Characterization of the tumor cell lamellipodia phosphoproteome
EMSL Project ID
11391
Abstract
This new collaborative project under PNNL’s NCRR Biomedical Technology Resource Center is to initiate the characterization of the tumor cell lamellipodia phosphoproteome leading to publication and collaborative funding opportunities.Overview: The overall goal of work in my laboratory 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. Recently, we developed a unique biochemical method to purify the very leading front (lamellipodia) of migrating cells (JCB 156:725. 2002). This breakthrough technology will 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 antiphosphotyrosine antibodies followed by mass spectrometry (MS) and sequence database 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:
1. To identify PY proteins and their specific sites of tyrosine phosphorylation in the leading front of migrating cells.
2. To functional 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.
3. To map the putative signaling cascades and develop functional relationships among the PY proteins using bioinformatics and computer modeling systems.
Methods for Specific Aim #1: An initial “bottom-up” PY protein analysis will be performed using PNNL’s high sensitivity and high resolution LC-MS/MS using immunopurified lamellipodia PY proteins in order to (1) create a mass and time (MT) tag database of the PY subproteome and (2) identify targets for specific aim #2 using either siRNA-directed knockdown approaches and/or site directed mutagenesis strategies. Immunopurified PY proteins will be subjected to tryptic digestion, peptide purification, and an off-line strong cation exchange separation coupled to LC-MS/MS analysis for the identification of PY proteins. To identify the specific tyrosine phosphorylation sites on the phosphoproteins, a portion of the tryptic digest from the PY proteins will be passed through an IMAC column to enrich for phosphopeptides present in the digest following methyl etherification of the peptides. This phosphopeptide enriched sample will then be analyzed by LC-MS/MS for determining (1) the identities of the phosphopeptides and (2) the specific locations of the phosphorylated residues which can then be targeted for achieving specific aim #2. Information gained from aims 1 and 2 will then be analyzed using bioinformatics and computer modeling to reveal potential phosphotyrosine networks that contribute to cancer cell metastasis.
Project Details
Project type
Exploratory Research
Start Date
2004-11-23
End Date
2007-06-28
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
“Profiling Signaling Polarity in Chemotactic Cells” Yingchun Wang, Shi-Jian Ding, Wei Wang, Jon M. Jacobs, Wei-Jun Qian, Ronald J. Moore, Feng Yang, David G. Camp II, Richard D. Smith, Richard L. Klemke (2007) Proc Natl Acad Sci USA 104, 8328-8333.