ENZYMOLOGY OF MITOSIS PROMOTING FACTOR (MPF)

描述：这是第一次有竞争力的赠款续期， 长期目标是描述生物化学事件的特征， 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Wee 1和Myt-1抑制细胞凋亡。 通过在Thr 14和Tyr 15上磷酸化cdc2来增强cdc2的活性。 当条件 变得适合有丝分裂，双特异性磷酸酶cdc25 使Thr 14和Tyr 15去磷酸化，从而激活cdc2， 磷酸化有丝分裂底物。 在这份提案中，邓菲博士 重点是潜在的机制，调节活动，这 临界磷酸酶CDC25。 他已经表明，cdc25的活性很低， 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， 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 一种名为Plx-1的新酶，Dunphy博士在1999年的研究中纯化并克隆了这种酶。 初始融资期。 在本申请中，他建议进行一项 利用非洲爪蟾对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（我）研究Plx-1在以下方面的作用： 有丝分裂控制，（ii）表征Plx-1的调节，（iii）鉴定 cdc25中被Plx-1磷酸化的特异性位点， 磷酸化对cdc25功能的影响，（iv）寻找其他 底物的Plx-1，最后（v）扩大分析范围， 检查可以调节CDC25活性的其他机制， 例如通过与结合蛋白如14 - 3 - 3的相互作用。

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.