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

被引:63
作者
Cho, SG
Dawson, G
机构
[1] Univ Chicago, Sch Med, Dept Pediat, Chicago, IL 60637 USA
[2] Univ Chicago, Sch Med, Dept Biochem, Chicago, IL 60637 USA
[3] Univ Chicago, Sch Med, Dept Mol Biol, Chicago, IL 60637 USA
关键词
palmitoyl protein thioesterase; apoptosis; neuroblastoma cells; p21(Ras); caspase; Akt;
D O I
10.1046/j.1471-4159.2000.0741478.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 P-0-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 C-2-ceramide or a phosphatidylinositol 3-kinase inhibitor (LY294002). Overexpression of PPT1 inhibited this C-2-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. p21(Ras) is known 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 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.
引用
收藏
页码:1478 / 1488
页数:11
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