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TAK1 is a ubiquitin-dependent kinase of MKK and IKK


TRAF6 is a signal transducer that activates IκB kinase (IKK) and Jun amino-terminal kinase (JNK) in response to pro-inflammatory mediators such as interleukin-1 (IL-1) and lipopolysaccharides (LPS)1,2,3,4. IKK activation by TRAF6 requires two intermediary factors, TRAF6-regulated IKK activator 1 (TRIKA1) and TRIKA2 (ref. 5). TRIKA1 is a dimeric ubiquitin-conjugating enzyme complex composed of Ubc13 and Uev1A (or the functionally equivalent Mms2). This Ubc complex, together with TRAF6, catalyses the formation of a Lys 63 (K63)-linked polyubiquitin chain that mediates IKK activation through a unique proteasome-independent mechanism5. Here we report the purification and identification of TRIKA2, which is composed of TAK1, TAB1 and TAB2, a protein kinase complex previously implicated in IKK activation through an unknown mechanism6,7. We find that the TAK1 kinase complex phosphorylates and activates IKK in a manner that depends on TRAF6 and Ubc13–Uev1A. Moreover, the activity of TAK1 to phosphorylate MKK6, which activates the JNK–p38 kinase pathway, is directly regulated by K63-linked polyubiquitination. We also provide evidence that TRAF6 is conjugated by the K63 polyubiquitin chains. These results indicate that ubiquitination has an important regulatory role in stress response pathways, including those of IKK and JNK.

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Figure 1: Identification of TRIKA2 as the TAK1–TAB1–TAB2 complex.
Figure 2: The TAK1 complex is both sufficient and necessary to activate IKK in conjunction with TRAF6 and Ubc13–Uev1A.
Figure 3: TAK1 is a ubiquitin-dependent kinase of the MKK–JNK pathway.
Figure 4: K63-linked polyubiquitination of TRAF6.

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We thank C. Pickart for providing expression constructs encoding ubiquitin lysine mutants and F. Lee for providing cDNAs encoding JNK, c-Jun and c-Jun (S63A/S73A). We also thank E. Spencer, L. Yang and A. Braun for technical support, and A. Tizenor and S. Johnson for graphic work. We are grateful to E. Olson, T. Maniatis and J. Jiang for critically reading the manuscript. This work was supported in part by grants from the National Institute of Health and the Welch Foundation. Z.J.C. is a Searle Scholar supported by The Chicago Community Trust.

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Correspondence to Zhijian J. Chen.

Supplementary information

Methods: Purification of IKK

HeLa cell extracts (S100) and the initial fractionation was carried out as described for TRIKA2, except that IKK was eluted from Q-Sepharose in Buffer A containing 0.3M NaCl following elution of TRIKA2 with Buffer A containing 0.21M NaCl. The eluted proteins were precipitated by 40% ammonium sulfate and dialyzed. IKK was then bound to ATP-Sepharose (Upstate Biotech Inc.) and eluted with 10 mM ATP according to the manufacture’s instruction. The partially purified IKK fractions were loaded onto a Heparin-Sepharose column (Applied Biosystems, Inc.) preequilibrated in Buffer D, and eluted with a 5 ml linear gradient of NaCl (0-0.5M) in Buffer D. The fractions containing IKK activity were pooled and loaded onto a MonoQ column using the SMART system (Pharmacia), and then eluted with a 3 ml linear gradient of NaCl (0.05-0.4M) in Buffer D. Silver staining and immunoblotting analyses of the IKK complex thus purified revealed that it contained predominantly IKKa, IKKb, and NEMO, in addition to some minor contaminants. In experiments requiring homogeneous IKK complex (Fig. 2b), the IKK-containing fraction was incubated with a Nemo-specific monoclonal antibody immobilized on Protein A/G-Sepharose for 2 hours at 4oC in the presence of Buffer E (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.25% NP40). Following extensive wash in Buffer E, the beads were used for kinase assays or eluted in SDS sample buffer for silver staining and immunoblotting analyses.

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Wang, C., Deng, L., Hong, M. et al. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412, 346–351 (2001).

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