Palmitoyl Protein Thioesterase 1 Protects Against Apoptosis Mediated by Ras—Akt—Caspase Pathway in Neuroblastoma Cells

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Abstract
Palmitoyl protein thioesterase (PPT) 1 is an enzyme involved in deacylation of palmitoylated proteins. A deficiency in PPT1 results in a genetic disease, infantile neuronal ceroid lipofuscinosis, associated with massive death of cortical neurons. The role of PPT1 in neuronal survival and apoptosis was studied in human neuroblastoma (LA‐N‐5) cells overexpressing PPT1. Overexpression of PPT1 was shown both by the 200‐350% increase in depalmitoylating activity over basal level (as determined by an in vitro PPT assay) and by western blot analysis of transiently expressed epitope‐tagged PPT1. Overexpressed PPT1 showed the same acidic pH optimum (pH 4.0) as the endogenous enzyme, when assayed with a Po‐derived octapeptide substrate, and reduced the growth rate by 30%. LA‐N‐5 cells underwent apoptosis, as evidenced by increased caspase 3‐like activity and increased DNA fragmentation, when challenged with either C2‐ceramide or a phosphatidylinositol 3‐kinase inhibitor (LY294002). Overexpression of PPT1 inhibited this C2‐ceramide‐ or LY294002‐mediated activation of caspase‐3 by 50%. There was also a concomitant decrease in DNA fragmentation and cell death. Consistent with increased resistance to apoptosis, we found increased phosphorylation of the antiapoptotic protein Akt (protein kinase B) in PPT1‐overexpressing cells. p21Ras is known to be dynamically palmitoylated and depalmitoylated and is involved in both growth and cell death. The C2‐ceramide‐induced membrane association of p21Ras was reduced by 30‐50% in PPT1‐overexpressing cells compared with control. PPT overexpression also led to reduced membrane association of another palmitoylated protein, GAP‐43, a neuron‐specific protein. Our studies suggest that protein palmitoylation could be a physiological regulator of apoptosis.