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J. Biol. Chem., Vol. 282, Issue 42, 30553-30561, October 19, 2007

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Mps1 Activation Loop Autophosphorylation Enhances Kinase Activity^*

Christopher P. Mattison, William M. Old, Estelle Steiner¹, Brenda J. Huneycutt², Katheryn A. Resing, Natalie G. Ahn^¶, and Mark Winey³

From the Molecular Cellular, and Developmental Biology, the Department of Chemistry and Biochemistry, and the ^¶Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309

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.

Received for publication, August 22, 2007

^* This work was supported by National Institutes of Health Grant GM51312 (to M. W.) and was aided by a Fellowship Award from the Colorado Tobacco Research Program (to C. P. M.) and Department of Defense Fellowship Award DAMD17-03-1-0404 (to C. P. M.). This work was also supported by National Institutes of Health Grant R01 CA87648 (to K. A. R.) and by National Institutes of Health Training Grant GM07135 (to B. J. H. and E. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of Biology, Niagara University, New York, NY, 14109.

² Present address: Finnegan, Henderson, Farabow, Garrett & Dunner, LLP, 901 New York Ave., NW, Washington, D.C., 20001.

³ To whom correspondence should be addressed. E-mail: mark.winey{at}colorado.edu.

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