but which is associated with the canonical pathway through which thymopoiesis proceeds is controversial. It is notable that some T cell development occurs in Ikarosdeficient mice. One possibility suggested by Yoshida et al. is that limited development of common lymphoid progenitor and common lymphoid progenitor 2 cells from the LMPP occurs. However, early T lineage progenitors are present at near-normal numbers in Ikaros-deficient mice10, making it likely that T cell development proceeds through a putative early lymphocyte progenitor–to–early T lineage progenitor pathway.
So, although it is known that Icarus is the son of Daedalus, it will be more challenging to establish parent-progeny relationships between early intermediates in the hematopoietic system. Nevertheless, it is reasonable to expect that understanding of early hematopoiesis and the function of Ikaros in this process will continue to increase. The study of Yoshida et al., using sophisticated flow cytometry analysis, state-of-the-art developmental assays and molecular genetics, provides a roadmap approach to meeting this expectation. 1. Yoshida, T., Ng, S.M., Zuniga-Pflucker, J.C. & Georgopoulos, K. Nat. Immunol. 7, 382–391
(2006). Adolfsson, J. et al. Cell 121, 295–306 (2005). Georgopoulos, K. et al. Cell 79, 143–156 (1994). Wang, J.H. et al. Immunity 5, 537–549 (1996). Nichogiannopoulou, A., Trevisan, M., Neben, S., Friedrich, C. & Georgopoulos, K. J. Exp. Med. 190, 1201–1213 (1999). 6. Klug, C.A. et al. Proc. Natl. Acad. Sci. USA 95, 657– 662 (1998). 7. Papathanasiou, P. et al. Immunity 19, 131–144 (2003). 8. Mackarehtschian, K. et al. Immunity 3, 147–161 (1995). 9. Igarashi, H. et al. Immunity 17, 117–130 (2002). 10. Allman, D. et al. Nat. Immunol. 4, 168–174 (2003). 11. Kondo, M., Weissman, I.L. & Akashi, K. Cell 91, 661– 672 (1997). 12. Martin, C.H. et al. Nat. Immunol. 4, 866–873 (2003).
2. 3. 4. 5.
CYLD: deubiquitination-induced TCR signaling Neil Lineberry & C Garrison Fathman Deubiquitinating enzymes remove polyubiquitin chains from and alter the fate of specific target proteins. The CYLD deubiquitinating enzyme regulates proximal T cell receptor signaling in thymocytes by selectively binding to and deubiquitinating the active form of the kinase Lck.
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entral tolerance ensures that peripheral T cells recognize and respond to foreign pathogens rather than to self antigens. In the thymus, developing T cells progress through a stepwise differentiation process until they reach the CD4+CD8+ double-positive (DP) stage, when they first express a newly generated αβ T cell receptor (TCR). TCRs that generate low survival signals after engagement with complexes of self peptide and major histocompatibility complex (MHC) are positively selected, and those that recognize self peptide–MHC complexes with high affinity are negatively selected. TCRs that fail to recognize self peptide–MHC complexes die by neglect. In this issue of Nature Immunology, Reiley and colleagues identify a new participant in the proximal TCR signaling cascade: the deubiquitinating enzyme CYLD1. In peripheral T cells, TCR engagement recruits the Src kinases Fyn and Lck, which phosphorylate the CD3 chains. After its recruitment to phosphorylated CD3 subunits, the Syk kinase Zap70 is phosphorylated and activated by Lck. The signal initiated by these core events is then disseminated through adaptor proteins such as Lat and SLP-76 and ultimately induces global changes in gene transcription and acquisition of effector functions. Responsibility for attenu-
Neil Lineberry and C. Garrison Fathman are in the Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, California 94305, USA. e-mail:
[email protected]
ating this powerful signaling cascade is held by two different regulatory networks. Proteins that remove phosphorylation residues, such as the SHP-1 phosphatase2, prevent activation-induced protein-protein interactions but do not degrade the central protein framework, thereby allowing rapid restoration of signaling. In contrast, proteins of the ubiquitin-proteasome system, such as the Cbl family3, degrade substrate proteins by attaching polymers of the small molecule ubiquitin, thereby terminating signaling until newly synthesized proteins are generated. The ubiquitin-proteasome system has evolved from its humble beginnings as a simple recycling system for old or nonfunctional proteins into a dynamic system also capable of regulating protein activation and subcellular localization. Covalent attachment of ubiquitin to its target, or substrate, is achieved by a stepwise process involving proteins of increasing number and specificity4. Ubiquitin monomers are first bound and activated by E1 activating enzymes. An E1 transfers the ubiquitin monomer to an E2 transferase, also called a ubiquitin carrier enzyme. The E2 transferase and its bound, activated ubiquitin are recruited to an E3 ligase. E3 ligases interact with target proteins and facilitate transfer of the ubiquitin to the ε-amino groups of target residues. Substrate specificity is determined by the ability of the E3 ligase to bind its substrate and the proper E2 transferase carrying an active ubiquitin molecule5. Multiple ubiquitin monomers can be linked in series via ubiquitin lysine residues to form polyubiquitin chains. The position of the ubiquitin lysine
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NEWS AND VIEWS
Figure 1 After engagement of the TCR by self peptide–MHC complexes, phosphorylated Lck is recruited to the CD3 complex, where it phosphorylates and activates Zap70, which then propagates the signal through adaptor proteins and second messengers. The deubiquitinating enzyme CYLD binds to and deubiquitinates active Lck and is required for Zap70 phosphorylation.
linkage (such as lysine 48 or lysine 63) and the overall number of ubiquitin monomers in the chain serve as contextual signals to determine the fate of the ubiquitinated substrate6. However, much like signaling via phosphorylation, ubiquitin conjugation is not irreversible, as deubiquitinating enzymes remove ubiquitins from substrates7. Experiments have suggested
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NEWS AND VIEWS that rather than indiscriminately removing any polyubiquitin linkage, individual deubiquitinating enzymes cleave specific varieties of intra-ubiquitin lysine linkages and can alter the fate of the ubiquitinated substrate, as seen in the pathway of transcription factor NF-κB8,9. Patients with cylindromatosis (also called ‘turban tumor syndrome’) have benign tumors on their face and neck arising from hair follicles and secreting cells of the sweat and scent glands. Linkage analysis and positional cloning has determined that this disease results from loss of heterozygosity at a single locus on chromosome 16q12-13, designated Cyld10. This gene encodes a deubiquitinating enzyme, and initial studies have suggested that CYLD deubiquitinating enzyme activity is pivotal in modulating tumor necrosis factor receptor (TNFR)–induced NF-κB activation11–13. TNFR engagement recruits the adaptor molecule TNFR-associated factor 2 (TRAF2), which autoubiquitinates (lysine 63–linked) and activates itself. TRAF2 activation ultimately leads to phosphorylation and degradation of the inhibitor IκBα, followed by the release and nuclear translocation of NF-κB. Those studies suggest a model in which CYLD is recruited to and removes lysine 63–linked polyubiquitin chains from activated TRAF2. In that model, the inhibitors of apoptosis can then bind and ubiquitinate TRAF2 by lysine 48–linked chains14. As a result, CYLD promotes the proteasomal degradation of TRAF2 and resultant attenuation of NF-κB signaling. One limitation of those landmark studies is their dependence on in vitro transfection experiments. Reiley and colleagues have generated Cyld–/– mice, and find, somewhat unexpectedly, no defect in TNFR-induced NF-κB signaling in Cyld–/– bone marrow–derived macrophages.
Instead, they note that CYLD-deficient mice contain few peripheral T cells and trace this defect to a thymocyte-intrinsic block in development at the DP–to–single-positive transition. The Cyld–/– thymic phenotype closely resembles the Zap70–/– thymic phenotype, and the authors note defective TCR-induced activation of Zap70, Lat and the kinase Erk in Cyld–/– DP thymocytes. However, activation of Lck, the tyrosine kinase responsible for phosphorylating and activating Zap70, is normal in DP Cyld–/– thymocytes. Immunoprecipitation experiments show reduced binding of active Lck to Zap70 in stimulated Cyld–/– thymocytes. This effect is not simply due to reduced total or active Lck, as those remain unchanged in stimulated Cyld–/– thymocytes. The authors have also sought to determine whether CYLD might function by promoting recruitment of active Lck to Zap70. Immunoprecipitation of endogenous CYLD from wild-type thymocytes indicates that after TCR stimulation, CYLD binds only the phosphorylated, active form of Lck. CYLD also interacts with a constitutively active mutant of Lck in 293 cells. This Lck mutant is spontaneously ubiquitinated, presumably by endogenous Cbl, in 293 cells, and cotransfection of wild-type CYLD attenuates Lck ubiquitination. By linking active Lck and Zap70, CYLD regulates the most proximal steps of TCR signaling (Fig. 1). Therefore, the Cyld–/– thymic phenotype most likely reflects attenuated positive selection of DP thymocytes. However, some open issues need to be resolved. It is not known where CYLD and Lck bind to each other. As CYLD and Cbl both ‘preferentially’ bind activated Lck, the kinetics of the ubiquitination and deubiquitination of Lck by Cbl and CYLD, respectively,
remain to be investigated. Do CYLD and Cbl bind Lck simultaneously, allowing CYLD to remove polyubiquitin chains added by Cbl in real time, or is it a sequential process? Also, this model unfortunately does not easily explain why the amount of active Lck is not changed in Cyld–/– thymocytes. If Cbl were able to ubiquitinate phosphorylated Lck unopposed by CYLD, an increased rate of active Lck turnover in Cyld–/– thymocytes might be expected. One explanation could be that Cbl ‘preferentially’ ubiquitinates active, Zap70-bound Lck. Active Lck in Cyld–/– DP thymocytes might reside in a cellular compartment inaccessible to Cbl, thus preventing any change in active Lck protein quantified by immunoblot. Reiley and colleagues have provided a seminal insight into the involvement of reversibility of polyubiquitination in thymocyte development. Further work investigating CYLD and other deubiquitinating enzymes is needed to more completely understand how the ubiquitin-proteasome system regulates T cell differentiation and function. 1. Reiley, W.W. et al. Nat. Immunol. 7, 411–417 (2006). 2. Zhang, J., Somani, A.K. & Siminovitch, K.A. Semin. Immunol. 12, 361–378 (2000). 3. Thien, C.B. & Langdon, W.Y. Biochem. J. 391, 153–166 (2005). 4. Pickart, C.M. Annu. Rev. Biochem. 70, 503–533 (2001). 5. Jackson, P.K. et al. Trends Cell Biol. 10, 429–439 (2000). 6. Pickart, C.M. & Fushman, D. Curr. Opin. Chem. Biol. 8, 610–616 (2004). 7. Nijman, S.M. et al. Cell 123, 773–786 (2005). 8. Boone, D.L. et al. Nat. Immunol. 5, 1052–1060 (2004). 9. Wertz, I.E. et al. Nature 430, 694–699 (2004). 10. Bignell, G.R. et al. Nat. Genet. 25, 160–165 (2000). 11. Brummelkamp, T.R., Nijman, S.M., Dirac, A.M. & Bernards, R. Nature 424, 797–801 (2003). 12. Trompouki, E. et al. Nature 424, 793–796 (2003). 13. Kovalenko, A. et al. Nature 424, 801–805 (2003). 14. Vaux, D.L. & Silke, J. Nat. Rev. Mol. Cell Biol. 6, 287– 297 (2005).
MyD88 beyond Toll Jiahuai Han The adaptor protein MyD88 is involved in interleukin 1 receptor and Toll-like receptor signaling. Unexpectedly, new evidence shows that MyD88 also participates in interferon-γ-induced cellular responses.
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ytoplasmic adaptor proteins couple ligandreceptor interactions to intracellular signaling events. The myeloid differentiation primary response gene 88 (MyD88) adaptor is involved in transmitting signals from Toll-like receptor
Jiahuai Han is in the Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA. e-mail:
[email protected]
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(TLR) and interleukin 1 receptor (IL-1R) family members (with the exception of TLR3)1–4. In this issue of Nature Immunology, Sun and Ding report that MyD88 is also involved in transmitting signals induced by interferon-γ (IFN-γ) in macrophages5. MyD88 contains an N-terminal death domain that is separated from a C-terminal Toll–IL-1R (TIR) domain by a short linker sequence. TLR ligation triggers recruitment of MyD88 to the receptor complex through TIR domain–TIR
domain interactions (Fig. 1). The death domain of MyD88 recruits a death domain–containing protein known as IL-1R-associated protein kinase (IRAK). Activation of IRAK leads to a series of ‘downstream’ signaling cascades that activate nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK) and other regulators of gene expression. Consequentially, proinflammatory gene transcription, protein translation and/or transcript stability are/is increased. Those changes in the expression of
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