Phosphorylation of the B-RAF kinase is an essential process in tumour induction and maintenance in several cancers. Herein the phosphorylation specificity of the activation segment of the wild type B-RAF kinase and the B-RAF(D594V), B-RAF(V600E) and B-RAF(K601E) mutants was examined by molecular dynamics (MD) simulations and GRID molecular interaction field analysis. According to our analysis, Thr599 and Ser602 were the only residues in the activation segment in B-RAF(WT) that were well exposed to ATP binding, which is in agreement with the experimental results, and provide a molecular basis of the observed phosphorylation. The phosphorylation specificity was altered significantly for each of the three different mutants studied due to the large conformational changes and subsequent alterations in the electrostatic forces between several residues for each of these mutants. Thus the analysis revealed limited phosphorylation potential of the non-active B-RAF(D594V) mutant and several potential ATP binding sites were identified for the highly active B-RAF(V600E) mutant. The Lys601 residue, which is specific to RAF and not present in the activation segment of other similar kinases, was identified to potentially be of major importance to the observed differences in the phosphorylation specificity of the mutants. Our results indicate that Lys601 might be a specific ATP coordinating residue, contributing to the B-RAF phosphorylation specificity. The overall results can be helpful for the understanding of the B-RAF phosphorylation processes on a molecular level. (c) 2009 Elsevier Inc. All rights reserved.