Figure 3
From: Lys63-linked polyubiquitination of BRAF at lysine 578 is required for BRAF-mediated signaling
BRAF with constitutively active mutation is modified by Lys63-linked polyubiquitination at Lys 578 within its kinase domain.
(A) Schematic view of BRAF full length (FL) and BRAF C-terminal (CT). (B) BRAF C-terminal (CT) with constitutively active mutation was modified with Lys63-linked polyubiquitination. The expression vector encoding FLAG-BRAF CT wild-type, constitutively active V600E or K601E mutant was co-transfected into HEK-293T cells with HA-Ub-K63-only expression vector. Lys63-linked polyubiquitinated FLAG-BRAF was detected by immunoprecipitation with anti-HA antibodies and immunoblotting with anti-FLAG antibodies. (C) BRAF sequence with the evolutionary conserved lysine residues within its C-terminal kinase domain amino acids indicated. (D) BRAF with K601E mutation was modified by Lys63-linked polyubiquitination at Lys 578. Expression vector encoding HA-Ub-K63-only was co-transfected into HEK-293T cells with control vector or FLAG-BRAF K601E or K601E plus other lysine to arginine mutants. Lys63-linked polyubiquitinated FLAG-BRAF was detected by immunoprecipitation with anti-FLAG antibodies and immunoblotting with anti-HA antibodies. (E) BRAF with V600E mutation was modified by Lys63-linked polyubiquitination at Lys 578. Expression vector encoding FLAG-BRAF WT, V600E or V600E plus K578R double mutant was co-transfected into HEK-293T cell with HA-Ub-K63-only vector. Lys63-linked polyubiquitinated FLAG-BRAF was detected by immunoprecipitation with anti-HA antibodies and immunoblotting with anti-FLAG antibodies. Cropped blots were used under the same experimental conditions.
