Abstract
TRAF6 is a ubiquitin ligase that is essential for the activation of NF-κB and MAP kinases in several signalling pathways, including those emanating from the interleukin 1 and Toll-like receptors1,2,3. TRAF6 functions together with a ubiquitin-conjugating enzyme complex consisting of UBC13 (also known as UBE2N) and UEV1A (UBE2V1) to catalyse Lys 63-linked polyubiquitination, which activates the TAK1 (also known as MAP3K7) kinase complex4,5. TAK1 in turn phosphorylates and activates IκB kinase (IKK), leading to the activation of NF-κB. Although several proteins are known to be polyubiquitinated in the IL1R and Toll-like receptor pathways, it is not clear whether ubiquitination of any of these proteins is important for TAK1 or IKK activation. By reconstituting TAK1 activation in vitro using purified proteins, here we show that free Lys 63 polyubiquitin chains, which are not conjugated to any target protein, directly activate TAK1 by binding to the ubiquitin receptor TAB2 (also known as MAP3K7IP2). This binding leads to autophosphorylation and activation of TAK1. Furthermore, we found that unanchored polyubiquitin chains synthesized by TRAF6 and UBCH5C (also known as UBE2D3) activate the IKK complex. Disassembly of the polyubiquitin chains by deubiquitination enzymes prevented TAK1 and IKK activation. These results indicate that unanchored polyubiquitin chains directly activate TAK1 and IKK, suggesting a new mechanism of protein kinase regulation.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Inoue, J., Gohda, J. & Akiyama, T. Characteristics and biological functions of TRAF6. Adv. Exp. Med. Biol. 597, 72–79 (2007)
Chen, Z. J. Ubiquitin signalling in the NF-κB pathway. Nature Cell Biol. 7, 758–765 (2005)
Krappmann, D. & Scheidereit, C. A pervasive role of ubiquitin conjugation in activation and termination of IκB kinase pathways. EMBO Rep. 6, 321–326 (2005)
Wang, C. et al. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412, 346–351 (2001)
Deng, L. et al. Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103, 351–361 (2000)
Singhirunnusorn, P., Suzuki, S., Kawasaki, N., Saiki, I. & Sakurai, H. Critical roles of threonine 187 phosphorylation in cellular stress-induced rapid and transient activation of transforming growth factor-β-activated kinase 1 (TAK1) in a signaling complex containing TAK1-binding protein TAB1 and TAB2. J. Biol. Chem. 280, 7359–7368 (2005)
Lamothe, B. et al. Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of IκB kinase activation. J. Biol. Chem. 282, 4102–4112 (2007)
Petroski, M. D. et al. Substrate modification with lysine 63-linked ubiquitin chains through the UBC13–UEV1A ubiquitin-conjugating enzyme. J. Biol. Chem. 282, 29936–29945 (2007)
Courtois, G. Tumor suppressor CYLD: negative regulation of NF-κB signaling and more. Cell. Mol. Life Sci. 65, 1123–1132 (2008)
Komander, D. et al. The structure of the CYLD USP domain explains its specificity for Lys63-linked polyubiquitin and reveals a B box module. Mol. Cell 29, 451–464 (2008)
Reyes-Turcu, F. E. et al. The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin. Cell 124, 1197–1208 (2006)
Kanayama, A. et al. TAB2 and TAB3 activate the NF-κB pathway through binding to polyubiquitin chains. Mol. Cell 15, 535–548 (2004)
Windheim, M., Stafford, M., Peggie, M. & Cohen, P. Interleukin-1 (IL-1) induces the Lys63-linked polyubiquitination of IL-1 receptor-associated kinase 1 to facilitate NEMO binding and the activation of IκBα kinase. Mol. Cell. Biol. 28, 1783–1791 (2008)
Conze, D. B., Wu, C. J., Thomas, J. A., Landstrom, A. & Ashwell, J. D. Lys63-linked polyubiquitination of IRAK-1 is required for interleukin-1 receptor- and toll-like receptor-mediated NF-κB activation. Mol. Cell. Biol. 28, 3538–3547 (2008)
Ea, C. K., Deng, L., Xia, Z. P., Pineda, G. & Chen, Z. J. Activation of IKK by TNFα requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol. Cell 22, 245–257 (2006)
Wu, C. J., Conze, D. B., Li, T., Srinivasula, S. M. & Ashwell, J. D. Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-κB activation. Nature Cell Biol. 8, 398–406 (2006)
Walsh, M. C., Kim, G. K., Maurizio, P. L., Molnar, E. E. & Choi, Y. TRAF6 autoubiquitination-independent activation of the NFκB and MAPK pathways in response to IL-1 and RANKL. PLoS One 3, e4064 (2008)
Yamamoto, M. et al. Key function for the Ubc13 E2 ubiquitin-conjugating enzyme in immune receptor signaling. Nature Immunol. 7, 962–970 (2006)
Chen, Z. J., Bhoj, V. & Seth, R. B. Ubiquitin, TAK1 and IKK: is there a connection? Cell Death Differ. 13, 687–692 (2006)
VanDemark, A. P., Hofmann, R. M., Tsui, C., Pickart, C. M. & Wolberger, C. Molecular insights into polyubiquitin chain assembly: crystal structure of the Mms2/Ubc13 heterodimer. Cell 105, 711–720 (2001)
Hofmann, R. M. & Pickart, C. M. Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell 96, 645–653 (1999)
Tokunaga, F. et al. Involvement of linear polyubiquitylation of NEMO in NF-κB activation. Nature Cell Biol. 11, 123–132 (2009)
Frias-Staheli, N. et al. Ovarian tumor domain-containing viral proteases evade ubiquitin- and ISG15-dependent innate immune responses. Cell Host Microbe 2, 404–416 (2007)
Gack, M. U. et al. TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature 446, 916–920 (2007)
Sadler, A. J. & Williams, B. R. Structure and function of the protein kinase R. Curr. Top. Microbiol. Immunol. 316, 253–292 (2007)
Zhang, F. et al. Binding of double-stranded RNA to protein kinase PKR is required for dimerization and promotes critical autophosphorylation events in the activation loop. J. Biol. Chem. 276, 24946–24958 (2001)
Li, X. et al. Mutant cells that do not respond to interleukin-1 (IL-1) reveal a novel role for IL-1 receptor-associated kinase. Mol. Cell. Biol. 19, 4643–4652 (1999)
You, J., Cohen, R. E. & Pickart, C. M. Construct for high-level expression and low misincorporation of lysine for arginine during expression of pET-encoded eukaryotic proteins in Escherichia coli. Biotechniques 27, 950–954 (1999)
Haystead, C. M., Gregory, P., Sturgill, T. W. & Haystead, T. A. γ-Phosphate-linked ATP-sepharose for the affinity purification of protein kinases. Rapid purification to homogeneity of skeletal muscle mitogen-activated protein kinase kinase. Eur. J. Biochem. 214, 459–467 (1993)
Pickart, C. M. & Raasi, S. Controlled synthesis of polyubiquitin chains. Methods Enzymol. 399, 21–36 (2005)
Acknowledgements
We thank C.-K. Ea for generating the HEK293 cell line stably expressing the TAP-tagged TAK1, J. Ashwell for the bacterial GST–Ub2 and GST–Ub3 expression plasmids, A. Garcia-Sastre for the expression vector encoding the viral OTU enzyme, CCHFV-L(1–169), and X. Li for the IRAK1-deficient HEK293 cells line. We also thank B. Skaug for critically reading the manuscript. This work was supported by grants from the National Institute of Health (RO1-AI09919 and RO1-GM63692) and the Robert Welch Foundation (I-1389). Z.J.C. is an Investigator of the Howard Hughes Medical Institute.
Author Contributions Z.-P.X., L.S. and Z.J.C. designed the experiments, which were performed by Z.-P.X. and L.S., with assistance from X.C. G.P., X.J., A.A. and W.Z. contributed reagents. The manuscript was written by Z.J.C. and Z.-P.X.
Author information
Authors and Affiliations
Corresponding author
Supplementary information
Supplementary Information
This file contains Supplementary Figures S1-S12 with Legends, Supplementary Notes (Results and Discussion) and Supplementary References. (PDF 1604 kb)
Rights and permissions
About this article
Cite this article
Xia, ZP., Sun, L., Chen, X. et al. Direct activation of protein kinases by unanchored polyubiquitin chains. Nature 461, 114–119 (2009). https://doi.org/10.1038/nature08247
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature08247
This article is cited by
-
Inhibition of TRAF6 improves hyperlipidemic acute pancreatitis by alleviating pyroptosis in vitro and in vivo rat models
Biology Direct (2023)
-
Effects of TmTak1 silencing on AMP production as an Imd pathway component in Tenebrio molitor
Scientific Reports (2023)
-
YEATS2 regulates the activation of TAK1/NF-κB pathway and is critical for pancreatic ductal adenocarcinoma cell survival
Cell Biology and Toxicology (2023)
-
Novel Therapeutic Avenues for Hypertrophic Cardiomyopathy
American Journal of Cardiovascular Drugs (2023)
-
The E3 ubiquitin ligase TRIM31 plays a critical role in hypertensive nephropathy by promoting proteasomal degradation of MAP3K7 in the TGF-β1 signaling pathway
Cell Death & Differentiation (2022)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.