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.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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)
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.
About this article
Cite this article
Xia, Z., Sun, L., Chen, X. et al. Direct activation of protein kinases by unanchored polyubiquitin chains. Nature 461, 114–119 (2009) doi:10.1038/nature08247
Analysis of the Zn-Binding Domains of TRIM32, the E3 Ubiquitin Ligase Mutated in Limb Girdle Muscular Dystrophy 2H
Oxidization of TGFβ-activated kinase by MPT53 is required for immunity to Mycobacterium tuberculosis
Nature Microbiology (2019)
Autophagy induction in atrophic muscle cells requires ULK1 activation by TRIM32 through unanchored K63-linked polyubiquitin chains
Science Advances (2019)
Physiological Reviews (2019)
In vitro and in vivo cancer cell apoptosis triggered by competitive binding of Cinchona alkaloids to the RING domain of TRAF6
Bioscience, Biotechnology, and Biochemistry (2019)