Figure 1.
Tax promotes TRAF6-dependent MCL-1 ubiquitination.
(A) Doxycycline (Dox)-induced expression of Tax wild-type (WT) and M22 in Jurkat Tet/On-Tax cells as shown by immunoblotting. (B) UbiScan analysis of Tax-induced MCL-1 ubiquitination at lysine 40 (K40). The relative intensity of peak (651.85 m/z) is marked on the top of each bar. (C–F) Tax induces the K63-linked polyubiquitination of MCL-1. Ubiquitination assay was performed with immunoprecipitates (IPs) derived from 293T cells transfected with Flag-MCL-1 with or without Tax (C), Jurkat Tet/On-Tax cells treated with Dox for 48 h (D), the indicated HTLV-1 transformed cell lines (E), and 293T cells transfected with Flag-MCL-1 together with shRNA (NS), TRAF6 shRNA 1 (T6 sh1), or TRAF6 shRNA 2 (T6 sh2) with or without Tax (F). “Env/Tax” indicates the Env-Tax fusion protein expressed in MT-2 cells (E). Asterisk (*) indicates immunoglobulin heavy chain (D and E). (G) Schematic of the TRAF6 binding motif of Tax. Highlighted in turquoise are consensus sequences among the indicated proteins. (H) Specific interaction of MCL-1 and TRAF6. Co-IP analysis was performed with Tax immunoprecipitates from lysates of 293T cells transfected with Tax together with Flag-TRAF2, TRAF3, TRAF5 or TRAF6. * indicates IgG heavy chain. (I) Mapping of the TRAF6 binding domain in Tax. Co-IP analysis was performed with Flag immunoprecipitates from lysates of 293T cells transfected with Flag-TRAF6 together with Tax WT or E345A. (J) Mapping of the Tax binding domain in TRAF6. Co-IP analysis was performed with Tax immunoprecipitates from lysates of 293T cells transfected with Tax together with Flag-TRAF6 WT, ΔRING (275–522 residues) lacking the RING finger domain, ΔZFs (113–522 residues) lacking zinc finger (ZF) domains, or ΔTRAF-C (1–351 residues) lacking the TRAF-C domain as shown. Western blot was performed with rabbit anti-Flag antibody.
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.
Figure 3.
Tax stabilizes MCL-1 in an IKK and TRAF6-dependent manner.
(A) Tax prolongs the half-life of MCL-1. CHX chase assay was performed with Jurkat Tet/On-Tax or M22 cells cultured in the presence or absence of Dox for 48 h and subsequently treated with CHX (100 µg/ml) for the indicated times. The half-life of MCL-1 was calculated from a linear least-square fit of the protein intensity. (B, C) MCL-1 is stabilized in Tax-expressing cell lines. CHX chase assays were performed by immunoblotting with whole cell lysates derived from Jurkat and HTLV-1 transformed cell lines including TL-OM1, MT-2 and MT-4 at the indicated times after CHX treatment (10 µg/ml). Band intensity was measured using Alpha Innotech gel imaging software. The relative intensity of MCL-1 bands was calculated compared to actin. (D, E) Depletion of Tax with shRNAs results in the loss of MCL-1 and apoptosis in HTLV-1 transformed cells. Immunoblotting was performed with the indicated antibodies using whole cell lysates derived from MT-2 (D) and MT-4 (E) cells cultured for 6 days after the lentiviral transduction of Tax shRNAs. (F) TRAF6 is essential for MCL-1 stabilization. CHX chase assay was performed by immunoblotting with whole cell lysates derived from Traf6+/+ and Traf6−/− MEFs at the indicated times after CHX treatment. (G) Tax requires TRAF6 for MCL-1 stabilization. Immunoblotting was performed using lysates from Traf6+/+ and Traf6−/− MEFs transfected with Tax. (H) Increased stabilization of MCL-1 by Tax and TRAF6. Immunoblotting was performed using lysates from 293 cells transfected with MCL-1, TRAF6 and Tax.
Figure 4.
Tax and TRAF6 protect MCL-1 from degradation induced by genotoxic stress stimuli.
(A) Tax protects MCL-1 from genotoxic stress-induced degradation. Immunoblotting was performed with whole cell lysates derived from Jurkat Tet/On-Tax cells cultured in the presence or absence of Dox for 48 h and subsequently treated with etoposide (Eto, 10 µg/ml) or sorafenib (Sora, 10 µM) for 12 h. (B) 293 cells transfected with Flag-MCL-1 and GFP together with Tax WT, M22 or E345A were treated with etoposide for 24 h. (C) Etoposide treatment enhances the interactions between endogenous TRAF6 and Tax proteins. TRAF6 immunoblotting was performed with Tax immunoprecipitates from lysates of Jurkat Tet/On-Tax cells incubated with Dox for 48 h (left panel) and MT-2 cells (right panel). The cells were treated with etoposide (10 µM) for 6 h before harvesting. Asterisk (*) indicates immunoglobulin heavy chain. (D) Tax-induced MCL-1 stabilization and protection is dependent on TRAF6-binding motif #3 of MCL-1. Immunoblotting was performed with whole cell lysates derived from 293 cells transfected with Flag-MCL-1 WT or 3PE/AA mutant together with empty vector or Tax and left untreated or treated with MG-132 (10 µM) for 24 h before harvesting. (E) Tax+ cell lines are protected from etoposide-induced MCL-1 degradation. HTLV-1 transformed and ATL cell lines (Tax+ or Tax-) cultured in the presence or absence of etoposide for 24 h. Immunoblotting was performed with the indicated antibodies. (F) TRAF6 protects MCL-1 from etoposide-induced degradation. Immunoblotting was performed with whole cell lysates derived from 293 cells transfected with Flag-MCL-1 and GFP together with Flag-TRAF6 WT, C70A or ΔTRAF-C, and then treated with etoposide for 24 h. (G) CD40L and LPS protect MCL-1 from etoposide-induced degradation in primary B cells. Immunoblotting was performed with whole cell lysates derived from primary murine splenic B cells cultured in the presence of CD40L or LPS for 24 h, and then treated with etoposide for an additional 24 h.
Figure 5.
Tax and TRAF6 promote MCL-1 K63-linked polyubiquitination on C-terminal lysines.
(A) Schematic of MCL-1 lysine mutants. The substituted arginine (R) residues are highlighted in blue. (B) Requirement of the four C-terminal lysine residues in Tax and TRAF6-induced MCL-1 stabilization. Immunoblotting was performed with whole cell lysates of 293 cells transfected with the indicated Flag-MCL-1 plasmids together with Tax or Flag-TRAF6. (C) Identification of C-terminal lysines required for MCL-1 stabilization. Immunoblotting was performed with whole cell lysates of 293 cells either transfected with the indicated Flag-MCL-1 plasmids together with Tax (top two panels) or treated with etoposide for 24 h after transfection (bottom two panels). (D) Ub-independent proteasomal degradation of MCL-1. Immunoblotting was performed with whole cell lysates of 293 cells transfected with Flag-MCL-1 WT or All-KR and then treated with etoposide and/or MG-132 (10 µM) for 24 h. (E) Tax and TRAF6 block the interaction between MCL-1 and the 20S proteasome. Immunoblotting was performed with Flag immunoprecipitates and whole cell lysates derived from 293 cells transfected with Flag-MCL-1 or MCL-1-All-KR together with Tax or His-TRAF6, and treated with the DSP cross-linker (2 µM) for 30 min prior to cell lysis. Asterisk (*) indicates immunoglobulin light chain. (F) Tax promotes the K63-linked polyubiquitination of the four C-terminal MCL-1 lysines. K63-Ub assay was performed with lysates from 293 cells transfected with WT 6×His-MCL-1 and the indicated MCL-1 mutants together with Tax. (G) Proposed molecular mechanism of Tax and TRAF6-induced stabilization of MCL-1.
Figure 6.
TRAF6 and MCL-1 are required for ATL cell survival and HTLV-1-mediated T-cell immortalization.
(A and B) Requirement of TRAF6 and MCL-1 for the viability of HTLV-1 transformed and ATL cell lines. (A) Cell viability assay was performed at 6 days after TL-OM1 and MT-2 cells were transduced with lentiviruses expressing the indicated shRNAs. Relative cell viability (%) was expressed as a percentage relative to the control cells. (B) Flow cytometric analysis of ATL cell lines transduced with shRNAs as described in (A). Cells were stained with both annexin-V-Alexa Fluor 488 and propidium iodide (PI). The distribution of cells is indicated as a percentage in each quadrant. (C) MCL-1 protein is overexpressed in HTLV-1-transformed T cells. Immunoblotting was performed with whole cell lysates of primary human CD4+ T cells (0 W), CD4+ T cells immortalized with HTLV-1 by co-culture with irradiated MT-2 cells for 12 weeks (12 W), Jurkat, ATL (TL-OM1) and HTLV-1 transformed cell lines (MT-2 and C8166). (D) TRAF6 and MCL-1 are essential for the immortalization of T cells by HTLV-1. At four and six weeks after the co-culture of shRNA-transduced PBMCs and irradiated MT-2 cells, viable cells were counted using the trypan blue exclusion method. Puromycin (5 µg/ml) was added at 4 weeks after the co-culture. The data are representative of two independent experiments.