How do cyclins drive the cell cycle?

描述（由申请人提供）：此更新申请将继续研究细胞周期蛋白如何驱动细胞周期。G1稳定剂Sic1和Cdh1多位点磷酸化的作用。由于Sid化学计量抑制剂的积累，以及由于Cdh1的高活性b型细胞周期蛋白水解，细胞周期的G1期对b型细胞周期蛋白依赖性激酶是难以耐受的。G1细胞周期蛋白对Sic1和Cdh1的多位点磷酸化被认为是G1退出的必要条件。然而，我们已经发现，来自内源性位点的不可磷酸化Sic1（所有9个Cdk位点突变）的表达导致了一个完全活的菌株，尽管G1延长。我们将类似地测试内源性启动子表达的不可磷酸化Cdh1的特性。这些实验将在生理水平上解决细胞周期蛋白- cdk复合物对G1调节因子的多位点磷酸化的动态后果。b型细胞周期蛋白与细胞周期执行机制的相互作用。虽然我们对控制细胞周期蛋白依赖性激酶和后期促进复合物水平的细胞周期振荡器了解很多，但对这些活动最终如何驱动细胞周期的实际事件（如DMA复制或纺锤体功能）知之甚少。我们最近的结果表明，6个酵母b型周期蛋白基因的高度冗余只是显而易见的：在缺乏周期蛋白特异性途径的情况下，非必要的“检查点”监视机制和其他监管保障措施变得必不可少。禁用这些监管保障措施可以关注由特定周期蛋白控制的细胞生物学途径。Cdc14：目标和监管机构。Cdc14是有丝分裂退出所必需的磷酸酶；它在有丝分裂结束前从核仁的隔离中释放出来。Cdc14可能会使Cdk靶点去磷酸化，从而帮助逆转有丝分裂状态，但目前尚不清楚Cdc14是否在体内对少数关键靶点具有特异性，或者是否会使大部分或全部Cdk底物去磷酸化。我们利用影响Cdc14定位的突变体来探索Cdc14靶点的光谱。在进一步的研究中，我们将在内源性表达水平上确定阻断Pds1降解的后果，以及cdk介导的有丝分裂退出激酶Dbf2磷酸化的功能意义。这些实验将探讨Clb激酶- cdc14磷酸酶拮抗在细胞周期调节中的动态影响。总的来说，我们对细胞周期动力学的调节和促进单个细胞周期事件的周期蛋白特异性途径感兴趣。

DESCRIPTION (provided by applicant): This renewal application is to continue work on how cyclins drive the cell cycle. The role of multi-site phosphorylation of the G1 stabilizers Sic1 and Cdh1. The G1 period of the cell cycle is refractory to B-type cyclin dependent kinase because of accumulation of the Sid stoichiometric inhibitor, and because of highly active B-type cyclin proteolysis due to Cdh1. Multi-site phosphorylation of both Sic1 and Cdh1 by G1 cyclins have been considered essential for exit from G1. We have found, though, that expression of unphosphorylatable Sic1 (all 9 Cdk sites mutated) from the endogenous locus results in a fully viable strain, though with a lengthened G1. We will similarly test the properties of unphosphorylatable Cdh1 expressed from the endogenous promoter. These experiments will address the dynamic consequences of multisite phosphorylation of G1 regulators by cyclin-Cdk complexes, at physiological levels. Interactions of B-type cyclins with cell cycle execution machinery. While a lot is known about the cell cycle oscillator controlling levels of cyclin-dependent kinase and anaphase-promoting complex, much less is known about how these activities eventually drive the actual events of the cell cycle such as DMA replication or spindle function. Our recent results indicate that the high degree of redundancy of the 6 yeast B-type cyclin genes is only apparent: non-essential 'checkpoint' surveillance mechanisms and other regulatory safeguards become essential in the absence of cyclin-specific pathways. Disabling these regulatory safeguards allows focus on cell biological pathways controlled by specific cyclins. Cdc14: targets and regulators. Cdc14 is a phosphatase required for exit from mitosis; it is released from sequestration in the nucleolus just before mitotic exit. Cdc14 probably dephosphorylates Cdk targets and thus helps reverse the mitotic state, but it is unresolved if Cdc14 is specific in vivo for a few critical targets, or alternatively dephosphorylates most or all Cdk substrates. We have exploited mutants affecting Cdc14 localization to explore the spectrum of Cdc14 targets. In additional studies, we will determine the consequences of blocking Pds1 degradation, at endogenous expression levels, and the functional significance of Cdk-mediated phosphorylation of the mitotic exit kinase Dbf2. These experiments will probe the dynamic consequences of Clb kinase-Cdc14 phosphatase antagonism in cell cycle regulation. Overall, we are interested in regulation of cell cycle dynamics, and in cyclin-specific pathways promoting individual cell cycle events.