PKC ζ is a molecular switch in signal transduction of TNF-α, bifunctionally regulated by ceramide and arachidonic acid

Abstract

Tumor necrosis factor (TNF-α) stimulates a number of signal transduction pathways in which phospholipases produce lipid second messengers. However, the immediate molecular targets of these messengers, in particular those of ceramide and arachidonic acid (AA) and their role in TNF signaling are not well defined. In this study we investigated the relationship of ceramide and AA in regulating an atypical PKC isozyme, PKC ζ, U937 cells responding to TNF-α treatment with NFKB activation displayed enhanced phosphorylation of PKC ζ, which is already detectable 30 s after stimulation, [14C] ceramide specifically binds to and regulates kinase activity of PKC ζ in a biphasic manner. Binding studies indicate high and low affinity binding with b(max) values of 60 and 600 nM and K(d) values of 7.5 and 320 nM respectively. At ceramide concentrations as low as 0.5 nM an up to 4-fold increase in autophosphorylation is obtained, which, at concentrations > 60 nM, again declines to basal levels. Interestingly, AA competes for ceramide binding and inhibits basal and ceramide-stimulated PKC ζ kinase activity at < 100 nM. Metabolism of [14C] ceramide in cells is slow and is inhibited in the presence of equimolar concentrations of lyso-phosphatidylcholine. Based on the bifunctional modulation of PKC ζ by the lipid messengers ceramide and AA, a model of TNF signal pathways is suggested in which PKC ζ takes a central position, acting as a molecular switch between mitogenic and growth inhibitory signals of TNF-α.