Abstract 338: Akt-Mediated Phosphorylation of EBP50 Regulates Skp2 Stability and Vascular Smooth Muscle Cell Proliferation

The PDZ domain-containing scaffolding protein, Ezrin-Radixin-Moesin-Binding Phosphoprotein 50 (EBP50) increases vascular smooth muscle cells (VSMC) proliferation and vascular remodeling following endoluminal vessel injury. Indeed, neointima formation is significantly reduced in EBP50 knockout mice. The mitogenic effect of EBP50 derives from the Akt-dependent stabilization of the S-phase kinase associating protein Skp2. However, the mechanisms by which Akt regulates EBP50-mediated cell proliferation are not known. We hypothesize that phosphorylation of EBP50 by Akt promotes its interaction with Skp2. This causes cytoplasmic re-localization and stabilization of Skp2 and VSMC proliferation. Using Akt inhibitors and siRNA inhibition of Skp2, we show that the mitogenic effect of EBP50 in VSMC is dependent on both Akt and Skp2. In vitro and in vivo phosphorylation assays show that Akt phosphorylates EBP50 at T156 in the second PDZ domain. Interestingly, NMR, fluorescence polarization and co-immunoprecipitation ...