How do cyclins drive the cell cycle?

这个续订申请是为了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。细胞的G1期 由于SID化学计量比的积累，Cycle对B型Cyclin依赖的激酶是难治性的 由于CDH1具有高活性的B型细胞周期蛋白水解酶。蛋白的多位点磷酸化 G1期细胞周期蛋白的SIC1和CDH1都被认为是退出G1期所必需的。不过，我们已经发现， 来自内源性基因座的不可磷酸化的SIC1(所有9个CDK位点都突变)的表达导致了 完全存活的菌株，尽管G1延长了。我们将类似地测试不可磷酸化的 CDH1由内源启动子表达。这些实验将解决动态 在生理水平上，细胞周期蛋白-CDK复合体对G1调节因子多位点磷酸化的影响。 B型细胞周期蛋白与细胞周期执行机制的相互作用。虽然人们对细胞有很多了解 周期振荡器控制周期蛋白依赖的激酶和后期促进复合体的水平，更少的是 了解这些活动最终如何驱动细胞周期的实际事件，如DNA复制 或纺锤功能。我们最近的结果表明，六个酵母B-型的高度冗余 细胞周期蛋白基因只是表象：非必要的‘检查点’监视机制和其他调控机制 在没有周期蛋白特异性通路的情况下，保障措施变得至关重要。禁用这些监管 保障措施允许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；它从 就在有丝分裂退出之前，核仁中的固存。CDC14可能使CDK靶标去磷酸化 因此有助于逆转有丝分裂状态，但如果CDC14在体内对几个关键靶点是特异的，或者 另一种方法是使大部分或全部CDK底物脱磷。我们已经利用了影响cdc14的突变体 本地化以探索CdC14靶的光谱。在进一步的研究中，我们将确定 在内源性表达水平上阻断Pds1降解的后果，以及功能性 CDK介导的有丝分裂退出激酶Dbf2磷酸化的意义。这些实验将探索 Clb激酶-CDC14磷酸酶拮抗在细胞周期调控中的动态后果。 总体而言，我们感兴趣的是细胞周期动力学的调节，以及促进细胞周期蛋白特异的通路 单个细胞周期事件。

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 DMAreplication or spindle function. Our recent results indicate that the high degree of redundancy of the six 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.