Cc. Franklin et al., MULTIPLE SIGNAL TRANSDUCTION PATHWAYS MEDIATE C-JUN PROTEIN-PHOSPHORYLATION, Cell growth & differentiation, 4(5), 1993, pp. 377-385
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.