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

Abstract

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 experiments show that Skp2 interacts with the first PDZ domain of EBP50, suggesting that T156 phosphorylation exerts allosteric effects on the interaction with Skp2. Expression of EBP50 in primary VSMC induces a re-localization of Skp2 from the nucleus to the cytoplasm, leading to its stabilization and VSMC proliferation. These effects were abrogated by mutations within the first PDZ domain or of T156 to Alanine. Notably, the interaction with EBP50 does not directly affect the function of Skp2 because mutation of the C-terminal Leu to Ala, which abrogates the interaction with EBP50, does not decrease the ability of Skp2 to degrade p21cip1 and p27kip1. Consistent with the observations in primary cells, the expression of Skp2 in injured femoral arteries was reduced in EBP50 knockout mice compared to WT mice. In conclusion, our studies identify a novel mechanism for the Akt-dependent regulation of cell proliferation by EBP50 during vascular remodeling. They indicate that inhibition of EBP50 may be useful in preventing neointima formation following angioplasty.