How do cyclins drive the cell cycle?

描述(申请人提供)：本次续签申请将继续研究细胞周期蛋白是如何驱动细胞周期的。G1稳定剂SIC1和CDH1多位点磷酸化的作用。由于SID化学计量比抑制物的积累，以及CDH1对B型细胞周期蛋白的高活性分解，细胞周期的G1期对B型细胞周期蛋白依赖的激酶来说是不容易的。G1期细胞周期蛋白对SIC1和CDH1的多位点磷酸化被认为是退出G1期所必需的。然而，我们已经发现，从内源基因座表达不能磷酸化的SIC1(所有9个CDK位点都突变)导致了一个完全活的菌株，尽管G1延长了。我们将类似地测试从内源启动子表达的不可磷酸化的CDH1的性质。这些实验将在生理水平上研究Cyclin-CDK复合体对G1调节器的多位点磷酸化的动态影响。B型细胞周期蛋白与细胞周期执行机制的相互作用。虽然关于细胞周期振荡器控制细胞周期依赖的激酶和后期促进复合体水平的了解很多，但关于这些活动如何最终驱动细胞周期的实际事件，如DMA复制或纺锤体功能，我们知道的要少得多。我们最近的结果表明，6个酵母B型细胞周期蛋白基因的高度冗余是显而易见的：在缺乏细胞周期蛋白特异性通路的情况下，非必要的“检查点”监视机制和其他调节保障变得必不可少。禁用这些调控保护措施可以将重点放在由特定周期蛋白控制的细胞生物学途径上。CDC14：目标和监管者。CDC14是一种退出有丝分裂所需的磷酸酶；它在有丝分裂退出之前从核仁中的隔离中释放出来。CDC14可能会去磷酸化CDK靶标，从而帮助逆转有丝分裂状态，但如果CDC14在体内对一些关键靶点具有特异性，或者替代地去磷酸化大部分或全部CDK底物，这一问题仍未解决。我们利用影响CDC14定位的突变体来探索CDC14靶标的光谱。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的磷酸化的功能意义。这些实验将探索Clb激酶-CDC14磷酸酶拮抗在细胞周期调节中的动态后果。总体而言，我们感兴趣的是细胞周期动力学的调节，以及促进单个细胞周期事件的周期蛋白特异性途径。

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.