MULTIPLE SIGNAL TRANSDUCTION PATHWAYS MEDIATE C-JUN PROTEIN-PHOSPHORYLATION

A variety of protein kinases, including pp42 and pp54 mitogen-activate d protein (MAP) kinases, p34cdc2, and a partially purified protein kin ase from 4beta-phorbol 12-myristate 13alpha-acetate (PMA)-treated U937 cells have been shown to phosphorylate the NH2-terminal activation do main of c-jun in vitro. To investigate the role of pp42 MAP kinase in mediating c-jun phosphorylation in vivo, we have treated U937 monocyti c leukemia cells with a variety of pharmacological agents, including P MA, cycloheximide, AlF4, and okadaic acid. Although all of these agent s stimulated c-Jun phosphorylation, cycloheximide and okadaic acid had no effect on pp42 MAP kinase phosphorylation, suggesting that MAP kin ase activation was not necessary for c-Jun phosphorylation in vivo. Be cause dominant-negative Ras(Asn17) has been shown to block the effects of PMA on pp42 MAP kinase phosphorylation, we assessed its effect on c-Jun phosphorylation by cotransfection with a truncated c-Jun constru ct (c-Jun234). We found that c-Jun234 was expressed only in the cytoso l and was inducibly phosphorylated with kinetics similar to those of e ndogenous nuclear c-Jun. Furthermore, we found that Ras(Asn17) had no effect on PMA-induced phosphorylation of c-Jun234. Because Ha-Ras requ ires isoprenylation for membrane binding, we examined the effect of th e isoprenylation inhibitors lovastatin and perillic acid on PMA-induce d c-Jun phosphorylation. Pretreatment of U937 cells with these agents had no effect on PMA-induced c-Jun or pp42 MAP kinase phosphorylation. These data suggest that there are multiple pathways mediating c-Jun N H2-terminal phosphorylation in vivo, some of which are independent of MAP kinase activation, and that phorbol ester-mediated c-Jun and pp42 MAP kinase. phosphorylation in U937 cells does not appear to be mediat ed by members of the Ras family.