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; 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）：从细胞内存储中排出Ca2+对于调节受精，分泌，肌肉收缩，神经元功能，免疫反应，程序性细胞死亡以及许多其他不同的细胞过程至关重要。特定受体/Ca2+离子通道（IP3R）的细胞内信使D-myositol 1,4,5-三磷酸（IP3）的结合介导了内部商店的Ca2+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-激酶的下游作用，在许多细胞环反应，包括细胞周期调节，调节中间代谢，细胞生存和转录调节的许多细胞反应中起着必不可少的介质作用。因此，IP3RS和AKT激酶在细胞内信号网络中的关键节点处。该赠款的起点是，IP3R的所有三种同工型都具有在C末端尾部Akt磷酸化的共有序列位点，而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。该建议的目的是研究Akt激酶与IP3RS之间相互作用的机制，并评估该磷酸化的生物学作用。拟议研究的具体目的是： I.为了研究IP3R通道Akt磷酸化的机制，将检查相对于IP3R和Akt激酶同工型的特异性。将研究PP1，PP2A和其他磷酸酶在IP3R磷酸化中的作用。免疫荧光和亚细胞分级将用于检验以下假设：大量Akt激酶激活发生在ER或AKT转移到ER膜上。 ii。确定Akt位点IP3R磷酸化的生物学后果。 COS细胞和DT-40 ZP3R敲除细胞将用于通过监测Ca2+通量来检查Akt磷酸化的功能后果。 AKT磷酸化抑制了细胞色素C与IP3R的结合的假说。表达Akt磷酸化位点IP3R突变体的稳定的DT -40细胞系将用于确定AKT磷酸化是否会影响细胞对CA2+依赖性基因激活或凋亡对IgM的凋亡的能力。该试点和可行性建议旨在评估IP3RS的Akt激酶磷酸化的生理意义。了解Ca2+信号传导和AKT途径之间的“串扰”可能会产生迄今无法识别的两种途径的见解。该应用中提出的研究很重要，因为两个信号系统在许多临床相关的过程中都起着重要作用，例如胰岛素和生长因子信号传导，血管生成和肿瘤发生。