Fig 1.
K27, K63 and M1 chains are involved in Tax-induced IKK activation.
(A) Jurkat cytosolic extracts were incubated with recombinant His6-Tax and ATP (2 mM) in the presence of ubiquitin mutants or HA-ubiquitin (50 μM). The reaction mixtures were analyzed by immunoblotting with the indicated antibodies. (B) Cytosolic extracts were prepared from HEK293T cells expressing a series of dominant-negative mutants of the E2 enzyme and subjected to cell-free analyses. (C) Cytosolic extracts were prepared from WT MEFs or Ubc13fl/fl MEFs expressing Cre and subjected to cell-free analyses with increasing amounts of Tax. The depicted results are representative of three independent experiments.
Fig 2.
LUBAC is required for Tax-induced IKK activation.
(A-C) Cytosolic extracts were prepared from HOIL-1L-deficient (A), Sharpin-deficient (B), HOIPΔlinear (C) and corresponding WT MEFs and subjected to cell-free analyses. (D) Sharpin-deficient and corresponding WT MEFs were mock-infected or were transduced with retroviruses expressing Tax. The MEFs were treated with MG132 (10 μM) for 1 h, and cell lysates were subjected to immunoblotting with the indicated antibodies. The depicted results are representative of three independent experiments.
Fig 3.
Tax recruits LUBAC to the IKK complex.
(A) Jurkat cytosolic extracts were incubated with recombinant His6-Tax or His6-M22 in the presence of ATP (2 mM). The reaction mixtures were subjected to immunoprecipitation with an anti-Tax antibody, followed by immunoblotting with the indicated antibodies. (B) Cytosolic extracts prepared from Jurkat cells expressing Flag-tagged NEMO were incubated with recombinant His6-Tax or His6-M22 in the presence of ATP (2 mM). The reaction mixtures were subjected to immunoprecipitation with an anti-Flag antibody, followed by immunoblotting with the indicated antibodies. (C) JPX-9 cells were untreated or treated with CdCl2 (20 μM) for 18 h. Cell lysates were subjected to immunoprecipitation with an anti-Tax antibody, followed by immunoblotting with the indicated antibodies. (D) Jurkat cytosolic extracts were incubated with recombinant His6-Tax and ATP (2 mM) in the presence of ubiquitin mutants or HA-ubiquitin (50 μM). The reaction mixtures were subjected to immunoprecipitation with an anti-Tax antibody, followed by immunoblotting with the indicated antibodies. (E) Recombinant His6-Tax was incubated with glutathione sepharose-bound recombinant GST, GST-HOIL-1L, GST-HOIP or GST-Sharpin. His6-Tax bound to GST-tagged protein was analyzed by immunoblotting with an anti-Tax antibody. Dots denote full-length GST fusion proteins. (F) HEK293T cells were transfected with expression plasmids encoding HOIL-1L-HA or various C-terminal HA-tagged HOIL-1L mutants together with a Tax expression plasmid. After 48 h, cell lysates were prepared and subjected to immunoprecipitation with an anti-HA antibody, followed by immunoblotting with an anti-Tax antibody (upper). A schematic representation of the various mutants of HOIL-1L (lower). (G) HEK293T cells were transfected with expression plasmids encoding Myc-HOIP or various N-terminal Myc-tagged HOIP mutants together with a Tax expression plasmid. After 48 h, cell lysates were prepared and subjected to immunoprecipitation with an anti-Myc antibody, followed by immunoblotting with an anti-Tax antibody (upper). A schematic representation of the various mutants of HOIP (lower). The depicted results are representative of three independent experiments.
Fig 4.
Tax-induced generation of IKK complex-associated K63/M1-linked hybrid chains is required for IKK activation.
(A) Jurkat cytosolic extracts were incubated with recombinant His6-Tax and ATP (2 mM), and the reaction mixtures were subjected to immunoprecipitation with an anti-NEMO antibody. The immunoprecipitates were left untreated or were treated with recombinant Otubain-1, AMSH, OTULIN or USP2 and analyzed by immunoblotting with anti-Ub (upper) and anti-M1 chain-specific (lower) antibodies. (B) Jurkat cytosolic extracts were incubated with or without recombinant His6-Tax and ATP (2 mM), and the reaction mixtures were subjected to immunoprecipitation with an anti-NEMO antibody. The immunoprecipitates were analyzed via the ubiquitin-AQUA method. The results are given as the mean ± SD (n = 3). (C) Cell-free reactions were performed in the presence of ubiquitin mutants or HA-ubiquitin (50 μM). The reaction mixtures were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-Ub antibody. (D) Cell-free reactions were performed in the presence of various DUBs. The depicted results are representative of three independent experiments.
Fig 5.
Tax induces formation of the macromolecular active IKK complex.
(A) Jurkat cytosolic extracts were incubated with recombinant His6-Tax or His6-MEKK1 together with ATP (2 mM) in the presence or absence of IsoT. (B) Cell-free reactions were performed in the presence of WT viral OTU or its catalytic inactive mutant 1A. (C) Jurkat cytosolic extracts were incubated with recombinant His6-Tax or its mutants together with ATP (2 mM). The reaction mixtures were subjected to immunoprecipitation with an anti-Tax antibody, followed by immunoblotting with an anti-Ub antibody. (D) Cytosolic extracts were prepared from NEMO-deficient MEFs reconstituted with human NEMO or its mutant and were subjected to cell-free analyses. (E) Cytosolic extracts were prepared from NEMO-deficient MEFs reconstituted with mouse NEMO or its mutants and were subjected to cell-free analyses. (F) Cell-free reactions were performed, and the reaction mixtures were subjected to Blue native-PAGE, followed by immunoblotting with anti-NEMO (left) and anti-p-IKKα/β (right) antibodies. The depicted results are representative of three independent experiments.
Fig 6.
LUBAC associates with Tax and is involved in NF-κB activation leading to target gene expression and cell proliferation in HTLV-1-infected cells.
(A) Cell lysates from HUT102 cells were subjected to immunoprecipitation with an anti-Tax antibody or a control antibody, followed by immunoblotting with the indicated antibodies. (B) MT-2 (left) or MT-4 (right) cells were infected with control or Tax shRNA expression lentivirus vector. Cell lysates were subjected to immunoprecipitation with an anti-Tax antibody, followed by immunoblotting with the indicated antibodies. (C) MT-2 (left) or MT-4 (right) cells were transfected with control or HOIP siRNA. Cell lysates were subjected to immunoblotting with an anti-pIKKα/β antibody. (D) MT-4 cells were transfected with control or HOIP siRNA as described in (C). The expression levels of IL6 (left), IL1 (middle) and MMP9 (right) were measured by quantitative real-time RT-PCR. The relative expression was calculated by dividing each expression value by that of a control siRNA treatment. (E) MT-2 (left) or MT-4 (right) cells were infected with HOIP shRNA expression lentivirus vector. The cells were seeded at 1.0×105 cells/well, and cell viability was assessed by trypan blue exclusion assay (upper). The whole cell lysates were subjected to immunoblotting with an anti-HOIP antibody to confirm the knockdown of HOIP (lower). The results shown in (D) and (E) are given as the mean ± SD (n = 3). The depicted results are representative of three independent experiments.
Fig 7.
A model illustrating HTLV-1 Tax-induced IKK activation.
Upon HTLV-1 infection, the Tax protein is translated from doubly spliced viral mRNA expressed from the provirus. Tax is able to bind to NEMO in the IKK complex and to HOIP and HOIL-1L of LUBAC, thereby forming the LUBAC/Tax/IKK complex, which further binds to the Ubc13/Uev1A E2 complex and unidentified K63 E3 enzymes (X) to generate an inactive “pre-Taxisome”. The K63/M1-linked hybrid polyubiquitin chains generated by LUBAC and X (K63 E3) interact with NEMO through its M1 chain-interacting UBAN domain and K63 chain-interacting NZF domain. This multivalent interaction between NEMO proteins and the hybrid polyubiquitin chains results in the oligomerization of the pre-Taxisome to form the macromolecular Taxisome, which allows close interactions between the IKK complexes, leading to the trans-autophosphorylation-mediated activation of the IKK complex. The K63/M1-linked hybrid polyubiquitin chains may contain recently identified branched chains [65].