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. 2009 Sep 3;461(7260):114-9.
doi: 10.1038/nature08247. Epub 2009 Aug 12.

Direct activation of protein kinases by unanchored polyubiquitin chains

Zong-Ping Xia  1 , Lijun Sun, Xiang Chen, Gabriel Pineda, Xiaomo Jiang, Anirban Adhikari, Wenwen Zeng, Zhijian J Chen
Affiliations

Direct activation of protein kinases by unanchored polyubiquitin chains

Zong-Ping Xia et al. Nature. .

Abstract

TRAF6 is a ubiquitin ligase that is essential for the activation of NF-kappaB and MAP kinases in several signalling pathways, including those emanating from the interleukin 1 and Toll-like receptors. 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 complex. TAK1 in turn phosphorylates and activates IkappaB kinase (IKK), leading to the activation of NF-kappaB. 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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Figures

Figure 1
Figure 1. In vitro reconstitution of TAK1 activation by TRAF6
a, Silver or Coomassie blue staining of purified proteins. b, Top: diagram of ubiquitin lysine mutants used in the assays. Bottom: purified TAK1 kinase complex was incubated with E1, Ubc13/Uev1A, TRAF6 and ubiquitin or its mutants in the presence of ATP for 1 hour at 30°C. Aliquots of the reaction products were analyzed by immunoblotting with an antibody against TAK1, p-TAK1 or ubiquitin. c, TAK1 activated by TRAF6-catalyzed ubiquitination as shown in (b) (lanes 3 &4) was treated with lamda protein phosphatase and then analyzed by immunoblotting.
Figure 2
Figure 2. Direct activation of TAK1 by unanchored polyubiquitin chains
a, Ubiquitination reaction was carried out in the presence of E1, Ubc13/Uev1A, TRAF6 and ubiquitin, then E1 and E2 were inactivated with NEM (step 1) before incubation with the TAK1 complex in the presence of ATP to measure TAK1 activation (step 2). b, Ubiquitination reactions were carried out as in step 1 in (a), then incubated with TAK1 and its substrate MKK6(K82A). c, Ubiquitination mixtures were incubated with nickel-NTA beads, then the bound and unbound fractions were tested for stimulation of TAK1. d, Top: procedure for purification of TAK1 activator; bottom: the Mono S fractions were analyzed for TAK1 activation and by silver staining and immunoblotting. e, Different concentrations of purified polyubiquitin chains (Mono S fraction 15 in panel d) were tested for TAK1 activation. f, K63- and K48-linked polyubiquitin chains were tested for TAK1 activation.
Figure 3
Figure 3. IL-1β induces the synthesis of unanchored polyubiquitin chains to activate TAK1
a, Purified polyUb chains were pre-incubated with the indicated enzymes and then tested for TAK1 activation. b, HEK293T/IL-1R cells were stimulated with IL-1β before cell lysates were immunoprecipitated with a TAB2 antibody. The precipitated materials were incubated with isopeptidase T and then analyzed by immunoblotting. c, A model of polyUb-induced TAK1 autophosphorylation. d, Purified polyUb was incubated with the wild-type TAK1 and/or TAK1(K63W) complexes in the presence of ATP. e, TAK1 complexes containing wild-type (WT) or C670A/C673A (C>A) mutant of TAB2 were incubated with polyUb and MKK6(K82A) to measure TAK1 activation. f, Similar to e, except that the TAK1 complexes contained wild-type (WT) or T187A mutant of TAK1.
Figure 4
Figure 4. Regulation of IKK by unanchored polyUb chains and CYLD
a. Ubiquitination reactions containing TRAF6 and Ubc13/Uev1A or Ubc5 were analyzed by immunoblotting with the indicated antibodies. b. Aliquots of reaction mixtures from (a) were denatured by heating in 1% SDS, then diluted to 0.1% SDS before immunoprecipitation with a TRAF6 antibody. c, PolyUb synthesized in the presence of TRAF6 and Ubc5 were pre-incubated with isopeptidase T or its mutant C335A, then incubated with the IKK complex and IκBα to measure IKK activation. d. Polyubiquitinated TRAF6 (Ubn-TRAF6) was synthesized in the presence of Ubc5, then incubated with the indicated DUBs before immunoblotting. e, Linear polyubiquitination of NEMO (Ubn-NEMO) was carried out in the presence of Ubc5 and HOIL-1L/HOIP (E3), then Ubn-NEMO was incubated with the indicated DUBs followed by immunoblotting.
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