Novel roles for palmitoylation of Ras in IL-1 beta-induced nitric oxide release and caspase 3 activation in insulin-secreting beta cells.
We recently demonstrated that functional inactivation of H-Ras results in significant reduction in interleukin 1 beta (IL-1 beta)-mediated effects on isolated beta cells. Since palmitoylation of Ras has been implicated in its membrane targeting, we examined the contributory roles of palmitoylation of Ras in IL-1 beta-induced nitric oxide (NO) release and subsequent activation of caspases. Preincubation of HIT-T15 or INS-1 cells with cerulenin (CER, 134 microM; 3 hr), an inhibitor of protein palmitoylation, significantly reduced (-95%) IL-1 beta-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 (-67%) IL-1 beta-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 [3H]palmitate into H-Ras and caused significant accumulation of Ras in the cytosolic fraction. CER-treatment also prevented IL-1 beta-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-beta-cyclodextrin, which also disrupts caveolar organization within the plasma membrane, abolished IL-1 beta-induced NO release suggesting that IL-1 beta-mediated Ras-dependent signaling in these cells involves the intermediacy of caveolae and their key constituents (e.g. caveolin-1) in isolated beta 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-1 beta-induced cytotoxic effects on the islet beta cell.
Chen
Hai-Qing
HQ
Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University and beta Cell Biochemistry Research Laboratory, John D. Dingell VA Medical Center, Detroit, MI 48201, USA.
Tannous
Marie
M
Veluthakal
Rajakrishnan
R
Amin
Rajesh
R
Kowluru
Anjaneyulu
A
D000215:Acylation; D000935:Antifungal Agents / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D002569:Cerulenin / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007375:Interleukin-1 / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000187:drug effects* / Q000378:metabolism; D008745:Methylation; D009569:Nitric Oxide / Q000378:metabolism*; D010084:Oxidation-Reduction; D010168:Palmitates / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism / Q000502:physiology*
D000935:Antifungal Agents; D007328:Insulin; D007375:Interleukin-1; D010168:Palmitates; D002569:Cerulenin; D009569:Nitric Oxide; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D018631:ras Proteins