Lys63-linked polyubiquitination of BRAF at lysine 578 is required for BRAF-mediated signaling

Abstract

The RAF kinase family is essential in mediating signal transduction from RAS to ERK. BRAF constitutively active mutations correlate with human cancer development. However, the precise molecular regulation of BRAF activation is not fully understood. Here we report that BRAF is modified by Lys63-linked polyubiquitination at lysine 578 within its kinase domain once it is activated by gain of constitutively active mutation or epidermal growth factor (EGF) stimulation. Substitution of BRAF lysine 578 with arginine (K578R) inhibited BRAF-mediated ERK activation. Furthermore, ectopic expression of BRAF K578R mutant inhibited anchorage-independent colony formation of MCF7 breast cancer cell line. Our studies have identified a previously unrecognized regulatory role of Lys63-linked polyubiquitination in BRAF-mediated normal and oncogenic signalings.

Figure 1. Activated BRAF is modified by Lys63-linked polyubiquitination in human cancer cell lines.

(A) Endogenous BRAF with constitutive active mutation is modified by Lys63-linked polyubiquitin chain. MCF10A cells (wild-type BRAF) and MDA-MB-231 cells (with KRAS G13D, BRAF G464V mutation) were lysed and immunoprecipitated with anti-normal IgG or BRAF antibodies followed by immunoblotting with anti-Ub-K63-specific antibodies to detect the presence of BRAF with Lys 63-linked polyubiquitination. (B) Hyperactivated RAS induces BRAF modification by Lys 63-linked polyubiquitination. HT29 (BRAF V600E mutation), A549 (with KRAS G12S mutation), and SW480 cell lines (with KRAS G12V mutation) were lysed and immunoprecipitated with anti-normal IgG or BRAF antibodies followed by immunoblotting with anti-Ub-K63-specific antibodies to detect the presence of Lys63-ubiquitinated endogenous BRAF. Cropped blots were used under the same experimental conditions.

Figure 2. BRAF with constitutively active mutation is linked with Lys63-linked polyubiquitin chains.

(A) BRAF with constitutively active mutation was linked with HA-Ub-K63-only. The expression vector encoding full length FLAG-BRAF WT or constitutively active V600E or K601E mutant was co-transfected into HEK-293T cells with HA-Ub-K63-only expression vector. Lys 63-polyubiquitinated FLAG-BRAF was detected by immunoprecipitation with anti-HA antibodies and immunoblotting with anti-FLAG antibodies. (B) BRAF with V600E constitutive active mutation was modified by Lys63- but not Lys48-linked polyubiquitination. Empty vector or expression vector encoding HA-Ub-K63R or -K48R was co-transfected into HEK-293T cells with FLAG-BRAF. Ubiquitinated FLAG-BRAF proteins were detected by immunoprecipitation with anti-HA antibodies and immunoblotting with anti-FLAG antibodies. (C) BRAF with constitutively active mutation was endogenously modified by Lys63-linked polyubiquitination. The expression vector encoding full length FLAG-BRAF WT, constitutively active V600E or K601E mutant was transfected into HEK-293T cells. Lys63-linked polyubiquitinated FLAG-BRAF was detected by immunoprecipitation with anti-FLAG antibodies and immunoblotting with anti-Ub-K63-specific antibodies. (D) BRAF ED mutant was modified by Lys63-linked polyubiquitin chains compared with BRAF WT or BRAF AA mutant. Expression vector encoding FLAG-BRAF WT, AA and ED were co-transfected into HEK-293T cells with HA-Ub-K63-only vector, respectively. 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.

Figure 3. 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.

Figure 4. EGF induces Lys63-linked BRAF polyubiquitination at Lys 578.

(A) BRAF was modified with Lys63-linked polyubiquitination after EGF treatment. Expression vector encoding FLAG-BRAF wild-type was co-transfected into HEK-293T cells with HA-Ub with Lys63- or Lys48-only expression vector. After 24 hours, cells were starved for 16 hours and treated with EGF (50 ng/ml) at time points indicated. Polyubiquitinated FLAG-BRAF was detected by immunoprecipitation with anti-FLAG antibodies and immunoblotting with anti-HA antibodies. (B) Endogenous BRAF was modified by Lys63-linked polyubiquitination after EGF treatment. HEK-293T cells were starved for 16 hours and treated with EGF (50 ng/ml) at time points indicated. Lys63-linked polyubiquitinated FLAG-BRAF was detected by immunoprecipitation with anti-BRAF antibodies and immunoblotting with anti-Ub-K63-specific antibodies. (C) EGF induced BRAF Lys63-linked polyubiquitination at Lys 578, which was required for EGF-induced ERK activation. Expression vector encoding empty vector, FLAG-BRAF wild-type or K578R mutant was co-transfected into HEK-293T cell with HA-Ub-K63-only expression vector. After 24 hours, cells were starved for 16 hours and treated with EGF (50 ng/ml) at time points indicated. Lys63-linked polyubiquitinated FLAG-BRAF was detected by immunoprecipitation with anti-FLAG antibodies and immunoblotting with anti-HA antibodies. (D) BRAF with K578R mutation blocked anchorage-independent growth of MCF7 breast cancer cells. MCF7 cells with stable expression of empty vector, FLAG-BRAF wild-type or K578 mutant was used for colony formation assay in triplicates. **p < 0.01. Cropped blots were used under the same experimental conditions.

Figure 5. Working models for the role of Lys63-linked polyubiquitination in BRAF activation.

(A) Normal BRAF activation. Upon EGF binding to its receptor, the Ras-GTP recruits BRAF and induces its phosphorylation at Thr 599-Ser 602 residues and Lys63-linked polyubiquitination at Lys 578 within its kinase domain, which in turn mediates the activation of MEK-ERK and regulates downstream gene expression. (B) Oncogenic BRAF activation. BRAF with constitutively active mutation gains its Lys63-linked polyubiquitination at Lys 578 to activate downstream effector gene independent of RAS activity.