PHOSPHATIDYLINOSITOL 4 KINASE (YEAST PIK1) REGULATION

磷脂酰肌醇 4 激酶（酵母 PIK1）调节

• 批准号: 2733324
• 负责人: BO QING WANG
• 金额: $ 3.05万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-19 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2733324
• 关键词: biological signal transduction; cell cycle; cytoskeleton; phosphatidylinositol 3 kinase; phosphatidylinositols; phospholipase C; phosphorylation; phosphotransferases; point mutation; protein kinase C; protein structure function; site directed mutagenesis; stress proteins

The goal of this research is to understand the function, regulation, and localization of phosphatidylinositol 4-kinase (PI4K) in yeast (yeast Pik1).PI4K catalyzes the first committed step in the biosynthesis of PI4,4-bisphosphate (PI4,5P2) and, thus, is likely to be highly regulated. Hydrolysis of PI4,5P2 by Phospholipase C in response to extracellular stimuli generates two second messengers, inositol 1,4,5- trisphosphate and diacylglycerol (DAG), which initiates intracellular signaling cascades that modulate cell proliferation and differentiation. A number of PIK-related kinases have recently been identified and their functions are being unveiled. The dysfunction of PIKs leads to physiological disorders such as loss of immunological function and cancer. Revealing the function of these PIKs will contribute to our understanding and the cure of cancer. The PIK1 gene encoding a soluble PI4-kinase (125 Kd, 1066 aa) from S. cerevisiae was cloned and shown to be an essential gene in Thorner lab. The 274-residue C-terminal segment of Pik1 shares the highest homology with other PIKs including several PI3-kinases and comprise the catalytic domain. The 792-residue N- terminal segment of Pik1 contains, near its N-terminal end, a second region of high similarity between Pik1 and PI3-kinases. Evidence suggests that the activity of Pik1 is probably regulated through the interaction of its putative N-terminal regulatory domain with some other cellular protein(s). Rabbit polyclonal antisera against the N-terminal segment of Pik1 as well as an epitope-tagged version of this domain have been made. Two-hybrid assay and co-immunoprecipitation are being used to search for proteins that interact with Pik1 and to characterize their interaction. The localization of Pik1 is being investigated using both subcellular fractionation and indirect immunofluorescence. The minimal functional domains of Pik1 will be determined by deletion mutagenesis.

本研究的目的是了解该系统的功能、调节和 磷脂酰肌醇4-激酶(PI4K)在酵母中的定位 PI4K催化了生物合成的第一步 PI4，4-二磷酸(PI4，5P2)，因此很可能是高度 受监管的。磷脂酶C对PI4，5P2的反应 细胞外刺激产生两个第二信使，肌醇1，4，5- 三磷酸和二酰甘油(DAG)，它启动细胞内 调节细胞增殖和分化的信号级联反应。 最近发现了许多与PIK相关的激酶，它们的 功能正在揭晓。PIKs功能障碍导致 生理紊乱，如免疫功能丧失和 癌症。揭示这些PIK的功能将有助于我们 对癌症的理解和治疗。PIK1基因编码一个可溶性的 从酿酒酵母中克隆了P4-激酶(125kD，1066aa)，并对其进行了鉴定。 是索纳实验室的必备基因。274位残基C-末端片段 与其他PIK的同源性最高，包括几个 PI3-激酶，并组成催化结构域。792个残基N- Pik1的终端段在其N端附近包含第二个 Pik1和PI3-激酶高度相似的区域。证据 提示Pik1的活性可能是通过 其N-末端调控结构域与其他结构域的相互作用 细胞蛋白(S)。兔抗N端多克隆抗血清 Pik1的片段以及该结构域的表位标记版本 已经做好了。目前正在使用双杂交检测和免疫共沉淀。 寻找与Pik1相互作用的蛋白质并鉴定其 互动。正在使用两种方法研究Pik1的本地化 亚细胞分离和间接免疫荧光。最小的 Pik1的功能结构域将通过缺失突变来确定。