Tax stimulates the catalytic activity of TGF-β-activating kinase 1. (A) 293 cells were either mock transfected (−) or transfected (+) with expression vectors encoding HA-tagged Tak1, Tax or both expression vectors. The IKK complex was isolated by immunoprecipitation (IP) using anti-IKKγ followed by in vitro kinase assay (KA) using glutathione S-transferase (GST)–IκBα (1–54) as substrate (panel 1). The kinase assay membrane was blotted with anti-IKKβ to detect the IKK protein in the immune complex (panel 2). The expression level of Tak1 and Tax in cell lysates was detected by immunoblotting (IB; panels 3,4). (B) 293 cells were transfected (in 24-well plates) with (+) or without (−) the indicated expression vectors along with a κB-luciferase reporter (100 ng) and a control Renilla luciferase reporter driven by the constitutive thymidine kinase promoter (pRL-tk-luc, 40 ng). The κB-specific luciferase activity was normalized on the basis of the control Renilla luciferase. Data are representative of three independent experiments. (C) 293 cells were transfected with (+) or without (−) HA–Tak1 or Tax as indicated. Tak1 was isolated by IP and subjected to in vitro kinase assays using MAP kinase kinase 6 (MKK6) as substrate (panel 1). The kinase assay membrane was subjected to IB to detect Tak1 protein (panel 2). IB was also carried out with the cell lysates to detect the expression levels of Tak1 and Tax (panels 3,4). (D) Tak1 was isolated by IP from the HTLV-negative SupT1 and the indicated Tax-expressing T cells transformed by either HTLV-I (C8166, SLB-1 and HUT102) or Tax (Tax1). Tak1 kinase assay (panel 1) and IB (panels 2–4) were carried out as in (B). (E) SupT1 cells were infected with retroviral vectors encoding either green fluorescent protein (GFP) or Tax followed by analysis of the Tak1 kinase activity and protein expression levels as in (B). HA, haemagglutinin; HTLV-I, human T-cell leukaemia virus type I; IKK, IκB kinase; Tak1, TGF-β-activating kinase 1.

Tax activation of nuclear factor-κB does not require the ubiquitin-binding function of IκB kinase. IKKγ-deficient Jurkat cells (JM4.5.2) were stably transfected with either wild-type (WT) IKKγ or the indicated IKKγ mutants defective in ubiquitin binding. These different cells were infected with retroviruses carrying either the pCLXSN(GFP) vector (−) or pCLXSN(GFP)-Tax. Approximately 30 h after infection, the cells were collected for preparation of nuclear and whole-cell extracts. Nuclear extracts were subjected to electrophoretic mobility shift assay using κB or NF-Y oligonucleotide probes, and the whole-cell extracts were analysed by IB to detect the indicated proteins. EMSA, electrophoretic mobility shift assay; GFP, green fluorescent protein; HA, haemagglutinin; IB, immunoblotting; IKK, IκB kinase.

TGF-β-activating kinase 1 is required for Tax-stimulated activation of IκB kinase and nuclear factor-κB. (A) Tak1^−/− and wild-type (Tak^+/+) MEFs were transfected with expression vectors encoding green fluorescent protein (pEGFP) or Tax (pCMV4-Tax). Around 36 h after transfection, nuclear extracts were isolated and subjected to electrophoretic mobility shift assay using NF-κB (upper panel) or NF-Y (lower panel) probes. (B) Cytoplasmic extracts (panels 1,2) and whole cell extracts (panels 3,4) from the cells used in (A) were subjected to immunoblotting (IB) using the indicated antibodies. (C) MEFs were transfected with expression vectors encoding either GFP or Tax. Around 36 h after transfection, RNA was prepared and subjected to semiquantitative reverse transcription–PCR to analyse the messenger RNA levels of tumour necrosis factor-α (TNF-α) and the housekeeping gene GAPDH. (D) The IκB kinase (IKK) complex was isolated from the cells used in (A), by immunoprecipitation using anti-IKKγ followed by in vitro kinase assays (KA) using glutathione S-transferase (GST)–IκBα (1–54) as substrate (panel 1). IB was carried out to monitor the expression level of IKKβ, Tax and the loading control tubulin (panels 2–4). (E) Whole-cell lysates prepared from the cells used in (A) were subjected to IB to detect the indicated proteins. GAPDH, glyceraldehyde phosphate dehydrogenase; MEF, mouse embryonic fibroblasts; Tak1, TGF-β-activating kinase 1.

Tax physically interacts with TGF-β-activating kinase 1 and induces the TGF-β-activating kinase 1–IκB kinase association. (A) 293 cells were transfected with Tax either in the presence (+) or absence (−) of haemagglutinin (HA)–Tak1. The Tak1 protein complex was isolated by immunoprecipitation (IP) using Tak1 followed by immunoblotting (IB) to detect the Tak1-associated Tax (panel 1) and level of precipitated HA–Tak1 (panel 2). IB was also carried out to detect the expression of Tax and HA–Tak1 in cell lysates (panels 3,4). The small amount of Tax precipitated by anti-Tak1 from HA–Tak1-negative cells (panel 1, lane 1) might be attributable to the binding of Tax to endogenous Tak1. (B) 293 cells were transfected with either an empty vector (−) or expression vectors encoding wild-type (WT) Tax or a mutant form of Tax (M22) known to be defective in IKK activation. Endogenous Tak1 was isolated by IP followed by IB to detect the co-precipitated Tax and M22 (panel 1) and the amount of precipitated Tak1 (panel 2). The expression level of Tax in cell lysates was also monitored by IB (panel 3). (C) 293 cells were transfected as in (B). Endogenous Tak1 was isolated by IP and subjected to kinase assays (KA) using MKK6 as substrate (panel 1). IB was carried out to detect the indicated proteins (panels 2–4). (D) JNK1 was isolated by IP from the transfected cells used in (B) and subjected to kinase assays using glutathione S-transferase (GST)–cJun (1–79) as substrate (top panel). The expression levels of JNK1 isoforms and Tax were analysed by IB (lower two panels). (E) Tak1 complex was isolated from either SupT1 or the HTLV-I-transformed SLB-1 cells followed by IB to detect the Tak1-associated IKKγ (panel 1) and Tax (panel 3) and also the precipitated Tak1 (panel 2). IB was also carried out with cell lysates to monitor the indicated proteins (panels 2–4). (F) 293 cells were transfected with Tak1 (+) either in the absence (−) or presence (+) of Tax or Tax M22. IKK complex was isolated by IP using anti-IKKγ followed by IB to detect the co-precipitated Tak1 (panel 1) and IKKγ (panel 2). The expression of transfected proteins was monitored by IB using the cell lysates (panels 3,4). HTLV-I, human T-cell leukaemia virus type 1; IKK, IκB kinase; JNK1, c-Jun amino-terminal kinase 1; Tak1, TGF-β-activating kinase 1.