Fig. 6
From: K27-linked ubiquitination of BRAF by ITCH engages cytokine response to maintain MEK-ERK signaling
Ubiquitination of BRAF disrupts the inhibitory interaction between BRAF and 14–3–3 proteins. a Ubiquitinated BRAF displayed reduced binding with 14–3–3 in vitro. In vitro ubiquitination assays using immunopurified Flag-BRAF proteins were performed followed by incubation of Flag-BRAF with 14–3–3. The anti-Flag immunoprecipitates (IP) were subjected to SDS-PAGE and immunoblot (IB) analysis. b 5KR-BRAF displayed stronger binding to 14–3–3 compared with WT-BRAF. c Binding between endogenous BRAF and 14–3–3 was reduced upon TNFα treatment. d S365A-BRAF and S729A-BRAF mutants exhibited a distinct affinity with 14–3–3 after ubiquitination. e Illustration of the proposed models for the disruption of BRAF-14–3–3 interaction by K27-linked ubiquitination. f PPP2CA and PPP2R2A subunits of the PP2A protein family specifically interacted with BRAF. g Ubiquitinated BRAF displayed increased binding with PPP2R2A in vitro. h In vitro phosphatase assay showing that compared with the catalytic PPP2CA subunit, the PP2A complex was more active in dephosphorylating p-S365-BRAF. Immunopurified Flag-BRAF proteins were incubated with the indicated PP2A phosphatases for the indicated time period before SDS-PAGE and IB analysis. i Phosphatase kinetics of PPP2CA and the PP2A complex in dephosphorylating BRAF. Initial rates were measured at various concentrations of Flag-BRAF protein using the continuous assay. The rates were replotted against substrate concentration and fit to the Michaelis–Menten equation. j In vitro phosphatase assay showing that ubiquitinated BRAF was a better substrate for the PP2A complex. Immunopurified Flag-BRAF were subjected to in vitro ubiquitination as described in Fig. 1c. Unmodified and ubiquitinated proteins were incubated with the indicated PP2A phosphatases for 60 min. The free phosphate was measured using the malachite green detection reagent, normalized, and calculated as mean ± SD (n = 3) from three independent experiments. *P < 0.05; Student’s t test. k, l IB analysis of WCL derived from WM3918 cells infected with the indicated shPPP2CA (k) or shPPP2R2A (l) lentiviral constructs. m IB analysis of WCL derived from WM3918 cells infected with shScr or shPPP2R2A lentiviral constructs. n IB analysis of WCL derived from HEK293 cells transfected with the indicated Flag-tagged WT-BRAF or BRAF mutants
