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Association of MEK1 with p21ras.GMPPNP is dependent on B-Raf
S A Moodie1, M J Paris, W Kolch
1Department of Cell Biology, Cleveland Clinic Foundation, Ohio 44195.
Abstract:
We have previously reported that immobilized p21ras forms a GMPPNP-dependent complex with a MEK activity. Furthermore, the association of the MEK activity was found to be independent of the presence of Raf-1. We have extended those observations to show that MEK1 is the MEK activity previously described to associate with immobilized p21ras.GMPPNP. The association between MEK1 and immobilized p21ras.GMPPNP increased its specific activity towards p42MAPK. We detected the specific association of B-Raf with immobilized p21ras.GMPPNP. In contrast to Raf-1-immunodepleted lysates, preclearance of the cytosolic B-Raf significantly reduced, by 96%, the amount of MEK1 activity associated with immobilized p21ras.GMPPNP. The decrease in MEK1 activity correlated with complete loss in the binding of both B-Raf and MEK1 proteins with immobilized p21ras.GMPPNP. These data suggest that the p21ras.GMPPNP-dependent activation of MEK1 in brain extracts is dependent on the presence of the B-Raf protein kinase.
Insights
Ras GTPase (p21ras) activation of MEK1 requires B-Raf. This study identifies MEK1 as the specific MEK activity associating with p21ras.GMPPNP, highlighting B-Raf
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Previous work showed immobilized p21ras forms a GMPPNP-dependent complex with MEK activity, independent of Raf-1.
- The specific MEK activity was not identified.
- The role of Raf family kinases in this complex was unclear.
Purpose of the Study:
- To identify the specific MEK activity that associates with immobilized p21ras.GMPPNP.
- To investigate the role of B-Raf and Raf-1 in the p21ras.GMPPNP-dependent activation of MEK1.
- To elucidate the mechanism of MEK1 activation by p21ras.GMPPNP in brain extracts.
Main Methods:
- Utilized immobilized p21ras.GMPPNP in binding assays.
- Measured MEK activity using p42MAPK as a substrate.
- Employed immunodepletion and preclearance techniques to remove specific proteins (Raf-1, B-Raf).
- Assessed protein binding to immobilized p21ras.GMPPNP.
Main Results:
- Identified MEK1 as the specific MEK activity associating with immobilized p21ras.GMPPNP.
- Demonstrated that MEK1 association with p21ras.GMPPNP enhances its activity towards p42MAPK.
- Showed specific association of B-Raf with immobilized p21ras.GMPPNP.
- Preclearing cytosolic B-Raf reduced MEK1 activity associated with p21ras.GMPPNP by 96%, with a concomitant loss of B-Raf and MEK1 binding.
- Raf-1 immunodepletion did not significantly affect MEK1 association.
Conclusions:
- MEK1 is the specific MEK activity that associates with immobilized p21ras.GMPPNP.
- The p21ras.GMPPNP-dependent activation of MEK1 in brain extracts is critically dependent on the presence of B-Raf.
- B-Raf acts as a necessary component in the signaling pathway linking p21ras to MEK1 activation.