Multi-kinase phosphorylation of the APC/C activator Cdh1 revealed by mass spectrometry

被引:30
作者
Hall, MC [1 ]
Warren, EN [1 ]
Borchers, CH [1 ]
机构
[1] Univ N Carolina, Sch Med, Dept Biochem & Biophys, Chapel Hill, NC 27599 USA
关键词
Cdh1; anaphase-promoting complex; cyclosome; phosphorylation; ubiquitin-dependent proteolysis; cyclin-dependent kinase; mass spectrometry;
D O I
10.4161/cc.3.10.1153
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Cdh1 contributes to proper exit from mitosis and maintenance of G(1) phase in eukaryotic cells by activating a large ubiquitin ligase called the anaphase-promoting complex, or cyclosome (APC/C). At the end of G(1), APC/C-Cdh1 is inhibited by cyclin-dependent kinase (CDK) phosphorylation of Cdh1. The specific Cdh1 phosphorylation sites used to regulate APC/C-Cdh1 activity have not been directly identified. Here, we used a mass spectrometric approach to identify the in vivo phosphorylation sites on yeast Cdh1. Surprisingly, in addition to several expected CDK phosphorylation sites, we discovered numerous non-CDK phosphorylation sites. In total, at least 19 serine and threonine residues on Cdh1 are phosphorylated in vivo. Seventeen of these sites are located in the N-terminal half of Cdh1, outside the highly conserved WD40 repeats. The pattern of phosphorylation was the same when Cdh1 was purified from yeast cultures arrested in S, early M and late M. Mutation of CDK consensus sequences eliminated detectable phosphorylation at many of the non-CDK sites. In contrast, mutation of non-CDK sites had no significant effect on CDK phosphorylation. We conclude that phosphorylation of CDK sites promotes the subsequent recognition of Cdh1 by at least one additional kinase. The function of non-CDK phosphorylation may differ from CDK phosphorylation because mutation of non-CDK sites did not result in constitutive activation of APC and consequent cell cycle arrest. These results suggest that phosphoregulation of APC/C-Cdh1 activity is much more complex than previously thought.
引用
收藏
页码:1278 / 1284
页数:7
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