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Functional relevance of particular residues within the BRAF kinase domain in anaplastic thyroid carcinoma

AutorGarcía-Rostan, Ginesa
Fecha de publicación2012
CitaciónAACR Annual Meeting 2012
ResumenThe BRAFV600E point mutation represents the most common oncogenic event in sporadic papillary thyroid cancer (PTC) pathogenesis. Although at a much lower frequency, different missense point mutations (K601E, G474R, G469R) and complex genetic alterations, such as rearrangements (AKAP9-BRAF) or frameshift mutations (V600E-K601del, V599ins, V600D-FLAGT601-605ins, T599I-VKSRdel), have also been reported in PTC. In contrast with PTC, no other alteration different from the V600E substitution has been shown in anaplastic thyroid carcinomas (ATC). In this study, we sought to determine the functional relevance of particular amino acid residues, within the BRAF kinase domain, that we found mutated in some ATC. Fifty-four ATC were investigated for the existence of mutations at the activation loop, the glicine-rich phosphate-binding loop and the AKT binding sites of BRAF by means of PCR-SSCP and direct sequence. The structural-conformational changes behind punctual, unreported BRAF mutations and their effect on BRAF catalytic activity, substrate binding and downstream MAPK signalling were evaluated. Besides the V600E mutation, two additional point mutations (G615E and G474R) were detected within the BRAF kinase domain. The prevalence of both mutations, G615E (11% = 6 ATC) and G474R (7.4% = 4 ATC), was much lower than that of V600E (20% = 11 ATC). Interestingly, structural biology studies showed that G615E and G474R induced profound conformational changes within the BRAF kinase domain that resulted in reduced catalytic activity, decreased substrate binding and impaired downstream signalling. The G474R mutant, by preventing the N lobe from folding into a well ordered beta sheet structure, caused a broken or distorted phosphorylation-loop (P-loop). Because the P-loop is pivotal for ATP coordination and phosphotransfer reactions, the kinase activity of the BRAF G474R mutant was severely compromised and BRAF was unable to phosphorylate the downstream substrate. The G615E mutant by disrupting the structural integrity of the activation segment in the C lobe could in addition inhibit substrate binding. Significantly, none of the ATC bearing the G615E or the G474R mutation expressed the phospho-p44/42 MAPK (Thr202/Tyr204), which specifically recognizes the dually phosphorylated and active forms of ERK1 and ERK2. This observation not only supported the existence of impaired BRAF kinase activity in vivo but also suggested the abrogation of intracellular MEK/ERK signalling in those tumours. In summary, this study demonstrates that G615E and G474R behave similarly to D594V and the kinase-dead BRAFK483M mutant, knocking-down BRAF catalytic activity and downstream MEK/ERK signalling. These results should significantly impact on the rationale of future treatment modalities for patients with deadly ATC. Clinical trials involving the inhibitor of threonine/serine kinases Sorafenib or kinase specific inhibitors targeting constitutively active BRAF will prove inefficient therapeutic strategies in patients with G615E or G474R mutations.
DescripciónResumen del trabajo presentado al 103rd Annual Meeting of the American Association for Cancer Research celebrado en Chicago (US) del 31 de marzo al 4 de abril de 2012.-- et al.
URIhttp://hdl.handle.net/10261/117235
Aparece en las colecciones: (IBGM) Comunicaciones congresos
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