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Post-translational modification of H-Ras is required for activation of, but not for association with, B-Raf
1Department of Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
Abstract:
B-Raf is regulated by Ras protein and acts as a mitogen-activated protein (MAP) kinase kinase kinase in PC12 cells and brain. Ras protein undergoes a series of post-translational modifications on its C-terminal CAAX motif, and the modifications are critical for its function. To elucidate the role of the post-translational modifications in interaction with, and activation of, B-Raf, we have analyzed a direct association between H-Ras and B-Raf, and constructed an in vitro system for B-Raf activation by H-Ras. By using methods based on inhibition of yeast adenylyl cyclase or RasGAP activity and by in vitro binding assays, we have shown that the segment of B-Raf corresponding to amino acid 1-326 binds directly to H-Ras with a dissociation constant (Kd) comparable to that of Raf-1 and that the binding is not significantly affected by the post-translational modifications. However, when the activity of B-Raf to stimulate MAP kinase was measured by using a cell-free system derived from rat brain cytosol, we observed that the unmodified form of H-Ras possesses an almost negligible activity to activate B-Raf in vitro compared to the fully modified form. H-RasSer-181,184 mutant, which was farnesylated but not palmitoylated, was equally active as the fully modified form. These results indicate that the post-translational modifications, especially farnesylation, are required for H-Ras to activate B-Raf even though they have no apparent effect on the binding properties of H-Ras to B-Raf.
Insights
Post-translational modifications of Ras protein, particularly farnesylation, are crucial for activating B-Raf kinase activity. These modifications enhance Ras protein
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- B-Raf acts as a mitogen-activated protein (MAP) kinase kinase kinase, regulated by Ras protein in cellular signaling pathways.
- Ras protein requires C-terminal CAAX motif post-translational modifications for its biological functions.
Purpose of the Study:
- To investigate the role of Ras protein post-translational modifications in its interaction with and activation of B-Raf.
- To construct an in vitro system for analyzing B-Raf activation by H-Ras.
Main Methods:
- In vitro binding assays to determine the direct association between H-Ras and B-Raf.
- Measurement of B-Raf's MAP kinase stimulating activity using a cell-free system derived from rat brain cytosol.
- Utilized methods based on inhibition of yeast adenylyl cyclase or RasGAP activity.
Main Results:
- The N-terminal segment (amino acids 1-326) of B-Raf directly binds H-Ras, with binding affinity unaffected by Ras post-translational modifications.
- Fully modified H-Ras exhibits significantly higher activity in activating B-Raf compared to unmodified H-Ras in a cell-free system.
- H-RasSer-181,184 mutant, farnesylated but not palmitoylated, showed activity comparable to fully modified H-Ras, highlighting farnesylation's importance.
Conclusions:
- Post-translational modifications of H-Ras, especially farnesylation, are essential for its ability to activate B-Raf.
- While modifications do not alter binding affinity, they are critical for the functional activation of B-Raf by H-Ras.