BIOCHEMISTRY OF CELL CYCLE REGULATION

细胞周期的正常运作及其对这两种反应的调节 内部和外部刺激对正常的生长、分裂、 以及细胞和组织的分化。直到最近的生物化学研究 这些过程中的任何一个都是困难的。遗传和生化 确定细胞周期蛋白/p34cdc2蛋白激酶复合体为关键 有丝分裂的诱导者已经打开了这一领域的分析。的活动 P34cdc2受调节蛋白、细胞周期蛋白和 抑制磷酸化和激活磷酸化。这些复杂的反应 越来越容易使用提纯的成分进行研究，以及 应该导致对细胞的更深层次的机械性洞察的方法 周而复始。 这里提出的工作的总体目标就是理解这一关键 细胞周期中的过渡。重点将放在如何开展 P34cdc2由反馈和细胞周期输入调节，以控制进入 转化为有丝分裂。长期的方法将是追踪监管机构 直接作用于p34cdc2的途径，通过维持 P34cdc2的磷酸化状态及其对启动的调节 信号。 该项目的具体目标是： 1)了解p34cdc2的磷酸化及其相互作用 P34cdc2与细胞周期蛋白和p13uc1共同调节其激酶活性。这个 预测在假定的ATP结合位点内的磷酸化 P34cdc2和细胞周期蛋白的缺失阻止p34cdc2与ATP结合 通过交联性研究进行了测试。细胞周期调控的存在 将探索蛋白质与p34cdc2的结合，以及p34cdc2- 控制p34cdc2磷酸化状态的结合蛋白p13uc1将 要下定决心。 2)研究CDC25磷酸酶的特异性和调控，并 克隆非洲爪哇的同源基因。CDC25专门去磷酸化一种酪氨酸 在p34cdc2中。这种去磷酸化是p34cdc2激活所必需的。 3)p34cdc2激活酶的鉴定、纯化和研究。 负责磷酸化p34cdc2的Thr-161，这是ITS所需的位点 活动。 4)确定六个磷酸化/去磷酸化事件中的哪一个 在p34cdc2上，在细胞周期中和不同细胞之间进行调节。 循环状态。将鉴定和研究受调控的酶以 确定这一周期控制的生化基础。

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.