Mps1 Activation Loop Autophosphorylation Enhances Kinase Activity

Abstract

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...

Autophosphorylation-dependent activation of human Mps1 is required for the spindle checkpoint

“…The efficient autophosphorylation of GST-Mps1 could be due to GST fusion proteins being intrinsically dimeric. During the course of our work, Mattison et al (23) showed that bacterially expressed Mps1 also underwent autophosphorylation at T676.…”

“…It is, however, unclear how Mps1 is activated during mitosis. Very recently, bacterially expressed human Mps1 has been shown to undergo autophosphorylation at an activation loop residue, T676 (23). The Mps1 T676A mutant has much lower kinase activity in vitro and, when overexpressed in human cells, fails to cause centrosome overduplication (23).…”

The Composition, Functions, and Regulation of the Budding Yeast Kinetochore

“…Mps1 has also been implicated in phosphorylation of Mad1 and Ndc80 (Hardwick et al 1996;Kemmler et al 2009), but the functions of these phosphorylation events have not been identified. Mps1 autophosphorylation is required for its activation and kinetochore localization in some organisms (Kang et al 2007;Mattison et al 2007;Xu et al 2009), although this has not been studied as much in budding yeast. It is unclear how many checkpoint substrates exist for Mps1, but its ability to halt the cell cycle in a kinetochore-independent manner suggests it likely has soluble targets downstream of the kinetochore that remain to be identified (Hardwick et al 1996;Fraschini et al 2001a;Poddar et al 2004).…”

Characterization of the Cellular and Antitumor Effects of MPI-0479605, a Small-Molecule Inhibitor of the Mitotic Kinase Mps1

“…Its expression and activity are regulated in a cell-cycle-dependent manner with maximal levels arising during mitosis. The localization of Mps1 to kinetochores during mitosis facilitates the trans-autophosphorylation and activation of Mps1 (2,3). Mps1 has multiple functions during mitosis.…”