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10. Fitzgerald, K.A. et al. Nat. Immunol. 4, 491–496 (2003).

11. Datta, S. et al. J. Immunol. 184, 1484–1491 (2010).

8. Hartupee, J., Liu, C., Novotny, M., Li, X. & Hamilton, T. J. Immunol. 179, 4135–4141 (2007).

9. Hartupee, J. et al. J. Immunol. 182, 1660–1666 (2009).

6. Swaidani, S. et al. J. Immunol. 182, 1631–1640 (2009).

7. Liu, C. et al. Sci. Signal. 2, ra63 (2009).

IKKα takes control of canonical NF-kB activationChristiane Pelzer & Margot Thome

The kinase IKKa has a well-described activating role in the noncanonical transcription factor NF-kB pathway. Evidence now suggests that IKKa also inhibits the canonical NF-kB pathway by phosphorylating the scaffold protein TAX1BP1 to promote the assembly of the A20 ubiquitin-editing complex.

Christiane Pelzer and Margot Thome are in

the Department of Biochemistry, University of

Lausanne, epalinges, Switzerland.

e-mail: margot.thomemiazza@unil.ch

NF-kB represents a family of transcrip-tion factors that are essential for efficient

immune responses. NF-kB is activated by receptors of the innate and adaptive immune systems and controls the expression of a variety of genes, including those encoding proinflam-matory cytokines, antiapoptotic proteins and cell-cycle regulators. Activation of NF-kB1 and NF-kB2 family members can be achieved by two pathways. The canonical (or ‘classical’) NF-kB pathway is triggered by receptors such as tumor necrosis factor receptor 1 (TNFR1), Toll-like receptors or the interleukin 1 (IL-1) recep-tor, which promote proinflammatory immune responses. In this pathway, entry of NF-kB1 into the nucleus is controlled by the IKK com-plex, composed of two active subunits (IKKα and IKKβ) and one regulatory subunit (IKKγ; also known as NEMO)1. The IKK complex phosphorylates and thereby tags the NF-kB1 inhibitor IkBα for proteasomal degradation. The noncanonical (or ‘alternative’) NF-kB path-way, in contrast, is triggered by receptors of the TNFR family, such as BAFFR and CD40 (ref. 2). Activation of NF-kB by those receptors requires only the IKK subunit IKKα, which phosphorylates the NF-kB2 precursor pro-tein p100 to induce its partial processing into the active p52 form. Together with the NF-kB subunit RelB, p52 regulates genes encoding molecules involved in B cell survival and lym-phoid organogenesis. Constitutive activation of NF-kB signaling has been associated with various cancers and autoimmune diseases; therefore, these signaling mechanisms must be tightly controlled to ensure transient signaling in response to receptor triggering. One level of control is achieved by the NF-kB-dependent expression of IkBα, which is part of a negative feedback loop that terminates canonical NF-kB signals. Another level of control is achieved

by the inducible expression and activation of deubiquitinases such as CYLD and A20, which terminate the signal by cleaving polyubiquitin chains that are added to signaling components during canonical NF-kB activation. In this issue of Nature Immunology, Shembade et al. have identified a mechanism that controls the A20-mediated negative regulation of the canonical NF-kB pathway3.

A20 was originally described as a ubiquitin-editing enzyme with dual effects on the TNFR signaling component RIP1, a serine-threonine kinase that becomes polyubiquitinated after TNFR triggering. A20 has a deubiquitinase activity that cleaves Lys63 (K63) polyubiquitin chains from RIP1, as well as an E3 ubiquitin ligase activity that subsequently assembles K48-linked ubiquitin chains onto RIP1 and thereby triggers its degradation4. It has also been shown that A20 inhibits NF-kB signaling by disrupting E2-E3 ubiquitin enzyme com-plexes, leading to the degradation of the E2 enzymes UBC13 and UbcH5c by the 26S pro-teasome5. Interestingly, A20 does not act alone but instead acts as part of the so-called A20 ubiquitin-editing complex that also includes the scaffold protein TAX1BP1 and the E3 ligases Itch and RNF11 (refs. 6,7).

The proposal of an essential role for TAX1BP1 as a component of the A20 ubiquitin-editing complex is supported by the observation that deficiency in TAX1BP1 leads to more NF-kB activation after stimulation with cytokines or lipopolysaccharide, due to enhanced K63-linked ubiquitination of RIP1 or the ubiq-uitin ligase TRAF6, respectively8,9. TAX1BP1 contains several structural domains: an N-terminal SKICH domain (skeletal muscle– and kidney-enriched inositol phosphatase carboxyl homology domain), followed by three coiled-coil domains and two C-terminal ubiquitin-binding zinc-finger domains, UBZ1 and UBZ2. These contain highly conserved PPXY motifs (Pro-Pro-X-Tyr, where ‘X’ is any amino acid), which are known to medi-ate binding to proteins that contain a WW

(Trp-Trp) domain10. One such protein, the HECT (homologous to E6 carboxyl termi-nus) domain–containing E3 ligase Itch, binds to TAX1BP1 through the PPXY motifs and thereby functions as an essential cofactor in the A20 ubiquitin-editing complex6. Another component that interacts with the A20-TAX1BP1-Itch complex and is necessary for the termination of NF-kB signaling is the E3 ligase RNF11 (ref. 7). The recognition of the substrates of the A20 ubiquitin-editing com-plex is probably mediated by the zinc-finger domains of TAX1BP1, which are important for the recruitment of RIP1 and TRAF6 and for the inhibitory effect of TAX1BP1 in TNF- or IL-1-induced signaling6,9.

Collectively, such studies support the idea of a critical role for the A20 ubiquitin-editing com-plex in the negative regulation of the canonical NF-kB pathway, but several important ques-tions have remained unanswered. What are the mechanisms that control the assembly of the A20 ubiquitin-editing complex? And how do receptors that trigger canonical NF-kB activa-tion concurrently initiate assembly of this com-plex to limit the signal in amplitude and time?

In this issue of Nature Immunology, Shembade et al. now identify IKKα-mediated phosphorylation of TAX1BP1 as the key event in the stimulation-induced assembly of the A20 ubiquitin-editing complex3. Through superb biochemical work, the authors show that TAX1BP1 is inducibly phosphorylated in an IKKα-dependent manner on Ser593 and addi-tionally on Ser624. Using phosphorylation-deficient mutants and mutants that mimic the phosphorylated protein (phospho-mimetic mutants) in combination with silencing app-roaches, the authors then demonstrate a crucial role for IKKα-mediated phosphorylation of TAX1BP1 in the assembly of the A20-TAX1BP1-RNF11-Itch complex and its recruitment to the ubiquitin ligases TRAF2 or TRAF6, which link the complex to the activated TNFR or IL-1 receptor complex, respectively (Fig. 1). How exactly TAX1BP1 phosphorylation promotes

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the present study3, together with the pheno-type of mice expressing a catalytically inactive form of IKKα11, suggest that IKKβ inhibitors with IKKα cross-reactivity might have com-promised efficiency and might potentially even exacerbate inflammatory responses.

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

1. Israël, A. Cold Spring Harb. Perspect. Biol. 2, a000158 (2010).

2. Häcker, H. & Karin, M. Sci. STKE 2006, re13 (2006).3. Shembade, N., Pujari, R., Harhaj, N.S., Abbott, D.w. &

Harhaj, e.w. Nat. Immunol. 12, 834–843 (2011).4. wertz, I.e. et al. Nature 430, 694–699 (2004).5. Shembade, N., Ma, A. & Harhaj, e.w. Science 327,

1135–1139 (2010).6. Shembade, N. et al. Nat. Immunol. 9, 254–262 (2008).7. Shembade, N., Parvatiyar, K., Harhaj, N.S. & Harhaj, e.w.

EMBO J. 28, 513–522 (2009).8. Shembade, N., Harhaj, N.S., Liebl, D.J. & Harhaj, e.w.

EMBO J. 26, 3910–3922 (2007).9. Iha, H. et al. EMBO J. 27, 629–641 (2008).10. verstrepen, L., verhelst, K., Carpentier, I. & Beyaert, R.

Trends Biochem. Sci. 36, 347–354 (2011).11. Lawrence, T., Bebien, M., Liu, G.Y., Nizet, v. & Karin, M.

Nature 434, 1138–1143 (2005).12. Li, Q. et al. Proc. Natl. Acad. Sci. USA 102, 12425–12430

(2005).13. Sun, S.C. & Yamaoka, S. Oncogene 24, 5952–5964

(2005).14. Lee, D.F. & Hung, M.C. Clin. Cancer Res. 14, 5656–5662

(2008).

intriguing question raised by this work is how exactly the receptors activate IKKα. Does this happen as part of the activation of the IKK complex, which comprises IKKα, IKKβ and IKKγ? The authors’ findings strongly chal-lenge this idea, as cells that lack expression of IKKβ or IKKγ show normal TAX1BP1 phos-phorylation and intact assembly of the A20 ubiquitin-editing complex. This suggests that there must be a signaling event that specifi-cally links the receptors to IKKα, which may become active as a monomer or dimer or as part of a larger complex containing additional components. Careful analysis of the size, composition and subcellular localization of IKKα-containing complexes in the presence and absence of IKKβ and IKKγ should give further molecular insight into the nature of this puzzling new pathway. Finally, these find-ings should also stimulate reflections on the desired specificities and possible side effects of inhibitors of IKKα and/or IKKβ to be used as anti- inflammatory drugs. Such inhibitors are now being tested in preclinical trials aimed at treating inflammation- mediated human dis-eases, including cancers with oncogenic acti-vation of the NF-kB pathway14. The results of

the formation of the ubiquitin-editing complex is not yet understood, but the authors propose a model in which phosphorylation of TAX1BP1 leads to exposure of its zinc fingers and PPXY motifs to allow the inducible recruitment of Itch, RNF11 and A20.

IKKα is a well-known component of the noncanonical NF-kB pathway; its inhibitory role in canonical NF-kB activation may there-fore seem surprising. However, two published studies of IKKα-deficient mice have already reported enhanced proinflammatory responses of IKKα-deficient macrophages, which sup-ports the idea of a role for IKKα in the limita-tion of inflammatory signals11,12. The authors of the present study also see a delay in IKKα-mediated phosphorylation of TAX1BP1 in embryonic fibroblasts deficient in the NF-kB-inducing kinase NIK, which suggests that both IKKα and NIK contribute to the negative regu-lation of the canonical NF-kB pathway3.

TAX1BP1 is also known for its ability to interact with the oncoprotein Tax of human T cell leukemia virus type I, a retrovirus that is causally linked to adult T cell leukemia and lymphoma. Tax induces persistent activation of NF-kB by constitutively associating with IKKγ to promote IKK-mediated phosphorylation of IkBα13. It is known that Tax can bypass nega-tive feedback inhibition of the NF-kB pathway by associating with TAX1BP1 and inhibiting A20 function5,6, but the underlying mecha-nism has remained elusive. Shembade et al. now show that Tax acts as a negative regulator of TAX1BP1 phosphorylation by suppressing IKKα-mediated TAX1BP1 phosphorylation3. This mechanism that underlies this function of Tax seems to make an essential contribu-tion to the NF-kB-promoting function of Tax, as a phospho-mimetic mutant of TAX1BP1 efficiently inhibits Tax-mediated activation of NF-kB. How exactly Tax prevents IKKα from phosphorylating TAX1BP1 remains unclear and should be further assessed; Tax may, for example, compete with IKKα for binding to TAX1BP1 or relocalize TAX1BP1 to a sub-cellular compartment that is inaccessible to IKKα-mediated phosphorylation.

In recent years, research on NF-kB sig-naling has made tremendous progress in elucidating the signaling events that control not only its onset but also the subsequent inactivation of NF-kB activation that is cru-cial for limiting the amplitude and duration of the proinflammatory signal. Although a critical role for the A20 ubiquitin-editing complex has already been established, this study now makes a substantial advance in the understanding of how NF-kB-inducing recep-tors trigger its formation and reveals a sur-prising role for IKKα in this process. A very

P P

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Figure 1 IKKα inhibits the canonical NF-kB pathway via phosphorylation of TAX1BP1. Triggering of TNFR or the receptor for IL-1 (IL-1R) induces the assembly of signaling complexes that promote activation of the IKK complex, which results in the phosphorylation (gray P) and subsequent degradation of IkB. The recruitment and activation of the IKK complex depend on K63-linked polyubiquitination of RIP1 and TRAF6, respectively. The A20 ubiquitin-editing complex, composed of A20, Itch, RNF11 and TAX1BP1, inhibits TNF- and IL-1-induced activation of NF-kB by cleaving the K63-linked polyubiquitin chains required for signal transduction. New work by Shembade et al.3 shows that TNF- and IL-1-induced activation of the IKK complex is time-restricted by a negative feedback mechanism that depends on IKKα-mediated phosphorylation of TAX1BP1, which promotes assembly of the active A20 ubiquitin-editing complex. The new work also shows that Tax inhibits IKKα-mediated phosphorylation of TAX1BP1, which prevents assembly of the A20 ubiquitin-editing complex and thereby favors persistent NF-kB activation. TRADD and MyD88 are adaptors.

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