Tumor necrosis factor induces phosphorylation and translocation of BAD through a phosphatidylinositide-3-OH kinase-dependent pathway

Tumor necrosis factor (TNF) induced the phosphorylation of BAD at serine 13 6 in HeLa cells under conditions that are not cytotoxic. BAD phosphorylatio n by TNF was dependent on phosphatidylinositide-3-OH kinase (PI3K) and was accompanied by the translocation of BAD from the mitochondria to the cytoso l. Blocking the phosphorylation of BAD and its translocation to the cytosol with the PI3K inhibitor wortmannin activated caspase-3 and markedly potent iated the cytotoxicity of TNF. Transient transfection with a PI3K dominant negative mutant or a dominant negative mutant of the serine threonine kinas e Akt, the downstream target of PI3K and the enzyme that phosphorylates BAD , similarly potentiated the cytotoxicity of TNF. By contrast, transfection with a constitutively active Akt mutant protected against the cytotoxicity of TNF in the presence of wortmannin. Phosphorylation of BAD prevents its i nteraction with the antiapoptotic protein Bcl-X-L. Transfection with a Bcl- X-L expression vector protected against the cytotoxicity of TNF in the pres ence of wortmannin. The mechanism by which the inhibition of the phosphoryl ation of BAD is likely linked to the induction of lethal mitochondrial dama ge in TNF-intoxicated cells is discussed.