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

doi:10.1074/jbc.m707063200

Christopher P Mattison, William M Old + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m707063200

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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 Thr(675), Thr(676), and Thr(686), but phosphatase-treated and reactivated enzyme was only phosphorylated on Thr(676). Mutation of Thr(676) in hMps1 or the corresponding Thr(591) 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.

Highlights

Hyperphosphorylation of human Mps1 (hMps1) Is Not Required for Activity— We isolated full-length recombinant hMps1 fused to both N-terminal GST and C-terminal hemagglutinin tags from E. coli to study the role of hMps1 autophosphorylation
The kinase dead enzyme isolated in the same manner showed only fast migrating forms of the kinase as seen previously (2, 3), strongly suggesting gel mobility retardation was due to autophosphorylation (Fig. 1A)
We found that hMps1-KD was phosphorylated, indicating that hMps1 autophosphorylation can occur through an intermolecular reaction (Fig. 1C)

Summary

Introduction

Mutation of Thr676 or Thr686 Reduces hMps1 Kinase Activity—Our mapping data suggested that Thr676 was an important site of autophosphorylation required for kinase activity, but it is possible that phosphorylation of Thr675 plays a role. Mutation of the T676A residue reduced kinase activity, causing a 7-fold reduction in MBP phosphorylation rate compared with wild type (Fig. 4).

Results

Conclusion