Overexpression of Akt (protein kinase B) confers protection against apoptosis and prevents formation of ceramide in response to pro-apoptotic stimuli
✍ Scribed by R. Goswami; J. Kilkus; S.A. Dawson; G. Dawson
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 169 KB
- Volume
- 57
- Category
- Article
- ISSN
- 0360-4012
No coin nor oath required. For personal study only.
✦ Synopsis
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.