CHARACTERIZATION OF THE TUMOR CELL LAMELLIPODIA PHOSPHOPROTEOME

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。我实验室的总体目标是了解控制肿瘤细胞迁移、侵袭和转移的分子信号机制。转移是疾病复发和患者生存率降低的主要原因。最近，我们开发了一种生化方法来纯化迁移细胞的前缘（板足）（JCB 156:725）。2002)。这项新技术将使我们能够识别促进板足形成的关键调节蛋白，这是负责介导细胞侵袭和转移的。我们将使用猴肾上皮细胞（COS-7）和转移性人乳腺腺癌细胞进行这些研究。初步分析显示，磷酸酪氨酸（PY）蛋白在这些细胞的板足高度活化。酪氨酸磷酸化的药理抑制抑制板足的形成，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，我们的目标是表征负责板足形成和癌细胞转移的PY蛋白（板足磷蛋白组）。Lamellipodia PY蛋白将使用抗PY抗体进行免疫纯化或使用IMAC柱进行磷酸化肽富集，然后使用NCRR高灵敏度，高分辨率LC-MS/MS进行分析，以识别关键蛋白并确定磷酸化残基的特定位置。然后将使用siRNA蛋白敲除和位点定向诱变进行功能测试，随后进行基于细胞的测定和在我们实验室建立的细胞迁移动物模型。从这些实验中获得的信息将使用生物信息学和计算机建模来分析，以揭示促进癌细胞转移的潜在磷酸酪氨酸网络。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。

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 my laboratory is to understand the molecular signaling mechanisms that control tumor cell migration, invasion, and metastasis. Metastasis is a major cause of disease relapse and decreased patient survival. Recently, we developed a biochemical method to purify the leading front (lamellipodia) of migrating cells (JCB 156:725. 2002). This novel technology will allow 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 (COS-7) and metastatic human breast adenocarcinoma cells for these studies. Initial analysis has revealed that phosphotyrosine (PY) proteins are highly activated in the lamellipodia of these cells. Pharmacological inhibition of tyrosine phosphorylation inhibits lamellipodia formation, indicating that complex signaling cascades control this process through modulation of tyrosine networks. Therefore, our objective is to characterize the PY proteins (lamellipodia phosphoproteome) responsible for lamellipodia formation and cancer cell metastasis. Lamellipodia PY proteins will be immunopurified with anti-PY antibodies or enriched for phosphopeptides using an IMAC column and then analyzed using the NCRR high sensitivity, high resolution LC-MS/MS to identify key proteins and determine the specific locations of the phosphorylated residues. Functional testing will then be performed using siRNA protein knockdown and site directed mutagenesis followed by cell-based assays and animal models of cell migration established in our laboratory. Information gained from these experiments will then be analyzed using bioinformatics and computer modeling to reveal potential phosphotyrosine networks that contribute to cancer cell metastasis. 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.