REGULATORS OF CDC2/CDK1

CDC2/CDK1 的调节因子

• 批准号: 7987211
• 负责人: JAMES E. FERRELL
• 金额: $ 38.2万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7987211
• 关键词: Accounting; Address; Affinity; Binding; Biological Process; CDC2 Protein Kinase; Cell Cycle; Cells; Complex; Cyclins; Embryo; Epitopes; Eukaryotic Cell; Family; Feedback; In Vitro; Interphase; Malignant Neoplasms; Maps; Meiosis; Mitosis; Mitotic; Modeling; Mutagenesis; Mutate; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Protein Dephosphorylation; Protein Isoforms; Protein Kinase; Protein phosphatase; Proteins; Regulation; Site; Source; System; Testing; Vertebrates; Work; Xenopus; Xenopus laevis; blastomere structure; chemotherapeutic agent; cyclin B1; egg; embryo cell; inhibitor/antagonist; inorganic phosphate; insight; interest; mutant; public health relevance; reconstitution; research study; response

DESCRIPTION (provided by applicant): The CDK1 protein kinase is the universal trigger of mitosis. In turn its activity is regulated by protein kinases of the Wee1 family and phosphoprotein phosphatases of the Cdc25 family. Here we propose three specific aims on the cooperative regulation of Wee1A by CDK1; the mechanisms of ultrasensitivity in the regulation of Cdc25C by CDK1; and the regulation of the phosphatases that oppose the phosphorylation of Wee1A and Cdc25C during the transition from interphase into M-phase. 1. For Wee1A, phosphorylation promotes CDK1 binding, which promotes more Wee1A phosphorylation. What is the mechanism for this? Our working hypothesis is that the p9 Cks2 protein acts as a phosphoepitope-binding subunit of the CDK1 complex, and is responsible for the increased affinity of CDK1-cyclin B1 for phospho-Wee1A compared to dephospho-Wee1. We plan to test this hypothesis through reconstitution, immunodepletion, and mutagenesis approaches. 2. Does Cdc25C phosphorylation promote CDK1-cyclin B1 binding, and if so does that explain Cdc25C's high intrinsic ultrasensitivity? The hyperphosphorylation of Cdc25C is a highly ultrasensitive function of the level of CDK1-cyclin B1 activity, well-approximated by a Hill function with a Hill coefficient of 11. Much of this ultrasensitivity can be seen in reconstituted systems composed of purified CDK1-cyclin B1 and Cdc25C. We plan to test the hypothesis that phosphorylation-induced CDK1-cyclin B1 binding is responsible for this intrinsic ultrasensitivity. 3. What phosphatases dephosphorylate Wee1A and Cdc25C during the transition into mitosis? Are they regulated? Does their regulation account for (or contribute to) the extrinsic ultrasensitivity of the Wee1A and Cdc25C responses? Recent work using model CDK1 substrates has implicated PP1 and/or PP2A-B554 as regulators of protein dephosphorylation during mitotic exit. Here we plan to use a panel of Wee1A and Cdc25C mutants to ask how the rates of dephosphorylation of various sites in these two proteins vary with the activity of CDK1 in Xenopus egg extracts; whether the sites behave differently or similarly to each other; what phosphatases are responsible for their dephosphorylation and whether these phosphatases are regulated in a switch-like, ultrasensitive fashion. PUBLIC HEALTH RELEVANCE: Cdc25 and Wee1 regulate mitosis in (as far as we know) all eukaryotic cells. From a fundamental perspective their own regulation is of interest because (1) mitotic entry is such a dramatic, important cell biological process; (2) the regulation of the Cdc25 and Wee1 has already yielded important general insight into the principles of protein regulation, and promises to continue to do so; (3) pharmacological Wee1 inhibitors hold promise as cancer chemotherapeutic agents.

描述（由申请人提供）：CDK1 蛋白激酶是有丝分裂的普遍触发因素。反过来，其活性受到 Wee1 家族的蛋白激酶和 Cdc25 家族的磷蛋白磷酸酶的调节。在此，我们提出了CDK1对Wee1A协同调控的三个具体目标； CDK1对Cdc25C的超敏调节机制；以及在从间期到 M 期的转变过程中对抗 Wee1A 和 Cdc25C 磷酸化的磷酸酶的调节。 1. 对于 Wee1A，磷酸化促进 CDK1 结合，从而促进更多的 Wee1A 磷酸化。 这其中的机制是什么？我们的工作假设是，p9 Cks2 蛋白作为 CDK1 复合物的磷酸表位结合亚基，与去磷酸-Wee1 相比，导致 CDK1-细胞周期蛋白 B1 对磷酸-Wee1A 的亲和力增加。我们计划通过重构、免疫耗竭和诱变方法来检验这一假设。 2. Cdc25C 磷酸化是否促进 CDK1-细胞周期蛋白 B1 结合，如果是的话，这是否可以解释 Cdc25C 的高内在超敏性？ Cdc25C 的过度磷酸化是 CDK1-细胞周期蛋白 B1 活性水平的高度超敏感函数，可通过 Hill 系数为 11 的 Hill 函数很好地近似。这种超敏感大部分可以在由纯化的 CDK1-细胞周期蛋白 B1 组成的重构系统中看到和 Cdc25C。我们计划检验这样的假设：磷酸化诱导的 CDK1-细胞周期蛋白 B1 结合是造成这种内在超敏性的原因。 3. 在有丝分裂过程中，哪些磷酸酶使 Wee1A 和 Cdc25C 去磷酸化？他们受到监管吗？它们的调节是否解释（或促成）Wee1A 和 Cdc25C 反应的外在超敏感性？最近使用模型 CDK1 底物的工作表明 PP1 和/或 PP2A-B554 作为有丝分裂退出期间蛋白质去磷酸化的调节剂。在这里，我们计划使用一组 Wee1A 和 Cdc25C 突变体来探究这两种蛋白质中各个位点的去磷酸化率如何随爪蟾卵提取物中 CDK1 的活性而变化；站点之间的行为是否不同或相似；哪些磷酸酶负责其去磷酸化，以及这些磷酸酶是否以类似开关的超灵敏方式受到调节。 公共健康相关性：Cdc25 和 Wee1 调节（据我们所知）所有真核细胞的有丝分裂。从基本角度来看，它们自身的调节是令人感兴趣的，因为（1）有丝分裂进入是一个如此戏剧性的、重要的细胞生物学过程； (2) Cdc25 和 Wee1 的调控已经对蛋白质调控原理产生了重要的普遍见解，并且有望继续这样做； (3) 药理学Wee1抑制剂有望成为癌症化疗药物。