Control of Mitotic Entry by Regulators of Cdc2

描述（由申请人提供）：由Cdc2/Cyclin B激酶催化进入有丝分裂，该激酶磷酸化多种蛋白以诱导有丝分裂特征的戏剧性细胞重排。当DNA复制受到抑制时，细胞周期检查点途径通过抑制Cdc2在Y15和T14位点的磷酸化来阻止有丝分裂。这种抑制包括Cdc2 Y15定向激酶Wee1的持续活性，以及Cdc2 Y15磷酸酶Cdc25的抑制。我们发现一种新的G2/M调节因子Hsl7促进核内Wee1降解，并且DNA复制检查点破坏Hsl7-Wee1相互作用。同时，Cdc25通过与14-3-3蛋白结合而受到抑制。从Cdc25中去除14-3-3是其有丝分裂激活所必需的，我们发现14-3-3-Cdc25复合体的解离需要Cdc25 T138的磷酸化（这降低了14-3-3对Cdc25的亲和力）和中间丝蛋白（然后结合14-3-3，从而作为丰富的14-3-3“汇”）。此外，我们发现一种先前描述的凋亡抑制剂Aven也可以调节有丝分裂进入，并且可能对dna反应性检查点操作很重要。本研究的目的是阐明dna反应性检查点通路的Wee1和cdc25调控臂，以充分理解M期进入的控制。为此，我们建议：1)阐明Hsl7在检查点介导的Wee1调控中的作用；2)阐明Cdc25调控14-3-3释放的机制；3)确定Aven调控有丝分裂进入的机制。

DESCRIPTION (provided by applicant): Entry into mitosis catalyzed by the Cdc2/Cyclin B kinase, which phosphorylates multiple proteins to induce the dramatic cellular rearrangements characteristic of mitosis. When DNA replication is inhibited, a cell cycle checkpoint pathway prevents mitosis through inhibitory phosphorylation of Cdc2 at Y15 and T14. This inhibition involves sustained activity of the Cdc2 Y15-directed kinase, Wee1, as well as suppression of the Cdc2 Y15 phosphatase, Cdc25. We have discovered that a novel G2/M regulator, Hsl7, promotes intranuclear Wee1 degradation, and that the DNA replication checkpoint disrupts Hsl7-Wee1 interactions. In parallel, Cdc25 is inhibited through binding to 14-3-3 protein. Removal of 14-3-3 from Cdc25 is required for its mitotic activation, and we have found that dissociation of the 14-3-3-Cdc25 complex requires phosphorylation of both Cdc25 T138 (which reduces the affinity of 14-3-3 for Cdc25) and intermediate filament proteins (which then bind 14-3-3, thereby serving as an abundant 14-3-3 "sink"). Additionally, we have discovered that a previously described apoptotic inhibitor, Aven, can also regulate mitotic entry and may be important for DNA-responsive checkpoint operation. The objective of this proposal to elucidate both the Wee1 and Cdc25-modulatory arms of DNA-responsive checkpoint pathways with the long term goal of fully understanding the control of M phase entry. Towards this end, we propose to I) Delineate the role of Hsl7 in checkpoint-mediated Wee1 regulation II) Elucidate the mechanism(s) which regulate 14-3-3 release from Cdc25 and III) Determine how Aven regulates mitotic entry.