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De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling


NF-κB transcription factors mediate the effects of pro-inflammatory cytokines such as tumour necrosis factor-α and interleukin-1β1. Failure to downregulate NF-κB transcriptional activity results in chronic inflammation and cell death, as observed in A20-deficient mice2. A20 is a potent inhibitor of NF-κB signalling, but its mechanism of action is unknown2. Here we show that A20 downregulates NF-κB signalling through the cooperative activity of its two ubiquitin-editing domains. The amino-terminal domain of A20, which is a de-ubiquitinating (DUB) enzyme of the OTU (ovarian tumour) family3, removes lysine-63 (K63)-linked ubiquitin chains from receptor interacting protein (RIP), an essential mediator of the proximal TNF receptor 1 (TNFR1) signalling complex4,5. The carboxy-terminal domain of A20, composed of seven C2/C2 zinc fingers6, then functions as a ubiquitin ligase by polyubiquitinating RIP with K48-linked ubiquitin chains, thereby targeting RIP for proteasomal degradation. Here we define a novel ubiquitin ligase domain and identify two sequential mechanisms by which A20 downregulates NF-κB signalling. We also provide an example of a protein containing separate ubiquitin ligase and DUB domains, both of which participate in mediating a distinct regulatory effect.

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  1. 1

    Dixit, V. & Mak, T. W. NF-kappaB signaling. Many roads lead to Madrid. Cell 111, 615–619 (2002)

  2. 2

    Lee, E. G. et al. Failure to regulate TNF-induced NF-kappaB and cell death responses in A20-deficient mice. Science 289, 2350–2354 (2000)

  3. 3

    Makarova, K. S., Aravind, L. & Koonin, E. V. A novel superfamily of predicted cysteine proteases from eukaryotes, viruses and Chlamydia pneumoniae. Trends Biochem. Sci. 25, 50–52 (2000)

  4. 4

    Ting, A. T., Pimentel-Muinos, F. X. & Seed, B. RIP mediates tumor necrosis factor receptor 1 activation of NF-kappaB but not Fas/APO-1-initiated apoptosis. EMBO J. 15, 6189–6196 (1996)

  5. 5

    Kelliher, M. A. et al. The death domain kinase RIP mediates the TNF-induced NF-kappaB signal. Immunity 8, 297–303 (1998)

  6. 6

    Opipari, A. W. Jr, Boguski, M. S. & Dixit, V. M. The A20 cDNA induced by tumor necrosis factor alpha encodes a novel type of zinc finger protein. J. Biol. Chem. 265, 14705–14708 (1990)

  7. 7

    Aravind, L., Dixit, V. M. & Koonin, E. V. Apoptotic molecular machinery: vastly increased complexity in vertebrates revealed by genome comparisons. Science 291, 1279–1284 (2001)

  8. 8

    Pickart, C. M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 503–533 (2001)

  9. 9

    Weissman, A. M. Themes and variations on ubiquitylation. Nature Rev. Mol. Cell Biol. 2, 169–178 (2001)

  10. 10

    Yeh, W. C. et al. Early lethality, functional NF-kappaB activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice. Immunity 7, 715–725 (1997)

  11. 11

    Lee, S. Y. et al. TRAF2 is essential for JNK but not NF-kappaB activation and regulates lymphocyte proliferation and survival. Immunity 7, 703–713 (1997)

  12. 12

    Kieser, A., Kaiser, C. & Hammerschmidt, W. LMP1 signal transduction differs substantially from TNF receptor 1 signaling in the molecular functions of TRADD and TRAF2. EMBO J. 18, 2511–2521 (1999)

  13. 13

    Tada, K. et al. Critical roles of TRAF2 and TRAF5 in tumor necrosis factor-induced NF-kappa B activation and protection from cell death. J. Biol. Chem. 276, 36530–36534 (2001)

  14. 14

    Hsu, H., Huang, J., Shu, H. B., Baichwal, V. & Goeddel, D. V. TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex. Immunity 4, 387–396 (1996)

  15. 15

    Zhang, S. Q., Kovalenko, A., Cantarella, G. & Wallach, D. Recruitment of the IKK signalosome to the p55 TNF receptor: RIP and A20 bind to NEMO (IKKgamma) upon receptor stimulation. Immunity 12, 301–311 (2000)

  16. 16

    Legler, D. F., Micheau, O., Doucey, M. A., Tschopp, J. & Bron, C. Recruitment of TNF receptor 1 to lipid rafts is essential for TNFalpha-mediated NF-kappaB activation. Immunity 18, 655–664 (2003)

  17. 17

    Micheau, O. & Tschopp, J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell 114, 181–190 (2003)

  18. 18

    Balakirev, M. Y., Tcherniuk, S. O., Jaquinod, M. & Chroboczek, J. Otubains: a new family of cysteine proteases in the ubiquitin pathway. EMBO Rep. 4, 517–522 (2003)

  19. 19

    Borodovsky, A. et al. Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family. Chem. Biol. 9, 1149–1159 (2002)

  20. 20

    Evans, P. C. et al. A novel type of deubiquitinating enzyme. J. Biol. Chem. 278, 23180–23186 (2003)

  21. 21

    Evans, P. C. et al. Zinc-finger protein A20, a regulator of inflammation and cell survival, has de-ubiquitinating activity. Biochem. J. 378, 727–734 (2004)

  22. 22

    Deng, L. et al. Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103, 351–361 (2000)

  23. 23

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

  24. 24

    Shi, C. S. & Kehrl, J. H. Tumor necrosis factor (TNF)-induced germinal center kinase-related (GCKR) and stress-activated protein kinase (SAPK) activation depends upon the E2/E3 complex Ubc13-Uev1A/TNF receptor-associated factor 2 (TRAF2). J. Biol. Chem. 278, 15429–15434 (2003)

  25. 25

    Wilkinson, K. D. Signal transduction: aspirin, ubiquitin and cancer. Nature 424, 738–739 (2003)

  26. 26

    Zhou, H. et al. Bcl10 activates the NF-kappaB pathway through ubiquitination of NEMO. Nature 427, 167–171 (2004)

  27. 27

    Hsu, H., Shu, H. B., Pan, M. G. & Goeddel, D. V. TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 84, 299–308 (1996)

  28. 28

    Devin, A. et al. The distinct roles of TRAF2 and RIP in IKK activation by TNF-R1: TRAF2 recruits IKK to TNF-R1 while RIP mediates IKK activation. Immunity 12, 419–429 (2000)

  29. 29

    Poyet, J. L. et al. Activation of the Ikappa B kinases by RIP via IKKgamma /NEMO-mediated oligomerization. J. Biol. Chem. 275, 37966–37977 (2000)

  30. 30

    Devin, A. et al. The alpha and beta subunits of IkappaB kinase (IKK) mediate TRAF2-dependent IKK recruitment to tumor necrosis factor (TNF) receptor 1 in response to TNF. Mol. Cell. Biol. 21, 3986–3994 (2001)

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The authors would like to thank R. Deshaies, T. Mayor, R. Feldman and members of the Dixit Lab for helpful discussions, T. Mayor and M. Petroski for reagents, D. Yansura for technical assistance, and K. Newton for editorial assistance. We acknowledge the contributions from colleagues whose work has been cited indirectly owing to space limitations. I.E.W. was supported in part by a PSTP fellowship from the University of California at Davis.

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Correspondence to Vishva M. Dixit.

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This document contains supplementary methods, supplementary figures 1-7 and supplementary references. (DOC 1395 kb)

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Figure 1: A20 catalyses in vitro ubiquitination.
Figure 2: A20 ubiquitinates and destabilizes RIP.
Figure 3: A20 de-ubiquitinates RIP.
Figure 4: A20-mediated RIP de-ubiquitination is a prerequisite for A20-induced degradation.


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