ENZYMOLOGY OF MITOSIS PROMOTING FACTOR (MPF)

描述：这是第一次竞争性续签赠款，其中 长期目标是确定构成生物化学事件的特征 有丝分裂促进因子(MPF)的调控，即CDC2-Cyclin B 很复杂。在间期，抑制激酶Wee1和MYT-1抑制 Thr14和Tyr15通过磷酸化cdc2的活性。When条件 变得适合有丝分裂，双特异性磷酸酶CDC25 使Thr14和Tyr15去磷酸化，从而激活cdc2并允许 使有丝分裂底物磷酸化。在这份提案中，邓菲博士 集中在调节这一活动的潜在机制上 关键磷酸酶，CDC25。他已经证明，CDC25的活性在 间期，但在G2/M相变时受到刺激，此时 它催化cdc2-cyClinB的去磷酸化和活化。 很复杂。CDC25的这种激活似乎是磷酸化的结果 至少由两种激酶催化，其中一种是cdc2-cyClinB，另一种是 一种名为PLX-1的新型酶，邓菲博士在 最初的资助期。在此应用程序中，他建议执行 利用非洲爪哇综合分析PLX-1的结构和功能 以卵母细胞为模型系统。他将(I)研究PLX-1在 有丝分裂控制，(Ii)表征PLX-1的调控，(Iii)鉴定 CDC25中被PLX-1磷酸化并决定 磷酸化对CDC25功能的影响，(Iv)寻找其他 PLX-1和最后(V)的底物将分析范围扩大到 研究CDC25活性可能被调节的其他机制， 例如通过与结合蛋白如14-3-3相互作用。

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.