title: Overexpression of Akt (protein kinase B) confers protection against apoptosis and prevents formation of ceramide in response to pro-apoptotic stimuli.
abstract: An immortalized dorsal root ganglion cell line F-11 exhibits many properties of spinal cord neurons and undergoes apoptosis in response to growth factor withdrawal and the exogenous addition of inhibitors of phosphatidylinositol-3-kinase (PI3K). To elucidate the mechanism of apoptosis we generated F-11 clones which overexpressed either the p110 subunit of PI3K, a constitutively active form of protein kinase B/Akt (Myristoylated Akt), or a dominant-negative form (c-Akt). The first two constructs were protective against apoptosis induced by PI3K inhibitors such as wortmannin and LY294002. Caspase-3 (CPP32) levels peaked at 4 hr to 6 hr in response to pro-apoptotic drugs, and this increase was attenuated by 50% in F-11 with constitutively active Akt. The Akt protection was confirmed by DNA fragmentation studies. Both neo-transfected and the c-Akt dominant-negative transfected F-11 cells showed increased ceramide formation (twofold) in response to staurosporine, wortmannin, or LY294002; whereas cells with a constitutively active Akt (Myr-Akt) showed no increase in ceramide when treated with staurosporine, wortmannin, or LY294002. Ceramide was a more potent activator of CPP32 and an inducer of apoptosis when added as the native form (hydroxy- or nonhydroxy-), rather than the more water-soluble C(2)-ceramide. Overexpression of PI3K (p110) and Akt protected cells against ceramide-induced apoptosis, suggesting that Ceramide action is upstream of Akt in these cells and suggesting that Akt might be a target for inhibition by ceramide. Both staurosporine and C(2)-ceramide activated the Jun kinase (JNK) cascade and C(2)-ceramide increased caspase-3 (CPP32) activity in cells expressing wild-type c-Jun, but not dominant-negative (TAM-67) c-Jun. We suggest that this pathway is also involved in apoptosis, consistent with the idea that ceramide has multiple kinase and kinase-modulating targets in the apoptotic pathway of neurons. J. Neurosci. Sci. 57:884-893, 1999.
authors.affiliation: Department of Pediatrics and Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: R
authors.initials: R
authors.lastname: Goswami
authors.forename: J
authors.initials: J
authors.lastname: Kilkus
authors.forename: S A
authors.initials: SA
authors.lastname: Dawson
authors.forename: G
authors.initials: G
authors.lastname: Dawson
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002518:Ceramides / Q000096:biosynthesis*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D051057:Proto-Oncogene Proteins c-akt; D019311:Staurosporine / Q000494:pharmacology
chemical_list: D002518:Ceramides; D004791:Enzyme Inhibitors; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine