Abstract 294: Protein interactions of PTEN and its cancer-associated G20E mutant compared by SILAC-based parallel affinity purification and mass spectrometry

The tumor suppressor PTEN (phosphatase and tensin homologue) negatively regulates the phosphatidyl-inositol-3-kinase (PI3K) pathway through its lipid phosphatase activity and is one of the most commonly lost tumor suppressors in human cancers. Though the tumor suppressive function involves PTEN9s lipid phosphatase-dependent and -independent activities, the mechanism leading to the phosphatase independent function of PTEN is poorly understood. Some PTEN mutants have lipid phosphatase activity but fail to suppress cell growth. Here we use a cancer associated mutant, G20E, to gain insight on the phosphatase independent function of PTEN by investigating protein-protein interactions using stable isotope labeling by amino acids in cell culture (SILAC). A strategy named parallel affinity purification (PAP) and mass spectrometry (MS) has been developed to allow prioritizing interactors acquired from SILAC pull-downs and the comparison of the interactions between wild-type and G20E PTEN. Liquid chromatography-MS analysis of tryptic digested SILAC-PAP pull-downs revealed known and new interactors. Hierarchical clustering of the heat map generated from the proteins with elevated SILAC ratios from PAP-MS analysis show three distinct clusters: 1) wild-type specific interactors, 2) those unique to the G20E mutant and 3) proteins common to wild-type and mutant. Pathway analyses indicate that the new interactors can be subdivided into apoptosis, cell migration, polarity and the Ca2+ signaling network. These finding contribute to a better understanding of the novel mechanisms of PTEN9s phosphatase dependent and independent functions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 294.