Mps1 activation loop autophosphorylation enhances kinase activity
Mps1 activation loop autophosphorylation enhances kinase activity
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DOI：
10.1074/jbc.m707063200
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发表时间：
2007-10-19
影响因子：
4.8
通讯作者：
Winey, Mark
中科院分区：
文献类型：
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作者：
Mattison, Christopher P.;Old, William M.;Winey, Mark
Mps 1蛋白激酶是有丝分裂纺锤体的正确组装、检查点信号传导以及细胞生长和分化的几个其他方面所必需的。mps 1的调节是通过细胞周期依赖性的转录和蛋白水平的变化介导的。在Mps 1的过度磷酸化有丝分裂形式和增加的激酶活性之间也存在强相关性。我们研究了自磷酸化在调节人Mps 1（hMps 1）蛋白激酶活性中的作用。在这里，我们报告说，过度磷酸化的hMps 1形式不是唯一的活性形式的激酶。然而，hMps 1在激活环内的自磷酸化是体外完全活性所必需的。大肠杆菌从头合成酶的质谱分析鉴定了在残基Thr（675）、Thr（676）和Thr（686）处的自磷酸化位点，但磷酸酶处理和再活化的酶仅在Thr（676）处磷酸化。hMps 1中的Thr（676）突变或酵母Mps 1中相应的Thr（591）残基的突变在体外降低激酶活性。我们发现hMps 1-T676 A突变的过表达抑制RPE 1细胞中的中心体复制。同样，携带mps 1-T591 A作为唯一MPS 1等位基因的酵母细胞也无法存活。我们的数据强烈支持的结论是，在激活环内的位点特异性Mps 1自磷酸化是所需的体外和体内的功能的全部活动。
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 hyper-phosphorylated 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.