CHARACTERIZATION OF THE TUMOR CELL 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。 该项目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。转移是疾病复发和降低患者存活率的主要原因。我们开发了一种生物化学方法来纯化迁移细胞的前锋(片状脂)(JCB 156：725)。2002)，这使我们能够确定促进板脂蛋白形成的关键调节蛋白，这是负责介导细胞入侵和转移的关键调控蛋白。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。初步分析表明，在这些细胞的片状脂膜中，磷酸酪氨酸(PY)蛋白高度激活。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，我们的目标是确定与片状脂体形成和癌细胞转移有关的PY蛋白(片状脂体磷化蛋白质组)。片状脂虫PY蛋白将用抗PY抗体免疫纯化或使用IMac柱富集磷酸肽，然后使用NCRR高灵敏度、高分辨率LC-MS/MS分析关键蛋白并确定磷酸化残基的特定位置。然后将使用siRNA蛋白敲除和定点突变进行功能测试，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。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进行分析，以揭示有助于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 this project 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. We have developed a biochemical method to purify the leading front (lamellipodia) of migrating cells (JCB 156:725. 2002) which 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 (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.