Multiple signal transduction pathways mediate c-Jun protein phosphorylation. Cell Growth Differ

A variety of protein kinases, including pp42 and pp54 mitogen-activated protein (MAP) kinases, p342, and a partially purified protein kinase from 4$-phorbol 12-myristate 13a-acetate (PMA)-treated U937 cells have been shown to phosphorylate the NH2-terminal activation domain of c-Jun in vitro. To investigate the role of pp42 MAP kinase in mediating c-Jun phosphorylation in vivo, we have treated U937 monocytic leukemia cells with a variety of pharmacological agents, including PMA, cycloheximide, AIF4, and okadaic acid. Although all of these agents stimulated c-Jun phosphorylation, cycloheximide and okadaic acid had no effect on pp42 MAP kinase phosphorylation, suggesting that MAP kinase activation was not necessary for c-Jun phosphorylation in vivo. Because dominant-negative Ras17 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 construct (c-Jun234). We found that c-Jun234 was expressed only in the cytosol and was inducibly phosphorylated with kinetics similar to those of endogenous nuclear c-Jun. Furthermore, we found that Ras17 had no effect on PMA-induced phosphorylation of c-Jun234. Because Ha-Ras requires isoprenylation for membrane binding, we examined the effect of the isoprenylation inhibitors lovastatin and perilhic acid on PMA-induced 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 NH2-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 mediated by members of the Ras family