Tumor Necrosis Factor Induces Phosphorylation and Translocation of BAD through a Phosphatidylinositide-3-OH Kinase-dependent Pathway

Abstract

mannin. The mechanism by which the inhibition of the phosphorylation of BAD is likely linked to the induction of lethal mitochondrial damage in TNF-intoxicated cells is discussed. Phosphatidylinositide-3-OH kinase (PI3K)1 is a member of a signaling cascade that eventuates in the phosphorylation of the proapoptotic protein BAD (1, 2). Such growth factors as NGF and interleukin-3 are presumed to promote cell survival as a consequence of the PI3K-mediated activation of the serinethreonine kinase Akt, which in turn phosphorylates BAD (3, 4). Whereas BAD can be phosphorylated at either the Ser-112 or Ser-136 site (5), Akt phosphorylates BAD specifically at Ser-136 (phospho-BAD-136) (2, 6). BAD is capable of forming heterodimers with the antiapoptotic proteins Bcl-X L or Bcl-2 (7). Importantly, phospho-BAD-136 cannot bind either Bcl-X L or Bcl-2 (5). It is postulated that by binding to Bcl-X L and Bcl-2 BAD antagonizes their antiapoptotic activity. Upon inducing its phosphorylation, growth factors lead to the dissociation of BAD from Bcl-X L and Bcl-2 and thereby promote cell survival by allowing the unhindered action of these proteins.The PI3K inhibitor wortmannin enhanced the activation of caspase-3 that was induced by TNF or anti-Fas (8). Here we demonstrate that TNF acts like other growth factors to promote the phosphorylation of BAD at Ser-136. As a result, there is the translocation of BAD from the mitochondria to the cytosol. Moreover, the phosphorylation of BAD by TNF occurs by a PI3K-dependent pathway and is necessary to prevent the cytotoxicity of this cytokine. Inhibition of PI3K prevents both the phosphorylation of BAD and its translocation from mitochondria to the cytosol, effects that are accompanied by substantially enhanced cell killing by TNF. EXPERIMENTAL PROCEDURESTissue Culture-HeLa cells (ATCC-CC-1, American Type Culture Collection) were maintained in 25 cm 2 polystyrene flasks (Corning Costar Corp., Oneonta, NY) with 5 ml of Dulbecco's modified Eagle's medium (DMEM) (high glucose, without pyruvate) (Life Technologies, Inc.) containing 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 10% heat-inactivated fetal bovine serum, all incubated under an atmosphere of 95% air and 5% CO 2 . For transient transfections, HeLa cells were plated at one-third of confluency (3.0 ϫ 10 4 cells/cm 2 ) in 24-well plates. After overnight incubation, the cells were washed twice in phosphate-buffered saline (PBS). Transfections were performed using Lipofectamine-Plus (Life Technologies, Inc.) according to the manufacturer's instructions. Transfection efficiencies of 25-30% were routinely obtained, as assessed by ␤-galactosidase staining. For experiments, cells were transfected with 0.5 g of pCDNA-LacZ and 5 g of either pCDNA, pCDNA3-myc-⌬p85 (referred to as pCDNA-PI3K(Ϫ)

Serum bioactive lysophospholipids prevent TRAIL-induced apoptosis via PI3K/Akt-dependent cFLIP expression and Bad phosphorylation

“…This lack of sensitivity may be attributed either to an unfavorable ratio of death to decoy receptors or to expression of resistant genes. 3,31 So far, the majority of normal human cells tested appear to be relatively TRAIL resistant; however, recent experiments have demonstrated that cultured human liver cells may be sensitive to TRAIL-induced apoptosis. 10 This discrepancy may be due to different serum concentrations used.…”

“…30 Although Bad is known to regulate intrinsic death machinery, 32 it has been demonstrated that Bad phosphorylation can prevent TNF-a-induced apoptosis. 31 Furthermore, the upregulation of Bad expression enhanced TRAIL-induced apoptosis, and the suppression of Bad phosphorylation by siRNA of PKA1, a kinase that phosphorylates Bad in some systems, prevented TRAILinduced apoptosis. 46 Consistent with these observations, our data showed that FBS and dBSA-LPA increased Bad phosphorylation, resulting in suppression of the translocation of cytosolic Bad to mitochondria and mitochondrial cytochrome c release ( Figure 5).…”

“…One possible mechanism by which Akt signaling suppresses apoptosis is associated with the inhibition of cytochrome c release by phosphorylating proapoptotic Bad and blocking its translocation into mitochondria from the cytosol. 30 Phosphorylation of Bad can couple survival signals to extrinsic death pathway 31 as well as intrinsic death machinery. 32 We therefore examined the effects of FBS and dBSA-LPA on intracellular localization of Bad and cytochrome c release by Western blot analysis.…”

Expression of the NF-κB Target Gene X-Ray-Inducible Immediate Early Response Factor-1 Short Enhances TNF-α-Induced Hepatocyte Apoptosis by Inhibiting Akt Activation

“…Thus, it may be reasonable to speculate that the proapoptotic effect of IEX-1S is mediated by the inhibition of antiapoptotic signal(s) activated by TNF-␣. In several types of cells such as HeLa cells and human endothelial cells, TNF-␣ appears to activate serine/threonine kinase Akt via PI3K (28, 34 -36), which protects cells from apoptosis (28,29,36,37). In hepatocytes, TNF-␣ activates the PI3K/Akt pathway which inhibits apoptosis mediated by TNF-␣ and this protective effect is independent of NF-B (8).…”

“…Mechanisms for the antiapoptotic effects of Akt have been reported previously. Pastorino et al (36) reported that TNF-␣ induces phosphorylation of a proapoptotic Bcl-2 family member Bad through the PI3K/Akt pathway and that phosphorylated Bad loses the ability to bind to Bcl-x L , which is known to act on mitochondria to block the apoptotic signaling cascade (38). However, phosphorylation of Bad was not observed in the TNF-␣-treated Hc cells (data not shown).…”