The Role of MIM in growth factor signaling and cell motility

描述（由申请人提供）：肿瘤扩散是细胞膜连续突起、侵入和迁移的结果，这些活动需要动态肌动蛋白细胞骨架重组。肌动蛋白在细胞前缘的组装是通过Arp 2/3复合物、WASP/WAVE家族蛋白和corpine的功能实现的，它们被Rho小GTP酶和蛋白酪氨酸激酶Src激活。我们最近的研究发现，corneumn结合的蛋白质产物的丢失在转移（MIM）基因，其表达经常丢失在一个子集的侵袭性肿瘤细胞和先进的人类膀胱细胞癌。MIM的过表达显著抑制肿瘤细胞或正常细胞由生长因子介导的运动，并抑制由组成性激活的Rac 1诱导的板状伪足形成，Rac 1是Rho GT3家族的成员。在细胞内，MIM与Rac 1共定位并能够与Rac 1共沉淀。在体外，MIM抑制WASP介导的肌动蛋白聚合。有趣的是，MIM经历了一个快速的酪氨酸磷酸化，同时短暂的相互作用与coronin响应生长因子。MIM抑制Rac 1需要两个酪氨酸残基的磷酸化。基于这些数据，我们假设MIM涉及一种新的信号通路，拮抗肌动蛋白细胞骨架组装中Rac的功能，该活性通过酪氨酸磷酸化进行调节。为了验证这一假设，我们提出了MIM拮抗Rac的机制。因此，我们将确定MIM是否直接与Rac相互作用，并检查直接关联是否是抑制Rac的必要和充分条件。我们还将分析Rac的效应子IRSp 53和WAVE在MIM功能中的作用，并测试Rac的抑制是否是MIM调节生长因子诱导的细胞运动的机制。我们将描述MIM磷酸化的信号通路，并检查Src是否是负责PDGF介导的MIM酪氨酸磷酸化的主要酪氨酸激酶，并分析Src敲除细胞和过表达温度敏感性v-Src的细胞中MIM的酪氨酸磷酸化。我们还将探讨酪氨酸磷酸化在MIM与Rac和coronin的相互作用以及细胞运动性的调节中的作用。最后，我们将研究MIM表达在低水平的肿瘤细胞的运动性的作用，通过沉默MIM在MIM表达细胞中使用RNA干扰，并研究是否MIM表达的抑制将导致增加在板状伪足的发展，Rac激活，肌动蛋白聚合在细胞和细胞运动性。预计该提案目标的实现将指导我们未来的努力，以了解MIM如何促进肿瘤进展。

DESCRIPTION (provided by applicant): Tumor spreading occurs as a consequence of continuous cell membrane protrusion, invasion and migration, the activities that require dynamic actin cytoskeleton reorganization. Actin assembly in the cell leading edge is achieved by the functions of Arp2/3 complex, WASP/WAVE family proteins and cortactin, which are activated by Rho small GTPases and protein tyrosine kinase Src. Our recent study has uncovered that cortactin binds to the protein product of missing in metastasis (MIM) gene, expression of which is frequently lost in a subset of aggressive tumor cells and advanced human bladder cell carcinomas. Overexpression of MIM inhibits markedly the motility of tumor cells or normal cells mediated by growth factors, and restrains the lamellipodia formation induced by constitutively activated Rac1, a member of the Rho GTPase family. Within cells MIM colocalizes with and is able to coprecipitate with Rac1. In vitro, MIM inhibits the actin polymerization mediated by WASP. Interestingly, MIM undergoes a rapid tyrosine phosphorylation in concurrent with transient interaction with cortactin in response to growth factors. Phosphorylation at two tyrosine residues is required for MIM to inhibit Rac1. Based on these data, we hypothesize that MIM is implicated in a novel signaling pathway antagonizing the function of Rac in the assembly of actin cytoskeleton, the activity that is subjected to a regulation through tyrosine phosphorylation. To test this hypothesis, we propose to characterize the mechanism for MIM to antagonize Rac. Therefore, we will determine whether MIM interacts directly with Rac and examine whether the direct association is necessary and sufficient to inhibit Rac. We will also analyze the role of IRSp53 and WAVE, the effectors of Rac, in the function of MIM, and test whether inhibition of Rac is the mechanism for MIM to modulate cell motility induced by growth factors. We will delineate the signaling pathway for MIM phosphorylation and examine whether Src is the primary tyrosine kinase responsible for the PDGF mediated tyrosine phosphorylation of MIM, and analyze tyrosine phosphorylation of MIM in Src knockout cells and the cells overexpressing temperature sensitive v-Src. We will also explore the role of tyrosine phosphorylation in the regulation of MIM with respect to interaction with Rac and cortactin, and cell motility. Finally, we will examine the role of MIM expression at low levels in the motility of tumor cells by silencing MIM using RNA interference in MIM expression cells, and examine whether suppression of MIM expression will lead to increase in lamellipodia development, Rac activation, actin polymerization in cells and cell motility. It is anticipated that achievement of the goal of this proposal will direct our future effort to understand how MIM contributes to tumor progression.