Patra D, Dunphy W G
Division of Biology, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125 USA.
Genes Dev. 1998 Aug 15;12(16):2549-59. doi: 10.1101/gad.12.16.2549.
Degradation of mitotic cyclins on exit from M phase occurs by ubiquitin-mediated proteolysis. The ubiquitination of mitotic cyclins is regulated by the anaphase-promoting complex (APC) or cyclosome. Xe-p9, the Xenopus homolog of the Suc1/Cks protein, is required for some step in mitotic cyclin destruction in Xenopus egg extracts. Specifically, if p9 is removed from interphase egg extracts, these p9-depleted extracts are unable to carry out the proteolysis of cyclin B after entry into mitosis and thus remain arrested in M phase. To explore the molecular basis of this defect, we depleted p9 from extracts that had already entered M phase and thus contained an active APC. We found that ubiquitin-mediated proteolysis of cyclin B was not compromised under these circumstances, suggesting that p9 is not directly required for ubiquitination or proteolysis. Further analysis of extracts from which p9 had been removed during interphase showed that, at the beginning of mitosis, these extracts are unable to carry out the hyperphosphorylation of the Cdc27 component of the APC, which coincides with the initial activation of the APC. p9 can be found in a complex with a small fraction of the Cdc27 protein during M phase but not interphase. The phosphorylation of the Cdc27 protein (either associated with the APC or in an isolated, bacterially expressed form) by recombinant Cdc2/cyclin B is strongly enhanced by p9. Our results indicate that p9 directly regulates the phosphorylation of the APC by Cdc2/cyclin B. These studies indicate that the Suc1/Cks protein modulates substrate recognition by a cyclin-dependent kinase.
有丝分裂周期蛋白在退出M期时的降解是通过泛素介导的蛋白水解作用实现的。有丝分裂周期蛋白的泛素化由后期促进复合物（APC）或细胞周期体调控。Xe-p9是Suc1/Cks蛋白的非洲爪蟾同源物，在非洲爪蟾卵提取物中，它是有丝分裂周期蛋白降解某些步骤所必需的。具体而言，如果从间期卵提取物中去除p9，这些缺乏p9的提取物在进入有丝分裂后就无法进行细胞周期蛋白B的蛋白水解，因此会停滞在M期。为了探究这种缺陷的分子基础，我们从已经进入M期且含有活性APC的提取物中去除p9。我们发现在这种情况下，泛素介导的细胞周期蛋白B的蛋白水解并未受损，这表明泛素化或蛋白水解并不直接需要p9。对间期已去除p9的提取物的进一步分析表明，在有丝分裂开始时，这些提取物无法对APC的Cdc27组分进行超磷酸化，而这与APC的初始激活是同时发生的。在M期而非间期，可以发现p9与一小部分Cdc27蛋白形成复合物。重组Cdc2/细胞周期蛋白B对Cdc27蛋白（无论是与APC相关还是以分离的、细菌表达的形式）的磷酸化作用在p9的作用下会大大增强。我们的结果表明，p9直接调控Cdc2/细胞周期蛋白B对APC的磷酸化作用。这些研究表明，Suc1/Cks蛋白可调节细胞周期蛋白依赖性激酶对底物的识别。
