MOLECULAR ASSOCIATIONS OF PHOSPHOLIPASE C

描述(改编自申请者摘要)：磷脂酰肌醇 4，5-二磷酸[PtdIns(4，5)P2]是一种非常重要的分子 参与细胞调控的真核信号转导通路 增殖、分化和致癌转化。PtdIns(4，5)P2 被磷脂酶C(PLC)水解以产生第二信使 肌醇1，4，5-三磷酸[Ins(1，4，5)P3]和二酰甘油(DG)。 INS(1，4，5)P3触发体内钙释放，从而 调节钙调节途径，而DG激活磷脂-和 依赖CA2的蛋白激酶C PtdIns(4，5)P2自身调节其他 重要的功能，如几种肌动蛋白结合蛋白的活性。 显然，PtdIns(4，5)P2‘S胞内浓度必须严格 监管，但对这一规定几乎一无所知。宽阔的， 长期目标是阐明调节细胞周期的机制 PtdIns(4，5)P2的动态平衡及其在细胞周期调控中的作用 信号转导。这项提案的具体目标采用了 双杂交筛选及酵母强大的实验优势 利用酿酒酵母鉴定与酵母相互作用的蛋白质 磷脂酶C(PLCLP)，并确定其意义 相互作用(S)为体内酶的功能。申请人有 进行了双杂交筛选，分离出了许多落入 至少6个不同的小组。申请人将完成这项工作，并将 通过测序确定相应的基因(SA1)。我们的计划是 通过以下方法证实PLCLP与这些基因编码的蛋白质之间的相互作用 自主生化技术(SA2)。其目的是确定 PLCLP负责互动的领域(S)并确定 这些相互作用在PLCLP(SA4)体内功能中的作用。

DESCRIPTION (Adapted from applicant's abstract): Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is a vitally important molecule in eukaryotic signal transduction pathways, involved in the control of cell proliferation, differentiation, and oncogenic transformation. PtdIns(4,5)P2 is hydrolyzed by phospholipase C (PLC) to produce the second messengers inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DG). Ins(1,4,5)P3 triggers release of calcium from internal stores and thus modulates Ca2+ -regulated pathways, while DG activates the phospholipid- and Ca2+ -dependent protein kinase C. PtdIns(4,5)P2 itself regulates other important functions, such as the activity of several actin binding proteins. Clearly, PtdIns(4,5)P2's intracellular concentration must be stringently regulated, but almost nothing is known about this regulation. The broad, long term objective is to elucidate the mechanisms regulating the homeostasis of PtdIns(4,5)P2 and to determine the role of this regulation in signal transduction. The specific aims of this proposal employ the two-hybrid screen and the powerful experimental advantages of the yeast Saccharomyces cerevisiae to identify proteins which interact with yeast phospholipase C (Plclp) and to determine the significance of the interaction(s) for the in vivo function of the enzyme. The applicant has performed the two-hybrid screen and isolated many plasmids which fall into at least 6 different groups. The applicant will complete this work and will identify the corresponding genes by sequencing (SA1). The plan is to confirm the interaction between Plclp and proteins encoded by these genes by independent biochemical techniques (SA2). The intent is to identify the domain(s) of Plclp responsible for the interactions (SA3) and to determine the roles of these interactions for the in vivo functions of Plclp (SA4).