Regulation of Cdc4 kinase activity in budding yeast

芽殖酵母中 Cdc4 激酶活性的调节

• 批准号: 6691359
• 负责人: David Randle
• 金额: $ 4.16万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6691359
• 关键词: autoradiography; enzyme activity; enzyme induction /repression; fungal proteins; mass spectrometry; phosphorylation; postdoctoral investigator; protein kinase; protein protein interaction; protein purification; site directed mutagenesis; transcription factor; yeasts

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.

描述（由申请人提供）： Polo样激酶（Plks）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病机制有关。在芽殖酵母中，酿酒酵母（Saccharomyces cerevisiae），Cdc 5是唯一的Plk，并且是有丝分裂退出所必需的。尽管Plks在有丝分裂中的重要性，激酶活性的调节仍然知之甚少。这里提出的研究将通过利用在芽殖酵母中纯化活性和非活性形式Cdc 5的能力来解决这个问题。首先，与活性Cdc 5特异性相关的蛋白质将通过质谱法鉴定，并在激酶测定和遗传实验中作为Cdc 5活性的调节剂进行测试。第二，将通过质谱法绘制活性Cdc 5上磷酸化的主要位点。这些位点的鉴定将能够产生一系列Cdc 5磷酸化位点突变体，这些突变体将在体外测试激酶活性，以及拯救Cdc 5 -1菌株的晚期有丝分裂停滞的能力。 最后，失活Cdc 5将被用作底物，通过标准色谱技术从野生型酵母提取物中纯化Cdc 5激活激酶。这些研究将允许识别和表征生理相关的调节蛋白，控制Cdc 5的活性。