Functional Dichotomy of Protein Kinase C (PKC) in Tumor Necrosis Factor-a (TNF-a) Signal Transduction in L929 Cells

Tumor necrosis factor-a (TNF-a) 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-a on PKC regulation, specifically on PKCa. In L929 murine fibroblasts, TNF-a (0.5‐ 5n M) caused potent inhibition of PKCa activity and induced translocation of PKCa from the cytosol to the membrane. Treatment of cells with TNF-a also induced dephosphorylation of PKCa 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 PKCa. Addition of TNF-a 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-a on PKCa activity, suggesting that ceramide production is necessary for the action of TNF-a. The diacylglycerol mimic phorbol 12-myristate 13-acetate was sufficient to cause translocation of PKCa, but not the mobility shift. Okadaic acid at 2 nM, a potent protein phosphatase inhibitor, blocked the effects of TNF-a on PKCa activity, but not on PKCa translocation, thus demonstrating that dephosphorylation and translocation are independent processes. These results demonstrate that PKCa acts as a downstream target for TNF-a and that different lipid-mediated pathways in TNF-a signaling lead to opposing signals in the regulation of PKCa activity.