Related Experiment Videos
4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase
S R von Manteuffel1, A C Gingras, X F Ming
1Friedrich Miescher Institute, Department of Growth Control, Basel, Switzerland.
Abstract:
It has previously been argued that the repressor of protein synthesis initiation factor 4E, 4E-BP1, is a direct in vivo target of p42mapk. However, the immunosuppressant rapamycin blocks serum-induced 4E-BP1 phosphorylation and, in parallel, p70s6k activation, with no apparent effect on p42mapk activation. Consistent with this finding, the kinetics of serum-induced 4E-BP1 phosphorylation closely follow those of p70s6k activation rather than those of p42mapk. More striking, insulin, which does not induce p42mapk activation in human 293 cells or Swiss mouse 3T3 cells, induces 4E-BP1 phosphorylation and p70s6k activation in both cell types. Anisomycin, which, like insulin, does not activate p42mapk, promotes a small parallel increase in 4E-BP1 phosphorylation and p70s6k activation. The insulin effect on 4E-BP1 phosphorylation and p70s6k activation in both cell types is blocked by SQ20006, wortmannin, and rapamycin. These three inhibitors have no effect on p42mapk activation induced by phorbol 12-tetradecanoate 13-acetate, though wortmannin partially suppresses both the p70s6k response and the 4E-BP1 response. Finally, in porcine aortic endothelial cells stably transfected with either the wild-type platelet-derived growth factor receptor or a mutant receptor bearing the double point mutation 740F/751F, p42mapk activation in response to platelet-derived growth factor is unimpaired, but increased 4E-BP1 phosphorylation is ablated, as previously reported for p70s6k. The data presented here demonstrate that 4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase.
Insights
The repressor of protein synthesis initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation is regulated by the FRAP-p70s6k pathway. This process is independent of mitogen-activated protein kinase (MAPK) signaling.
Area of Science:
- Cellular and Molecular Biology
- Signal Transduction Pathways
- Protein Kinase Signaling
Background:
- The repressor of protein synthesis initiation factor 4E-binding protein 1 (4E-BP1) is a key regulator of cap-dependent translation.
- Previous studies suggested that p42mapk directly phosphorylates 4E-BP1 in vivo.
- The role of MAPK signaling in 4E-BP1 regulation requires further investigation.
Purpose of the Study:
- To investigate the signaling pathways regulating 4E-BP1 phosphorylation.
- To determine the relationship between 4E-BP1 phosphorylation and the activation of p70s6k and p42mapk.
- To elucidate whether p42mapk is the primary kinase responsible for 4E-BP1 phosphorylation.
Main Methods:
- Cell culture (human 293 cells, Swiss mouse 3T3 cells, porcine aortic endothelial cells).
- Stimulation with insulin, anisomycin, and phorbol 12-tetradecanoate 13-acetate.
- Inhibition studies using rapamycin, SQ20006, and wortmannin.
- Analysis of protein phosphorylation and activation of signaling molecules (4E-BP1, p70s6k, p42mapk).
- Use of cells with wild-type and mutant platelet-derived growth factor receptors.
Main Results:
- Rapamycin inhibited serum-induced 4E-BP1 phosphorylation and p70s6k activation without affecting p42mapk activation.
- The kinetics of 4E-BP1 phosphorylation mirrored p70s6k activation, not p42mapk activation.
- Insulin and anisomycin induced 4E-BP1 phosphorylation and p70s6k activation independently of p42mapk activation.
- Inhibitors of the FRAP-p70s6k pathway blocked insulin-induced 4E-BP1 phosphorylation, while having minimal effect on p42mapk activation.
- Platelet-derived growth factor-induced 4E-BP1 phosphorylation was abolished in cells with a mutated PDGF receptor, despite unimpaired p42mapk activation.
Conclusions:
- 4E-BP1 phosphorylation is primarily mediated by the FRAP-p70s6k signaling pathway.
- Mitogen-activated protein kinase (MAPK) is not the direct kinase responsible for 4E-BP1 phosphorylation in response to the studied stimuli.
- These findings clarify the distinct signaling cascades regulating translation initiation factors.