Insights into the molecular function of the inactivating mutations of B-Raf involving the DFG motif.
BRAF gene mutations have been associated with human cancers. Among the naturally occurring mutations, two that involve amino acids of the conserved DFG motif in the activation loop (D594V and G596R), appear to be inactivating. Aim of this study was to analyze the molecular mechanisms involved in the loss of function of B-Raf inactivating mutation G596R. Furthermore, the ability of the B-Raf DFG motif mutants to generate heterodimers with C-Raf and the possible functional consequences of the B-Raf/C-Raf heterodimer formation was examined. Wet molecular experiments in HEK293T cells demonstrate that B-Raf(G596R) is a kinase-impaired mutant. Molecular dynamics simulations show that the loss of function of B-Raf(G596R) depends on a restraining effect of Arg596 on the catalytic residue Asp594, which results in the loss of the appropriate spatial localization and/or conformation of the latter necessary for anchoring ATP to the enzyme. Exploration of B-Raf/C-Raf heterodimer formation indicates the occurrence of functioning heterodimers in the case of all the DFG B-Raf mutants, independently from the expected differences in spatial conformation of the activation loop, although the transforming activity of the mutants appear negligible. In conclusion, this study delivers novel information on the functional properties of the B-Raf DFG motif inactivating mutants and on the mechanisms driving B-Raf/C-Raf heterodimerization and consequent C-Raf transactivation.
Moretti
Sonia
S
Dipartimento di Medicina Interna, Università di Perugia, Perugia, Italy.
De Falco
Valentina
V
Tamburrino
Anna
A
Barbi
Flavia
F
Tavano
Maria
M
Avenia
Nicola
N
Santeusanio
Fausto
F
Santoro
Massimo
M
Macchiarulo
Antonio
A
Puxeddu
Efisio
E
D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D051379:Mice; D020125:Mutation, Missense; D041681:NIH 3T3 Cells; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism
C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf