PKC TARGETING INTERACTIONS

了解细胞内信号是如何特异性地从 细胞膜上不同的细胞内目标仍然是一个艰巨的任务， 挑战，鉴于大量的多肽致力于该过程 真核细胞内的信号转导。现在很明显， 将这些多肽限制于局部作用位点是一种 调节过程的功能，以影响这些特异性 信号酶虽然项目的总体重点是 了解亚细胞靶向相互作用， 两类关键的蛋白激酶：cAMP依赖性蛋白激酶（PKA） 和Ca 2 +/磷脂依赖性蛋白激酶C（PKC），项目将 重点放在划分两个关键类别的蛋白激酶：cAMP 依赖蛋白激酶（PKA）和钙/磷脂依赖蛋白 激酶C（PKC），项目2将重点放在两个区室化 通过与一种叫做gravin的常见锚定蛋白的结合， Gravin含有与PKA和PKC相关的酶结合位点， 将gravin信号传导支架引导到 膜细胞骨架目的1研究gravin蛋白激酶C相互作用， 激酶活性与牛顿博士（项目）合作，我们将 确定这两个区域是否参与酶结合，或者是否 在Gravin上存在多个PKC结合位点。表面等离子体共振 和自旋荧光技术将用于测量结合 建立了Gravin/PKC的作用机制 通过筛选由Newton博士开发的PKC突变体家族， （项目3）。与詹宁斯博士的合作企业（项目4）将 采用多维NMR来确定溶液的结构， 与PKC β II复合时的PKC结合肽。目标2侧重于 gravin靶向结构域。免疫荧光数据表明，gravin是 靶向膜细胞骨架，并在丝状伪足中富集 贴壁细胞，生化和共沉淀方法将是 用于确定gravin是否是肌动蛋白结合蛋白， 与其他细胞骨架成分结合。Gravin诱导 表达伴随着粘附表型的出现， 某些细胞类型功能研究（与亚当斯博士和Ellisman， 成像核心），以确定是否正确的PKA或PKC gravin的锚定和/或膜-细胞骨架靶向是必需的， 保持粘附表型。

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.