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Epidermal growth factor stimulates phosphorylation of RAF-1 independently of receptor autophosphorylation and
M Baccarini1, G N Gill, E R Stanley
1Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, New York, New York 10461.
Abstract:
Phosphorylation of the RAF-1 protooncogene product and activation of its associated serine/threonine kinase are common features of the response of cells to peptide growth factors. We have used wild-type and mutant epidermal growth factor (EGF) receptors to investigate mechanisms of RAF-1 phosphorylation. In vivo EGF treatment rapidly stimulated phosphorylation of RAF-1 exclusively on serine residues. Stimulation of RAF-1 phosphorylation occurred at 37 degrees C but not at 4 degrees C and persisted after dissociation of EGF from its receptor. EGF-induced RAF-1 serine phosphorylation required the intrinsic tyrosine kinase activity of the EGF receptor but was independent of EGF receptor self-phosphorylation and of ligand-induced receptor internalization. Down-regulation of protein kinase C did not affect the EGF-induced increase in RAF-1 phosphorylation. These data suggest that the activated tyrosine kinase activity of the EGF receptor enhances serine phosphorylation of RAF-1 via an intermediary molecule(s).
Insights
Epidermal growth factor (EGF) rapidly stimulates RAF-1 phosphorylation on serine residues. This process requires EGF receptor tyrosine kinase activity, suggesting an intermediary molecule is involved.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Oncogene research
Background:
- Peptide growth factors, such as epidermal growth factor (EGF), trigger cellular responses involving the phosphorylation and activation of RAF-1 kinase.
- Understanding the precise mechanisms of RAF-1 phosphorylation is crucial for deciphering cellular signal transduction and cancer development.
Purpose of the Study:
- To investigate the mechanisms by which epidermal growth factor (EGF) receptors mediate the phosphorylation of RAF-1.
- To determine the specific requirements for EGF-induced RAF-1 serine phosphorylation.
Main Methods:
- Utilized wild-type and mutant EGF receptors in cellular experiments.
- Administered EGF in vivo and analyzed RAF-1 phosphorylation status at different temperatures.
- Assessed the role of EGF receptor tyrosine kinase activity, self-phosphorylation, and internalization.
- Investigated the involvement of protein kinase C by down-regulation experiments.
Main Results:
- EGF treatment rapidly induced RAF-1 phosphorylation exclusively on serine residues.
- Phosphorylation occurred at 37°C but not 4°C and persisted after EGF dissociation.
- EGF-induced RAF-1 serine phosphorylation was dependent on EGF receptor tyrosine kinase activity.
- This phosphorylation was independent of EGF receptor self-phosphorylation, internalization, and protein kinase C activity.
Conclusions:
- The activated tyrosine kinase activity of the EGF receptor is essential for enhancing serine phosphorylation of RAF-1.
- An intermediary molecule(s) likely mediates the signal from the activated EGF receptor to RAF-1.
- These findings elucidate a key step in growth factor-mediated cellular signaling.