Abstract 362: Akt-Dependent Interaction with EBP50 Regulates Skp2 Localization and Stability

Abstract

The PDZ domain-containing scaffolding protein, Ezrin-Radixin-Moesin-Binding Phosphoprotein 50 (EBP50) regulates vascular stenosis following endoluminal vessel injury. Its expression in vascular smooth muscle cells (VSMC) increases after wire injury, and neointima formation is significantly reduced in EBP50 knockout mice. We previously reported that the mitogenic effect of EBP50 is Akt-dependent and mediated by the S-phase kinase-associated protein 2 (Skp2), a component of the E3 ligase complex that degrades the cell cycle inhibitor p21cip1. The mechanisms by which Akt directly increases Skp2 stability are controversial. We hypothesize that EBP50 regulates Akt-dependent stabilization of Skp2. The C-terminal four amino acids of Skp2 (PSCL) are a canonical PDZ-binding sequence. Co-immunoprecipitation and in-gel overlay assays demonstrated the direct interaction between EBP50 and Skp2. Mutation of the C-terminal Leu to Ala (L424A-Skp2) abrogated the interaction with EBP50. Akt potentiated the association between EBP50 and Skp2. Sequence analysis indicated the presence of an Akt phosphorylation motif (RPRLCT) in EBP50 between residues 151 and 156. Indeed, in vitro assays using purified EBP50 and constitutively active Akt showed that Akt phosphorylates EBP50. This effect was abrogated by a naturally occurring mutation R153Q in EBP50 that disrupts the Akt consensus sequence. EBP50 was expressed exclusively in the cytoplasm of VSMC, where it associated with the cytoskeleton. Cell fractionation and fluorescence microscopy studies showed that expression of EBP50, but not of R153Q, induced relocalization of Skp2 from the nucleus to the cytoplasm. EBP50 expression increased Skp2 stability and, consequently, VSMC proliferation. Collectively, these experiments show that Akt-mediated EBP50 phosphorylation increases the interaction with Skp2, regulates Skp2 cellular localization and increases Skp2 stability. These studies identify a novel function for EBP50 in the direct regulation of the cell cycle and provide the mechanistic basis for the effect of this scaffolding protein on VSMC proliferation and neointima formation.