Biochemical and molecular characterization of the novel BRAF V599Ins mutation detected in a classic papillary thyroid carcinoma

Activating mutations of the BRAF gene are the most common genetic alterations in papillary thyroid carcinomas (PTCs) and the T1799A transversion, resulting in BRAF^V600E, appeared virtually unique in this cancer type. Here, we report on the identification in a classic PTC of a novel BRAF mutation, namely a 1795GTT insertion, resulting in BRAF^V599Ins, and describe its biochemical and molecular characterization. Kinase assays carried out on BRAF^V599Ins and BRAF^V600E revealed a three- to five-fold increase in the enzymatic activity of both mutants with respect to BRAF^WT. Similarly, evaluation of BRAF-induced phosphorylation of MEK, MAPK and RSK revealed a significant MAPK cascade activation in cells expressing BRAF^V599Ins or BRAF^V600E, but not in cells expressing BRAF^WT. Molecular dynamic simulations showed a destabilization of the inactive conformation of the enzyme in both BRAF^V599Ins and BRAF^V600E mutants, but not in BRAF^WT. The analysis of the interaction energies inside the catalytic site allowed to demonstrate the presence of repulsive electrostatic forces acting on the activation loop and moving from inward to outward of the mutant enzymes. Finally, focus assays in NIH-3T3 cells confirmed a high transformation rate in the cells transfected either with BRAF^V599Ins or BRAF^V600E. In conclusion, this study demonstrated that BRAF^V599Ins, as BRAF^V600E, is a 'gain of function' mutation, characterized by a constitutive catalytic activation, which accounts for its causative role in the studied PTC.