Fig. 8.
The X2 isoform displays a faster decay due to increased proteosomal-mediated degradation. a Schematic representation of the chimerical protein derived from the fusion of EGFP coding sequence with the CR3 domain of BRAFV600E-ref, X1, and X2 within the pEGFP-C1 plasmid. The asterisk indicates the presence of the V600E mutation. b-e Upon the transient transfection of pEGFP-C1 empty (pEGFP-empty), pEGFP-CR3-ref, pEGFP-CR3-X1, and pEGFP-CR3-X2 plasmids in A375 cells, real-time PCR performed with primers for EGFP and for total BRAF indicates that the chimerical mRNAs are all transcribed at similar levels (b), but western blot (c), flow cytometry (d) and confocal microscopy analysis (e) indicate that, when fused with CR3-X2, EGFP protein is expressed at lower levels. The dotted box shows a higher exposure of the anti-EGFP antibody. f-h When PIG-BRAFV600E-ΔCterm plasmid, which lacks the nucleotides encoding for the X2-specific C-terminal domain (f), is transiently transfected in HEK293T cells, not only BRAF mRNA (g), but also BRAF protein is detectable (h). e: empty PIG-NotI; X1: PIG-BRAFV600E-X1 (used as positive control); X2: PIG-BRAFV600E-X2; ΔCterm: PIG-BRAFV600E-ΔCterm. i-j Upon the transient transfection of pEGFP-empty, pEGFP-CR3-ref, pEGFP-CR3-X1, and pEGFP-CR3-X2 plasmids, A375 cells were treated with 100 ug/ml cicloheximide (CHX) (i) or 20 uM MG132 (j) for 8 h. The CHX treatment indicates that the decay rate of CR3-X2 is faster than that of CR3-ref and CR3-X1, while the MG132 treatment suggests that this is due to higher degradation rate through the ubiquitin-proteasome pathway. k The prediction of potential proteasomal cleavage sites using 3 different algorithms retrieves the indicated X2-specific consensus peptide. (l) The mutagenesis of Lys739 into a proteasome-insensitive Arg rescues the expression of the X2 isoform of BRAF protein. e: empty PIG-NotI; X1: PIG-BRAFV600E-X1 (used as positive control); X2: PIG-BRAFV600E-X2; X2K739R: PIG-BRAFV600E-X2 in which Lys(K)739 has been substituted with Arg(R) (AAA to AGA triplet change). m Cartoon that summarizes the main findings of this article (details in the text). The pictures are taken from 1 out of 3 independent experiments performed, all with comparable outcome. The graphs represent the mean ± SEM of 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001