Subject(s)
Cell Cycle Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , p21-Activated Kinases/metabolism , Amino Acid Sequence , Animals , Cell Cycle Proteins/chemistry , Epidermal Growth Factor/pharmacology , Guanine Nucleotide Exchange Factors/chemistry , Humans , Mass Spectrometry , Mice , Molecular Sequence Data , Neoplasms/enzymology , Phosphorylation/drug effects , Protein Binding/drug effects , Rho Guanine Nucleotide Exchange Factors , p21-Activated Kinases/chemistryABSTRACT
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.
Subject(s)
Cell Movement/genetics , Proteins/analysis , Proteomics/methods , Signal Transduction/genetics , Adaptor Proteins, Signal Transducing/metabolism , Blotting, Western , Cell Cycle Proteins/metabolism , Cell Line , Chromatography, Liquid/methods , Humans , Immunoprecipitation , Mass Spectrometry/methods , Paxillin/metabolism , Protein Serine-Threonine Kinases/metabolism , p21-Activated KinasesABSTRACT
Protein kinase C epsilon is an oncogenic, actin nucleating protein that coordinately regulates changes in cell growth and shape. Cells constitutively expressing PKCepsilon spontaneously acquire a polarized morphology and extend long cellular membrane protrusions. Here we report that the regulatory C1 domain of PKCepsilon contains an actin binding site that is essential for the formation of elongate invadopodial-like structures, increased pericellular metalloproteinase activity, in vitro invasion of a Matrigel barrier, and the invasion and metastasis of tumors grown in vivo by PKCepsilon-transformed NIH3T3 fibroblasts in nude mice. While removing this small actin binding motif caused a dramatic reversion of tumor invasion, the deletion mutant of PKCepsilon remained oncogenic and tumorigenic in this experimental system. We propose that PKCepsilon directly interacts with actin to stimulate polymerization and the extension of membrane protrusions that transformed NIH3T3 cells use in vivo to penetrate and degrade surrounding tissue boundaries.