C-JUN IS A DOWNSTREAM TARGET FOR CERAMIDE-ACTIVATED PROTEIN PHOSPHATASE IN A431 CELLS

Stimulation of [H-3]serine-labeled A431 cells with tumor necrosis fact or-alpha (TNF alpha) or bacterial sphingomyelinase (SMase) resulted in a rapid decrease (similar to 50% by 15 min) in cellular [H-3]sphingom yelin content and generation of the lipid moiety [H-3]ceramide, which remained elevated 60 min later, Sphingomyelin hydrolysis in response t o TNF alpha or bacterial SMase resulted in a time-dependent decrease i n the phosphorylation state of c-Jun protein, an effect that was also observed in cells treated with the membrane-permeable ceramide analogu e N-hexanoylsphingosine (C-6-ceramide). The rapid dephosphorylation of the c-Jun gene product in response to TNF alpha, SMase, or C-6-cerami de was not observed in A431 cells treated with the serine-threonine ph osphatase inhibitor okadaic acid. After the initial steps of previousl y described methods for the purification of a ceramide-activated prote in phosphatase termed CAPP (Dobrowsky, R, T., Kamibayashi, C,, Mumby, M, C., and Hannun, Y. A, (1993) J, Biol, Chen. 268, 15523-15530), we o btained a cytosolic fraction from A431 cells that specifically dephosp horylated P-32(i)-labeled c-Jun protein used as substrate in an immuno complex phosphatase assay, Phosphatase activity in vitro was apparent only in the presence of ceramide (5 mu m) and was specifically abrogat ed when okadaic acid (1 nM) was included in the immunocomplex phosphat ase assay, These results provide strong evidence for c-Jun as a downst ream target for CAPP activated in response to post-TNF signaling in A4 31 cells.