The Role of MIM in growth factor signaling and cell motility

描述(申请人提供)：肿瘤扩散是细胞膜持续突出、侵袭和迁移的结果，这些活动需要肌动蛋白细胞骨架的动态重组。肌动蛋白在细胞前沿的组装是通过Arp2/3复合体、WASP/WAVE家族蛋白和皮质蛋白的功能实现的，这些蛋白由Rho小GTP酶和蛋白酪氨酸激酶Src激活。我们最近的研究发现，Cortactin与MIM基因的蛋白产物结合，MIM基因在侵袭性肿瘤细胞和晚期人类膀胱癌中经常表达缺失。MIM的过表达显著抑制了肿瘤细胞或正常细胞由生长因子介导的运动，并抑制了Rho GTP酶家族成员rac1诱导的片状脂肪的形成。在细胞内，MIM与rac1共定位，并能够与rac1共沉淀。在体外，MIM抑制WASP介导的肌动蛋白聚合。有趣的是，MIM经历了快速的酪氨酸磷酸化，同时伴随着与皮质素的短暂相互作用，以响应生长因子。MIM需要在两个酪氨酸残基上进行磷酸化才能抑制rac1。基于这些数据，我们假设MIM参与了一种新的信号通路，拮抗RAC在肌动蛋白细胞骨架组装中的功能，该活性受到酪氨酸磷酸化的调节。为了验证这一假设，我们建议表征MIM拮抗RAC的机制。因此，我们将确定MIM是否与RAC直接相互作用，并研究直接关联是否必要且足以抑制RAC。我们还将分析RAC的效应因子IRSP53和WAVE在MIM功能中的作用，并检验抑制RAC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，并研究Src是否是PDGF介导的MIM酪氨酸磷酸化的主要酪氨酸激酶，并分析在Src基因敲除细胞和过表达温敏性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调节MIM中与RAC和Cortactin的相互作用以及细胞运动中的作用。最后，我们将通过在表达MIM的细胞中使用RNA干扰沉默MIM来研究低水平的MIM表达在肿瘤细胞运动中的作用，并检测抑制MIM表达是否会导致片状脂体发育、Rac激活、细胞内肌动蛋白聚合和细胞运动的增加。预计这项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.