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setting will probably induce T cells with a range of intermediate phenotypes. Nevertheless, the classical idea that T cells can become terminally differentiated predicts that key regulatory cytokines could generate T cells with a relatively stable phenotype. Whether the IL-9–IL-10–producing cells represent a transient T cell phenotype or a terminally differentiated phenotype remains to be
determined. Scientists are driven to empirically categorize what is observed. Perhaps it would be better to follow the example that which is being studied and be more flexible in viewing T cell differentiation and function. 1. O’Shea, J.J. et al. Nat. Immunol. 9, 450–453 (2008). 2. Murphy, E. et al. J. Exp. Med. 183, 901–913 (1996).
3. Veldhoen, M. et al. Nat. Immunol. 9, 1341–1346 (2008). 4. Dardalhon, V. et al. Nat. Immunol. 9, 1347–1355 (2008). 5. Wei, J. et al. Proc. Natl. Acad. Sci. USA 104, 18169– 18174 (2007). 6. Mantel, P.Y. et al. PLoS Biol. 5, e329 (2007). 7. Zhou, L. et al. Nature 453, 236–240 (2008).. 8. Izcue, A. et al Immunity 28, 559–570 (2008). 9. Mucida, D. et al. Science 317, 256–260 (2007). 10. Cliffe, L.J. et al. Adv. Parasitol. 57, 255–307 (2004).
Self-termination of the terminator David Wallach & Andrew Kovalenko The protein kinase NIK is regulated by a complex of ubiquitin ligases that destroys it. When NIK-activating receptors are triggered, the ubiquitin ligase complex self-destructs.
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embers of the tumor necrosis factor– nerve growth factor (TNF-NGF) receptor ‘superfamily’ mediate their numerous effects on cell function by initiating changes in gene expression, for which they signal by activating protein kinases, or by inducing cell death, which is mediated by caspase activation. Activation of caspases by the receptors occurs through direct binding of the caspases to receptor-associated adaptor proteins. In contrast, the protein kinases by which the receptors signal for gene regulation are not directly affected by the receptors; instead, their activation occurs through the intermediary functions of proteins with ubiquitin-ligase activity. Of those ubiquitinligase proteins, several belong to the TNF receptor–associated factor (TRAF) family, and two, cIAP1 and cIAP2, belong to the family of inhibitor of apoptosis proteins (IAPs), which associate with the receptors by binding to TRAF2. How this intermediary function is exerted is still poorly understood. The studies of Karin and Cheng and their collaborators in this issue of Nature Immunology1,2 provide new information on the function of those ubiquitin ligases in regulating the expression and function of NIK, a serinethreonine kinase structurally related to the MAP3 kinases that is crucial to many of the regulatory effects of the receptors on lymphocyte development and function. Like some other protein kinases that mediate gene regulation through TNF-NGF receptors, NIK binds directly to TRAFs. Its
David Wallach and Andrew Kovalenko are in the Department of Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel. e-mail:
[email protected]
carboxy-terminal region contains a cryptic TRAF2-binding motif3, and its amino-terminal region binds TRAF3 (ref. 4). However, in contrast to the activation of other protein kinases that signal for TNF-NGF receptor effects, the binding of NIK to TRAF3 is apparently constitutive, and the activity of NIK seems to be suppressed by the TRAFs, as deficiency of either TRAF2 or TRAF3 results in chronic activation of NIK. NIK is also spontaneously activated when expression of both cIAP1 and cIAP2 is blocked. The activation of NIK resulting from deficiency of these various ubiquitin ligases is associated with higher NIK expression5. In mice, knockout of either TRAF2 or TRAF3 results in perinatal death associated with lymphoid abnormalities and the generation of inflammatory mediators. The studies of Karin and of Cheng show that on a NIKdeficient background, this pathology fails to develop, which suggests that it arises mainly if not entirely because of chronic activation of NIK. Such activation also occurs in tumor cells deficient in TRAF2 or TRAF3 or both cIAP1 and cIAP2 (ref. 6). It is also found in B cells expressing a NIK mutant deficient in its TRAF3-binding domain and has been found to facilitate their growth and survival7. How can deficiency of different ubiquitin ligases result in higher expression of the same protein? The inverse relationship between the expression of NIK and of TRAF2 or TRAF3 is particularly unexpected because the ubiquitin linkage dictated by these two TRAFs is not of the lysine 48 (K48) type but rather is of the K63 type, and such polyubiquitination is usually related to functions other than proteasomal degradation, such as protein activation. On the basis of their findings
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and those of others, both Karin and Cheng suggest that the different ligases act together in a multiprotein complex in which each of these proteins makes a distinct functional contribution. As TRAF2 and TRAF3 can form heterodimers, the association of TRAF3 with the amino-terminal region of NIK allows it to recruit TRAF2 to the kinase along with its associated cIAP1 or cIAP2. The recruited cIAP1 and cIAP2 then catalyze NIK’s ubiquitination and degradation (Fig. 1a). Studies have shown that several receptors of the TNF-NGF family that activate NIK also enhance its expression4. In the case of CD40 ligand and B cell–activation factor, enhancement of NIK expression is associated with induced proteasome-mediated degradation of TRAF2 and TRAF3, whereas in cells treated with the ligand of the TNF family, TWEAK, it is associated with induced lysosomal degradation of TRAF2 and cIAP1 (refs. 5,8). Findings presented in the study by Karin and colleagues suggest that TRAF2, beyond participating in the ‘hinge’ that links cIAP1 and cIAP2 to NIK, also serves as a conduit for signals that regulate NIK degradation and that it does so by imposing on cIAP1 and cIAP2 a change in their target specificity2. Triggering by CD40 endows TRAF2 with the ability to direct the K48-linked ubiquitinating activity of cIAP2 against TRAF3 and probably also against TRAF2, causing their degradation. This effect of TRAF2 on the K48-linked ubiquitinating activity of cIAP2 correlates with more K63-linked polyubiquitination of the latter. Thus, whereas in unstimulated cells the NIK-associated complex of ubiquitin ligases acts as a continuous terminator of NIK (Fig. 1a), after triggering by CD40, this complex redirects its destructive activity toward
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Figure 1 Regulation of NIK expression and function. (a–c) The ‘brake pedal’. (a) In unstimulated cells, the ubiquitin ligase cIAP1 or cIAP2 destroys NIK by mediating its K48-linked ubiquitination (K48(Ub)n). A molecular ‘hinge’ comprising the ubiquitin ligases TRAF2 and TRAF3, by binding to cIAP1 and cIAP2, brings those last two molecules into close contact with NIK. (b,c) In response to receptor stimulation, TRAF2, apparently by mediating K63-linked ubiquitination of cIAP1 and/or cIAP2, imposes a change in the latter’s substrate specificity. Thus, rather than ubiquitinating NIK, cIAP1 and/or cIAP2 ubiquitinates TRAF3 and then TRAF2. This receptor-induced self-termination of the terminating complex of NIK facilitates a considerable increase in cellular NIK. (d–f) The ‘gas pedal’. In the regulation of various other IAPs, self-destruction serves to control only the amount of the target protein, not the triggering of its activity. A possible mechanism of NIK activation is presented here. (d) NIK forms a closed structure in which its amino (N) and carboxyl (C) termini associate. (e,f) ‘Opening’ the NIK molecules allows them to self-associate and then activate each other by transphosphorylation. The open structure of NIK exposes a cryptic TRAF2-binding site at its carboxyl terminus, which raises the possibility that TRAF2 controls the transition of NIK to the active form. (g) NIK-mediated signaling. Once activated, NIK phosphorylates the kinase IKK1, which in turn phosphorylates the p100 precursor form of NF-κB1 (p100, which associates with NIK), thus targeting p100 for partial proteasomal degradation (the alternative NF-κB pathway). This results in the generation of dimers of the NF-κB proteins p52 and RelB (p52-RelB), as well as the release of p50RelA and p50-RelB dimers that until then were kept inactive by their association with p100 (ref. 16). The receptors that stimulate NIK also use NIK to signal the generation of p50-RelA by activating IKK2, which in turn dictates ubiquitination and degradation of the NF-κB inhibitory protein IκB (the canonical NF-κB pathway14). NEMO, NF-kB essential modifier (also called IKKγ).
self-termination, thereby allowing cellular NIK to increase (Fig. 1b,c). The ability of scientists to analyze the mechanisms that regulate NIK function is restricted by several technical limitations at present. In most cells, the amount of NIK present before stimulation is below the limit of detection. This makes it difficult to monitor NIK or to isolate the signaling complexes in which it participates at the times of greatest interest: before stimulation, when it supposedly interacts with proteins that dictate its degradation, and shortly after stimulation, when its activity is triggered. Moreover, the assays available for the function distinctive of NIK—triggering of the alternative transcription factor NF-κB pathway (Fig. 1)—are rather insensitive, so the exact times of its initiation and termination cannot be determined. Finally, attempts to generate functional recombinant NIK have so far failed; thus, it is not yet possible to reconstitute the
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NIK-containing molecular complexes from pure protein preparations. It is therefore hard to tell for sure, for example, whether the cIAP1-cIAP2–induced ubiquitination of NIK occurs directly or through another ubiquitin ligase as yet unidentified. Because of the limitations noted above, the model proposed by both Karin and Cheng is, like most knowledge of NIK, based on acellular (that is, in vitro) tests with impure protein preparations and on the use of cells transfected with NIK cDNA or treated with a proteasomal inhibitor or stimulated by ligands of the TNF family to enhance NIK expression. Because the knowledge is based on test systems that correspond only partially to reality, it cannot be expected to provide more than only a partial view of the mechanisms involved. Indeed, the Karin-Cheng model cannot easily account for some findings reported in connection with NIK function. As an example, the role suggested for
TRAF2 in transmitting the effect of CD40 on cIAP function is unlikely to account for NIK activation by B cell–activation factor receptor, a receptor that seems incapable of binding TRAF2 and that probably initiates NIK activation by binding TRAF3. Data showing that the TRAF3 RING-finger domain is required for maintaining NIK at a low cellular concentration are also inconsistent with the idea that TRAF3 serves only to link NIK to TRAF2 (ref. 9). When knowledge of a mechanism is at such a primordial stage, much might be gained from comparing it with other, evolutionarily related ones. Many ubiquitin ligases, including other IAPs, self-terminate by inducing their own destruction. However, in those cases, the outcome of the self-termination process is not thought to be enhancement of activity of a particular target protein but rather an increase in total amount of the protein(s) targeted by the ubiquitin ligases. Regulation of the drosophila death-inducing caspase Dronc by the IAP Diap1, for example, is sometimes illustrated by comparison with the way driving speed is controlled by the combined action of the gas and brake pedals of a car. In the case of Dronc, too, the IAP acts as the terminator that (at times of celldeath induction) self-terminates. However, although it alleviates the restraint imposed on the expression of Dronc, self-destruction of Diap1 does not trigger Dronc’s activity. The ‘gas’ that sparks this activation is provided by Dark, a specific allosteric regulator of Dronc. Orthologous ‘gas and brake’ elements also control the action of caspases in the mammalian intrinsic cell-death pathway10,11. Given the ‘gas and brake’ mode of action of other IAPs, it is particularly worth noting that although complete ablation of the ubiquitin-ligase complex that affects NIK both enhances the expression and triggers the activation of this kinase, there is no evidence that the magnitude of increase in NIK expression obtained in response to receptor stimulation alone suffices to initiate NIK activity. The spontaneous activation of NIK when subjected to enforced overexpression does not necessarily correspond to the way such activation occurs after receptor stimulation; in fact, many proteins whose activation in response to triggering of cell surface receptors occurs by allosteric regulation can be artificially activated merely by their overexpression. Nor does the fact that activation of the alternative pathway depends on new protein synthesis necessarily indicate, as both Karin and Cheng suggest, that this activation depends on newly synthesized NIK. There is evidence to suggest that such activation might
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n e w s and v i e w s rather reflect dependence on synthesis of new NF-κB1 molecules12. The idea that NIK may well also be subject to allosteric regulation is indicated by findings that suggest it can attain either a ‘closed’, inactive form in which its amino and carboxyl termini bind to each other or an ‘open’ form in which two adjacent NIK molecules apparently activate each other by transphosphorylation13. Notably, binding of TRAF2 to NIK is enhanced considerably when NIK is prevented from attaining its ‘closed’ form by deletion of its amino terminus3, which suggests that TRAF2 is involved in regulating the structural dynamics of NIK (Fig. 1d–f) as well as the dynamics of NIK’s associations shortly after stimulation14. It may well be that, as in the case of cytokine-induced activation of the STAT1 and STAT3 signaling molecules15, NIK
activation is induced in two phases: initially by allosteric or covalent modification and, above a certain cellular amount of NIK, independently of those modifications. The fact that NIK activity can become seriously harmful once the function of any one of various ubiquitin ligases is arrested poses a challenge to both basic and translational scientists. It indicates the need to develop drugs that can inhibit NIK function and thus provide protection from the pathological effect of mutations or drugs (such as cIAP antagonists) that interfere with the function of these ligases. It also emphasizes the need to overcome the difficulties involved in studying the function and regulation of this protein kinase at its very low endogenous concentration. 1. Zarnegar, B.J. et al. Nat. Immunol. 9, 1371–1378 (2008).
2. Vallabhapurapu, S. et al. Nat. Immunol. 9, 1364–1370 (2008). 3. Malinin, N.L., Boldin, M.P., Kovalenko, A.V. & Wallach, D. Nature 385, 540–544 (1997). 4. Liao, G., Zhang, M., Harhaj, E.W. & Sun, S.C. J. Biol. Chem. 279, 26243–26250 (2004). 5. Varfolomeev, E. & Vucic, D. Cell Cycle 7, 1511–1521 (2008). 6. Xiao, G. et al. Cytokine Growth Factor Rev. 17, 281–293 (2006). 7. Sasaki, Y. et al. Proc. Natl. Acad. Sci. USA 105, 10883– 10888 (2008). 8. Ashwell, J.D. J. Cell Biol. 182, 15–17 (2008). 9. He, J.Q. et al. J. Biol. Chem. 282, 3688–3694 (2007). 10. Vaux, D.L. & Silke, J. Nat. Rev. Mol. Cell Biol. 6, 287– 297 (2005). 11. Steller, H. Cell Death Differ. 15, 1132–1138 (2008). 12. Mordmuller, B. et al. EMBO Rep. 4, 82–87 (2003). 13. Xiao, G. & Sun, S.C. J. Biol. Chem. 275, 21081–21085 (2000). 14. Ramakrishnan, P., Wang, W. & Wallach, D. Immunity 21, 477–489 (2004). 15. Stark, G.R. Cytokine Growth Factor Rev. 18, 419–423 (2007). 16. Basak, S. et al. Cell 128, 369–381 (2007).
RIG-I-like antiviral protein in flies Osamu Takeuchi & Shizuo Akira The function of gene expression in the response of drosophila to viral infection is poorly understood. A report now demonstrates that the helicase Dicer-2 controls antiviral gene expression in addition to RNA interference–mediated gene silencing.
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he immune system senses invasion by viruses and mounts antiviral responses for eliminating the infection using patternrecognition receptors that discriminate self and viral components1,2. In contrast to mammals, in which the type I interferon system is essential for antiviral immunity, insects such as Drosophila melanogaster do not encode a counterpart to type I interferon in their genome. Instead, insects and plants have developed RNA interference as an antiviral mechanism3. Long viral double-stranded RNA (dsRNA) is recognized by members of the Dicer-like endonuclease family, which generate small interfering RNA (siRNA) 21–24 nucleotides in length. These RNA molecules guide specific antiviral silencing by argonaute (AGO) proteins in the RNA-induced silencing complex. In drosophila, the helicase Dicer-2 is essential for the generation of siRNA, whereas Dicer-1 cleaves double-stranded micro-RNA precursors. The drosophila-specific protein r2d2, with tanOsamu Takeuchi and Shizuo Akira are in the Department of Host Defense, World Premier International Immunology Frontier Research Center, and the Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan. e-mail:
[email protected]
dem dsRNA-binding domains, is critical for efficient loading of siRNA onto AGO2 in the RNA-induced silencing complex. It has been shown that Dicer-2-mediated gene silencing is essential for antiviral host defense against viruses such as flock house virus (FHV) and cricket paralysis virus4,5. However, in addition to eliciting silencing of the viral genome, virus infection is known to activate the expression of various genes in drosophila similar to those in mammals. Moreover, the mechanism of gene expression as well as the function of virusinducible proteins are not well understood. In this issue of Nature Immunology, Deddouche et al. demonstrate that Vago, which encodes a small cysteine-rich polypeptide, is expressed in a Dicer-2-dependent way in the fat body in response to infection with drosophila C virus (DCV)6. The Vago protein expressed controls viral propagation in the fat body in DCVinfected flies. Vago was identified as a DCV-inducible gene by microarray analysis. Vago encodes a 160– amino acid protein with a CX20CX4CX10–11 CX7–9CX13–14CCX4C motif (where ‘X’ is any amino acid) that is conserved in invertebrates and whose function has not been identified7. Vago-mutant fly lines are viable and fertile, which indicates that the Vago protein is not involved in the developmental process6. Vago-
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mutant flies infected with DCV have a higher viral load in the fat body than that of infected control wild-type flies. Expression of a Vago transgene in Vago-mutant flies ‘rescues’ the impaired clearance of DCV, which indicates that Vago protein has antiviral activity in response to DCV infection. The authors find that Vago protein has some sequence similarity to granularin, a molluscan polypeptide that opsonizes parasites. However, the mechanism of Vago-mediated control of DCV infection is yet to be clarified. The authors also investigate the molecules responsible for Vago expression and find that Vago expression is considerably impaired in flies lacking Dicer-2. Notably, Vago expression is not impaired in r2d2- or AGO2-deficient flies. Therefore, the impaired Vago expression is not due to silencing mediated by the RNA-induced silencing complex, and Dicer-2 may trigger an unknown signaling pathway to inducing Vago upregulation. Dicer-2 is composed of an amino-terminal DExD/Hbox helicase domain and a carboxy-terminal RNase III domain. The RNase III domain is essential for the generation of siRNA, whereas mutations in the sequence encoding the helicase domain impaired but did not abrogate processing of dsRNA8. A missense mutation in the sequence encoding the Dicer-2 helicase
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