DYNAMIC PROTEOMICS OF TUMOR CELL MIGRATION AND THE LAMELLIPODIA PHOSPHOPROTEOME

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 该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存活率降低的主要原因。 我们已经开发了一种独特的生化方法来纯化迁移细胞的最前沿（片状伪足）（2）。 这项突破性的技术使我们能够确定促进片状伪足形成的关键调控蛋白，这是负责介导细胞侵袭和转移。 我们将使用猴肾上皮细胞（COS-7）和转移性人乳腺癌细胞（市售）进行这些研究。 从这些细胞纯化的板状伪足的初步分析表明，磷酸酪氨酸（PY）蛋白是高度活化的领先板状伪足。 酪氨酸磷酸化的药理学抑制抑制片状伪足的形成，表明复杂的信号传导级联通过酪氨酸网络的调节来控制该过程。 因此，我们的主要目标是表征PY蛋白（板状伪足磷酸蛋白质组）负责板状伪足的形成和癌细胞转移使用免疫亲和纯化与抗磷酸酪氨酸抗体，然后通过蛋白质组分析，以确定感兴趣的蛋白质。 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： 具体目标1。目的：鉴定迁移细胞前沿的PY蛋白及其酪氨酸磷酸化的特异位点。 具体目标2。通过siRNA蛋白敲低和通过MS鉴定的关键磷酸酪氨酸位点的定点诱变，然后通过基于细胞的测定和我们实验室建立的细胞迁移动物模型，对鉴定的PY蛋白进行功能性检测。 具体目标3。为了确定含酪氨酸蛋白质的时间和空间分布，磷酸化和非磷酸化，以及它们的相对丰度，在休眠与活跃迁移细胞的时间过程研究中，应用脉冲稳定同位素标记与细胞体和富集板状伪足的亚细胞分级分离相结合。 具体目标4。利用生物信息学和计算机模拟系统对PY蛋白的信号通路进行定位，并建立PY蛋白之间的功能关系。

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.