ROLES OF PH DOMAINS IN MAST CELL SIGNAL TRANSDUCTION

说明(改编自调查人员摘要)：本报告的目的 研究建议是确定蛋白质-蛋白质和 Pleckstrin同源(PH)结构域介导的蛋白质-脂质相互作用 信号转导。这项研究的结果有望对以下方面有所启发 肥大细胞生理学的新方面。调查员最近的研究 蛋白激酶C(PKC)是一种PH结构域结合蛋白，并显示 与BTK结合的PKC是一种Tec家族酪氨酸激酶，它使BTK磷酸化，并 抑制其酶活性。1)为了阐明其作用机制， PKC对BTK(及其相关EMT)的调控，(A)PKC结合部位 在BTK的PH结构域上；和(B)PKC上的PH结构域结合位点将是 已映射。(C)BTK(和EMT)上PKC磷酸化位点的测定 将有助于理解PKC对BTK(和EMT)的监管。 研究人员有证据表明，膜中的PKC-Beta1是 酪氨酸在FceRI交联物上的磷酸化和酶调节 BTK体外培养。因此，(D)他将以生化和生物为特征 PKC-β酪氨酸磷酸化的作用。鉴于最近的 磷脂酰肌醇-4，5-二磷酸(PIP2)与PH结构域结合的研究 并且该脂类在PH域上的结合位置与 初步确定的PKC结合部位，(E)PIP2对PKC-PH的影响 我们将分析域绑定。2)调查员的初步 实验已经确定肌动蛋白是另一种PH结构域结合蛋白。 由于肌动蛋白是许多生理学中最重要的细胞骨架蛋白 在细胞方面，PH结构域将被表征为肌动蛋白结合 模块。3)PKC在细胞信号的各个方面发挥着关键作用。 包括BTK结构域在内的几个PH结构域的C-末端部分与 异源三聚体G蛋白的β/伽马复合体，参与 许多信号转导系统。因此，一些有趣的可能性 都被抚养长大。其一是单个PH结构域可能同时与PKC和 G蛋白β/γ，因此PKC可能影响细胞的活性 G蛋白β/伽马或反之亦然。另一种可能性是PKC和 G蛋白β/γ相互竞争BTK的PH域。(A) 这些可能性将通过转染异基因细胞来检验。 以及相关的表达载体。(B)同样，BTK之间的互动 和肌动蛋白的特征。(C)因为有几个PH 结构域结合分子，单个结合分子的不同作用 将使用相互作用特定的抑制剂或PH域进行研究 BTK的突变体已失去与一个或一个子集相互作用的能力 PH结构域结合分子。

DESCRIPTION (Adapted from Investigator's abstract): The purpose of this research proposal is to determine the roles of protein-protein and protein-lipid interactions mediated by pleckstrin homology (PH) domains in signal transduction. Results of this study are expected to shed light on new aspects of mast cell physiology. The investigator's recent study identified protein kinase C (PKC) as a PH domain-binding protein and showed that PKC bound to Btk, a Tec family tyrosine kinase, phosphorylates Btk and inhibits its enzymatic activity. 1) In order to elucidate the mechanism for the Btk (and its relative Emt) regulation by PKC, (a) the PKC binding site on the PH domain of Btk and (b) the PH domain-binding site on PKC will be mapped. (c) Determination of the PKC phosphorylation site on Btk (and Emt) will be informative for understanding the Btk (and Emt) regulation by PKC. The investigator has evidence that PKC-beta1 in membranes is tyrosine-phosphorylated upon FceRI cross-linking and enzymatically regulated by Btk in vitro. Hence, (d) he will characterize biochemical and biologic effects of PKC-betaI tyrosine phosphorylation. In light of the recent findings that PH domains bind to phosphatidylinositol 4,5bisphosphate (PIP2) and that the binding site of this lipid on the PH domain overlaps that of the tentatively assigned PKC binding site, (e) effects of PIP2 on the PKC-PH domain binding will be analyzed. 2) The investigator's preliminary experiments have identified actin as another PH domain-binding protein. Since actin is the most important cytoskeletal protein in many physiologic aspects of the cell, PH domains will be characterized as an actin-binding module. 3) PKC plays key roles in various aspects of cellular signaling. The C-terminal portions of several PH domains including that of Btk bind to the beta/gamma complex of heterotrimeric G-proteins, complexes involved in many signal transduction systems. Therefore, some interesting possibilities are raised. One is that a single PH domain might interact with both PKC and G-protein beta/gamma and hence PKC might affect the activity of the G-protein beta/gamma or vice versa. Another possibility is that PKC and G-protein beta/gamma compete with each other for the PH domain of Btk. (a) These possibilities will be tested by transfection of heterologous cells with relevant expression vectors. (b) Similarly, interactions between Btk and actin will be characterized. (c) Since there are several PH domain-binding molecules, distinct roles of the individual binding molecules will be investigated using interaction-specific inhibitors or PH domain mutants of Btk that have lost the capacity to interact with one or a subset of the PH domain-binding molecules.