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, 9 (1), 16014

SQSTM-1/p62 Potentiates HTLV-1 Tax-mediated NF-κB Activation Through Its Ubiquitin Binding Function

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SQSTM-1/p62 Potentiates HTLV-1 Tax-mediated NF-κB Activation Through Its Ubiquitin Binding Function

Aurélien Schwob et al. Sci Rep.

Abstract

The NF-κB pathway is constitutively activated in adult T cell leukemia, an aggressive malignancy caused by Human T Leukemia Virus type 1 (HTLV-1). The viral oncoprotein Tax triggers this constitutive activation by interacting with the ubiquitin-rich IKK complex. We previously demonstrated that Optineurin and TAX1BP1, two members of the ubiquitin-binding, Sequestosome-1 (SQSTM-1/p62)-like selective autophagy receptor family, are involved in Tax-mediated NF-κB signaling. Here, using a proximity-dependent biotinylation approach (BioID), we identify p62 as a new candidate partner of Tax and confirm the interaction in infected T cells. We then demonstrate that p62 knock-out in MEF cells as well as p62 knock-down in HEK293T cells significantly reduces Tax-mediated NF-κB activity. We further show that although p62 knock-down does not alter NF-κB activation in Jurkat T cells nor in infected T cells, p62 does potentiate Tax-mediated NF-κB activity upon over-expression in Jurkat T cells. We next show that p62 associates with the Tax/IKK signalosome in cells, and identify the 170-206 domain of p62 as sufficient for the direct, ubiquitin-independent interaction with Tax. However, we observe that this domain is dispensable for modulating Tax activity in cells, and functional analysis of p62 mutants indicates that p62 could potentiate Tax activity in cells by facilitating the association of ubiquitin chains with the Tax/IKK signalosome. Altogether, our results identify p62 as a new ubiquitin-dependent modulator of Tax activity on NF-κB, further highlighting the importance of ubiquitin in the signaling activity of the viral Tax oncoprotein.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Functional validation of the BirA*-Tax fusion protein and identification of p62 as a new candidate partner of Tax. (a) Lysates from HEK293T cells transfected with the indicated plasmids for 24 h and then treated overnight with biotin or left untreated were analyzed by western blot. (b) U2OS cells transiently expressing Tax-His or Myc-BirA*-Tax-His and treated overnight with biotin were analyzed by epifluorescence microscopy after staining with Streptavidin (Strept., green) and His-specific antibodies (red). Nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bar = 10 μm. The arrows indicate perinuclear accumulation of Tax reminiscent of the Tax/IKK signalosome. (c) HEK293T cells were transfected with Myc-BirA* or Myc-BirA*-Tax-His, together with an NF-κB-luc construct. Luciferase activity was measured and normalized over the “Myc-BirA*” condition. The graph shows the result from a representative experiment. (d) Lysates from HEK293T cells transiently expressing Myc-BirA* or Myc-BirA*-Tax-His were submitted to a His-specific Ni-NTA pulldown in denaturing conditions before western blot analyses. WCL, whole cell lysate. (e) SQSTM-1/p62 is a BirA*-Tax-specific biotinylated protein identified by mass spectrometry. (f) Lysates from HTLV-1 chronically infected cells (C8166, HuT102 or C91PL cells) were immunoprecipitated with a p62-specific or Tax-specific antibody, or with control Ig (IP Ig). Samples were then analyzed by western blot. WCL, whole cell lysates. Full-length blots are presented in Supplementary Fig. S4.
Figure 2
Figure 2
p62 potentiates Tax-dependent NF-κB activation upstream of IKK activation. (a) Wild type (WT) and p62−/− MEF cells were transfected with Tax-His, together with an NF-κB-luc construct. Luciferase activity was measured and normalized over the corresponding Tax-negative condition. The graph shows results from at least 3 independent experiments. (b) WT and p62−/− MEF cells were transfected with Tax-His. After fractionation of cell nuclei, transcriptionally active p65 was quantified by ELISA. (c) Lysates from WT and p62−/− MEF cells transfected with Tax-His were analyzed by western blot. (d) WT and p62−/− MEF cells were transfected with Tax-His. After RNA extraction and conversion to cDNAs, Il6 and gapdh cDNAs were amplified by PCR (left panel). The normalized Il6 signal intensities were calculated and are shown relative to the corresponding Tax-negative conditions on the graph (results from 2 independent experiments). Cell lysates were also analyzed by western blot (right panel). (e) HEK293T cells were transfected with control (siCTRL) or p62-specific (sip62) siRNA and Tax-His, together with an NF-κB-luc construct. Luciferase activity was measured and normalized over the corresponding Tax-negative condition. The graph shows results from at least 3 independent experiments. (f) Lysates from HEK293T cells transfected with siCTRL or sip62 and Tax-His were analyzed by western blot. (g) Jurkat cells were transfected with increasing amounts of Myc-p62 and an NF-κB-luc construct, followed by transduction with an empty or Flag-Tax-encoding lentivector. Luciferase activity was measured and normalized to the corresponding Tax-negative condition. Values obtained with endogenous p62 were set to 1 and other values are shown as fold change over the “endogenous p62” condition. The graph shows results from 3 independent experiments. (h) Lysates from Jurkat cells were analyzed by western blot. ***p < 0.001; **p < 0.01; *p < 0.05 (one-way ANOVA with Bonferroni post-hoc test). Full-length blots and gels are presented in Supplementary Fig. S4.
Figure 3
Figure 3
p62 associates with Tax/IKK signalosomes in peri-golgian structures. (a,b) Jurkat cells transiently expressing Tax-His and HTLV-1 chronically infected C91PL cells were analyzed by confocal microscopy after staining with His- or Tax- (green), p62- (red), and GM130- (a, white) and IKKγ-specific (b, white) antibodies. Nuclei were counterstained with DAPI (blue). Representative images are shown. Tax, p62, GM130 and IKKγ signals were quantified along the segments represented on the merge panels and plotted on the histogram. Scale bar = 10 μm. (c) Lysates from HeLa cells transiently expressing Tax-His and/or IKKγ-FLAG were submitted to a FLAG-immunoprecipitation followed by a His-specific Ni-NTA purification and western blot analyses. Full-length blots are presented in Supplementary Fig. S4.
Figure 4
Figure 4
p62 does not allow Tax autophagic degradation at the steady-state. (a) HeLa cells stably expressing GFP-LC3 were transfected with Tax-His and analyzed by confocal microscopy after staining with His- (white) and p62- or OPTN-specific (red) antibodies. Representative images are shown. Tax, p62 or OPTN and GFP-LC3 signals were quantified along the segment represented on the merge panel and plotted on the histogram. Scale bar = 10 μm. (b,c) HeLa cells transiently expressing Tax-His were treated with lysosomal inhibitors (b, E64D/Pep.) or starved (c, HBSS) before lysis, western blot analyses and quantification. Full-length blots are presented in Supplementary Fig. S4. Blots and graphs show results representative of at least 3 experiments.
Figure 5
Figure 5
p62 directly interacts with Tax through its 170-206 domain. (a) GST and GST-tagged p62 were expressed in bacteria and used for GST pulldown of in vitro translated 35S-labeled Tax. Inputs as well as eluates were run on SDS-PAGE gels and autoradiography was performed. (b) Domain organization of p62 constructs with different deletion used for GST and MBP pulldown assays. The results of the pulldown assays shown in (c,d) indicate Tax binding ability and are indicated on the right. PB1, Phox and Bem1 domain; ZZ, zinc finger domain; MIR, multiple protein interaction region; LIR, LC3-interacting region; KIR, KEAP1 interacting region; UBA, ubiquitin-associated domain. (c,d) GST pulldown (c) and MBP pulldown assays (d) were performed with different p62 constructs. The percentage of input radioactively labeled-Tax bound to p62 was quantified from three independent experiments. CBB, Coomassie brilliant blue-stained SDS-PAGE gel.
Figure 6
Figure 6
p62 binding to ubiquitin is required for p62 potentiation of Tax-mediated NF-κB activation. (a) Jurkat cells were transfected with full-length Myc-p62 (My-p62 FL) or p62 mutants in which the Tax-interacting region (Myc-p62 Δ170-221) or the ubiquitin-binding domain (Myc-p62 ΔUBA) were deleted, and an NF-κB-luc construct, followed by transduction with an empty or Flag-Tax-encoding lentivector. Luciferase activity was measured and normalized to the corresponding Tax-negative condition. Values obtained with full-length ectopic p62 were set to 1 and other values are shown as fold change over the “p62 FL” condition. The graph shows results from 3 independent experiments. (b) Lysates from p62−/− MEF cells transiently expressing Tax-His and either Myc-tagged full-length p62 (p62FL) or p62 Δ170-221 were immunoprecipitated with a Myc-specific antibody followed by western blot analyses. (c) Lysates from p62−/− MEF cells transiently expressing Tax-His and either Myc-tagged full-length p62 (p62FL) or p62 ΔUBA were immunoprecipitated with a Myc-specific antibody followed by western blot analyses. (d) HeLa cells were transfected with control (−) or p62-specific (+) siRNA and Tax-His. Cell lysates were submitted to a His-specific Ni-NTA pulldown in denaturing conditions before western blot analyses. (e) Lysates from WT and p62−/− MEF cells transiently expressing Tax-His- and FLAG-IKKγ were immunoprecipitated with a FLAG-specific antibody followed by western blot analyses. **p < 0.01; ns, p > 0.05 (one-way ANOVA with Bonferroni post-hoc test). Full-length blots are presented in Supplementary Fig. S4.
Figure 7
Figure 7
Involvement of ubiquitin binding by p62 in Tax-induced NF-κB activation: working model. p62 associates with Tax in peri-golgian structures, downstream of the formation of Tax/IKK complexes, and participates in the efficient activation of the IKK complex by a mechanism requiring binding to ubiquitin chains. See text for further details. T1BP1: TAX1BP1. Artwork is a derivative of Servier Medical Art (https://smart.servier.com/), used under CC BY 3.0 FR (https://creativecommons.org/licenses/by/3.0/), and includes modifications in shapes, colors and disposition of the original material.

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