STRUCTURE AND FUNCTION OF PAXILLIN

桩蛋白是一种多结构域的68 kDa磷蛋白，在培养的哺乳动物细胞和体内定位于专门的肌动蛋白膜附着位点。 我们将测试的假设，桩蛋白作为一个支架/适配器蛋白质在质膜，从而促进粘附和生长因子衍生的信号，导致肌动蛋白细胞骨架的重组和基因表达的调制之间的相互作用。 目的1将鉴定和表征最近鉴定的桩蛋白结合蛋白p42（actin binding protein）和PKL（ARF-GAP）的功能结构域，所述桩蛋白结合蛋白p42是肌动蛋白结合蛋白; PKL是将桩蛋白连接到包括PIX（a p21 Cdc 42/Rac GEF）和p21活化激酶（PAK）的蛋白复合物的ARF-GAP。 新的PKL和p42结合蛋白将被鉴定和表征。 克隆p42家族成员。 目的2将评估桩蛋白与这些结合蛋白的相互作用在调节肌动蛋白细胞骨架重组中的重要性，所述肌动蛋白细胞骨架重组与细胞粘附、可溶性生长因子或p21 Rho或p21 ARF GTf 3家族成员的共表达刺激的培养细胞中的p21 Rho和p21 ARF家族成员的活化相关。 这些突变体对细胞运动性的影响将在改良的Boyden室测定和延时视频显微镜中测定。 目的3将研究桩蛋白、PKL和p42如何影响与细胞粘附和生长因子刺激相关的生化信号级联。 分析将包括使用GST-p21-GTdR效应蛋白结合结构域监测p21 GTdR活性的变化。 将使用磷酸特异性抗体评价粘着斑蛋白桩蛋白、PKL、FAK和CAS的磷酸酪氨酸含量的变化以及p38/JNK和ERK MAPK活化的变化。 预计这些研究将提供深入了解细胞与细胞外环境相互作用和交流以调节正常和肿瘤生长以及细胞运动的分子机制。

Paxillin is a multi-domain, 68kDa phosphoprotein that localizes to specialized actin-membrane attachment sites in cultured mammalian cells and in vivo. We will test the hypothesis that paxillin functions as a scaffold/adapter protein at the plasma membrane, thereby facilitating the interplay between adhesion- and growth factor-derived signals that result in reorganization of the actin cytoskeleton and modulation of gene expression. Aim 1 will identify and characterize the functional domains of the recently identified paxillin binding proteins p42, an actin binding protein; and PKL, an ARF-GAP that links paxillin to a complex of proteins including PIX (a p21 Cdc42/Rac GEF) and the p21 activated kinase (PAK). Novel PKL and p42 binding proteins will be identified and characterized. Cloning of p42 family members is proposed. Aim 2 will evaluate the importance of paxillin interactions with these binding proteins in regulating reorganization of the actin cytoskeleton associated with activation of p21Rho- and p21ARF-family members in cultured cells stimulated by cell adhesion, soluble growth factors, or by co- expression of p21Rho or p21ARF GTPase family members. The effect of these mutants on cell motility will be determined in modified Boyden chamber assays and time-lapse video microscopy. Aim 3 will examine how paxillin, PKL and p42 impact on the biochemical signaling cascades associated with cell adhesion and growth factor stimulation. Analysis will include monitoring of changes in p21 GTPase activities using GST-p21-GTPase effector protein binding domains. Changes in phosphotyrosine content of focal adhesion proteins paxillin, PKL, FAK and CAS as well as changes in p38/JNK and ERK MAPK activation will be evaluated using phosphospecific antibodies. It is anticipated that these studies will provide insight into the molecular mechanisms by which cells interact and communicate with the extracellular environment to regulate normal and neoplastic growth, and cell motility.