Accurate chromosome segregation depends on proper assembly and function of the kinetochore and the mitotic spindle. In the budding yeast, Saccharomyces cerevisiae, the highly conserved protein kinase Mps1 has well-characterized roles in spindle pole body (SPB, yeast centrosome equivalent) duplication and the mitotic checkpoint. However, an additional role for Mps1 is suggested by phenotypes of MPS1 mutations that include genetic interactions with kinetochore mutations and meiotic chromosome segregation defects and also by the localization of Mps1 at the kinetochore, the latter being independent of checkpoint activation. We have developed a new MPS1 allele, mps1-as1, that renders the kinase specifically sensitive to a cell-permeable ATP analog inhibitor, allowing us to perform high-resolution execution point experiments that identify a novel role for Mps1 subsequent to SPB duplication. We demonstrate, by using both fixed- and live-cell fluoresence techniques, that cells lacking Mps1 function show severe defects in mitotic spindle formation, sister kinetochore positioning at metaphase, and chromosome segregation during anaphase. Taken together, our experiments are consistent with an important role for Mps1 at the kinetochore in mitotic spindle assembly and function.

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. The mono-polar spindle-1 (MPS1) gene was identified in Saccharomyces cerevisiae in a screen for mitotic spindle defective mutants (1) and was subsequently shown to encode an essential dual specificity, autophosphorylating protein kinase (2, 3). MPS1 is conserved (4, 5) 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 (6 -13). Recent evidence also suggests that human Mps1 (hMps1) 4 is involved in a DNA damage checkpoint, functioning upstream of Chk2 (14). In S. cerevisiae, duplication of spindle pole bodies requires MPS1 at multiple steps (reviewed in Ref. 15). Similarly, centrosome duplication in mice and humans has been shown to require Mps1 (5, 16), but there is conflicting data on this point (9, 10). Roles for Mps1 in development and in the response to stress have been demonstrated in yeast, Drosophila, and Zebrafish (11,(17)(18)(19)(20)(21).Highly controlled regulation of Mps1 kinase activity is essential for growth. For example, overexpression of MPS1 in S. cerevisiae results in inappropriate checkpoint activation (12, 22), whereas too little Mps1 activity is lethal (2). Mps1 is regulated at both the transcription level, in response to cell cycle progression and cell differentiation (3,4,11,23), and by changes in protein stability (5, 24, 25). The activity of hMps1 rises to an extent greater than what can be explained by the increase in protein level alone during the G 2 /M transition (9, 23). Furthermore, checkpoint activation with nocodazole treatment of cells results in a ϳ30-fold increase in hMps1 activity, whereas the protein level remains similar to untreated mitotic cells (9). Increased hMps1 activity is correlated with more slowly electrophoretically migrating forms thought to be the result of phosphorylation (7, 9). These observations suggest that Mps1 is also regulated by changes in phosphorylation state.Many kinases are activated when phosphorylated within the activation loop (reviewed in Refs. 26 -28). In some cases, this can be catalyzed by autophosphorylation. For example, ERK8 autophosphorylates in vitro on both Thr and Tyr residues for activation, and this is also the likely method for activation in vivo (29). Although it is not clear which other kinases or regulatory s...

Duplication of the Saccharomyces cerevisiae spindle pole body (SPB) once per cell cycle is essential for bipolar spindle formation and accurate chromosome segregation during mitosis. We have investigated the role that the major yeast cyclin-dependent kinase Cdc28/Cdk1 plays in assembly of a core SPB component, Spc42, to better understand how SPB duplication is coordinated with cell cycle progression. Cdc28 is required for SPB duplication and Spc42 assembly, and we found that Cdc28 directly phosphorylates Spc42 to promote its assembly into the SPB. The Mps1 kinase, previously shown to regulate Spc42 phosphorylation and assembly, is also a Cdc28 substrate, and Cdc28 phosphorylation of Mps1 is needed to maintain wild-type levels of Mps1 in cells. Analysis of nonphosphorylatable mutants in SPC42 and MPS1 indicates that direct Spc42 phosphorylation and indirect regulation of Spc42 through Mps1 are two overlapping pathways by which Cdc28 regulates Spc42 assembly and SPB duplication during the cell cycle.

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The yeast spindle pole body (SPB) component Spc110p (Nuf1p) undergoes specific serine/threonine phosphorylation as the mitotic spindle apparatus forms, and this phosphorylation persists until cells enter anaphase. We demonstrate that the dual-specificity kinase Mps1p is essential for the mitosis-specific phosphorylation of Spc110p in vivo and that Mps1p phosphorylates Spc110p in vitro. Phosphopeptides generated by proteolytic cleavage were identified and sequenced by mass spectrometry. Ser 60 , Thr 64 , and Thr 68 are the major sites in Spc110p phosphorylated by Mps1p in vitro, and alanine substitution at these sites abolishes the mitosis-specific isoform in vivo. This is the first time that phosphorylation sites of an SPB component have been determined, and these are the first sites of Mps1p phosphorylation identified. Alanine substitution for any one of these phosphorylated residues, in conjunction with an alanine substitution at residue Ser 36 , is lethal in combination with alleles of SPC97, which encodes a component of the Tub4p complex. Consistent with a specific dysfunction for the alanine substitution mutations, simultaneous mutation of all four serine/threonine residues to aspartate does not confer any defect. Sites of Mps1p phosphorylation and Ser 36 are located within the Nterminal globular domain of Spc110p, which resides at the inner plaque of the SPB and binds the Tub4p complex.