HTLV-1 Tax Stabilizes MCL-1 via TRAF6-Dependent K63-Linked Polyubiquitination to Promote Cell Survival and Transformation
Figure 2
Tax activates TRAF6 and enhances its interaction with MCL-1 in the mitochondria.
(A) Tax requires NEMO and TRAF6 binding to activate TRAF6. Ubiquitination assay was performed with Flag immunoprecipitates from lysates of 293T cells transfected with Flag-TRAF6 along with empty vector (EV), Tax and Tax point mutants M22 and E345A. (B) Tax requires NEMO and TRAF6 binding to promote MCL-1 K63-linked polyubiquitination. Ubiquitination assay was performed with Flag immunoprecipitates from lysates of 293T cells transfected with Flag-MCL-1 together with Tax WT, M22 and E345A. (C, D) Tax induces the mitochondrial localization of TRAF6. (C) Immunoblotting was performed with the indicated fractions derived from 293 cells transfected with Flag-TRAF6 together with Tax WT, M22 or E345A. Fifty-fold excess of mitochondrial extracts compared to total cell homogenates were loaded onto the gel for normalization. Voltage-dependent anion channel 1 (VDAC1) and lactate dehydrogenase (LDH) were used as markers for mitochondria and cytosol, respectively. Mitochondrial TRAF6 was quantitated using Alpha Innotech gel imaging software. Relative intensity of mitochondrial TRAF6 bands normalized to VDAC1 was calculated by dividing by the normalized band intensity of total TRAF6. (D) Immunofluorescence assay was performed with HeLa cells transfected with Flag-TRAF6 along with Tax WT, M22 or E345A and incubated with MitoTracker Red for 30 min prior to fixation. Nuclei were counterstained with DAPI (blue) before mounting coverslips. (E) Endogenous TRAF6 is ubiquitinated and localized in the mitochondria in HTLV-1 transformed cells. Immunoblotting was performed with the indicated fractions derived from TL-OM1 and MT-2 cells. Fifty fold excess of mitochondrial extracts compared to total cell homogenates and cytosol were loaded onto the gel for normalization. VDAC1 and LDH served as markers for mitochondria and cytosol, respectively. (F) Tax induces TRAF6 and MCL-1 interactions. Immunoblotting was performed with MCL-1 immunoprecipitates from lysates of 293T cells transfected with Flag-MCL-1 together with Flag-TRAF2, TRAF5 or TRAF6, or HA-TRAF3, with or without Tax. Asterisk (*) indicates immunoglobulin heavy chain. (G) Three identified TRAF6 binding motifs (T6BMs) in the MCL-1 PEST domain. Consensus sequences of the T6BMs in blue were substituted for alanine in red as shown in the diagram. (H) Mapping of the T6BMs of MCL-1. GST pull-down assay was performed using purified Flag-TRAF6 and GST-MCL-1 WT or mutants as indicated. (I) Proposed model of Tax, IKK and TRAF6 regulation of MCL-1 K63-linked polyubiquitination.