ENZYMOLOGY OF MITOSIS PROMOTING FACTOR (MPF)

描述：这是第一个竞争性续签赠款，其中 长期目标是表征潜在的生化事件 有丝分裂促进因子 (MPF) 的调节，即 cdc2-cyclin B 复杂。 在间期，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。 当条件 变得适合有丝分裂，双特异性磷酸酶 cdc25 Thr14 和 Tyr15 去磷酸化，从而激活 cdc2 并允许 它使有丝分裂底物磷酸化。 在此建议中，邓菲博士 重点关注这一活动的监管机制 关键磷酸酶，cdc25。 他表明 cdc25 活性较低 间期，但在 G2/M 相变时受到刺激，此时 它催化 cdc2-cyclinB 的去磷酸化和激活 复杂。 cdc25 的这种激活似乎是磷酸化的结果 由至少两种激酶催化，其中一种是 cdc2-cyclinB，另一种是 邓菲博士在 初始资助期。 在这份申请中，他建议开展一项 利用 Xenopus 全面分析 Plx-1 的结构和功能 卵母细胞作为模型系统。 他将 (I) 检查 Plx-1 在 有丝分裂控制，(ii) 表征 Plx-1 的调节，(iii) 确定 cdc25 中被 Plx-1 磷酸化的特定位点并确定 磷酸化对 cdc25 功能的影响，(iv) 寻找其他 Plx-1 的底物，最后 (v) 扩大分析范围 检查 cdc25 活动可能受到调节的其他机制， 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.