STRUCTURE AND FUNCTION OF PAXILLIN

Paxlin是一种多结构域的68 kDa磷酸蛋白，定位于培养的哺乳动物细胞和体内特定的肌动蛋白-膜附着部位。我们将检验这一假设，即paxlin作为质膜上的支架/适配蛋白，从而促进黏附和生长因子衍生的信号之间的相互作用，导致肌动蛋白细胞骨架的重组和基因表达的调节。目的1鉴定和鉴定最近发现的Paclin结合蛋白P42和PKL的功能结构域，P42是一种肌动蛋白结合蛋白；PKL是一种ARF-GAP，将PIX(一种p21CDC42/RAC gef)和p21激活的激酶(PAK)等蛋白质的复合体连接起来。新的PKL和P42结合蛋白将被鉴定和鉴定。建议克隆P42家族成员。目的评价在细胞黏附、可溶性生长因子或p21Rho或p21ARF GTPase家族成员的共同表达的刺激下，与这些结合蛋白相互作用的Paxlin在调节与p21Rho-和p21ARF-家族成员激活相关的肌动蛋白细胞骨架重组中的重要性。这些突变体对细胞运动的影响将在改进的博伊登小室分析和延时视频显微镜中确定。目的3将研究如何影响与细胞黏附和生长因子刺激相关的生化信号级联的巴西林，PKL和P42。分析将包括利用GST-p21-GTP酶效应蛋白结合域监测p21GTP酶活性的变化。焦点黏附蛋白Paxlin、PKL、FAK和CAS的磷酸化酪氨酸含量的变化以及p38/JNK和ERK MAPK活性的变化将使用磷酸化特异性抗体来评估。预计这些研究将提供对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的洞察。

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.