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Activation of c-Raf-1 by Ras and Src through different mechanisms: activation in vivo and in vitro
1Onyx Pharmaceuticals, Richmond, CA 94806, USA. dhstokoe@onyx-pharm.com
Abstract:
The c-Raf-1 protein kinase plays a critical role in intracellular signaling downstream from many tyrosine kinase and G-protein-linked receptors. c-Raf-1 binds to the proto-oncogene Ras in a GTP-dependent manner, but the exact mechanism of activation of c-Raf-1 by Ras is still unclear. We have established a system to study the activation of c-Raf-1 in vitro. This involves mixing membranes from cells expressing oncogenic H-RasG12V, with cytosol from cells expressing epitope-tagged full-length wild-type c-Raf-1. This results in a fraction of the c-Raf-1 binding to the membranes and a concomitant 10- to 20-fold increase in specific activity. Ras was the only component in these membranes required for activation, as purified recombinant farnesylated K-Ras.GTP, but not non-farnesylated K-Ras.GTP or farnesylated K-Ras.GDP, was able to activate c-Raf-1 to the same degree as intact H-RasG12V membranes. The most potent activation occurred under conditions in which phosphorylation was prohibited. Under phosphorylation-permissive conditions, activation of c-Raf-1 by Ras was substantially inhibited. Consistent with the results from other groups, we find that the activation of c-Raf-1 by Src in vivo occurs concomitant with tyrosine phosphorylation on c-Raf-1, and in vitro, activation of c-Raf-1 by Src requires the presence of ATP. Therefore we propose that activation of c-Raf-1 by Ras or by Src occurs through different mechanisms.
Insights
Ras protein activates c-Raf-1 kinase through a distinct mechanism compared to Src. Ras-mediated activation is most effective without phosphorylation, unlike Src activation which requires it.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- c-Raf-1 protein kinase is crucial for intracellular signaling pathways, mediating signals from tyrosine kinase and G-protein-linked receptors.
- The precise mechanism by which Ras, a proto-oncogene, activates c-Raf-1 in a GTP-dependent manner remains incompletely understood.
Purpose of the Study:
- To establish an in vitro system for studying the activation mechanism of c-Raf-1 by Ras.
- To elucidate the distinct pathways of c-Raf-1 activation by Ras and Src.
Main Methods:
- An in vitro system was developed by combining membranes from cells expressing oncogenic H-RasG12V with cytosol from cells expressing wild-type c-Raf-1.
- Purified recombinant K-Ras.GTP (farnesylated) was used to assess Ras's role in activation.
- Experiments were conducted under both phosphorylation-permissive and prohibitory conditions to evaluate the role of phosphorylation.
Main Results:
- Mixing H-RasG12V membranes with c-Raf-1 cytosol resulted in a 10- to 20-fold increase in c-Raf-1 specific activity.
- Purified farnesylated K-Ras.GTP, but not non-farnesylated or GDP-bound forms, effectively activated c-Raf-1.
- Ras-mediated c-Raf-1 activation was most potent when phosphorylation was prohibited and significantly inhibited under phosphorylation-permissive conditions.
- Src-mediated c-Raf-1 activation in vitro requires ATP and is associated with tyrosine phosphorylation in vivo, contrasting with Ras activation.
Conclusions:
- Ras and Src activate c-Raf-1 through fundamentally different molecular mechanisms.
- Ras activation of c-Raf-1 is independent of phosphorylation, whereas Src activation is dependent on it.