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. 2008 May 30;283(22):15127-33.
doi: 10.1074/jbc.M704831200. Epub 2008 Jan 26.

In vitro activation of the IkappaB kinase complex by human T-cell leukemia virus type-1 Tax

Sohini Mukherjee  1 , Veera S Negi, Gladys Keitany, Yuetsu Tanaka, Kim Orth
Affiliations

In vitro activation of the IkappaB kinase complex by human T-cell leukemia virus type-1 Tax

Sohini Mukherjee et al. J Biol Chem. .

Abstract

Human T-cell leukemia virus type-I expresses Tax, a 40-kDa oncoprotein that activates IkappaB kinase (IKK), resulting in constitutive activation of NFkappaB. Herein, we have developed an in vitro signaling assay to analyze IKK complex activation by recombinant Tax. Using this assay in combination with reporter assays, we demonstrate that Tax-mediated activation of IKK is independent of phosphatases. We show that sustained activation of the Tax-mediated activation of the NFkappaB pathway is dependent on an intact Hsp90-IKK complex. By acetylating and thereby preventing activation of the IKK complex by the Yersinia effector YopJ, we demonstrate that Tax-mediated activation of the IKK complex requires a phosphorylation step. Our characterization of an in vitro signaling assay system for the mechanism of Tax-mediated activation of the IKK complex with a variety of mutants and inhibitors results in a working model for the biochemical mechanism of Tax-induced activation.

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Figures

FIGURE 1.
FIGURE 1.
Recombinant Tax and TRAF6. Wild type and mutant forms of Tax (lanes 1–5) were expressed as His6-tagged proteins in BL21-DE3 cells and purified from BL21-DE3 cells using standard protocols. Recombinant TRAF6 (lane 6) was purified from Sf9 cells as previously described (16). Sample of each recombinant protein was resolved on SDS-PAGE and stained with Coomassie Blue (A) or subjected to Western blotting using anti-Tax antibody (B). Thin lines denote where lanes have been deleted from the scanned image.
FIGURE 2.
FIGURE 2.
In vitro activation of NFκB pathway by recombinant Tax and TRAF6. A, IKK activation by wild type (wt) Tax and TRAF6 using in vitro signaling assay. Lane 1, lysate control; lanes 2 and 3, addition of 2 μm Tax and 1 μm TRAF6, respectively, to lysate. B, concentration dependence of Tax-mediated IκB phosphorylation. Lane 1, lysate control; lanes 2–4, increasing amounts of Tax (0.8–9.0 μm) added to lysate. Thin lines denote where lanes have been deleted from the scanned image. For all experiments, activation of IKK complex was detected by immunoblotting with anti-phospho-IκB antibody. C, in vitro kinase assay of complexes isolated with anti-IKKγ and anti-Tax from unstimulated (lanes 1, 2, and 4) and Tax-stimulated (lanes 3 and 5) lysates. Immuno-isolated IKK complexes were incubated with [γ-32P]ATP and GST-IκBα-(1–52), separated by SDS-PAGE, and analyzed by autoradiography. The data shown are representative of three independent experiments.
FIGURE 3.
FIGURE 3.
Activation of the NFκB pathway by wild type and mutant Tax. A, activation of a NFκB luciferase reporter gene by Tax. HEK293 cells were transfected with an empty vector (V), wild type Tax (wt), or the different Tax mutants (M22, H41Q, H43Q, and K85N), 5xNFκB luciferase reporter, and pRSV-Renilla (to serve as the internal standard control) for 24 h. The luciferase assay was performed using Fluostar Optima. Error bars denote mean ± S.D. of triplicates; results shown are representative of four independent experiments. B, HEK293 cells were transfected with empty vector (lane 1), wild type Tax (wt; lane 2), M22 (lane 3), H41Q (lane 4), H43Q (lane 5), or K85N (lane 6) and FLAG-IκB. Activation of IKK complex, Tax expression, and FLAG-IκB were detected by immunoblotting with anti-phospho-IκB antibody, anti-IκB antibody, anti-Tax antibody, and anti-FLAG antibody, respectively. The data shown are representative of three independent experiments.
FIGURE 4.
FIGURE 4.
In vitro activation of NFκB pathway by Tax is independent of phosphatases. A, activation of the IKK complex by buffer (lane 1) wild type (wt) Tax (lane 2), M22 (lane 3), H41Q (lane 4), H43Q (lane 5), or K85N (lane 6) using the in vitro signaling assay. Activation of IKK complex was detected by immunoblotting with anti-phospho-IκB antibody. B, in vitro activation of the IKK complex by buffer (lanes 1 and 2), Tax (lanes 3 and 4), or TRAF6 (lanes 5 and 6) in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of 10 nm okadaic acid (OA). Activation of the IKK complex was detected by immunoblotting with anti-phospho-IκB antibody. To confirm equal amounts of IκBin all lanes, the same lysates were blotted with α-IκB antibody. C, activity of PP2A in S100 lysate in the presence or absence of 10 nm OA. PP2A activity was measured using pNPP as a substrate. The data shown are representative of three independent experiments. Error bars denote mean ± S.D. of triplicates; results shown are representative of four independent experiments.
FIGURE 5.
FIGURE 5.
Inhibition of Tax-mediated activation of the NFκB pathway by geldanamycin. A, in vitro activation of the IKK complex by buffer (lanes 1 and 2), Tax (lanes 3 and 4), or TRAF6 (lanes 5 and 6) in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of geldanamycin (GA). Thin lines denote where lanes have been deleted from the scanned image. Activation of the IKK complex was detected by immunoblotting with anti-phospho-IκB antibody. B, cell-free lysates (1 mg/ml) were immunodepleted with Hsp90 by performing two successive rounds of immunoprecipitation with anti-Hsp90 antibody (1:100). The levels of Hsp90 and IKKβ in these lysates were assessed by immunoblotting against anti-Hsp90 (upper panel) and anti-IKKβ antibodies. C, lysates immunodepleted with Hsp90 (lanes 4–6) or not (lanes 1–3) were incubated with 2 μm Tax and 1 μm TRAF6 for 10 min at 37 °C. Samples were subjected to SDS-PAGE followed by immunoblotting with anti-phospho-IκB antibody. The data shown are representative of three independent experiments.
FIGURE 6.
FIGURE 6.
Activation of the NFκB pathway by Tax is inhibited by the acetyltransferase YopJ. A, activation of an NFκB luciferase reporter gene by Tax. HEK293 cells were transfected with an empty vector (Vector) or wild type Tax (pcDNA3-Tax) in the presence of an empty vector (V), YopJ (J), or catalytically inactive YopJC172A (C/A), the 5xNFκB luciferase reporter, and pRSV-Renilla (to serve as the internal standard control) for 24 h. The luciferase assay was performed using Fluostar Optima. B, HEK293 cells were cotransfected with Tax and either empty vector (V), YopJ (J), or catalytically inactive YopJC172A (C/A) in the presence or absence of FLAG-IκB followed by immunoblotting cell lysates with anti-phospho-IκB antibody and anti-IκB antibody. The asterisk indicates transfected FLAG-IκB. C, in vitro activation of the IKK complex by Tax (+, 0.8 μm; ++, 1.6 μm) in lysates isolated from cells containing empty vector (V), YopJ (J), or catalytically inactive YopJC172A (C/A). Activation of IKK complex was detected by immunoblotting with anti-phospho-IκB antibody. The data shown are representative of three independent experiments.
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