pp60c-Src Phosphorylates and Activates Vacuolar Protein Sorting 34 to Mediate Cellular Transformation

Vacuolar protein sorting 34 (VPS34) contributes to the regulation of the mammalian target of rapamycin complex 1/S6 kinase 1 pathway downstream of nutrient signaling. However, intracellular mechanisms leading to VPS34 activation remain unclear. Here, we report that Src directly phosphorylates VPS34, and that this phosphorylation activates VPS34 lipid kinase activity, leading to Src-Y527F-mediated cellular transformation. Silencing endogenous VPS34 specifically inhibits Src-Y527F-induced colony formation in soft agar, but not Ras-G12V-induced colony formation. We have identified two novel hVPS34 mutations, which either eliminate lipid kinase activity (kinase-dead mutant) or reduce tyrosine phosphorylation by Src-Y527F. When kinase-dead mutant of hVPS34 is stably expressed in Src-Y527F-transformed cells, transformation activities are blocked, indicating that the lipid kinase activity of hVPS34 is essential for Src-mediated cellular transformation. Furthermore, stable expression of this hVPS34 kinase-dead mutant causes an increased number of binucleate and multinucleate cells, suggesting that the kinase activity of hVPS34 is also required for cytokinesis. Moreover, when the hVPS34 mutant that has reduced tyrosine phosphorylation by Src is stably expressed in SrcY527F- transformed cells, Src-Y527F-stimulated colony formation is also reduced. Data presented here provide important evidence that VPS34 lipid kinase activity could be positively regulated by Src-mediated tyrosine phosphorylation in mammalian cells. This finding highlights a previously unappreciated relationship between VPS34, a class III phosphatidylinositol-3-kinase, and Src non-receptor tyrosine kinase. Additionally, we find that the levels of VPS34 expression and tyrosine phosphorylation are correlated with the tumorigenic activity of human breast cancer cells, indicating that Src to VPS34 signaling warrants further investigation as a pathway contributing to the development and progression of human cancers. Cancer Res; 70(14); 5974-83. (C) 2010 AACR.