Phosphorylation of Eukaryotic Translation Initiation Factor 2B by Glycogen Synthase Kinase-3 Regulates -Adrenergic Cardiac Myocyte Hypertrophy

Glycogen synthase kinase 3 (GSK-3 ) negatively regulates cardiac hypertrophy. A potential target mediating the antihypertrophic effect of GSK-3 is eukaryotic translation initiation factor 2B (eIF2B ). Overexpression of GSK-3 increased the cellular kinase activity toward GST-eIF2B in neonatal rat cardiac myocytes, whereas LiCl (10 mmol/L) or isoproterenol (ISO) (10 mol/L), a treatment known to inhibit GSK-3 , decreased it. Immunoblot analyses using anti-S535 phosphospecific eIF2B antibody showed that S535 phosphorylation of endogenous eIF2B was decreased by LiCl or ISO, suggesting that GSK-3 is the predominant kinase regulating phosphorylation of eIF2B -S535 in cardiac myocytes. Decreases in eIF2B -S535 phosphorylation were also observed in a rat model of cardiac hypertrophy in vivo. Overexpression of wild-type eIF2B alone moderately increased cell size ( 31 11%; P 0.05 versus control), whereas treatment of eIF2B -transduced myocytes with LiCl ( 73 22% versus eIF2B only; P 0.05) or ISO ( 84 33% versus eIF2B only; P 0.05) enhanced the effect of eIF2B . Overexpression of eIF2B -S535A, which is not phosphorylated by GSK-3 , increased cell size ( 107 35%) as strongly as ISO ( 95 25%), and abolished antihypertrophic effects of GSK-3 , indicating that S535 phosphorylation of eIF2B critically mediates antihypertrophic effects of GSK-3 . Furthermore, expression of eIF2B F259L, a dominantnegative mutant, inhibited ISO-induced hypertrophy, indicating that eIF2B is required for -adrenergic hypertrophy. Interestingly, expression of eIF2B -S535A partially increased cytoskeletal reorganization, whereas it did not increase expression of atrial natriuretic factor gene. These results suggest that GSK-3 is the predominant kinase mediating phosphorylation of eIF2B -S535 in cardiac myocytes, which in turn plays an important role in regulating cardiac hypertrophy primarily through protein synthesis. (Circ Res. 2004;94:●●●-●●●.)