Novel roles for palmitoylation of Ras in IL-1β-induced nitric oxide release and caspase 3 activation in insulin-secreting β cells (original) (raw)

2003, Biochemical Pharmacology

We recently demonstrated that functional inactivation of H-Ras results in significant reduction in interleukin 1b (IL-1b)-mediated effects on isolated b cells. Since palmitoylation of Ras has been implicated in its membrane targeting, we examined the contributory roles of palmitoylation of Ras in IL-1b-induced nitric oxide (NO) release and subsequent activation of caspases. Preincubation of HIT-T15 or INS-1 cells with cerulenin (CER, 134 mM; 3 hr), an inhibitor of protein palmitoylation, significantly reduced (À95%) IL-1b-induced NO release from these cells. 2-Bromopalmitate, a structurally distinct inhibitor of protein palmitoylation, but not 2-hydroxymyristic acid, an inhibitor of protein myristoylation, also reduced (À67%) IL-1b-induced NO release from HIT cells. IL-induced inducible nitric oxide synthase gene expression was markedly attenuated by CER. Further, CER markedly reduced incorporation of [ 3 H]palmitate into H-Ras and caused significant accumulation of Ras in the cytosolic fraction. CER-treatment also prevented IL-1b-induced activation of caspase 3 in these cells. Moreover, N-monomethyl-L-arginine, a known inhibitor of inducible nitric oxide synthase, markedly inhibited IL-induced activation of caspase 3, thus establishing a link between IL-induced NO release and caspase 3 activation. Depletion of membrane-bound cholesterol using methyl-b-cyclodextrin, which also disrupts caveolar organization within the plasma membrane, abolished IL-1binduced NO release suggesting that IL-1b-mediated Ras-dependent signaling in these cells involves the intermediacy of caveolae and their key constituents (e.g. caveolin-1) in isolated b cells. Confocal light microscopic evidence indicated significant colocalization of Ras with caveolin-1. Taken together, our data provide the first evidence to indicate that palmitoylation of Ras is essential for IL-1b-induced cytotoxic effects on the islet b cell.