Tumor necrosis factor-␣ (TNF-␣) 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-␣ on PKC regulation, specifically on PKC␣. In L929 murine fibroblasts, TNF-␣ (0.5-5 nM) caused potent inhibition of PKC␣ activity and induced translocation of PKC␣ from the cytosol to the membrane. Treatment of cells with TNF-␣ also induced dephosphorylation

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... f PKC␣ 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 PKC␣. Addition of TNF-␣ led to accumulation of both ceramide and diacylglycerol. Fumonisin B 1 , an inhibitor of ceramide synthase, and glutathione, an inhibitor of neutral sphingomyelinase, both reversed the effect of TNF-␣ on PKC␣ activity, suggesting that ceramide production is necessary for the action of TNF-␣. The diacylglycerol mimic phorbol 12-myristate 13-acetate was sufficient to cause translocation of PKC␣, but not the mobility shift. Okadaic acid at 2 nM, a potent protein phosphatase inhibitor, blocked the effects of TNF-␣ on PKC␣ activity, but not on PKC␣ translocation, thus demonstrating that dephosphorylation and translocation are independent processes. These results demonstrate that PKC␣ acts as a downstream target for TNF-␣ and that different lipid-mediated pathways in TNF-␣ signaling lead to opposing signals in the regulation of PKC␣ activity.