Palmitoyl protein thioesterase 1 protects against apoptosis mediated by Ras-Akt-caspase pathway in neuroblastoma cells

Palmitoyl protein thioesterase (PPT) 1 is an enzyme involved in deacylation of palmitoylated proteins. A deficiency in PPT1 results in a genetic disea se, infantile neuronal ceroid lipofuscinosis, associated with massive death of cortical neurons. The role of PPT1 in neuronal survival and apoptosis w as studied in human neuroblastoma (LA-N-5) cells overexpressing PPT1. Overe xpression of PPT1 was shown both by the 200-350% increase in depalmitoylati ng activity over basal level (as determined by an in vitro PPT assay) and b y western blot analysis of transiently expressed epitope-tagged PPT1. Overe xpressed PPT1 showed the same acidic pH optimum (pH 4.0) as the endogenous enzyme, when assayed with a P-0-derived octapeptide substrate, and reduced the growth rate by 30%. LA-N-5 cells underwent apoptosis, as evidenced by i ncreased caspase 3-like activity and increased DNA fragmentation, when chal lenged with either C-2-ceramide or a phosphatidylinositol 3-kinase inhibito r (LY294002). Overexpression of PPT1 inhibited this C-2-ceramide- or LY2940 02-mediated activation of caspase-3 by 50%. There was also a concomitant de crease in DNA fragmentation and cell death. Consistent with increased resis tance to apoptosis, we found increased phosphorylation of the antiapoptotic protein Akt (protein kinase B) in PPT1-overexpressing cells. p21(Ras) is k nown to be dynamically palmitoylated and depalmitoylated and is involved in both growth and cell death. The C-2-ceramide-induced membrane association of p21(Ras) was reduced by 30-50% in PPT1-overexpressing cells compared wit h control. PPT overexpression also led to reduced membrane association of a nother palmitoylated protein, GAP-43, a neuron-specific protein. Our studie s suggest that protein palmitoylation could be a physiological regulator of apoptosis.