A different function for a critical tryptophan in c-Raf and Hck

The similarity of the catalytic domains of Raf and Src family members sugge sts that functions of homologous residues may be similar in both kinase fam ilies. A tryptophan residue, W260, in the WEI region of the Src family kina se Hck has an important role in regulating ATP binding. We tested the hypot hesis that the tryptophan, W342, in the WEI region of c-Raf may have a simi lar role to the W260 of Hck. Mutation of W260 to A in Hck activates kinase activity, but me found that mutation of W342 to A in c-Raf inactivates the kinase activity. Mutating W342 to aspartate (D), lysine (K) or histidine (H ) also inactivated c-Raf whether assayed as a purified immunoprecipitate or when recruited to the plasma membrane. A constitutively active c-Raf can b e generated by mutating two regulatory tyrosines to aspartate. When placed into this active c-Raf mutant, mutation of W342 to D, K or H enabled phosph orylation and activation of the c-Raf substrate MEK at the plasma membrane but not in an immunoprecipitation assay. We conclude that (1) Tryptophan ha s a different role in the WEI regions of c-Raf and Hck, (2) W342 is not dir ectly involved in MEK binding as both positive and negative residues at 342 are permissive for MEK activation at the membrane in a constitutively acti ve c-Raf mutant, (3) Factors at the membrane are capable of potentiating ac tivation of c-Raf containing mutated W342 in a hyperactivated c-Raf, but no t in a wild type c-Raf and (4) There is a stringent structural requirement for W at residue 342 in c-Raf.