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首页> 外文期刊>Cell cycle >Multi-kinase phosphorylation of the APC/C activator Cdh1 revealed by mass spectrometry.
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Multi-kinase phosphorylation of the APC/C activator Cdh1 revealed by mass spectrometry.

机译：质谱显示APC / C激活剂Cdh1的多激酶磷酸化。

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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 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.
机译：Cdh1通过激活一种大的泛素连接酶（称为后期促进复合物或环体（APC / C）），有助于从真核细胞中有丝分裂的适当退出和G（1）期的维持。在G（1）的末尾，APC / C（Cdh1）被Cdh1的细胞周期蛋白依赖性激酶（CDK）磷酸化抑制。用于调节APC / C（Cdh1）活性的特定Cdh1磷酸化位点尚未直接鉴定。在这里，我们使用质谱法来鉴定酵母Cdh1上的体内磷酸化位点。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上的至少19个丝氨酸和苏氨酸残基在体内被磷酸化。这些位点中的十七个位于Cdh1的N末端一半，在高度保守的WD40重复序列之外。当从停在S，早期M和晚期M的酵母培养物中纯化Cdh1时，磷酸化的模式是相同的。CDK共有序列的突变消除了许多nonCDK位点可检测到的磷酸化。相反，非CDK位点的突变对CDK磷酸化没有明显影响。我们得出结论，CDK位点的磷酸化促进了至少一种其他激酶对Cdh1的后续识别。 nonCDK磷酸化的功能可能不同于CDK磷酸化，因为nonCDK位点的突变不会导致APC的组成性激活和随后的细胞周期停滞。这些结果表明，APC / C（Cdh1）活性的磷酸化比以前认为的要复杂得多。

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