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. 2005 Dec;25(23):10391-406.
doi: 10.1128/MCB.25.23.10391-10406.2005.

Exclusive ubiquitination and sumoylation on overlapping lysine residues mediate NF-kappaB activation by the human T-cell leukemia virus tax oncoprotein

Isabelle Lamsoul  1 , Julie Lodewick, Sylvie Lebrun, Robert Brasseur, Arsène Burny, Richard B Gaynor, Françoise Bex
Affiliations

Exclusive ubiquitination and sumoylation on overlapping lysine residues mediate NF-kappaB activation by the human T-cell leukemia virus tax oncoprotein

Isabelle Lamsoul et al. Mol Cell Biol. 2005 Dec.

Abstract

The transcription factor NF-kappaB is critical for the induction of cancer, including adult T-cell leukemia, which is linked to infection by human T-cell leukemia virus type 1 and the expression of its regulatory protein Tax. Although activation of the NF-kappaB pathway by Tax involves its interaction with the regulatory subunit of the IkappaB kinase (IKK) complex, NEMO/IKKgamma, the mechanism by which Tax activates specific cellular genes in the nucleus remains unknown. Here, we demonstrate that the attachment of SUMO-1 to Tax regulates its localization in nuclear bodies and the recruitment of both the RelA subunit of NF-kappaB and free IKKgamma in these nuclear structures. However, this sumoylation step is not sufficient for the activation of the NF-kappaB pathway by Tax. This activity requires the prior ubiquitination and colocalization of ubiquitinated Tax with IKK complexes in the cytoplasm and the subsequent migration of the RelA subunit of NF-kappaB to the nucleus. Thus, the ubiquitination and sumoylation of Tax function in concert to result in the migration of RelA to the nucleus and its accumulation with IKKgamma in nuclear bodies for activation of gene expression. These modifications may result in targets for the treatment of adult T-cell leukemia.

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Figures

FIG. 1.
FIG. 1.
Differential intracellular partitioning of colocalized Tax and SUMO or Tax and ubiquitin molecules. 293T cells were transfected with vectors expressing (A) Tax alone or (B) Tax in combination with HA-Ub or HA-SUMO-1 and stained by immunofluorescence with (A) an anti-Tax MAb or (B) the anti-Tax MAb (IgG2a), an anti-HA rabbit polyclonal antibody, and a MAb (IgG1) recognizing either the Golgi matrix marker GM130 (upper panels) or the promyelocytic leukemia protein (lower panels). The cells were analyzed by laser scanning confocal microscopy, with one confocal section being depicted in panel A and the projection of Z-series confocal images depicted in panel B. Diagrams depict the intensity of the fluorescence for each staining along lines drawn on the overlay images with the boundaries of the nuclei (NE, nuclear envelope), the Tax NB, the Golgi apparatus (G), and the PML bodies (PML) indicated. The percentages of cells having Tax in nuclear bodies only (NB), in the cytoplasm and in the nuclear bodies (Cy + NB), or in the cytoplasm only (Cy) with or without overexpression of HA-ubiquitin or HA-SUMO-1 are depicted in panels A and C. DIC, differential inference contrast.
FIG. 2.
FIG. 2.
Tax is modified by conjugation to SUMO and ubiquitin. (A) 293T cells were transfected with vectors expressing wild-type or mutant Tax-6His. The cells extracts were purified by Ni-NTA pulldown assay followed by Western blotting with anti-SUMO-1 or anti-Tax rabbit polyclonal antibodies. (B) Schematic representation of the Tax proteins with the positions of the lysines replaced in the different Tax mutants used in this work. (C and D) 293T cells were cotransfected with vectors expressing wild-type or mutant Tax-6His and either (C) HA-SUMO-1 or (D) HA-Ub. Proteins were revealed with anti-HA or anti-Tax rabbit polyclonal antibodies. (E) Quantitation was performed on the anti-HA immunoblots in panels C and D after subtraction of the background in lanes 1 and normalization to an equal amount of the unmodified 40-kDa Tax species on the anti-Tax immunoblots. The values representing the percentages of modified forms of Tax mutants relative to wild-type Tax are reported in the diagram for both SUMO-modified (black bars) and ubiquitin-modified (white bars) Tax species. Error bars for quantitation of the SUMO-modified forms of the Tax mutants R7-8K and R4-5/7-8K represent the standard deviation in three independent experiments. (F) 293T cells either not transfected or transfected with a vector expressing Tax in combination with vectors expressing either HA-Ub or HA-SUMO-1 (HA-S1) were fractionated, and the cytoplasmic (C) and nuclear (N) fractions were immunoblotted with anti-Tax, anti-HA, or anti-GM130 antibodies. (G) HUT102 HTLV-1-transformed T lymphocytes were lysed under highly denaturing conditions and immunoblotted with anti-Tax rabbit polyclonal serum in parallel with extracts from 293T cells or Jurkat T lymphocytes transfected with a vector expressing wild-type Tax-6His with or without vectors for the expression of HA-Ub or HA-SUMO-1 that were revealed with anti-Tax, anti-HA, or anti-SUMO-1 antibodies. The position of the tri-sumoylated Tax species is indicated as well as the mono-, di-, and tri-ubiquitinated forms (*). Molecular mass markers (in kilodaltons) are noted at the left of the blots.
FIG. 3.
FIG. 3.
Lysines K7 and K8 are critical for the ability of Tax to localize in nuclear bodies. 293T cells were transfected with vectors expressing wild-type Tax or the Tax mutants as indicated. The cells were stained by dual immunofluorescence with an anti-Tax MAb and an anti-RelA rabbit polyclonal antibody. Diagrams depict the intensity of the fluorescence for each staining along lines drawn on the overlay images with the boundaries of the nuclei (NE) and the nuclear bodies (NB) indicated. Cells were considered positive for the translocation of RelA to the nucleus when the intensity of the fluorescence in the nuclei was at least twice that found in cells that did not express Tax. The percentages of cells displaying Tax-containing nuclear bodies or the translocation of RelA to the nucleus are indicated.
FIG. 4.
FIG. 4.
Lysines K7 and K8 are critical for Tax activation of gene expression via the NF-κB pathway. (A) 293T cells or (B) Jurkat T lymphocytes were cotransfected with either the HIV-1 LTR or the HTLV-1 LTR-luciferase reporter plasmids and vectors expressing wild-type or mutant Tax. Luciferase activities are represented as percentages of the activity seen with wild-type Tax. The values are averages from at least six independent experiments, with the standard deviations indicated. The 293T cell extracts from the luciferase assays were immunoblotted with the anti-Tax and antiactin antibodies (A), which indicates similar levels of wild-type and mutant Tax proteins in each extract.
FIG.5.
FIG.5.
Phenotypes of Tax mutants fused to ubiquitin or SUMO. (A) 293T cells were transfected with a vector expressing either wild-type or mutant K4-8R or mutant R4-6K Tax-6His not fused or fused to either ubiquitin or SUMO-1. The proteins were purified by Ni-NTA pulldown assay and immunoblotted with anti-Tax or anti-SUMO-1 rabbit polyclonal antibodies or an anti-Ub MAb. WT-S1, K4-8R-S1, and R4-6K-S1, WT-SUMO-1, K4-8R-SUMO-1, and R4-6K-SUMO-1, respectively. (B) 293T cells were transfected with the vector expressing wild-type or mutant Tax fusions to ubiquitin or SUMO-1 and stained by dual-immunofluorescence staining with an anti-Tax MAb and an anti-RelA rabbit polyclonal antibody. Diagrams depict the intensity of the fluorescence for each staining along lines drawn on the overlay images with the boundaries of the nuclei (NE, nuclear envelope) and nuclear speckles (NS) or nuclear bodies indicated. The percentages of cells displaying Tax-containing nuclear bodies or the translocation of RelA to the nucleus is indicated. (C and D) 293T cells were cotransfected with either (C) the HIV-1 LTR luciferase or (D) the HTLV-1 LTR-luciferase reporter plasmids and vectors expressing wild-type or mutant ubiquitin or SUMO-1 Tax fusions. Luciferase activities are represented as percentages of the activities obtained with the homologous wild-type Tax or the Tax-Ub or Tax-SUMO-1 fusions. The luciferase activities of the WT-Ub and WT-SUMO-1 fusions relative to that of nonfused wild-type Tax are presented on top of the bars. The values shown represent averages from at least six independent experiments, with the standard deviations indicated.
FIG. 6.
FIG. 6.
Tax colocalizes with IKK complexes in the cytoplasm and with free IKKγ in the nucleus. (A) 293T cells were cotransfected with the vector for expression of either Flag-IKKα or Flag-IKKβ, alone or in combination with wild-type Tax or the Tax mutant K4-8R. The cells were submitted to triple-immunofluorescence staining with the anti-Tax MAb (IgG2a), an anti-Flag MAb (IgG1), and an anti-IKKγ rabbit polyclonal antibody. (B) 293T cells were transfected as described for panel A, except that HA-Ub was expressed instead of the IKK subunits and an anti-HA MAb (IgG1) was used during the triple-immunofluorescence staining. The images represent projections of Z-series confocal images. A fivefold enlargement of a field in the overlay images (Zoom) and diagrams of the intensity of the fluorescence for each of the staining along lines crossing cellular structures (S, cytoplasmic speckles; F, cytoplasmic fibers; NE, nuclear envelope) are shown.
FIG. 7.
FIG. 7.
Schematic diagram depicting a model for concerted ubiquitin and SUMO conjugation of the HTLV-1 Tax protein in Tax-mediated activation of the NF-κB pathway. In the cytoplasm, ubiquitin-conjugated Tax activates the IKK signalosomes, resulting in the translocation of the RelA subunit of NF-κB to the nucleus. Deubiquitinated Tax migrates to the nucleus, in which it is sumoylated. Sumoylated Tax assembles nuclear bodies that include the RelA subunit of NF-κB and free IKKγ along with other transcription and splicing factors, leading to the activation of specific Tax-responsive target genes. Tax can further be desumoylated, which reveals its nuclear export signal, leading to the exit of Tax from the nucleus.
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