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Multiple signal transduction pathways mediate c-Jun protein phosphorylation
C C Franklin1, T Unlap, V Adler
1Division of Hematology/Oncology, University of Alabama, Birmingham 35294.
Abstract:
A variety of protein kinases, including pp42 and pp54 mitogen-activated protein (MAP) kinases, p34cdc2, and a partially purified protein kinase from 4 beta-phorbol 12-myristate 13 alpha-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 RasAsn17 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 RasAsn17 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 perillic 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Mitogen-activated protein (MAP) kinase activation is not required for c-Jun phosphorylation in vivo. Pharmacological agents and dominant-negative RasAsn17 did not affect c-Jun phosphorylation, suggesting alternative signaling pathways are involved.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- c-Jun protein phosphorylation is crucial for its transcriptional activity.
- Mitogen-activated protein (MAP) kinases, including pp42 MAP kinase, are known to phosphorylate c-Jun in vitro.
- The in vivo role of pp42 MAP kinase in mediating c-Jun phosphorylation remains unclear.
Purpose of the Study:
- To investigate the role of pp42 MAP kinase in c-Jun phosphorylation in U937 monocytic leukemia cells.
- To determine if MAP kinase activation is necessary for in vivo c-Jun phosphorylation.
- To explore the involvement of Ras signaling and isoprenylation in c-Jun phosphorylation.
Main Methods:
- U937 cells were treated with phorbol 12-myristate 13-acetate (PMA), cycloheximide, AIF4, and okadaic acid.
- Cotransfection with dominant-negative RasAsn17 and a truncated c-Jun construct (c-Jun234) was performed.
- Inhibitors of isoprenylation, lovastatin and perillic acid, were used to assess Ha-Ras involvement.
Main Results:
- PMA, cycloheximide, AIF4, and okadaic acid stimulated c-Jun phosphorylation.
- Cycloheximide and okadaic acid did not affect pp42 MAP kinase phosphorylation.
- Dominant-negative RasAsn17 did not inhibit PMA-induced c-Jun phosphorylation.
- Isoprenylation inhibitors had no effect on PMA-induced c-Jun or pp42 MAP kinase phosphorylation.
Conclusions:
- MAP kinase activation is not essential for in vivo c-Jun phosphorylation in U937 cells.
- The Ras signaling pathway and Ha-Ras isoprenylation do not appear to mediate PMA-induced c-Jun phosphorylation.
- Alternative signaling pathways likely regulate c-Jun phosphorylation in response to PMA and other stimuli.