BIOCHEMISTRY OF CELL CYCLE REGULATION

细胞周期的正常运作及其对两者的调节 内部和外部刺激对于正常生长，分裂， 以及细胞和组织的分化。 直到最近的生化研究 这些过程是困难的。 基因和生物化学 细胞周期蛋白/p34 cdc 2蛋白激酶复合物的鉴定是关键 有丝分裂的诱导剂已经打开了这一领域的分析。 的活性 p34 cdc 2受调节蛋白cyclin的结合调节， 抑制和激活磷酸化。 这些复杂的反应 越来越适合使用纯化的成分进行研究， 这种方法应该会导致对细胞更深入的机械见解， 周期 这里提出的工作的总体目标是理解这个关键 细胞周期的转变。 重点将是如何活动的 p34 cdc 2通过反馈和细胞周期输入来调节，以控制进入 进入有丝分裂 长期的方法是追踪监管机构 从直接作用于p34 cdc 2的途径，通过维持 p34 cdc 2磷酸化状态及其调节，对启动 信号了 该项目的具体目标是： 1)了解p34 cdc 2的磷酸化及其与蛋白质的相互作用 p34 cdc 2与cyclin和p13 suc 1一起调节其激酶活性。 的 预测，在假定的ATP结合位点的磷酸化， p34 cdc 2和细胞周期蛋白的缺乏阻止了p34 cdc 2结合ATP， 通过交联研究测试。 细胞周期调控的存在 蛋白质结合p34 cdc 2将被探索和一个p34 cdc 2的作用， 结合蛋白p13 suc 1在控制p34 cdc 2磷酸化状态时， 下定决心。 2)研究cdc 25磷酸酶的特异性和调节， 克隆爪蟾的同源基因cdc 25特异性地使酪氨酸去磷酸化 在p34 cdc 2中。 这种去磷酸化是p34 cdc 2激活所必需的。 3)为了鉴定、纯化和研究p34 cdc 2激活激酶， 负责磷酸化p34 cdc 2的Thr-161，这是其磷酸化所需的位点。 活动 4)为了确定六个磷酸化/去磷酸化事件中 p34 cdc 2在细胞周期中以及不同细胞之间的表达受调控。 循环状态。 将鉴定和研究受调节的酶， 确定这种循环控制的生化基础。

Proper functioning of the cell cycle and its regulation in response to both internal and external stimuli is essential for the normal growth, division, and differentiation of cells and tissues. Until recently biochemical study of these processes was difficult. The genetic and biochemical identification of the cyclin/p34cdc2 protein kinase complex as the key inducer of mitosis has opened this area to analysis. The activity of p34cdc2 is regulated by the binding of a regulatory protein, cyclin, and by both inhibitory and activating phosphorylations. These complex reactions are becoming increasingly amenable to study using purified components, an approach that should lead to deeper mechanistic insights into the cell cycle. The overall objective of the work proposed here is to understand this key transition in the cell cycle. The focus will be on how the activity of p34cdc2 is regulated by feedback and cell cycle inputs to control entry into mitosis. The long term approach will be to trace the regulatory pathways from direct effects on p34cdc2, through the enzymes that maintain the p34cdc2 phosphorylation state and their regulation, to the initiating signal. The Specific Aims of this project are: 1) To understand how the phosphorylation of p34cdc2 and the interactions of p34cdc2 with cyclin and with p13suc1 regulate its kinase activity. The prediction that phosphorylation within the putative ATP-binding site of p34cdc2 and the absence of cyclin prevent p34cdc2 from binding ATP will be tested by crosslinking studies. The existence of cell-cycle regulated protein binding to p34cdc2 will be explored and the role of one p34cdc2- binding protein, p13suc1, in controlling p34cdc2 phosphorylation state will be determined. 2) To study the specificity and regulation of the cdc25 phosphatase, and to clone the Xenopus homolog. cdc25 specifically dephosphorylates a tyrosine in p34cdc2. This dephosphorylation is required for p34cdc2 activation. 3) To identify, purify, and study the p34cdc2 activating kinase, the enzyme responsible for phosphorylating Thr-161 of p34cdc2, a site required for its activity. 4) To determine which of the six phosphorylation/dephosphorylation events on p34cdc2 are regulated during the cell cycle and between different cell cycle states. Regulated enzymes will be identified and studied to determine the biochemical basis for this cycle control.