REGULATORS OF CDC2/CDK1

CDC2/CDK1 的调节因子

• 批准号: 8518351
• 负责人: JAMES E. FERRELL
• 金额: $ 45.82万
• 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/8518351
• 关键词: Accounting; Address; Affinity; Binding; Biological Process; CDC2 Protein Kinase; CDC2 gene; 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 2A Regulatory Subunit PR53; 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通用触发因素。反过来，其活性受Cdc25家族的WEE1家族和磷酸蛋白磷酸酶的蛋白激酶的调节。在这里，我们提出了CDK1对WEE1A的合作调节的三个具体目标。通过CDK1调节CDC25C的超敏机制；以及在从相间向M相过渡过程中与WEE1A和CDC25C磷酸化相反的磷酸酶的调节。 1。对于WEE1A，磷酸化促进CDK1结合，从而促进更多的WEE1A磷酸化。 这是什么机制？我们的工作假设是，P9 CKS2蛋白充当CDK1复合物的磷光蛋白质结合亚基，并且与Dephospho-wee1相比，CDK1-CYCLIN B1对CDK1-CYCLIN B1的亲和力增加。我们计划通过重组，免疫部门和诱变方法来检验这一假设。 2。cdc25c磷酸化是否会促进CDK1-CYCLIN B1结合，如果可以的话，是否可以解释CDC25C的高内在超敏？ Cdc25c的高磷酸化是CDK1-循环B1活性水平的高度超敏感的功能，该功能通过丘陵功能（山丘系数为11）充分x. hill功能。在由纯化的CDK1-Cyclin B1组成的重组系统中，可以看到这种超抑制。和CDC25C。我们计划检验以下假设：磷酸化诱导的CDK1-CYCLIN B1结合负责这种内在的超敏性。 3。在过渡到有丝分裂过程中，哪些磷酸酶去磷酸化酶的WEE1A和CDC25C？他们受到监管吗？他们的法规是否解释了WEE1A和CDC25C响应的外在超敏性（或促进）？使用Model CDK1底物的最新工作已将PP1和/或PP2A-B554视为有丝分裂出口期间蛋白质去磷酸化的调节剂。在这里，我们计划使用一系列WEE1A和CDC25C突变体来询问这两种蛋白质中各种位点的去磷酸化速率如何随CDK1在Xenopus卵提取物中的活性而变化；这些站点的行为是不同的还是彼此相似的；哪些磷酸酶是导致其去磷酸化的原因，以及这些磷酸酶是否以开关状的超敏感方式调节。 公共卫生相关性：CDC25和WEE1调节（据我们所知）所有真核细胞的有丝分裂。从基本的角度来看，他们自己的调节是有意义的，因为（1）有丝分裂的进入是如此引人注目的重要细胞生物学过程； （2）CDC25和WEE1的调节已经对蛋白质调节原理产生了重要的一般见解，并有望继续这样做； （3）药理学WEE1抑制剂作为癌症化学治疗剂有望。