STRUCTURE AND FUNCTION OF PAXILLIN

Paxillin是一种多结构域，68kDa的磷酸化蛋白，在培养的哺乳动物细胞和体内定位于特定的肌动蛋白膜附着位点。我们将验证paxillin作为质膜支架/适配器蛋白的功能，从而促进粘附和生长因子衍生信号之间的相互作用，从而导致肌动蛋白细胞骨架的重组和基因表达的调节。目的1将鉴定和表征最近鉴定的paxillin结合蛋白p42（一种肌动蛋白结合蛋白）的功能域；PKL是一种ARF-GAP，它将帕西林与包括PIX （p21 Cdc42/Rac GEF）和p21活化激酶（PAK）在内的蛋白质复合物连接起来。新的PKL和p42结合蛋白将被鉴定和表征。建议克隆p42家族成员。Aim 2将评估paxillin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，这些细胞骨架与p21Rho-和p21ARF家族成员的激活有关，在受细胞粘附、可溶性生长因子或p21Rho或p21ARF GTPase家族成员的共同表达刺激的培养细胞中。这些突变体对细胞运动的影响将通过改进的博伊登室试验和延时视频显微镜来确定。目的3将研究paxillin、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反应。分析将包括使用GST-p21-GTPase效应蛋白结合域监测p21 GTPase活性的变化。使用磷酸特异性抗体评估局灶黏附蛋白paxillin、PKL、FAK和CAS中磷酸酪氨酸含量的变化以及p38/JNK和ERK MAPK活化的变化。预计这些研究将为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提供深入的见解。

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.