Related Experiment Videos
A different function for a critical tryptophan in c-Raf and Hck.
R A McPherson1, M M Taylor, E D Hershey
1Department of Clinical Pharmacology and Howard Hughes Medical Institute, University of Virginia Health Sciences Center, Charlottesville 22908-0577, USA.
Oncogene
|August 22, 2000
Summary
Tryptophan at residue 342 in c-Raf kinase has a different role than in Hck, with mutations inactivating c-Raf activity. Membrane factors can activate mutated c-Raf, suggesting a stringent structural requirement for this residue.
Area of Science:
- Molecular Biology
- Protein Kinase Research
- Cell Signaling
Background:
- The catalytic domains of Raf and Src kinases share similarities, suggesting conserved functions for homologous residues.
- A tryptophan at W260 in the Src family kinase Hck regulates ATP binding.
- The role of the homologous tryptophan (W342) in c-Raf kinase activity was investigated.
Purpose of the Study:
- To test if W342 in c-Raf has a similar regulatory role to W260 in Hck.
- To investigate the impact of W342 mutations on c-Raf kinase activity and substrate (MEK) activation.
- To explore the influence of the plasma membrane environment on c-Raf activity with W342 mutations.
Main Methods:
- Site-directed mutagenesis of W342 in c-Raf to alanine, aspartate, lysine, and histidine.
- Assaying c-Raf kinase activity using purified immunoprecipitates and plasma membrane recruitment.
- Evaluating MEK phosphorylation and activation in both wild-type and constitutively active c-Raf mutants.
Main Results:
- Mutation of W342 to alanine inactivated c-Raf kinase activity, unlike the activating effect in Hck.
- Mutations of W342 to aspartate, lysine, or histidine also inactivated c-Raf kinase activity.
- In a constitutively active c-Raf mutant, W342 mutations allowed MEK activation at the plasma membrane but not in vitro.
Conclusions:
- W342 plays a distinct role in c-Raf compared to W260 in Hck, with a stringent structural requirement for tryptophan at this position.
- W342 is not directly involved in MEK binding, as various residues at this site permit MEK activation in a membrane-anchored, hyperactivated c-Raf.
- The plasma membrane environment can potentiate the activation of c-Raf mutants with altered W342, but only in a hyperactivated context, not wild-type c-Raf.