PKC TARGETING INTERACTIONS

了解细胞内信号是如何从 识别细胞内靶点的膜仍然令人望而生畏 挑战，因为这一过程中有大量的多肽 真核细胞内的信号转导。现在很明显， 这些多肽对局部作用部位的限制是 影响这些产品的专一性的监管过程 信号酶。虽然计划项目的总体重点是 了解划分的亚细胞靶向相互作用 两类关键的蛋白激酶：cAMP依赖的蛋白激酶(PKA) 以及钙/磷脂依赖的蛋白激酶C(PKC)，项目将 专注于区分两类关键的蛋白激酶：cAMP 依赖蛋白激酶(PKA)与钙/磷脂依赖蛋白 激酶C(PKC)，项目2将重点放在两者的划分上 通过与一种名为Gravin的常见锚定蛋白的关联来激活蛋白。 Gravin含有与PKA和PKC相关的酶结合部位，以及 几个目标基序将Gravin信号支架定向到 膜-细胞骨架。目标1专注于Gravin PKC相互作用和抑制 激活酶活性。通过与牛顿博士(项目)的合作，我们将 确定这两个区域是否都参与了酶结合，或者 在Gravin上有多个PKC结合位点。表面等离子激元共振 并将使用自旋荧光技术来测量结合 亲和力相互作用与Gravin/PKC机制的建立 筛选牛顿博士开发的PKC突变体家族的相互作用 (项目3)。与Jennings博士的合作项目(项目4)将 用多维核磁共振确定该化合物的溶液结构 当与PKCbetaII络合时，PKC结合肽。目标2侧重于 Gravin目标域。免疫荧光数据表明Gravin是 靶向于膜-细胞骨架，并富含在丝状伪足中 贴壁细胞、生化和共沉淀方法将是 用来确定Gravin是否是肌动蛋白结合蛋白以及它是否 与其他细胞骨架组件相关联。诱导坟墓 表达伴随着粘附性表型的开始 某些细胞类型。功能研究(与亚当斯博士和埃利斯曼， 成像核心)将被启动以确定正确的PKA或PKC Gravin的锚定和/或膜细胞骨架靶向是必要的 保持一种附着的表型。

Understanding how intracellular signals are specifically relayed from the membranes to distinct intracellular targets still remains a daunting challenge, given the large number of polypeptides devoted to the process of signal transduction within a eukaryotic cell. It is now apparent that the restriction of these polypeptides to localized sites of action is a regulatory process that functions to influence the specificity pf these signaling enzymes. While the overall focus of the program project is to understand the subcellular targeting interactions that compartmentalize two key classes of protein kinase: the cAMP dependent protein kinase (PKA) and the Ca2+/phospholipid dependent protein kinase C (PKC), project will focus on the compartmentalize two key classes of protein kinase: the cAMP dependent protein kinase (PKA) and the Ca2+/phospholipid dependent protein kinase C (PKC), project 2 will focus on the compartmentalization of both kinases through association with a common anchoring protein called gravin. Gravin contains enzyme binding sites that associate with PKA and PKC, and several targeting motifs that direct the gravin signaling scaffold to the membrane-cytoskeleton. Aim 1 focuses on gravin PKC interaction and inhibit kinase activity. In collaboration with Dr. Newton (project) we will establish whether both regions participate in enzyme binding or whether there are multiple PKC-binding sites on gravin. Surface plasmon resonance and spin fluorescence techniques will be used to measure the binding affinity of interaction and establish the mechanism of gravin/PKC interaction by screening a family of PKC mutants developed by Dr. Newton (Project 3). Collaborative ventures with Dr. Jennings (Project 4) will employ multi-dimensional NMR to determine the solution structure of the PKC binding peptides when complexed with PKCbetaII. Aim 2 focuses on the gravin targeting domains. Immunofluorescence data suggest that gravin is targeted to the membrane-cytoskeleton and is enriched in the filopodia of adherent cells, biochemical and co-sedimentation approaches will be utilized to determine whether gravin is an actin binding protein and if it associates with other cyoskeletal components. Induction of gravin expression is concomitant with the onset of an adherent phenotype in certain cell-types. Functional studies (with Dr. Adams and Ellisman, imaging core) will be initiated to determine whether correct PKA or PKC anchoring and/or membrane-cytoskeleton targeting of gravin is necessary to maintain an adherent phenotype.