IP3 Receptor Phosphorylation by Akt Kinase

描述(由申请人提供)：细胞内钙离子的释放对受精、分泌、肌肉收缩、神经功能、免疫反应、程序性细胞死亡和许多其他不同的细胞过程的调节至关重要。细胞内信使D-肌醇-1，4，5-三磷酸(IP3)与特定的受体/钙离子通道(IP3R)结合，介导细胞内钙离子的动员。多功能丝氨酸/苏氨酸蛋白激酶Akt位于磷脂酸肌醇3-激酶的下游，在细胞周期调控、中间代谢调控、细胞存活和转录调控等多种细胞反应中发挥重要的调节作用。因此，IP3Rs和Akt激酶在细胞内信号网络中处于关键节点。这项授权的起点是IP3Rs的三种异构体都在C末端有一个共同的Akt磷酸化序列位点，我们的初步数据表明，该位点在体外和体内都可以被Akt激酶磷酸化。这项建议的目的是研究Akt激酶和IP3Rs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： 为了研究IP3R通道Akt磷酸化的机制，我们将检测IP3R和Akt激酶亚型的特异性。PP1、PP2A和其他磷酸酶在IP3R磷酸化中的作用将被研究。免疫荧光和亚细胞分级将被用来检验这样的假设，即大量的Akt激酶激活发生在内质网，或者Akt转位到ER膜。II.确定IP3R在Akt位点的磷酸化的生物学后果。CoS细胞和DT-40 ZP3R基因敲除细胞将被用来通过监测钙离子流动来检测Akt磷酸化的功能后果。细胞色素c与IP3R结合被Akt磷酸化抑制的假设将得到验证。稳定表达Akt磷酸化位点IP3R突变体的DT-40细胞株将被用来确定Akt磷酸化是否影响细胞对IgM的钙依赖基因激活或凋亡的能力。这项中试和可行性建议旨在评估Akt激酶磷酸化IP3Rs的生理意义。了解Ca~(2+)信号和Akt通路之间的‘串扰’可能会对这两个通路产生迄今为止尚未被认识到的见解。由于这两个信号系统在胰岛素和生长因子信号转导、血管生成和肿瘤形成等许多临床相关过程中发挥着重要作用，因此在这一应用中提出的研究具有重要意义。

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.