Functional dichotomy of protein kinase C (PKC) in tumor necrosis factor-alpha (TNF-alpha ) signal transduction in L929 cells. Translocation and inactivation of PKC by TNF-alpha.
Tumor necrosis factor-alpha (TNF-alpha) 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-alpha on PKC regulation, specifically on PKC alpha. In L929 murine fibroblasts, TNF-alpha (0.5- 5 nm) caused potent inhibition of PKC alpha activity and induced translocation of PKC alpha from the cytosol to the membrane. Treatment of cells with TNF-alpha also induced dephosphorylation of PKC alpha 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 alpha. Addition of TNF-alpha 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-alpha on PKC alpha activity, suggesting that ceramide production is necessary for the action of TNF-alpha. The diacylglycerol mimic phorbol 12-myristate 13-acetate was sufficient to cause translocation of PKC alpha, but not the mobility shift. Okadaic acid at 2 nm, a potent protein phosphatase inhibitor, blocked the effects of TNF-alpha on PKC alpha activity, but not on PKC alpha translocation, thus demonstrating that dephosphorylation and translocation are independent processes. These results demonstrate that PKC alpha acts as a downstream target for TNF-alpha and that different lipid-mediated pathways in TNF-alpha signaling lead to opposing signals in the regulation of PKC alpha activity.
Lee
J Y
JY
Department of Medicine, Duke University Medical Center,Durham, North Carolina 27710, USA.
Hannun
Y A
YA
Obeid
L M
LM
D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005347:Fibroblasts; D007527:Isoenzymes / Q000502:physiology*; D051379:Mice; D011493:Protein Kinase C / Q000502:physiology*; D051571:Protein Kinase C-alpha; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology / Q000502:physiology*
D007527:Isoenzymes; D014409:Tumor Necrosis Factor-alpha; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha