Direct activation of protein kinases by unanchored polyubiquitin chains

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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.

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Figure 1: In vitro reconstitution of TAK1 activation by TRAF6.
Figure 2: Direct activation of TAK1 by unanchored polyubiquitin chains.
Figure 3: IL1β induces the synthesis of unanchored polyubiquitin chains to activate TAK1.
Figure 4: Regulation of IKK by unanchored polyUb chains and CYLD.

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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.

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Correspondence to Zhijian J. Chen.

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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

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