (69) Google Scholar, 11Poss K.D. Nechiporuk A. Hillam A.M. Johnson S.L. Keating M.T. Development. 2002; 129: 5141-5149Crossref PubMed Google Scholar, 12Weiss E. Winey M. J. Cell Biol. 1996; 132: 111-123Crossref PubMed Scopus (359) Google Scholar, 13Jones M.H. Huneycutt B.J. Pearson C.G. Zhang C. Morgan G. Shokat K. Bloom K. Winey M. Curr. Biol. 2005; 15: 160-165Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Recent evidence also suggests that human Mps1 (hMps1) 4The abbreviations used are: h, human; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; MBP, myelin basic protein; LC, liquid chromatography; MS/MS, tandem mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; ha, hemagglutinin; GFP, green fluorescent protein. is involved in a DNA damage checkpoint, functioning upstream of Chk2 (14Wei J.H. Chou Y.F. Ou Y.H. Yeh Y.H. Tyan S.W. Sun T.P. Shen C.Y. Shieh S.Y. J. Biol. Chem. 2005; 280: 7748-7757Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In S. cerevisiae, duplication of spindle pole bodies requires MPS1 at multiple steps (reviewed in Ref. 15Jaspersen S.L. Winey M. Annu. Rev. Cell Dev. Biol. 2004; 20: 1-28Crossref PubMed Scopus (237) Google Scholar). Similarly, centrosome duplication in mice and humans has been shown to require Mps1 (5Fisk H.A. Winey M. Cell. 2001; 106: 95-104Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 16Fisk H.A. Mattison C.P. Winey M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14875-14880Crossref PubMed Scopus (157) Google Scholar), but there is conflicting data on V.M. Sillje H.H. Arnaud L. Nigg E.A. EMBO J. 2002; 21: 1723-1732Crossref PubMed Scopus (212) Google Scholar, 10Stucke V.M. Baumann C. Nigg E.A. Chromosoma. 2004; 113: 1-15Crossref PubMed Scopus (69) Google Scholar). for Mps1 in and in the to been in yeast, and K.D. Nechiporuk A. Hillam A.M. Johnson S.L. Keating M.T. Development. 2002; 129: 5141-5149Crossref PubMed Google Scholar, Winey M. Mol. Biol. Cell. PubMed Scopus Google Scholar, Curr. Biol. 2005; 15: Full Text Full Text PDF PubMed Scopus Google Scholar, K.D. Keating M.T. 2002; PubMed Scopus Google Scholar, K.D. Nechiporuk A. C. Keating M.T. Dev. 2004; PubMed Scopus Google Scholar, S. U. Curr. Biol. 2004; 14: Full Text Full Text PDF PubMed Scopus Google Scholar). regulation of Mps1 kinase activity is essential for growth. overexpression of MPS1 in S. cerevisiae in checkpoint activation E. Winey M. J. Cell Biol. 1996; 132: 111-123Crossref PubMed Scopus (359) Google Scholar, Weiss E. Luca F.C. Winey M. 1996; PubMed Scopus Google Scholar), Mps1 activity is (2Lauze' E. Stoelcker B. Luca F.C. Weiss E. Schutz A. Winey M. EMBO Eur. Mol. Biol. Organ. J. 1995; 14: 1655-1663Crossref PubMed Scopus (132) Google Scholar). Mps1 is at the transcription in to cell and cell O. Schwob E. Fraipont F. Camasses A. Bordonne' R. Martin R. Mol. Gen. Genet. 1994; 243: 641-653Crossref PubMed Scopus (26) Google Scholar, G.B. Schmandt R. McGill M. Amendola A. Hill M. Jacobs K. May C. Rodricks A. Campbell S. Hogg D. J. Biol. Chem. 1992; 267: 16000-16006Abstract Full Text PDF PubMed Google Scholar, 11Poss K.D. Nechiporuk A. Hillam A.M. Johnson S.L. Keating M.T. Development. 2002; 129: 5141-5149Crossref PubMed Google Scholar, D. C. D. Amendola A. T. J. Schmandt R. G. 1994; Google Scholar), and by changes in protein (5Fisk H.A. Winey M. Cell. 2001; 106: 95-104Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, S.L. Huneycutt B.J. Winey M. Dev. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, S.L. Winey M. PubMed Scopus Google Scholar). The activity of hMps1 to an by the in protein during the V.M. Sillje H.H. Arnaud L. Nigg E.A. EMBO J. 2002; 21: 1723-1732Crossref PubMed Scopus (212) Google Scholar, D. C. D. Amendola A. T. J. Schmandt R. G. 1994; Google Scholar). checkpoint activation of cells in a in hMps1 the protein to mitotic cells V.M. Sillje H.H. Arnaud L. Nigg E.A. EMBO J. 2002; 21: 1723-1732Crossref PubMed Scopus (212) Google Scholar). hMps1 activity is forms to the of S.T. Chan G.K. Hittle J.C. Fujii G. Lees E. Yen T.J. Mol. Biol. Cell. 2003; 14: 1638-1651Crossref PubMed Scopus (144) Google Scholar, 9Stucke V.M. Sillje H.H. Arnaud L. Nigg E.A. EMBO J. 2002; 21: 1723-1732Crossref PubMed Scopus (212) Google Scholar). that Mps1 is also by changes in are phosphorylated within the activation loop (reviewed in M. D. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Annu. Rev. Cell Dev. Biol. PubMed Scopus Google Scholar, 1998; PubMed Google Scholar). In by in vitro on and residues for and is also the for activation in M. S. P. J. PubMed Scopus Google Scholar). is not other or a role in Mps1 is that Mps1 in vitro (2Lauze' E. Stoelcker B. Luca F.C. Weiss E. Schutz A. Winey M. EMBO Eur. Mol. Biol. Organ. J. 1995; 14: 1655-1663Crossref PubMed Scopus (132) Google Scholar, G.B. Schmandt R. McGill M. Amendola A. Hill M. Jacobs K. May C. Rodricks A. Campbell S. Hogg D. J. Biol. Chem. 1992; 267: 16000-16006Abstract Full Text PDF PubMed Google Scholar, 10Stucke V.M. Baumann C. Nigg E.A. Chromosoma. 2004; 113: 1-15Crossref PubMed Scopus (69) Google Scholar, D. C. D. Amendola A. T. J. Schmandt R. G. 1994; Google Scholar, K. L. T. J.C. Mol. Cell Biol. 1992; PubMed Scopus Google Scholar, T. Google Scholar, Winey M. Mol. Biol. Cell. 1998; PubMed Scopus Google Scholar). and of a active hMps1 Escherichia coli that Thr676 in the activation loop is a for autophosphorylation T. Google Scholar). In we to and the role of autophosphorylation in Mps1 activity. We find that yeast and human Mps1 in the activation loop at a conserved Mutation of residue in human and the corresponding Thr591 in kinase activity in vitro and inhibits function in vivo. that Mps1 autophosphorylation is essential for and of and of protein been H.A. Mattison C.P. Winey M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14875-14880Crossref PubMed Scopus (157) Google Scholar). The of not kinase of hMps1 not kinase activity. We that E. coli has activity of myelin basic protein and for is the activity of the yeast enzyme yeast, we to for myelin basic protein and for by DNA and and the to mutants by of hMps1 in the H.A. Mattison C.P. Winey M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14875-14880Crossref PubMed Scopus (157) Google Scholar) and of the by and the in a to and and cells in and of and hMps1 in was the H.A. Mattison C.P. Winey M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14875-14880Crossref PubMed Scopus (157) Google Scholar). DNA was and cells and kinase of hMps1 in kinase MBP, for at hMps1 to was in the of of at for h, by in to the and a in kinase kinase activity. kinase the and to to for in and kinase activity. Mps1 protein was on and to in and the or and or the the or or the the was as in Ref. 16Fisk H.A. Mattison C.P. Winey M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14875-14880Crossref PubMed Scopus (157) Google the The for centrosome the of in at cells for by Mass E. coli was at for h, at in the for h, and at the a mass a a for was and was was used to the a at in B. of the was the and the and was was required for in of at a of a to the a The was at a of to in in and for The was by in B. by by a to the 2005; PubMed Scopus Google Scholar). In the by in an to a for the a mass of to and to the of the was a of a in was for and mass mass of was and used as an for of a a and for that on the a data of including the of the mass was and was to and at and for and of Mps1 was a in Ref. E. Stoelcker B. Luca F.C. Weiss E. Schutz A. Winey M. EMBO Eur. Mol. Biol. Organ. J. 1995; 14: 1655-1663Crossref PubMed Scopus (132) Google Scholar) in yeast cells a was cell by to glutathione The on an and to and the protein was the C. L. P. 1992; Google Scholar). and and by The in and to the the was and the was in The was and a for of hMps1 for hMps1 to and E. coli to the role of hMps1 multiple of in and are and the kinase was phosphorylated and is changes in hMps1 in mitotic cells S.T. Chan G.K. Hittle J.C. Fujii G. Lees E. Yen T.J. Mol. Biol. Cell. 2003; 14: 1638-1651Crossref PubMed Scopus (144) Google Scholar, 9Stucke V.M. Sillje H.H. Arnaud L. Nigg E.A. EMBO J. 2002; 21: 1723-1732Crossref PubMed Scopus (212) Google Scholar, 10Stucke V.M. Baumann C. Nigg E.A. Chromosoma. 2004; 113: 1-15Crossref PubMed Scopus (69) Google Scholar). In the kinase enzyme in the only forms of the kinase as (2Lauze' E. Stoelcker B. Luca F.C. Weiss E. Schutz A. Winey M. EMBO Eur. Mol. Biol. Organ. J. 1995; 14: 1655-1663Crossref PubMed Scopus (132) Google Scholar, 3Poch O. Schwob E. Fraipont F. Camasses A. Bordonne' R. Martin R. Mol. Gen. Genet. 1994; 243: 641-653Crossref PubMed Scopus (26) Google Scholar), strongly was to autophosphorylation that the of hMps1 was to we hMps1 increased and a to that of the kinase protein also and hMps1 E. coli autophosphorylation in we kinase hMps1 active We that was that hMps1 autophosphorylation an In the kinase protein was phosphorylated a of of that a residue was We the of by hMps1 that of hMps1 to the of autophosphorylation on hMps1 kinase activity. phosphatase-treated hMps1 kinase activity the hMps1 and for the of the kinase phosphatase-treated hMps1 in the and activity of and and data not hMps1 autophosphorylation not required for activity and other in vivo. phosphorylated residues for activity by the or phosphorylated by hMps1 at sites not by the or we We to by hMps1 autophosphorylation sites and for kinase activity. hMps1 on Thr675, Thr676, and within the kinase activity of phosphatase-treated hMps1 was that of the autophosphorylation was for as has been V.M. Baumann C. Nigg E.A. Chromosoma. 2004; 113: 1-15Crossref PubMed Scopus (69) Google Scholar). we autophosphorylation sites on E. and by We used a for is in Ref. 2005; PubMed Scopus Google we of the protein E. of residues in the activation loop between kinase and are to for activation of several M. D. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), and we analysis on autophosphorylation sites within for the of of hMps1 in the of that the activation residues and of by mass for the of and subsequently for and The was only in a phosphorylated the phosphorylated of the was of of identified in an a of in the protein but in a phosphorylated that the sites we in the hMps1 protein to phosphorylated residues within at residues Thr675, Thr676, and was by the of to and sites in was at or Thr676 on of corresponding to by and phosphorylated forms of not in of the of on sites also on phosphatase-treated enzyme that been to in the or of autophosphorylation sites in the reactivated enzyme not a but are to autophosphorylation sites required for activity. In the we not sites in of the activation loop on the was in the of and Thr676 was the of The of was that was We to in the in the reactivated The of on to of to at require in or to de novo protein In of the residues within the hMps1 activation loop we find that Thr675, Thr676, and by However, on the was only on de novo synthesized protein E. at and not on reactivated suggests that of residue is to for activity. In of the was at in the de novo and reactivated and Thr676 by on but Thr676 was the Mutation of Thr676 or hMps1 data that Thr676 was an of autophosphorylation required for kinase but is that of also plays a we hMps1 activation loop residues to the on kinase activity. We changes at of the phosphorylated activation loop residues Thr675, Thr676, and we not at or we also changes at sites within the activation loop as for Mutation of the residue kinase a in The mutation not the autophosphorylation only a mutation of in a in autophosphorylation and The and in in kinase activity the of not kinase mutation of the residue kinase activity a in the and a in the autophosphorylation The corresponding residue is conserved in M. D. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, T. J. 1995; PubMed Scopus Google Scholar), and the of suggests that residue to residues for and in the active E.A. J.A. Sci. 1994; PubMed Scopus Google Scholar). we that the in activity by the mutation is to role in a of at Our and mutation analysis of hMps1 that of Thr676 within the hMps1 activation loop is for full kinase activity. hMps1 Thr676 for the of at Thr676 in we mutation of residue has on centrosome We also the and has that overexpression of the kinase protein centrosome duplication H.A. Mattison C.P. Winey M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14875-14880Crossref PubMed Scopus (157) Google Scholar). RPE1 cells or of hMps1 to and in the by the of for The of was by as in Ref. 16Fisk H.A. Mattison C.P. Winey M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14875-14880Crossref PubMed Scopus (157) Google of the kinase or the hMps1 in the of centrosome duplication in of RPE1 a cells or in of cells not but was in cells in of cells of We to by the in a cell the duplication in the cell has been shown to to of the protein for H.A. Mattison C.P. Winey M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14875-14880Crossref PubMed Scopus (157) Google Scholar). we that only of cells the or hMps1 not only of cells hMps1 or not centrosome duplication are in RPE1 cell and data H.A. Mattison C.P. Winey M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14875-14880Crossref PubMed Scopus (157) Google Scholar). The of cells the protein was also We find between cells or and of cells for hMps1 activity on the that cells are to centrosome in the V.M. Sillje H.H. Arnaud L. Nigg E.A. EMBO J. 2002; 21: 1723-1732Crossref PubMed Scopus (212) Google Scholar). to H.A. Mattison C.P. Winey M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14875-14880Crossref PubMed Scopus (157) Google Scholar), we find that overexpression of hMps1 the in of a The activity in and a in In only of cells hMps1-T676A or in vitro kinase activity data and that the active protein centrosome overexpression in but of cells is to cells the kinase allele in only of cells as H.A. Mattison C.P. Winey M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14875-14880Crossref PubMed Scopus (157) Google Scholar). Mps1 Thr591 and for in analysis of hMps1 that Thr676 is an autophosphorylation for activity. We sites in the yeast Mps1 enzyme to of Mps1 activation loop residues is an protein in Ref. E. Stoelcker B. Luca F.C. Weiss E. Schutz A. Winey M. EMBO Eur. Mol. Biol. Organ. J. 1995; 14: 1655-1663Crossref PubMed Scopus (132) Google Scholar) was S. data (2Lauze' E. Stoelcker B. Luca F.C. Weiss E. Schutz A. Winey M. EMBO Eur. Mol. Biol. Organ. J. 1995; 14: 1655-1663Crossref PubMed Scopus (132) Google Scholar), the protein in a kinase that is and analysis of Mps1 in and identified the activation loop in at of Thr591 and not data that yeast and human Mps1 are phosphorylated within the of the activation activation loop was not in residues within the yeast Mps1 activation loop are for we the or Thr676 residue in and residue in a for the essential the or to but harboring the and not in are hMps1 and that the and Mps1 kinase activity. We the kinase activity of and that the protein of and the protein activity In on we to of yeast Mps1 by or of the and a for the essential and the conclusion that autophosphorylation of residue is for activity. the mutation not the that mutation activity cerevisiae Mps1 activation loop autophosphorylation is required for kinase in vitro kinase activity protein not in vitro kinase activity protein in vitro kinase activity protein in vitro kinase activity protein not in a of Mps1 in yeast is to a spindle assembly checkpoint Weiss E. Luca F.C. Winey M. 1996; PubMed Scopus Google Scholar). of the or the of an not a checkpoint suggests that the or not increased activity. We also the and in but in active not However, the mutation only a in kinase activity the by the Our yeast Mps1 activation loop hMps1 and that residues in the human and yeast Mps1 are required for activity. of human Thr676 and yeast Thr591 is essential for full kinase the hMps1 and yeast Mps1 residues a role for activity. are by several as or changes in protein in to activity by a variety of including the of or the of changes in and by upstream or the Mps1 protein is to by changes in transcription and protein that during cell O. Schwob E. Fraipont F. Camasses A. Bordonne' R. Martin R. Mol. Gen. Genet. 1994; 243: 641-653Crossref PubMed Scopus (26) Google Scholar, G.B. Schmandt R. McGill M. Amendola A. Hill M. Jacobs K. May C. Rodricks A. Campbell S. Hogg D. J. Biol. Chem. 1992; 267: 16000-16006Abstract Full Text PDF PubMed Google Scholar, 5Fisk H.A. Winey M. Cell. 2001; 106: 95-104Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 11Poss K.D. Nechiporuk A. Hillam A.M. Johnson S.L. Keating M.T. Development. 2002; 129: 5141-5149Crossref PubMed Google Scholar, D. C. D. Amendola A. T. J. Schmandt R. G. 1994; Google Scholar, S.L. Huneycutt B.J. Winey M. Dev. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, S.L. Winey M. PubMed Scopus Google Scholar), the of Mps1 activity is not has been shown to for Mps1 in Xenopus Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) and kinase activity in vitro V.M. Baumann C. Nigg E.A. Chromosoma. 2004; 113: 1-15Crossref PubMed Scopus (69) Google Scholar), there are or upstream for in Mps1 autophosphorylation are to for Mps1 activity in to cell and checkpoint We report that autophosphorylation of a conserved activation loop residue in human and yeast Mps1 is required for full kinase activity. is to kinase for activation loop residue autophosphorylation is the of activation in M. S. P. J. PubMed Scopus Google Scholar). However, the that of Mps1 in by other protein kinase at within the activation loop in but the to activation in requires by kinase Jones J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). We that the in hMps1 was phosphorylated on de novo and reactivated and data are in a report that a of hMps1 is phosphorylated on Thr676 T. Google Scholar). we also at Thr675, Thr676 was the of on data that a on is phosphorylated by suggests Thr676 as the of autophosphorylation required for increased hMps1 activity. In support of mutation of residue to in hMps1 in vitro activity at and in cells an of hMps1 centrosome duplication Similarly, mutation of the corresponding residue in yeast, also activity and function in In hMps1 the Thr675, was also phosphorylated at a but mutation of residue not centrosome the mutation in an in activity in that of residue kinase activity. There are several for but that we is that mutation of residue to increased autophosphorylation at the Thr676 in enzyme activity. There for at the and Thr676 that at of sites at the other on to the in a phosphorylated and the data is phosphorylated at a at Thr676 is required for proper of that at not the active required for increased activity. However, activity a hMps1 mutation is the mutation not that in the of Thr676 activity. The in hMps1 and in yeast Mps1 also kinase activity. We identified as a of autophosphorylation in de novo synthesized hMps1 is not to find that sites for the kinase sites are at F. Mol. Cell Full Text Full Text PDF PubMed Scopus Google Scholar), and sites in as the of kinase activation R. M. F. M. D. P. F. D. B. C. R. Mol. Cell Full Text Full Text PDF PubMed Scopus Google Scholar). However, the was not hMps1 was to that of is not required for activity. to at of at the residue not to kinase activity. that mutation of in the of the residue is conserved in M. D. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, T. J. 1995; PubMed Scopus Google Scholar), and the corresponding in the of is to for proper of a conserved residue for and in the loop E.A. J.A. Sci. 1994; PubMed Scopus Google Scholar). there is evidence for of the corresponding residue in several other including a kinase to the hMps1 but mutation activity J. U. J. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, M. Mol. Cell Biol. PubMed Scopus Google Scholar, D. X. J. B. EMBO J. PubMed Scopus Google Scholar). the other not the function in and a of active Mps1 to and to a that of residue at is for kinase activity. Our data that autophosphorylation of Thr676 in the hMps1 activation loop for a in activity in vitro the in hMps1 activity during the or mitotic of cells V.M. Sillje H.H. Arnaud L. Nigg E.A. EMBO J. 2002; 21: 1723-1732Crossref PubMed Scopus (212) Google Scholar, D. C. D. Amendola A. T. J. Schmandt R. G. 1994; Google Scholar). has been that hMps1 cells has a activity V.M. Baumann C. Nigg E.A. Chromosoma. 2004; 113: 1-15Crossref PubMed Scopus (69) Google Scholar). of hMps1 in E. coli proper protein In yeast, Mps1 requires the for kinase activity E. Winey M. J. Cell Biol. PubMed Scopus Google Scholar), and is that to hMps1 autophosphorylation by proper during a between the and hMps1 that is required for increased hMps1 activity and centrosome function M. Mattison C.P. Winey M. K. Google Scholar). The kinase of hMps1 has been shown to and kinase activity in vitro V.M. Baumann C. Nigg E.A. Chromosoma. 2004; 113: 1-15Crossref PubMed Scopus (69) Google Scholar). that in to Mps1 regulation by proper hMps1 and kinase activity. multiple forms of regulation for the and site-specific activation of hMps1 at within the cell of Mps1 activation several organisms that residues we identified as for kinase activity are also is conserved in of the Mps1 we and Thr676 is conserved in several Mps1 including Zebrafish, and that in there is only in the yeast enzyme a in autophosphorylation Thr591 in the S. cerevisiae Mps1 Thr676 in that enzyme activation the to is that the of phosphorylated residues in the human and Thr676, to activity. The of and Thr676 on hMps1 enzyme activity suggests a for an to kinase activity. there is a strong correlation between hyperphosphorylated forms of Mps1 and kinase activity (2Lauze' E. Stoelcker B. Luca F.C. Weiss E. Schutz A. Winey M. EMBO Eur. Mol. Biol. Organ. J. 1995; 14: 1655-1663Crossref PubMed Scopus (132) Google Scholar, 7Liu S.T. Chan G.K. Hittle J.C. Fujii G. Lees E. Yen T.J. Mol. Biol. Cell. 2003; 14: 1638-1651Crossref PubMed Scopus (144) Google Scholar, 9Stucke V.M. Sillje H.H. Arnaud L. Nigg E.A. EMBO J. 2002; 21: 1723-1732Crossref PubMed Scopus (212) Google Scholar, Winey M. Mol. Biol. Cell. 1998; PubMed Scopus Google Scholar), data that of the autophosphorylation is for activity. We find that hMps1 activity and subsequently at Thr676 for the of in function as proper or protein or as a for protein There is evidence in yeast that Mps1 kinase activity and is for protein S.L. Huneycutt B.J. Winey M. Dev. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, S.L. Winey M. PubMed Scopus Google Scholar). of the data the of active has that autophosphorylation of conserved activation loop residues within Mps1 is essential for kinase activity. is that as activity in vivo. We and for of

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