@article{b302eb908a3e4db98b516c9961f00516,
title = "The kinase IKKα inhibits activation of the transcription factor NF-κB by phosphorylating the regulatory molecule TAX1BP1",
abstract = "In response to stimulation with proinflammatory cytokines, the deubiquitinase A20 inducibly interacts with the regulatory molecules TAX1BP1, Itch and RNF11 to form the A20 ubiquitin-editing complex. However, the molecular signal that coordinates the assembly of this complex has remained elusive. Here we demonstrate that TAX1BP1 was inducibly phosphorylated on Ser593 and Ser624 in response to proinflammatory stimuli. The kinase IKKα, but not IKKβ, was required for phosphorylation of TAX1BP1 and directly phosphorylated TAX1BP1 in response to stimulation with tumor necrosis factor (TNF) or interleukin 1 (IL-1). TAX1BP1 phosphorylation was pivotal for cytokine-dependent interactions among TAX1BP1, A20, Itch and RNF11 and downregulation of signaling by the transcription factor NF-κB. IKKα therefore serves a key role in the negative feedback of NF-κB canonical signaling by orchestrating assembly of the A20 ubiquitin-editing complex to limit inflammatory gene activation.",
author = "Noula Shembade and Rajeshree Pujari and Harhaj, {Nicole S.} and Abbott, {Derek W.} and Harhaj, {Edward W.}",
note = "Funding Information: M. Karin (University of California San Diego) for Ikka−/−, Ikbkb−/− and Ikbkg−/− MEFs; E. Androphy (University of Massachusetts) for Flag-TAX1BP1 amino acids 1–789; and J. Leszyk for mass spectrometry and analysis. Supported by the US National Institutes of Health (RO1GM083143 and RO1CA135362 to E.W.H.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute, National Institute of General Medical Sciences or the US National Institutes of Health. Funding Information: Cells and reagents. Tax1bp1+/− and Tax1bp1−/− MEFs have been described26,29; Ikka−/−, Ikbkb−/− and Ikbkg−/− MEFs were provided by M. Karin; MEFs doubly deficient in both Akt1 and Akt2 were provided by M. Birnbaum; 293T cells were from American Type Culture Collection; BMDMs were made as described26; TL-OM1 cells were provided by M. Maeda48; C8166 and MT-2 cells have been described49; and Jurkat Tax Tet-on cells were a gift from W. Greene50. Plasmids Flag-TAX1BP1 and pCMV4-Tax have been described35. The Flag-tagged TAX1BP1 deletion mutants consisting of amino acids 1–696, 1–650, 1–504, 1–360 and 546–747 were generated by PCR-assisted subcloning into p3x-Flag-CMV-7.1 (Sigma). Flag-TAX1BP1 1–789 was a gift from E. Androphy51. TAX1BP1 amino acids 1–204, 205–599 and 600–747 fused to GST were generated by cloning of PCR-amplified TAX1BP1 fragments into pGEX4T3 (GE Healthcare Life Sciences). Flag-tagged point mutants TAX1BP1(S593A), TAX1BP1(S593E), TAX1BP1(S624A), TAX1BP1(S624E), TAX1BP1(S641A) and TAX1BP1(S652A) were generated with the QuikChange site-directed mutagenesis kit (Stratagene). Primer sequences are in the Supplementary Methods. Hemagglutinin-tagged IKKα, IKKα(K44M), IKKβ and IKKγ (NEMO), and IκBα amino acids 1–54 fused to GST, were provided by S.C. Sun. The siGENOME SMARTpool siRNA specific for IKKα and scrambled control siRNA were from Dharmacon–Thermo Scientific. ON-TARGETplus siRNA for mouse TAX1BP1 was from Dharmacon–Thermo Scientific. The sequence of the TAX1BP1-specific siRNA was 5′-GAACGAUGCUUCAAUAAAU-3′. Recombinant active IKKα fused to GST and histidine-tagged IKKβ were from Millipore. Recombinant TAX1BP1 amino acids 1–204, 205–599 and 600–747 fused to GST were expressed and purified from BL21 Escherichia coli competent cells with a GST Spin Trap kit (GE Healthcare Life Sciences). The following antibodies were used: anti-TAX1BP1 (ab22049; Abcam), anti-β-actin (AC15; Abcam), anti-actin (A2103; Sigma), anti-RNF11 (ab57180; Abcam), anti-cIAP1 (ab2399; Abcam), anti-UbcH5c (ab58251; Abcam), anti-Flag (M2; Sigma), antihemagglutinin (12CA5; Roche), anti-IκBα (C-21; Santa Cruz Biotechnology), antibody to phosphorylated IκBα (14D4; Cell Signaling), anti-TRAF2 (C-20; Santa Cruz Biotechnology), anti-TRAF6 (H-274; Santa Cruz Biotechnology), anti-IKKα (B-8; Santa Cruz Biotechnology), anti-GST (Z-5; Santa Cruz Biotechnology), anti-Jnk (56G8; Cell Signaling), antibody to phosphorylated Jnk (9251; Cell Signaling), anti-RIP1 (38; BD Biosciences Pharmingen), anti-A20 (E5-1619; BD Biosciences Pharmingen), anti-A20 (H-100; Santa Cruz Biotechnology), anti-Itch (32; BD Biosciences Pharmingen), anti-Ubc13 (clone 4E11; Invitrogen) and antibody specific for ubiquitin Lys63 (HWA4C4; Millipore). Anti-Tax35was prepared from a Tax hybridoma (168B17-46-34) from the AIDS Research and Reference Program of the National Institute of Allergy and Infectious Diseases (US National Institutes of Health). Recombinant TNF and IL-1 were from R&D Systems; LPS was from Sigma; and calf intestinal alkaline phosphatase was from New England Biolabs.",
year = "2011",
month = sep,
doi = "10.1038/ni.2066",
language = "English (US)",
volume = "12",
pages = "834--843",
journal = "Nature Immunology",
issn = "1529-2908",
publisher = "Nature Publishing Group",
number = "9",
}