Functional dichotomy of protein kinase C (PKC) in tumor necrosis factor-α (TNF-α) signal transduction in L929 cells. Translocation and inactivation of PKC by TNF-α

Tumor necrosis factor-α (TNF-α) is capable of inducing a variety of biologic responses through multiple signaling pathways. Because of the potential role of protein kinase C (PKC) in apoptosis, we examined the effects and mechanisms of TNF-α on PKC regulation, specifically on PKCα. In L929 murine fibroblasts, TNF-α (0.5-5 nM) caused potent inhibition of PKCα activity and induced translocation of PKCα from the cytosol to the membrane. Treatment of cells with TNF-α also induced dephosphorylation of PKCα as detected by a mobility shift on SDS-polyacrylamide gel and inhibition of PKC phosphorylation as probed by anti-phospho-PKC antibodies. Since PKC is activated directly by diacylglycerol and inactivated indirectly by ceramide, we next examined the roles of these lipid mediators in the regulation of PKCα. Addition of TNF-α led to accumulation of both ceramide and diacylglycerol. Fumonisin B₁, an inhibitor of ceramide synthase, and glutathione, an inhibitor of neutral sphingomyelinase, both reversed the effect of TNF-α on PKCα activity, suggesting that ceramide production is necessary for the action of TNF-α. The diacylglycerol mimic phorbol 12-myristate 13-acetate was sufficient to cause translocation of PKCα, but not the mobility shift. Okadaic acid at 2 nM, a potent protein phosphatase inhibitor, blocked the effects of TNF-α on PKCα activity, but not on PKCα translocation, thus demonstrating that dephosphorylation and translocation are independent processes. These results demonstrate that PKCα acts as a downstream target for TNF-α and that different lipid-mediated pathways in TNF-α signaling lead to opposing signals in the regulation of PKCα activity.