Abstract 5680: Serum and Glucocorticoid-Inducible Kinase 1 Regulates Sirtuin 3, the Mitochondrial Deacetylase via Phosphorylation and Sumoylation Mechanisms in Rat Aortic Smooth Muscle Cells

Abstract

Serum and glucocorticoid-inducible kinase 1 (SGK1) is a serine/threonine kinase whose activity and expression is up-regulated in vascular neointimal lesions in association with restenosis. Our previous studies show that vascular smooth muscle cell (VSMC) proliferation is augmented upon activation of SGK1. However, the molecular mechanism(s) was not fully examined. Thus, to identify proteins that participate in SGK1-mediated stimulation of VSMC proliferation, we performed a kinase substrate protein array with activated SGK1. We identified sirtuin3 (Sirt3), a class III histone deacetylase as a novel putative substrate for SGK1 in this screen. Preliminary studies from two-dimensional SDS-PAGE indicate that expression of active SGK1 (S422D-SGK1) is associated with a shift in Sirt3 to a more acidic profile, consistent with an increase in phosphorylation. Moreover, S422D-SGK1 expression resulted in a 2- and 1.6-fold increase in Sirt3 mRNA and protein; respectively, compared to empty vector or a kinase-defective SGK1 mutant (S422A-SGK1). To determine whether Sirt3 expression is regulated by growth factors, we performed timecourse studies with PDGF-BB, a potent inducer of VSMC proliferation as well as SGK1. Quantitative real-time PCR analysis, revealed a modest reduction in Sirt3 mRNA (~25%) after 4 hours of stimulation. However, under the same conditions, PDGF-BB augments cytoplasmic Sirt3 protein expression more than 32-fold, suggesting a posttranslational method of regulation. Notably, sumoylation is known to regulate the activity, stability or subcellular distribution of its substrates and our analysis indicates that Sirt3 contains the core SUMO consensus motif. Thus, we assessed whether Sirt3 can be sumoylated and tested the hypothesis that PDGF acting through SGK1 signaling modulates sumoylation of Sirt3 in VSMC. Indeed, results obtained from an in vitro sumoylation assay indicate that Sirt3 is a target for SUMO-1. Lastly, we found a higher level of SUMO-1 conjugation in Sirt3 immunoprecipitates from VSMC expressing S422D-SGK1 compared to empty vector- or S422A-SGK1-expressing cells. Altogether, our findings implicate Sirt3 as novel substrate and potential mediator of the growth effects of SGK1 in VSMC.