PROTEIN-KINASE-C-ZETA ISOFORM IS CRITICAL FOR KAPPA-B-DEPENDENT PROMOTER ACTIVATION BY SPHINGOMYELINASE

Recent evidence demonstrates that the protein kinase C zeta (zeta PKC) isoform is required for the activation of nuclear factor kappa B (NF- kappa B) and mitogenic signaling in Xenopus oocytes and mammalian cell s. The mechanism whereby zeta PKC regulates NF-kappa B most probably i nvolves the activation of a putative I kappa B kinase of molecular mas s similar to 50 kDa, which phosphorylates and inactivates I kappa B. T umor necrosis factor alpha (TNF alpha) and interleukin-1 besides activ ating the phospholipase C-mediated breakdown of phosphatidylcholine, a lso generate ceramide, which is produced by stimulation of sphingomyel in hydrolysis. We show here that exogenous addition of sphingomyelinas e (SMase) to NIH-3T3 fibroblasts transactivates a kappa B-dependent ch loramphenicol acetyltransferase reporter plasmid, to an extent similar to that produced by TNF alpha or phosphatidylcholine/phospholipase C. More importantly, the ability of SMase to stimulate this parameter is severely impaired by transfection of a zeta PKC kinase defective domi nant negative mutant, which suggests a critical role of zeta PKC in SM ase signaling. In keeping with this notion, we also demonstrate here t hat zeta PKC is acti vated in vitro by ceramide and in vivo by treatme nt of NIH-3T3 fibroblasts with SMase.