Cell Cycle Regulation of Glc7p Protein Phosphatase

真核细胞分裂的有序步骤部分是通过及时的蛋白质降解和可逆的蛋白质磷酸化来调节的。蛋白磷酸酶-1(PP1)酶在细胞周期去磷酸化反应中起重要作用。萌芽酵母酿酒酵母提供了已知的最简单的PP1系统，因为它只有一个PP1基因，GLC7(基因产物是GlcTp)。Glc7p对酵母细胞周期中的两个转变至关重要的蛋白质进行去磷酸化。第一个时期，G1期到S期是由细胞的营养状态调节的。第二阶段，在后期之前，似乎使用GlcTp来监测有丝分裂纺锤体组装，这是忠实的染色体分离所必需的。目前尚不清楚控制细胞周期的GlcTp去磷酸化蛋白。最近的研究结果表明，Glc7p蛋白水平在细胞周期中振荡。Glc7p的峰值出现在G1期晚期和S期。相反，在依赖泛素的蛋白分解破坏G1或有丝分裂周期蛋白的细胞周期间隔中，检测到低水平的表达。因此，他们假设Glc7p是泛素依赖的蛋白质分解的目标，泛素连接酶复合体可以破坏细胞周期蛋白。这个项目的第一个目标是分析潜在的依赖泛素的Glc7p蛋白分解。这项建议的第二个目的是定义Glc8p在其调控中的作用，即Glc7p的一个非催化亚基，看看它是否保护Glc7p在G1期间免受泛素化和破坏，从而允许Glc7p去磷酸化底物蛋白。在Glc7p缺失的细胞中发现高的细胞周期蛋白依赖性蛋白激酶活性，这表明当Glc7p活性降低时，S期细胞周期蛋白或有丝分裂周期蛋白没有被适当破坏。这一发现进一步表明，被称为后期促进复合体(APC)的泛素连接酶可能被Glc7p去磷酸化激活。最后一个目标是确定作为Glc7P底物和APC调节者的负责调节的磷蛋白。尽管几种磷蛋白在细胞分裂中发挥重要作用，但控制其磷酸化状态的蛋白激酶和磷酸酶在很大程度上是未知的。细胞分裂的控制是一个复杂的过程，涉及多个参与者。初步分析表明，Glc7p蛋白磷酸酶在这一过程中发挥了作用。该项目的目标是将Glc7p功能与细胞分裂的其他已知方面相结合。

The orderly steps of eukaryotic cell division are regulated in part by timely protein degradation and reversible protein phosphorylation. Protein phosphatase-1 (PP1) enzymes are important for cell cycle dephosphorylation reactions. The budding yeast, Saccharomyces cerevisiae, offers the simplest known PP1 system because it has only a single gene for PP1, GLC7 (gene product is GlcTp). Glc7p dephosphorylates proteins critical for two transitions in the cell cycle of yeast. The first, G1 to S-phase is regulated by the nutritional status of the cells. The second transition, prior to anaphase, appears to use GlcTp to monitor the mitotic spindle assembly, which is necessary for faithful chromosome segregation. The proteins dephosphorylated by GlcTp, which control the cell cycle, are unknown.Recent results of this investigator show that Glc7p protein levels oscillate during the cell cycle. Peak levels of Glc7p were found in late G1 and S-phases. Conversely, low levels were detected during cell cycle intervals in which G1 or mitotic cyclins are destroyed by ubiquitin-dependent proteolysis. Therefore, they hypothesize that Glc7p is a target of ubiquitin-dependent proteolysis by the ubiquitin ligase complexes that destroy cyclins. The first aim of this project is to analyze potential ubiquitin-dependent Glc7p proteolysis. The second aim of this proposal defines the role of Glc8p, a noncatalytic subunit of Glc7p, in its regulation, to see if it shields Glc7p from ubiquitination and destruction during G1 to allow Glc7p to dephosphorylate substrate proteins . The high cyclin-dependent protein kinase (CDK) activity found in Glc7p-depleted cells suggests that S-phase or mitotic cyclins are not destroyed appropriately when Glc7p activity is diminished. This finding further implies the ubiquitin ligase known as the anaphase-promoting complex (APC) may be activated by Glc7p dephosphorylation. The last aim seeks to identify the responsible regulatory phosphoproteins that are Glc7p substrates and APC regulators.Although several phosphoproteins play important roles in cell division, the protein kinases and phosphatases that control their phosphorylation state are largely unknown. The control of cell division is a complicated process, which involves many participants. Preliminary analysis indicates that Glc7p protein phosphatase plays a part in this process. The goal of this project is to integrate Glc7p function with other known aspects of cell division.