BIOCHEMISTRY OF CELL CYCLE REGULATION

细胞周期的正常运作及其对两者的反应的调节 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 这些过程很困难。 遗传和生化 鉴定周期蛋白/p34cdc2蛋白激酶复合物是关键 有丝分裂诱导剂开启了这一领域的分析。 的活动 p34cdc2 通过调节蛋白、细胞周期蛋白的结合进行调节 抑制性和激活性磷酸化。 这些复杂的反应 越来越适合使用纯化的成分进行研究， 这种方法应该能够对细胞有更深入的机制了解 循环。 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。 重点将在于如何开展活动 p34cdc2 受反馈和细胞周期输入调节以控制进入 进入有丝分裂。 长期的方法将是追踪监管 直接影响 p34cdc2 的途径，通过维持的酶 p34cdc2 磷酸化状态及其调节，以启动 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 与细胞周期蛋白以及 p13suc1 一起调节其激酶活性。 的 预测 ATP 结合位点内的磷酸化 p34cdc2 和细胞周期蛋白的缺失会阻止 p34cdc2 结合 ATP 通过交联研究进行测试。 细胞周期调控的存在 将探索与 p34cdc2 的蛋白质结合，以及一个 p34cdc2- 的作用 结合蛋白 p13suc1 控制 p34cdc2 磷酸化状态 被确定。 2）研究cdc25磷酸酶的特异性和调控， 克隆爪蟾同源物。 cdc25 特异性地使酪氨酸去磷酸化 在 p34cdc2 中。 p34cdc2 激活需要这种去磷酸化。 3) 鉴定、纯化和研究 p34cdc2 激活激酶 负责磷酸化 p34cdc2 的 Thr-161，这是其磷酸化所需的位点 活动。 4) 确定六种磷酸化/去磷酸化事件中的哪一种 p34cdc2 在细胞周期和不同细胞之间受到调节 循环状态。 将鉴定和研究受调节的酶 确定此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.