IP3 Receptor Phosphorylation by Akt Kinase

Akt 激酶对 IP3 受体进行磷酸化

• 批准号: 6913966
• 负责人: SURESH K JOSEPH
• 金额: $ 20.35万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6913966
• 关键词: CHO cells; biological signal transduction; calcium channel; calcium flux; cytochrome c; endoplasmic reticulum; immunofluorescence technique; immunoprecipitation; inositol phosphates; intracellular transport; phosphomonoesterases; phosphorylation; protein isoforms; protein structure function; serine threonine protein kinase; tissue /cell culture

DESCRIPTION (provided by applicant): The discharge of Ca2+ from intracellular stores is crucial to the regulation of fertilization, secretion, muscle contraction, neuronal function, immune responses, programmed cell death and many other diverse cellular processes. Binding of the intracellular messenger D-myo-inositol 1,4,5-trisphosphate (IP3) by a specific receptor/Ca2+ ion channel (IP3R) mediates the mobilization of Ca2+ from internal stores. The multifunctional serine/threonine protein kinase Akt acts downstream of the lipid kinase phosphoinositide 3-kinase and functions as an essential mediator in many cellular responses including cell-cycle regulation, modulation of intermediary metabolism, cell survival and transcriptional regulation. Thus IP3Rs and Akt kinase are at key nodes in intracellular signaling networks. This grant has as its starting point that all three isoforms of IP3Rs have a consensus sequence site for Akt phosphorylation in the C-terminal tail and our preliminary data indicate that this site can be phosphorylated by Akt kinase in vitro and in vivo. The objective of this proposal is to study the mechanism of the interaction between Akt kinase and IP3Rs and to assess the biological role of this phosphorylation. The specific aims of the proposed study are: I. To study the mechanism of Akt phosphorylation of IP3R channels The specificity with respect to IP3R and Akt kinase isoforms will be examined. The role of PP1, PP2A and other phosphatases in IP3R phosphorylation will be investigated. Immunofluorescence and subcellular fractionation will be used to test the hypothesis that a substantial amount of Akt kinase activation occurs at the ER or that Akt translocates to the ER membrane. II. To determine the biological consequences of IP3R phosphorylation at the Akt site. COS cells and DT-40 ZP3R knock-out cells will be used to examine the functional consequences of Akt phosphorylation by monitoring Ca2+ fluxes. The hypothesis that cytochrome c binding to the IP3R is inhibited by Akt phosphorylation will be tested. Stable DT-40 cell lines expressing IP3R mutants of the Akt phosphorylation site will be used to determine if Akt phosphorylation influences the ability of the cells to undergo Ca2+ -dependent gene activation or apoptosis in response to IgM. This pilot and feasibility proposal is intended to assess the physiological significance of Akt kinase phosphorylation of IP3Rs. Understanding the 'crosstalk' between Ca2+ signaling and the Akt pathways may yield hitherto unrecognized insights into both pathways. The studies proposed in this application are significant since both signaling systems play important roles in many clinically relevant processes such as insulin and growth factor signaling, angiogenesis and oncogenesis.

描述（由申请人提供）：从细胞内储存释放Ca 2+对于受精、分泌、肌肉收缩、神经元功能、免疫应答、程序性细胞死亡和许多其他不同细胞过程的调节至关重要。细胞内信使D-肌肌醇1，4，5-三磷酸（IP 3）通过特异性受体/Ca 2+离子通道（IP 3R）的结合介导Ca 2+从内部储存的动员。多功能丝氨酸/苏氨酸蛋白激酶Akt在脂质激酶磷酸肌醇3-激酶的下游起作用，并在许多细胞反应中起重要的介体作用，包括细胞周期调节、中间代谢的调节、细胞存活和转录调节。因此，IP 3R和Akt激酶在细胞内信号网络中处于关键节点。该授权的出发点是IP 3R的所有三种亚型在C-末端尾中具有Akt磷酸化的共有序列位点，并且我们的初步数据表明该位点可以在体外和体内被Akt激酶磷酸化。该提案的目的是研究Akt激酶和IP 3Rs之间相互作用的机制，并评估这种磷酸化的生物学作用。拟议研究的具体目标是： I.为了研究IP 3R通道Akt磷酸化的机制，将检查关于IP 3R和Akt激酶同种型的特异性。将研究PP 1、PP 2A和其他磷酸酶在IP 3R磷酸化中的作用。免疫荧光和亚细胞分级分离将被用来测试的假设，大量的Akt激酶激活发生在ER或Akt易位到ER膜。二.确定Akt位点IP 3R磷酸化的生物学后果。COS细胞和DT-40 ZP 3R敲除细胞将用于通过监测Ca 2+通量来检查Akt磷酸化的功能后果。将检验Akt磷酸化抑制细胞色素c与IP 3R结合的假设。表达Akt磷酸化位点的IP 3R突变体的稳定DT-40细胞系将用于确定Akt磷酸化是否影响细胞响应IgM而经历Ca 2+依赖性基因活化或凋亡的能力。该试验性和可行性提案旨在评估IP 3Rs的Akt激酶磷酸化的生理意义。理解Ca 2+信号传导和Akt通路之间的“串扰”可能会产生迄今为止未被认识到的对这两种通路的见解。本申请中提出的研究是重要的，因为两种信号传导系统在许多临床相关过程中发挥重要作用，如胰岛素和生长因子信号传导、血管生成和肿瘤发生。