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 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 P0-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.
Cho
S
S
Department of Pediatrics, University of Chicago, Illinois, USA.
Dawson
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G
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000201:enzymology; D002470:Cell Survival / Q000502:physiology; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D009447:Neuroblastoma; D009474:Neurons / Q000166:cytology* / Q000201:enzymology*; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D010170:Palmitoyl-CoA Hydrolase / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; C064769:N-acetylsphingosine; D011518:Proto-Oncogene Proteins; D010171:Palmitoyl Coenzyme A; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D010170:Palmitoyl-CoA Hydrolase; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D013110:Sphingosine