Tax ubiquitylation and sumoylation control critical cytoplasmic and nuclear steps of NF-κB activation by Rihab Nasr, Estelle Chiari, Marwan El-Sabban, Renaud Mahieux, Youmna Kfoury, Maher Abdulhay, Victor Yazbeck, Olivier Hermine, Hugues de Thé, Claudine Pique, and Ali Bazarbachi Blood Volume 107(10):4021-4029 May 15, 2006 ©2006 by American Society of Hematology

Tax C-terminal lysines are critical for NF-κB, but not CREB, activation. Tax C-terminal lysines are critical for NF-κB, but not CREB, activation. (A) Schematic representation of the lysine-to-arginine Tax mutants. (B) 293T cells were transfected with the various Tax-6His plasmids and proteins purified by denaturing Ni-NTA pull-down were blotted with mabs to poly-ubiquitin chains (top panel) or to Tax (middle and bottom panels). (C) Representation of Tax mutants in which lysines 7 and/or 8 were reintroduced to the lysineless protein K1-10R-6His. (D) 293T cells were transfected with Tax-6His, the lysineless Tax K1-10R-6His, or the indicated reverse mutants in the presence of an HA-Ub plasmid to favor Tax ubiquitylation. Proteins purified by denaturing Ni-NTA pull-down were blotted with mabs to HA (top panel) or to Tax (middle and bottom panels). (E) Jurkat cells were cotransfected with the NF-κB-Luc (top panel) or the HTLV-LTR-Luc (bottom panel) reporter plasmids, and the plasmids encoding for Tax or Tax mutants. For normalization, cells were cotransfected with renilla luciferase expression plasmid. Activity of the wild-type protein was set to 100%. The results represent the mean plus or minus standard deviation of at least 3 different experiments. (F) Jurkat cells were cotransfected with Tax or Tax mutants. NF-κB DNA-binding activity was assessed by electrophoretic mobility shift assay using a consensus oligonucleotide for NF-κB. Specificity of the NF-κB complex was determined by the addition of an excess of an unlabeled consensus (cold) or mutated (mutant) oligonucleotides. Rihab Nasr et al. Blood 2006;107:4021-4029 ©2006 by American Society of Hematology

Tax C-terminal lysines are critical for IKK binding, IKK activation, and nuclear translocation of RelA. Tax C-terminal lysines are critical for IKK binding, IKK activation, and nuclear translocation of RelA. (A) HeLa cells were transfected with Tax or Tax mutants. Lysates were immunoprecipitated with anti-Tax mab and recovered proteins were blotted using anti-Tax, anti-IKKα, anti-IKKβ, or anti-IKKγ antibodies, as indicated. (B) HeLa cells were transfected as in panel A. Lysates were immunoprecipitated using anti-IKKγ antibodies and recovered proteins were blotted using anti-Tax, anti-IKKα, anti-IKKβ,or anti-IKKγ antibodies, as indicated. (C) Jurkat cells were transfected with Tax or Tax mutants. Activity of immunoprecipitated IKK was assessed by kinase assay using GST-IκB-α as substrate (top panel). Immunoprecipitates were probed with anti-IKKα antibody to determine the amounts of precipitated kinase and with anti–GST-IκB-α to determine the amount of substrate. Equal amounts of lysates were determined by blotting with anti-GAPDH antibody. (D) HeLa cells were transfected with either Tax plasmid and stained with anti-Tax and anti-RelA antibodies. The percentage of nuclear translocation of RelA in Tax-positive cells is indicated. Rihab Nasr et al. Blood 2006;107:4021-4029 ©2006 by American Society of Hematology

Tax ubiquitylation mediates IKK binding and nuclear translocation of RelA. Tax ubiquitylation mediates IKK binding and nuclear translocation of RelA. (A) HeLa cells were transfected with wild-type Tax, Tax-Ub, Tax K4-8R, or Tax K4-8R-Ub. After immunoprecipitation with anti-Tax mab, recovered proteins were blotted with anti-Tax, anti-IKKα, anti-IKKβ, or anti-IKKγ antibodies, as indicated. (B) HeLa cells were transfected as in panel A and stained by dual immunofluorescence with anti-Tax (green) and anti-RelA (red) antibodies. The percentage of nuclear translocation of RelA in Tax-positive cells is indicated. (C) Jurkat cells were cotransfected with the NF-κB-Luc (▪) or the HTLV-LTR-Luc (▨) reporters, renilla luciferase, and either Tax plasmid. Activity of the wild-type protein was set to 100%. Rihab Nasr et al. Blood 2006;107:4021-4029 ©2006 by American Society of Hematology

Tax is SUMO-conjugated in the nucleus. Tax is SUMO-conjugated in the nucleus. (A-D) 293T (A-C) or Jurkat (D) cells were transfected with the plasmids encoding for Tax-6His or Tax mutants in the presence of HA-SUMO1 (A,D), HA-SUMO2 (B,D), or HA-SUMO3 (C,D) plasmids. Proteins purified by denaturing Ni-NTA pull-down were blotted with anti-HA (top panel) or anti-Tax (bottom panel) mabs. (E) HeLa cells were cotransfected with Tax and either Ub-HA and/or HA-SUMO3 plasmids. After immunoprecipitation with anti-Tax mab, recovered proteins were blotted with anti-Tax or anti-HA antibodies. Cell fractionation was performed as described in “Materials and methods.” Total lysates (left panels), nuclear extracts (lamin positive, tubulin negative; middle panels), and cytoplasmic extracts (lamin negative, tubulin positive; right panels) are displayed. Lysates were blotted with anti-HA and antitubulin antibodies to ensure equal loading. Rihab Nasr et al. Blood 2006;107:4021-4029 ©2006 by American Society of Hematology

Tax sumoylation is involved in the formation of Tax nuclear bodies and transcriptional activation. Tax sumoylation is involved in the formation of Tax nuclear bodies and transcriptional activation. (A-B) HeLa cells were transfected with either Tax plasmid together with HA-Ub (A) or HA-SUMO3 (B), and stained by dual immunofluorescence with anti-Tax (green) or anti-HA (red) mabs. (C-D) HeLa cells were transfected with a control plasmid or the Tax plasmid and stained with anti-SUMO, anti-Tax (green), anti-PML, or anti-p300 (red) antibodies. (E) HeLa cells were transfected with Tax K6-8R or Tax K6-8R-SUMO1 plasmids and stained with anti-Tax antibodies. (F) Jurkat cells were cotransfected with the NF-κB-Luc reporter, renilla luciferase, and the plasmid encoding wild-type Tax, Tax-SUMO1, Tax K6-8R, or Tax K6-8R-SUMO1. Activity of the wild-type protein was set to 100%. Rihab Nasr et al. Blood 2006;107:4021-4029 ©2006 by American Society of Hematology

Endogenous Tax is ubiquitylated. Endogenous Tax is ubiquitylated. (A) HuT-102 or Jurkat cells were either directly lysed in Laemli buffer or processed for immunoprecipitation with anti-Tax mab and blotting with anti-Tax sera. Lysates were also blotted with anti-GAPDH antibody. (B) C8166 cells were cotransfected with the NF-κB-LacZ (▪) or the HTLV-LTR-LacZ (□) reporter plasmids, and increasing doses of either Tax plasmid. The amount of transfected DNA was maintained at 3 μg using the PSG5 plasmid. Results correspond to β-gal activity after subtraction of the signal obtained in absence of LacZ plasmid and normalization to the amount of total proteins. Activity of the wild-type Tax protein was set to 100%. Rihab Nasr et al. Blood 2006;107:4021-4029 ©2006 by American Society of Hematology

Proposed model for the role of Tax ubiquitylation and sumoylation in NF-κB activation. . Rihab Nasr et al. Blood 2006;107:4021-4029 ©2006 by American Society of Hematology