Characterization of regulatory mechanisms by V600EBRAF
thesisposted on 12.03.2013, 10:41 by Maria Montserrat Aguilar Hernández
More than 90% of BRAF mutations in human cancer are represented by a valine to glutamic acid mutation at residue 600 known as the V600EBRAF mutation. Our laboratory has generated mouse models expressing a conditional knock-in allele of BrafV600E and in previous work we have shown that expression of the oncogene in a range of tissues including embryonic fibroblasts (MEFs), lung tissue, melanocytes and gastrointestinal crypts induces cell proliferation followed by senescence. Previous work from our laboratory showed that in the transition from proliferation to senescence, a decrease in Braf protein levels occurred in lung adenomas from BrafV600E mice. I have further investigated the mechanisms underpinning this behavior in two different cellular systems: primary mice embryonic fibroblasts conditionally expressing endogenous V600EBraf and HEK293T cells transiently expressing V600EBRAF. My findings show that in a similar way to BrafV600E lung adenomas, a reduction in Braf expression is observed in both cellular systems in addition to a decrease in Craf protein levels. I found through qRTPCR experiments that Braf and Craf mRNA levels are not downregulated in BrafV600E MEFs. However, a reduction in the stability of BRAF and CRAF proteins in the soluble fraction and a simultaneous accumulation of these proteins in the insoluble fractions was observed in both cellular systems. Inhibition of the Erk pathway at the level of Braf and Mek rescued Braf and Craf expression in BrafV600E MEFs indicating that the expression of these proteins is dependent on Braf and Mek kinase activities. LC-MS/MS analysis of ectopic WTCRAF when co-expressed with ectopic V600EBRAF showed that the accumulation of ectopic WTCRAF in the insoluble fraction was associated with the phosphorylation of six novel serine residues in CRAF. The mutation of these phospho-residues to non-phosphorylatable alanines rescued ectopic WTCRAF expression in the soluble fraction and prevented the accumulation of this protein in the insoluble fraction. Although phosphorylation of Ser675 in ectopic V600EBRAF was also specifically detected in the insoluble fraction, mutation of this site did not alter the distribution of this protein. Thus, I propose that the post-translational regulation of BRAF and CRAF expression is an alternative mechanism for the regulation of the MAPK pathway output in cells expressing the V600EBRAF oncogene.