ENZYMOLOGY OF MITOSIS PROMOTING FACTOR (MPF)

有丝分裂促进因子（MPF）的酶学

• 批准号: 6179747
• 负责人: William G Dunphy
• 金额: $ 23.04万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 2001-12-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6179747
• 关键词: CD2 molecule; Xenopus; Xenopus oocyte; affinity chromatography; autoradiography; cell cycle proteins; cell growth regulation; cyclins; enzyme activity; enzyme substrate; immunoprecipitation; molecular cloning; nucleic acid sequence; oligonucleotides; phosphoprotein phosphatase; phosphorylation; polymerase chain reaction; protein kinase; protein sequence; protein structure function; tissue /cell culture

DESCRIPTION: This is the first competitive renewal of a grant in which the long-term goal is to characterize the biochemical events that underlie the regulation of Mitosis-Promoting Factor (MPF), i.e., the cdc2-cyclin B complex. During interphase, inhibitory kinases Wee1 and Myt-1 suppress the activity of cdc2 by phosphorylating it on Thr14 and Tyr15. When conditions become appropriate for mitosis, the dual specificity phosphatase cdc25 dephosphorylates both Thr14 and Tyr15, thereby activating cdc2 and allowing it to phosphorylate mitotic substrates. In this proposal Dr. Dunphy focusses on the mechanisms underlying the regulation of the activity of this critical phosphatase, cdc25. He has shown that cdc25 activity is low in interphase but is stimulated at the G2/M phase transition, the time at which it catalyzes the dephosphorylation and activation of the cdc2-cyclinB complex. This activation of cdc25 appears to result from phosphorylation catalyzed by at least two kinases, one of which is cdc2-cyclinB, the other a novel enzyme termed Plx-1 that Dr. Dunphy purified and cloned during the initial funding period. In this application he proposes to carry out a comprehensive analysis of the structure and function of Plx-1, using Xenopus oocytes as the model system. He will (I) examine the role of Plx-1 in mitotic control, (ii) characterize the regulation of Plx-1, (iii) identify specific sites in cdc25 that are phosphorylated by Plx-1 and determine the effects of phosphorylation on the function of cdc25, (iv) search for other substrates of Plx-1 and finally (v) broaden the scope of the analysis to examine other mechanisms by which the activity of cdc25 may be regulated, for example through interaction with binding proteins such as 14-3-3.

描述：这是赠款的第一个竞争续约 长期目标是表征构成的生化事件 调节有丝分裂促进因子（MPF），即Cdc2-cyclin b 复杂的。 在相间期间，抑制性激酶WEE1和MYT-1抑制 通过在Thr14和Tyr15上磷酸化Cdc2的活性。 何时条件 变得适合有丝分裂，双重特异性磷酸酶CDC25 将Thr14和Tyr15脱磷酸化，从而激活CDC2并允许 它可以磷酸化有丝分裂底物。 在这个建议中，邓菲博士 重点关注该活动调节的机制 临界磷酸酶CDC25。 他表明CDC25活性很低 相间但在G2/m相变刺激的时间 它催化Cdc2-cyclinb的去磷酸化和激活 复杂的。 CDC25的这种激活似乎是由磷酸化引起的 至少由两个激酶催化，一种是cdc2-cyclinb，另一个是a 新颖的酶称为dunphy博士在纯化和克隆的酶中被称为PLX-1。 初始资金期。 在此申请中，他建议执行 使用Xenopus对PLX-1的结构和功能进行全面分析 卵母细胞作为模型系统。 他将（i）检查PLX-1在 有丝分裂对照，（ii）表征PLX-1的调节，（iii）识别 Cdc25中的特定位点被PLX-1磷酸化并确定 磷酸化对CDC25，（iv）搜索其他功能的影响 PLX-1的底物，最后（V）扩大了分析范围 检查其他可能调节Cdc25活性的机制， 例如，通过与结合蛋白（如14-3-3）相互作用。