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.
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