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Differential Raf requirement for activation of mitogen-activated protein kinase by growth factors, phorbol esters,
T S Chao1, D A Foster, U R Rapp
1Ben May Institute, University of Chicago, Illinois 60637.
Abstract:
Although a pathway that requires sequential activation of Ras, Raf, and MAP kinase kinase has been proposed as the major mechanism for stimulation of mitogen-activated protein kinase (MAP kinase), alternative pathways also exist. A wide variety of extracellular stimuli have been shown to activate MAP kinase; however, the precise mechanisms by which these stimuli mediate the signaling events have not been elucidated. Using a Balb/c-derived cell line expressing a dominant-negative mutant of Raf, we determined whether Raf is required for the activation of MAP kinase by growth factors, phorbol esters, and calcium. Insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), and phorbol 12,13-dibutyrate activated Ras in both mutant and control cells. However, stimulation of MAP kinase by IGF-I was nearly abolished in the dominant-negative Raf mutant. Stimulation of MAP kinase by the Ca2+ mobilizer thapsigargin was also inhibited in the presence of the Raf mutant. In contrast, EGF and phorbol 12,13-dibutyrate remained potent stimulators of MAP kinase in the dominant-negative Raf cells. The activation of MAP kinase by these stimuli can be further distinguished by differential requirements for Ca2+ and protein kinase C. These results suggest that Raf is required for the activation of MAP kinase by IGF-I and calcium, whereas EGF and possibly phorbol esters may employ alternative Raf-independent pathways for MAP kinase activation.
Insights
Raf is crucial for mitogen-activated protein kinase (MAP kinase) activation by IGF-I and calcium. However, epidermal growth factor and phorbol esters activate MAP kinase via Raf-independent pathways.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Signal transduction
Background:
- Mitogen-activated protein kinase (MAP kinase) activation is essential for cellular responses.
- While the Ras-Raf-MAP kinase kinase pathway is a major route, alternative MAP kinase activation mechanisms exist.
- The precise signaling mechanisms for various extracellular stimuli remain incompletely understood.
Purpose of the Study:
- To investigate the role of Raf in MAP kinase activation by different extracellular stimuli.
- To determine if Raf is essential for MAP kinase activation mediated by growth factors, phorbol esters, and calcium.
Main Methods:
- Utilized a Balb/c-derived cell line expressing a dominant-negative Raf mutant.
- Compared MAP kinase activation in mutant and control cells following stimulation with IGF-I, EGF, phorbol 12,13-dibutyrate, and thapsigargin.
- Assessed Ras activation in both cell types.
Main Results:
- IGF-I and thapsigargin-induced MAP kinase activation were significantly inhibited in dominant-negative Raf cells.
- EGF and phorbol 12,13-dibutyrate potently stimulated MAP kinase even in the presence of the Raf mutant.
- Ras activation occurred in both cell types, indicating stimulus-specific downstream signaling.
Conclusions:
- Raf is required for MAP kinase activation by Insulin-like Growth Factor I (IGF-I) and calcium signaling.
- Epidermal Growth Factor (EGF) and potentially phorbol esters activate MAP kinase through Raf-independent pathways.
- This highlights the existence of divergent signaling routes for MAP kinase activation.