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...

In many organisms, including yeasts and humans, meiotic recombination is initiated preferentially at a limited number of sites in the genome referred to as recombination hotspots. Predicting precisely the location of most hotspots has remained elusive. In this study, we tested the hypothesis that hotspots can result from multiple different sequence motifs. We devised a method to rapidly screen many short random oligonucleotide sequences for hotspot activity in the fission yeast Schizosaccharomyces pombe and produced a library of $500 unique 15-and 30-bp sequences containing hotspots. The frequency of hotspots found suggests that there may be a relatively large number of different sequence motifs that produce hotspots. Within our sequence library, we found many shorter 6-to 10-bp motifs that occurred multiple times, many of which produced hotspots when reconstructed in vivo. On the basis of sequence similarity, we were able to group those hotspots into five different sequence families. At least one of the novel hotspots we found appears to be a target for a transcription factor, as it requires that factor for its hotspot activity. We propose that many hotspots in S. pombe, and perhaps other organisms, result from simple sequence motifs, some of which are identified here.

The MPS1 gene from Saccharomyces cerevisiae encodes an essential protein kinase required for spindle pole body (SPB) duplication and for the mitotic spindle assembly checkpoint. Cells with the mps1-1 mutation fail early in SPB duplication and proceed through monopolar mitosis with lethal consequences. We identified CDC37 as a multicopy suppressor of mps1-1 temperature-sensitive growth. Suppression is allele specific, and synthetic lethal interactions occur between mps1 and cdc37 alleles. We examined the cdc37-1 phenotype for defects related to the SPB cycle. The cdc37-1 temperature-sensitive allele causes unbudded, G1 arrest at Start (Reed, S.I. 1980. Genetics. 95: 561–577). Reciprocal shifts demonstrate that cdc37-1 arrest is interdependent with α-factor arrest but is not a normal Start arrest. Although the cells are responsive to α-factor at the arrest, SPB duplication is uncoupled from other aspects of G1 progression and proceeds past the satellite-bearing SPB stage normally seen at Start. Electron microscopy reveals side-by-side SPBs at cdc37-1 arrest. The outer plaque of one SPB is missing or reduced, while the other is normal. Using the mps2-1 mutation to distinguish between the SPBs, we find that the outer plaque defect is specific to the new SPB. This phenotype may arise in part from reduced Mps1p function: although Mps1p protein levels are unaffected by the cdc37-1 mutation, kinase activity is markedly reduced. These data demonstrate a requirement for CDC37 in SPB duplication and suggest a role for this gene in G1 control. CDC37 may provide a chaperone function that promotes the activity of protein kinases.

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.

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