A novel mechanism for the regulation of cellular apoptosis mediated by posttranslational N-myristoylation of cytoskeletal proteins.

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Protein N-myristoylation has been recognized as a cotranslational protein modification. Recently, it was demonstrated that protein N-myristoylation could also occur posttranslationally, as in the case of the pro-apoptotic protein BID and cytoskeletal actin. Our previous study showed that the N-terminal 9 residues of the C-terminal caspase-cleavage product of human gelsolin, an actin-regulatory protein, efficiently direct the protein N-myristoylation.To analyze the posttranslational N-myristoylation of gelsolin during apoptosis, metabolic labeling of gelsolin and its caspase-cleavage products expressed in COS-1 cells with [^3H]myristic acid was performed. It was found that the C-terminal caspase-cleavage product of human gelsolin (tGelsolin) expressed in COS-1 cells was efficiently N-myristoylated. When COS-1 cells transiently transfected with cDNA coding for full-length gelsolin were treated with etoposide or staurosporine, apoptosis-inducing agents, N-myristoylated tGelsolin was gener … More ated, as demonstrated by in vivo metabolic labeling. The caspase-mediated generation of posttranslationally N-myristoylated tGelsolin during apoptosis was also observed on endogenous gelsolin expressd in Hela cells. Immunofluorescence staining (coupled with MitoTracker staining) and subcellular fractionation revealed that exogenously expressed tGelsolin did not localize to mitochondria, but rather was diffusely distributed in the cytoplasm.To study the role of this modification in the anti-apoptotic activity of tGelsolin, we constructed the bicistronic expression plasmid tGelsolin-IRES-EGFP capable of overexpressing tGelsolin concomitantly with EGFP. Overexpression of N-myristoylated tGelsolin in COS-1 cells using the plasmid tGelsolin-IRES-EGFP significantly inhibited etoposide-induced apoptosis, whereas overexpression of the non-myristoylated tGelsolinG2A mutant did not cause resistance to apoptosis.These results indicate that posttranslational N-myristoylation of tGelsolin does not direct mitochondrial targeting, but this modification is involved in the anti-apoptotic activity of tGelsolin. Less