Control of Mitotic Entry by Regulators of Cdc2

描述(申请人提供)：在所有真核细胞中，进入有丝分裂是由细胞周期蛋白依赖的激酶CDC2控制的。Cdc2/Cyclin B复合体受抑制激酶Myt1和Wee1和激活磷酸酶CDC25控制。虽然已经确定了一些CDC25和Myt1/Wee1调控通路，但这些通路是如何相互连接的，以及哪些上游调节子影响这些通路还不完全清楚。为了了解生理刺激和检查点控制通路如何调控G2/M转变，我们必须了解这些不同调控因子之间的因果联系和调控相互作用。该方案利用非洲爪哇卵提取物提供的强大的有丝分裂进入体外重建系统来研究CDC25和Wee1的调控。在这个提案中，我们解决了G21M调控中的几个悬而未决的问题，包括CDC25在有丝分裂时通过去除结合的14-3-3蛋白而被激活的机制，CDC25直接激活的磷酸酶是否可能受到细胞周期的调控，以及CDC25的不同亚型可能如何受到差异调控。我们还研究了Wee1的几个候选调节因子，包括激酶Pix和甲基转移酶Hsl7。我们的长期目标是从机制上了解控制进入有丝分裂的分子事件。

DESCRIPTION (provided by applicant): In all eukaryotic cells, entry into mitosis is controlled by the Cyclin-dependent kinase, Cdc2. Cdc2/Cyclin B complexes are controlled by the inhibitory kinases, Myt1 and Wee1 and by the activating phosphatase, Cdc25. Although a number of Cdc25 and Myt1/Wee1-regulatory pathways have been identified, how these pathways are interconnected and which upstream modulators impinge on these pathways is not yet entirely clear. To understand how physiological stimuli and checkpoint control pathways regulate the G2/M transition, it is essential that we understand the causal connections and regulatory interplays between these diverse regulators. This proposal employs the powerful in vitro reconstitution system for mitotic entry provided by Xenopus egg extracts to investigate Cdc25 and Wee1 regulation. In this proposal we address several outstanding issues in G21M regulation, including the mechanism by which Cdc25 is activated at mitosis through removal of bound 14-3-3 proteins, whether Cdc25-directed activating phosphatases may be cell cycle regulated and how different isoforms of Cdc25 may be differentially regulated. We also investigate several candidate regulators of Wee1, including the kinase PIx and the methyltransferase, Hsl7. Our long-term objective is to obtain a mechanistic understanding of the molecular events that control entry into mitosis.