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.
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References
Barman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H et al. (2000). Nucleic Acids Res 28: 235–242.
Bennasroune A, Gardin A, Aunis D, Cremel G, Hubert P . (2004). Crit Rev Oncol Hematol 50: 23–38.
Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S, Karplus M . (1983). J Comp Chem 4: 187–217.
Carta C, Moretti S, Passeri L, Barbi F, Avenia N, Cavaliere A et al. (2006). Clin Endocrinol 64: 105–109.
Ciampi R, Knauf JA, Kerler R, Gandhi M, Zhu Z, Nikiforova MN et al. (2005). J Clin Invest 115: 94–101.
Cichowski K, Jacks T . (2001). Cell 104: 593–604.
Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S et al. (2002). Nature 417: 949–954.
Dibb NJ, Dilworth SM, Mol CD . (2004). Nat Rev Cancer 4: 718–727.
Fiser A, Sali A . (2003). Methods Enzymol 374: 461–491.
Greene FL, Page DL, Fleming ID, Fritz AG, Balch CM, Haller DG et al. (eds). (2002). American Joint Committee on Cancer 2002 AJCC Cancer Staging Manual. Springer-Verlag: New York Berlin, Heidelberg, pp 77–87.
Grubmüller H . (1996). Solvate v1.0 Theoretical Biophysics Group, Institut für Medizinische Optik. Ludwig-Maximilians-Universität München: München, Germany.
Jerome L, Shiry L, Leyland-Jones B . (2003). Endocr Relat Cancer 10: 561–578.
Kalé L, Skeel R, Bhandarkar M, Brunner R, Gursoy A, Krawetz N et al. (1999). J Comp Phys 151: 283–312.
Malumbres M, Barbacid M . (2003). Nat Rev Cancer 3: 459–465.
Melillo RM, Castellone MD, Guarino V, De Falco V, Cirafici AM, Salvatore G et al. (2005). J Clin Invest 115: 1068–1081.
Oler G, Ebina KN, Michaluart Jr P, Kimura ET, Cerutti J . (2005). Clin Endocrinol 62: 509–511.
Puxeddu E, Moretti S, Elisei R, Romei C, Pascucci R, Martinelli M et al. (2004). J Clin Endocrinol Metab 89: 2414–2420.
Santoro M, Melillo RM, Carlomagno F, Vecchio G, Fusco A . (2004). Endocrinology 145: 5448–5451.
Tartaglia M, Niemeyer CM, Shannon KM, Loh ML . (2004). Curr Opin Hematol 11: 44–50.
Trovisco V, Soares P, Preto A, de Castro IV, Lima J, Castro P et al. (2005a). Virchows Arch 446: 589–595.
Trovisco V, Soares P, Soares R, Magalhaes J, Sa-Couto P, Sobrinho-Simoes M . (2005b). Hum Pathol 36: 694–697.
Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM et al. (2004). Cell 116: 855–867.
Xing M . (2005). Endocr Relat Cancer 12: 245–262.
Acknowledgements
This study was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) and by the Fondazione Cassa di Risparmio di Perugia.
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Moretti, S., Macchiarulo, A., De Falco, V. et al. Biochemical and molecular characterization of the novel BRAFV599Ins mutation detected in a classic papillary thyroid carcinoma. Oncogene 25, 4235–4240 (2006). https://doi.org/10.1038/sj.onc.1209448
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DOI: https://doi.org/10.1038/sj.onc.1209448
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