Biochemical and molecular characterization of the novel BRAFV599Ins mutation detected in a classic papillary thyroid carcinoma

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Abstract
Activating mutations of the BRAF gene are the most common genetic alterations in papillary thyroid carcinomas (PTCs) and the T1799A transversion, resulting in BRAFV600E, 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 BRAFV599Ins, and describe its biochemical and molecular characterization. Kinase assays carried out on BRAFV599Ins and BRAFV600E revealed a three- to five-fold increase in the enzymatic activity of both mutants with respect to BRAFWT. Similarly, evaluation of BRAF-induced phosphorylation of MEK, MAPK and RSK revealed a significant MAPK cascade activation in cells expressing BRAFV599Ins or BRAFV600E, but not in cells expressing BRAFWT. Molecular dynamic simulations showed a destabilization of the inactive conformation of the enzyme in both BRAFV599Ins and BRAFV600E mutants, but not in BRAFWT. 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 BRAFV599Ins or BRAFV600E. In conclusion, this study demonstrated that BRAFV599Ins, as BRAFV600E, is a 'gain of function' mutation, characterized by a constitutive catalytic activation, which accounts for its causative role in the studied PTC.