DYNAMIC PROTEOMICS OF TUMOR CELL MIGRATION AND THE LAMELLIPODIA PHOSPHOPROTEOME

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这个项目的总体目标是了解控制肿瘤细胞迁移、侵袭和转移到体内不同部位的分子信号机制。细胞转移是疾病复发和患者生存下降的主要原因。我们开发了一种独特的生化方法来纯化非常领先的迁移细胞前沿(片状脂)(2)。这项突破性的技术使我们能够识别促进片状脂体形成的关键调控蛋白，片状脂体的形成负责介导细胞的入侵和转移。我们将使用猴肾上皮细胞(COS-7)和转移性人乳腺腺癌细胞(商业上可获得)进行这些研究。从这些细胞中提纯的片状脂体的初步分析表明，磷酸酪氨酸(PY)蛋白在主要的片状脂体中高度激活。药物抑制酪氨酸磷酸化抑制片状脂体的形成，表明复杂的信号级联通过调节酪氨酸网络来控制这一过程。因此，我们的主要目标是用抗磷酸酪氨酸抗体进行免疫亲和纯化，然后进行蛋白质组分析，以确定与片状脂虫形成和癌细胞转移有关的PY蛋白(片状脂体磷酸蛋白质组)的特征。我们的研究结果将为控制细胞迁移和转移的信号提供有价值的信息，并为癌症进展的治疗干预提供靶点。我们的具体目标是： 具体目的1.鉴定PY蛋白及其在迁移细胞前沿酪氨酸磷酸化的特异性部位。 具体目的2.利用siRNA蛋白敲除和MS鉴定的关键磷酸酪氨酸位点的定点突变，对鉴定出的PY蛋白进行功能测试，然后进行基于细胞的分析和我们实验室建立的细胞迁移的动物模型。 具体目的3.应用脉冲稳定同位素标记结合胞体亚细胞分离和富集层脂膜的方法，在研究休眠和活跃迁移细胞的时间过程中，确定含酪氨酸蛋白的时间和空间分布，以及它们的相对丰度。 具体目的4.利用生物信息学和计算机模拟系统绘制PY蛋白之间可能的信号级联，并建立其功能关系。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of this project is to understand the molecular signaling mechanisms that control tumor cell migration, invasion, and metastasis to distinct sites in the body. Cell metastasis is the major cause of disease relapse and decreased patient survival. We have developed a unique biochemical method to purify he very leading front (lamellipodia) of migrating cells(2). This breakthrough technology allows us to identify the key regulatory proteins that facilitate lamellipodia formation which is responsible for mediating cell invasion and metastasis. We will use monkey kidney epithelial cells (COS-7) and metastatic human breast adenocarcinoma cells (commercially available) for these studies. Initial analysis of purified lamellipodia from these cells has revealed that phosphotyrosine (PY) proteins are highly activated in the leading lamellipodia. Pharmacological inhibition of tyrosine phosphorylation inhibits lamellipodia formation indicating that complex signaling cascades operate to control this process through modulation of tyrosine networks. Therefore, our major objective is to characterize the PY proteins (lamellipodia phosphoproteome) responsible for lamellipodia formation and cancer cell metastasis using immunoaffinity purification with anti-phosphotyrosine antibodies followed by proteome analysis to identify proteins of interest. Results from our study will provide valuable information on the signals that control cell migration and metastasis, and provide targets for therapeutic intervention of cancer progression. Our specific aims are: Specific Aim 1. To identify PY proteins and their specific sites of tyrosine phosphorylation in the leading front of migrating cells. Specific Aim 2. To functionally test identified PY proteins using siRNA protein knockdown and site directed mutagenesis of key phosphotyrosine sites identified by MS followed by cell-based assays and animal models of cell migration established in our laboratory. Specific Aim 3. To determine the temporal and spatial distribution of tyrosine-containing proteins, phosphorylated and non-phosphorylated, and their relative abundance in a time course investigation of dormant vs actively migrating cells applying a combination of pulsed stable isotopic labeling with subcellular fractionation of cell bodies and of enriched lamellipodia. Specific Aim 4. To map the putative signaling cascades and develop functional relationships among the PY proteins using bioinformatics and computer modeling systems.