Abstract P197: Reversible Acetylation of GSK3β Regulates the Development of Cardiac Hypertrophy

Abstract

Glycogen synthase kinase 3β (GSK3β) is a critical regulator of diverse cellular functions involved in the maintenance of cellular structure, function and survival. In the heart, GSK3β has been shown to play a key role in antagonizing the development of cardiac hypertrophy. It has been shown that GSK3β activity is inhibited by phosphorylation at Ser9 during the development of cardiac hypertrophy. However, recent studies indicated that blocking the Ser9 phosphorylation alone is not sufficient to protect GSK3β activity, suggesting that alternative mechanisms might be involved in regulation of GSK3β activity. In the present study, we tested the hypothesis whether GSK3β is acetylated in established hypertrophy, which leads to inhibition of GSK3β activity, and thereby promoting progression and maintenance of cardiac hypertrophy. We examined acetylation, phosphorylation and catalytic activity of GSK3β in pressure overload model of cardiac hypertrophy at different stages of hypertrophy development. The results showed that GSK3β catalytic activity was significantly down-regulated throughout the course of hypertrophy development. However, the inhibitory phosphorylation of GSK3β was observed only at initial stages, but not at later stages of pressure overload (4 weeks). Interestingly, we observed a significantly increased acetylation of GSK3β, which corresponded to the reduced activity at this later stage of hypertrophy. We then determined the impact of GSK3β acetylation on its catalytic activity, by measuring the ability of GSK3β to phosphorylate the substrate glycogen synthase. The results showed that acetylation decreased the catalytic activity of the GSK3β by almost 3 fold. Next, we performed mass spectrometry to find out the target lysine residues of GSK3β. Proteomic analysis revealed that multiple lysine residues in different regions of GSK3β were acetylated. By using site directed mutagenesis, we found that acetylation inhibits the catalytic activity of GSK3β by suppressing its substrate binding ability, which is independent of inhibitory-Ser 9 phosphorylation. These studies for the first time show a phosphorylation independent mechanism regulating GSK3β activity during progression and maintenance of cardiac hypertrophy.