Identification of protein networks associated with the PAK1-betaPIX-GIT1-paxillin signaling complex by mass spectrometry.
The process of cell motility involves coordinate signaling events among proteins associated in interactive integrin-linked networks. Mass spectrometric analysis of immunoprecipitation-derived protein mixtures have provided efficient means of identifying proteomes. In this study, we investigate strategies to enhance the detection of interactome proteins for the known signaling module: PAK1, betaPIX, GIT1, and paxillin. Our results indicate that near-endogenous expression levels of bait protein enhances the identification of associated proteins, and that phosphatase inhibition augments the detection of specific protein interactions. Following the analysis of a large pool of spectral data, we have identified and mapped clusters of proteins that either share common interactions among the four bait proteins of interest or are exclusive to single bait proteins. Taken together, these data indicate that biochemical manipulations can enhance the ability for LC-MS/MS to identify interactome proteins, and that qualitative screening of multiple samples leads to the compilation of proteins associated with a known plexus.
Mayhew
Mark W
MW
Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908, USA. mwm9r@virginia.edu
Webb
Donna J
DJ
Kovalenko
Mykola
M
Whitmore
Leanna
L
Fox
Jay W
JW
Horwitz
Alan F
AF
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D002465:Cell Movement / Q000235:genetics*; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry / Q000379:methods; D051419:Paxillin / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction / Q000235:genetics*; D054462:p21-Activated Kinases
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; C485647:GIT1 protein, human; D051419:Paxillin; D011506:Proteins; C515903:PAK1 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases