Mps1 activation loop autophosphorylation enhances kinase activity

Christopher P. Mattison, William M. Old, Estelle Steiner, Brenda J. Huneycutt, Katheryn A. Resing, Natalie G. Ahn, Mark Winey

Research output: Contribution to journalArticle

61 Scopus citations

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. Mass spectrometry analysis of de novo synthesized enzyme in Escherichia coli identified autophosphorylation sites at residues Thr675, Thr 676, and Thr686, but phosphatase-treated and reactivated enzyme was only phosphorylated on Thr676. Mutation of Thr 676 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.

Original languageEnglish (US)
Pages (from-to)30553-30561
Number of pages9
JournalJournal of Biological Chemistry
Volume282
Issue number42
DOIs
StatePublished - Oct 19 2007
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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  • Cite this

    Mattison, C. P., Old, W. M., Steiner, E., Huneycutt, B. J., Resing, K. A., Ahn, N. G., & Winey, M. (2007). Mps1 activation loop autophosphorylation enhances kinase activity. Journal of Biological Chemistry, 282(42), 30553-30561. https://doi.org/10.1074/jbc.M707063200