Phosphorylation of eukaryotic translation initiation factor 2Bε by glycogen synthase kinase-3β regulates β-adrenergic cardiac myocyte hypertrophy

Glycogen synthase kinase 3beta (GSK-3beta) negatively regulates cardiac hypertrophy. A potential target mediating the antihypertrophic effect of GSK-3beta is eukaryotic translation initiation factor 2Bepsilon (eIF2Bepsilon). Overexpression of GSK-3beta increased the cellular kinase activity toward GST-eIF2Bepsilon in neonatal rat cardiac myocytes, whereas LiCl (10 mmol/L) or isoproterenol (ISO) (10 mumol/L), a treatment known to inhibit GSK-3beta, decreased it. Immunoblot analyses using anti-S535 phosphospecific eIF2Bepsilon antibody showed that S535 phosphorylation of endogenous eIF2Bepsilon was decreased by LiCl or ISO, suggesting that GSK-3beta is the predominant kinase regulating phosphorylation of eIF2Bepsilon-S535 in cardiac myocytes. Decreases in eIF2Bepsilon-S535 phosphorylation were also observed in a rat model of cardiac hypertrophy in vivo. Overexpression of wild-type eIF2Bepsilon alone moderately increased cell size (+31+/-11%; P < 0.05 versus control), whereas treatment of eIF2B epsilon-transduced myocytes with LiCl (+73 +/- 22% versus eIF2B epsilon only; P < 0.05) or ISO (+84+/-33% versus eIF2Bepsilon only; P < 0.05) enhanced the effect of eIF2B epsilon. Overexpression of eIF2B epsilon-S535A, which is not phosphorylated by GSK-3 beta, increased cell size (+107 +/- 35%) as strongly as ISO (+95 +/- 25%), and abolished antihypertrophic effects of GSK-3 beta, indicating that S535 phosphorylation of eIF2B epsilon critically mediates antihypertrophic effects of GSK-3 beta. Furthermore, expression of eIF2B epsilon-F259L, a dominant-negative mutant, inhibited ISO-induced hypertrophy, indicating that eIF2B epsilon is required for beta-adrenergic hypertrophy. Interestingly, expression of eIF2B epsilon-S535A partially increased cytoskeletal reorganization, whereas it did not increase expression of atrial natriuretic factor gene. These results suggest that GSK-3 beta is the predominant kinase mediating phosphorylation of eIF2B epsilon-S535 in cardiac myocytes, which in turn plays an important role in regulating cardiac hypertrophy primarily through protein synthesis.