ROLES OF PH DOMAINS IN MAST CELL SIGNAL TRANSDUCTION

描述（改编自研究者摘要）：本研究的目的 研究建议是确定蛋白质-蛋白质的作用， 蛋白质-脂质相互作用介导的普列克底物蛋白的同源性（PH）结构域， 信号转导 这项研究的结果有望揭示 肥大细胞生理学的新方面。 调查员最近的研究 鉴定蛋白激酶C（PKC）为PH结构域结合蛋白，并显示 PKC与Tec家族酪氨酸激酶Btk结合，使Btk磷酸化， 抑制其酶活性。 1)为了阐明 PKC对Btk（及其相关Emt）的调节，（a）PKC结合位点 （B）PKC上的PH结构域结合位点将是 映射。 (c)Btk（和Emt）上PKC磷酸化位点的测定 将为理解PKC对Btk（和EMT）的调节提供信息。 研究者有证据表明，膜中的PKC-β 1 在FceRI交联后酪氨酸磷酸化，并且酶促调节 Btk in vitro. 因此，（d）他将描述生物化学和生物学 PKC-β I酪氨酸磷酸化的影响。 鉴于最近的 发现PH结构域与磷脂酰肌醇4，5二磷酸（PIP 2）结合 并且这种脂质在PH结构域上的结合位点与 初步指定的PKC结合位点，（e）PIP 2对PKC-PH的影响 将分析域结合。 2)调查员的初步报告 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过程中最重要的细胞骨架蛋白， 在细胞的各个方面，PH结构域将被表征为肌动蛋白结合蛋白。 module. 3)PKC在细胞信号传导的各个方面起着关键作用。 几个PH结构域的C-末端部分，包括Btk的C-末端部分，结合至 异源三聚体G蛋白的β/γ复合物， 许多信号转导系统。 因此，一些有趣的可能性 被抬高了 一个是一个单一的PH结构域可能与PKC和 G-蛋白β/γ和PKC可能会影响细胞的活性， G蛋白β/γ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结构域。（一） 这些可能性将通过转染异源细胞进行测试 相关的表达载体。 (b)同样，Btk之间的相互作用 和肌动蛋白将被表征。 (c)因为有几个PH值 结构域结合分子，单个结合分子的不同作用 将使用相互作用特异性抑制剂或PH结构域进行研究 Btk的突变体已经失去了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.