Hall Mark C, Warren Erin N, Borchers Christoph H
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.
Cell Cycle. 2004 Oct;3(10):1278-84. doi: 10.4161/cc.3.10.1153. Epub 2004 Oct 6.
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 nonCDK 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 nonCDK sites. In contrast, mutation of nonCDK 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 nonCDK phosphorylation may differ from CDK phosphorylation because mutation of nonCDK 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.
Cdh1通过激活一种名为后期促进复合物或细胞周期体（APC/C）的大型泛素连接酶，有助于真核细胞正确退出有丝分裂并维持G1期。在G1期末期，Cdh1被细胞周期蛋白依赖性激酶（CDK）磷酸化而抑制APC/C（Cdh1）。用于调节APC/C（Cdh1）活性的特定Cdh1磷酸化位点尚未得到直接鉴定。在此，我们采用质谱方法鉴定酵母Cdh1的体内磷酸化位点。令人惊讶的是，除了几个预期的CDK磷酸化位点外，我们还发现了许多非CDK磷酸化位点。总共，Cdh1上至少有19个丝氨酸和苏氨酸残基在体内被磷酸化。其中17个位点位于Cdh1的N端一半，在高度保守的WD40重复序列之外。当从停滞在S期、早期M期和晚期M期的酵母培养物中纯化Cdh1时，磷酸化模式相同。CDK共有序列的突变消除了许多非CDK位点的可检测磷酸化。相反，非CDK位点的突变对CDK磷酸化没有显著影响。我们得出结论，CDK位点的磷酸化促进了至少一种额外激酶对Cdh1的后续识别。非CDK磷酸化的功能可能与CDK磷酸化不同，因为非CDK位点的突变不会导致APC的组成型激活和随之而来的细胞周期停滞。这些结果表明，APC/C（Cdh1）活性的磷酸化调节比以前认为的要复杂得多。
