Regulation of Cdc4 kinase activity in budding yeast

描述(由申请人提供)： Polo-like kinase(Plk)是有丝分裂进程的重要调节因子，PLK功能异常与不同类型肿瘤的发生密切相关。在发芽酵母中，CDC5是唯一的PLK，是有丝分裂退出所必需的。尽管Plks在有丝分裂中很重要，但对激酶活性的调节仍然知之甚少。这里提出的研究将通过开发在萌芽酵母中纯化活性和非活性形式的Cdc5的能力来解决这一问题。首先，与活性CDC5特定相关的蛋白质将通过质谱学进行鉴定，并在激酶分析和遗传实验中被测试为CDC5活性的调节器。其次，利用质谱学方法对活性CdC5上的主要磷酸化位点进行定位。这些位点的鉴定将使一系列cdc5亚磷酸盐突变体的产生，这些突变体将在体外测试激酶活性，并检测拯救cdc5-1菌株晚期有丝分裂停滞的能力。最后，以失活的CdC5为底物，采用标准的层析技术从野生型酵母提取物中纯化出CdC5激活蛋白(S)。这些研究将允许识别和表征控制CDC5活性的生理相关调节蛋白。

DESCRIPTION (provided by applicant): Polo-like kinases (Plks) are important regulators of mitotic progression, and abnormal Plk function has been implicated in the pathogenesis of different tumor types. In the budding yeast, Saccharomyces cerevisiae, Cdc5 is the only Plk, and is essential for mitotic exit. Despite the importance of Plks in mitosis, the regulation of kinase activity remains poorly understood. The research proposed here will address this problem by exploiting the ability to purify both active and inactive forms of Cdc5 in budding yeast. First, proteins that specifically associate with active Cdc5 will be identified by mass spectrometry, and tested as regulators of Cdc5 activity in kinase assays and genetic experiments. Second, the major sites of phosphorylation on active Cdc5 will be mapped by mass spectrometry. Identification of these sites will enable a series of Cdc5 phosphosite mutants to be generated, which will be tested for kinase activity in vitro, and for the ability to rescue the late mitotic arrest of the cdc5-1 strain. Finally, inactive Cdc5 will be used as a substrate to purify Cdc5-activating kinase(s) from extracts of wild-type yeast by standard chromatographic techniques. These studies will permit the identification and characterization of physiologically relevant regulatory proteins that control the activity of Cdc5.