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CYLD Tidies Up Dishevelled Signaling
Molecular Cell
Previews CYLD Tidies Up Dishevelled Signaling David Komander1,* 1Medical Research Council Laboratory of Molecular Biology, Protein and Nucleic Acid Chemistry Division, Hills Road, Cambridge CB2 0QH, UK *Correspondence: [email protected] DOI 10.1016/j.molcel.2010.02.017
An increasing number of cellular signaling pathways utilize protein ubiquitination for activation of signaling cascades. In this issue of Molecular Cell, Maurice and colleagues demonstrate that nondegradative Lys63-linked polyubiquitin chains modulate Dishevelled (Dvl) activity upstream in the Wnt/b-catenin signaling pathway. In many cellular signaling pathways, activation of membrane receptors by extracellular ligands leads to assembly of large cytoplasmic protein complexes that activate downstream signaling, either by activating effectors or by inhibiting inhibitors of the pathway. The Wnt signaling pathway is a good example for the latter mechanism. Wnt signaling is key to embryonic development, affecting cell proliferation, cell polarity, and cell fate. Wnt signaling is also tightly linked to disease, and mutations in pathway components can drive cancer. Canonical Wnt/b-catenin signaling leads to the activation of b-catenin and involves recruitment of Dvl and Axin to the Frizzled/LRP6 coreceptor complex (MacDonald et al., 2009). The adaptor molecule Dvl polymerizes, recruiting the b-catenin inhibitory complex consisting of Axin, adenomatous polyposis coli (APC), casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3) to the plasma membrane, which results in inhibition of GSK3. This leads to accumulation of b-catenin because the GSK3-mediated constitutive degradation of b-catenin is now switched off, and b-catenin can act as a transcriptional coactivator (MacDonald et al., 2009). Lys63 Polyubiquitin Chains Activate Wnt/b-Catenin Signaling Tauriello et al. identify the deubiquitinating enzyme CYLD as a new negative regulator of Dvl-mediated signaling (Tauriello et al., 2010). A siRNA screen against ubiquitin-specific protease (USP) family deubiquitinases indicated that CYLD depletion leads to elevated b-catenin accumulation and Wnt target gene transcription. Subsequent epistasis analysis
to identify the point of CYLD interference with Wnt signaling suggested a role for the deubiquitinase upstream of b-catenin activation, at the level of Dvl. Knockdown of CYLD led to hyperubiquitination of overexpressed and endogenous Dvl, and by using ubiquitin mutants and mass spectroscopy, these ubiquitin chains were found to be primarily Lys63 linked. CYLD is best known for inhibiting NFkB activation; however, the authors find no evidence for NFkB-mediated effects on Wnt signaling. Interestingly, CYLD seems to act on upstream components of the signaling cascades in both pathways. Lys63-linked ubiquitin chains have prominent upstream roles in NFkB activation (Sun, 2010). The CYLD deubiquitinase domain is endowed with Lys63 specificity and mediates removal of these chains. CYLD is constitutively active and may function as a general downregulator of Lys63 ubiquitination events; however, its activity can be regulated. For example, phosphorylation can inhibit CYLD, and as a result, Lys63 chains accumulate on CYLD targets (Sun, 2010). The identification of CYLD leads to the question of how Lys63-linked ubiquitination positively modulates Dvl signaling. Tauriello et al. mapped ubiquitination sites on Dvl, and it was found that seven conserved Lys residues in the DIX domain are ubiquitinated. The DIX domain of Dvl undergoes reversible head-to-tail polymerization in vitro, and overexpressed polymerization-competent Dvl forms highly dynamic puncta in cells (SchwarzRomond et al., 2005, 2007). Polymerization-deficient Dvl mutants are dominant negative and interfere with Wnt signaling (Bilic et al., 2007). Tauriello et al. find that ubiquitination of these mutants is
decreased. Mutation of seven Lys residues in the DIX domain leads to a 2-fold reduction in Dvl-mediated signaling and a slight reduction in observed ubiquitination of Dvl. DIX domain ubiquitination does not seem to be required for DIXmediated puncta formation because Dvl still forms puncta in cells when DIX domain Lys residues are mutated. DIX domains adopt a ubiquitin-like fold with resemblance to PB1 domains (Schwarz-Romond et al., 2007), and therefore ubiquitination of DIX polymers would lead to a branched, heterogeneous meshwork of DIX and ubiquitin chains that might stabilize Dvl protein assemblies. A signalosome-stabilizing role for polyubiquitination has recently also been suggested for TNF receptor complexes (Haas et al., 2009). It will be interesting to analyze whether ubiquitination affects the dynamic behavior of Dvl selfassembly. In order to achieve its signaling function, Dvl binds to and inhibits the b-catenin inhibitory complex (Axin, APC, CK1, GSK3), yet how this is performed is still unclear (MacDonald et al., 2009). The new data suggest that ubiquitinated Dvl is more efficient in inhibiting this complex, and it is tempting to speculate that Lys63linked ubiquitin chains attract the b-catenin inhibitory complex. Yet, none of the mentioned proteins have discernible ubiquitin-binding domains, and whether they bind to ubiquitin has not been tested. Wnt Signaling as a Contributor to Cylindromatosis? Functional loss of the tumor suppressor gene CYLD gives rise to familial cylindromatosis, a rare skin condition in which patients develop benign tumors in hair
Molecular Cell 37, March 12, 2010 ª2010 Elsevier Inc. 589
Molecular Cell
Previews follicles and sweat glands of head and neck (Bignell et al., 2000). Tauriello et al. show that b-catenin levels are increased in some cells of cylindroma tumor samples, indicative of Wnt pathway activation and consistent with a role of CYLD in Wnt/ b-catenin signaling. The tumor suppressor function of CYLD has been linked to its role in NFkB signaling, but it is possible that Wnt/b-catenin signaling contributes to cylindromatosis. In addition to roles in NFkB activation, recent data have implicated CYLD also in cell-cycle regulation and a number of cell-type-specific pathways (Sun, 2010). It is, however, surprising that, despite the important pathways that CYLD was suggested to affect, knockout mouse models of CYLD have resulted in relatively mild phenotypes, and these mice do not develop tumors spontaneously (Sun, 2010). However, cylindromatosis arises from loss of heterozygosity and truncation of the second allele, suggesting potential dominant-negative roles of CYLD fragments. Mouse models with patientderived mutations would be informative to shed further light on this disease. Activating versus Inhibitory Roles of Lys63-Linked Polyubiquitin in Wnt Signaling Lys63-linked ubiquitin chains seem to upregulate b-catenin activity due to modulating upstream signaling events. This is intriguing because, further downstream, the same chain type was reported to dampen Wnt/b-catenin signaling (Tran et al., 2008). Subsequent to b-catenin activation, Lys63-linked ubiquitin chains on APC have been suggested to inhibit the TCF/b-catenin transcription complex, and here, another deubiquitinase with
Lys63 specificity, TRABID, was suggested to act as a positive regulator of Wnt signaling (Tran et al., 2008). Another study has recently found that loss of the E2 ubiquitin-conjugating enzyme Ubc13, which is Lys63 specific and attributed to most functions of this chain type, leads to accumulation of b-catenin and increased Wnt target gene transcription, consistent with an inhibitory role for Lys63-linked ubiquitin chains in b-catenin activation (Wu et al., 2009). Indeed, the positive modulation further upstream, mediated by Dvl ubiquitination, is also dependent on Ubc13 (Tauriello et al., 2010), leading to an apparent disagreement between these recent reports. It appears that the same type of signaling ubiquitin chain mediates positive as well as negative effects. However, because the negative effects of increased Lys63linked chains are further downstream, they are epistatic and overrule their positive modulation of the pathway upstream in the cascade. How do the deubiquitinases CYLD and TRABID achieve their specificity for different stages of signaling? The answer may lie in the subcellular localization of the deubiquitinases. CYLD is excluded from the nucleus, whereas TRABID is found throughout the cell, including in the nucleus, where it might modulate b-catenin activity. Alternatively, selective substrate interaction may provide the required specificity. What Is the Ligase? Three independent reports have now identified Lys63-linked ubiquitin chains as modulators of Wnt/b-catenin signaling, and the Lys63-specific enzymes CYLD, TRABID, and Ubc13 have been implicated in this pathway. What is not known
590 Molecular Cell 37, March 12, 2010 ª2010 Elsevier Inc.
is the identity of the E3 ligase(s) that mediate(s) Lys63-linked ubiquitination. So far, the focus has been on the degradative roles of ubiquitination in Wnt/b-catenin signaling, yet in order to understand this intricate regulatory network, nondegradative ubiquitination events also need to be taken into account. ACKNOWLEDGMENTS I would like to thank Mariann Bienz for valuable comments and discussions. REFERENCES Bignell, G.R., Warren, W., Seal, S., Takahashi, M., Rapley, E., Barfoot, R., Green, H., Brown, C., Biggs, P.J., Lakhani, S.R., et al. (2000). Nat. Genet. 25, 160–165. Bilic, J., Huang, Y.L., Davidson, G., Zimmermann, T., Cruciat, C.M., Bienz, M., and Niehrs, C. (2007). Science 316, 1619–1622. Haas, T.L., Emmerich, C.H., Gerlach, B., Schmukle, A.C., Cordier, S.M., Rieser, E., Feltham, R., Vince, J., Warnken, U., Wenger, T., et al. (2009). Mol. Cell 36, 831–844. MacDonald, B.T., Tamai, K., and He, X. (2009). Dev. Cell 17, 9–26. Schwarz-Romond, T., Merrifield, C., Nichols, B.J., and Bienz, M. (2005). J. Cell Sci. 118, 5269–5277. Schwarz-Romond, T., Fiedler, M., Shibata, N., Butler, P.J.G., Kikuchi, A., Higuchi, Y., and Bienz, M. (2007). Nat. Struct. Mol. Biol. 14, 484–492. Sun, S.C. (2010). Cell Death Differ. 17, 25–34. Tauriello, D.V.F., Haegebarth, A., Kuper, I., Edelmann, M.J., Henraat, M., Canninga-van Dijk, M.R., Kessler, B.M., Clevers, H., and Maurice, M.M. (2010). Mol. Cell 37, this issue, 607–619. Tran, H., Hamada, F., Schwarz-Romond, T., and Bienz, M. (2008). Genes Dev. 22, 528–542. Wu, X., Yamamoto, M., Akira, S., and Sun, S.C. (2009). Proc Natl. Acad. Sci. USA, in press. Published online November 19, 2009. 10.1073/pnas. 0906547106.
Previews CYLD Tidies Up Dishevelled Signaling David Komander1,* 1Medical Research Council Laboratory of Molecular Biology, Protein and Nucleic Acid Chemistry Division, Hills Road, Cambridge CB2 0QH, UK *Correspondence: [email protected] DOI 10.1016/j.molcel.2010.02.017
An increasing number of cellular signaling pathways utilize protein ubiquitination for activation of signaling cascades. In this issue of Molecular Cell, Maurice and colleagues demonstrate that nondegradative Lys63-linked polyubiquitin chains modulate Dishevelled (Dvl) activity upstream in the Wnt/b-catenin signaling pathway. In many cellular signaling pathways, activation of membrane receptors by extracellular ligands leads to assembly of large cytoplasmic protein complexes that activate downstream signaling, either by activating effectors or by inhibiting inhibitors of the pathway. The Wnt signaling pathway is a good example for the latter mechanism. Wnt signaling is key to embryonic development, affecting cell proliferation, cell polarity, and cell fate. Wnt signaling is also tightly linked to disease, and mutations in pathway components can drive cancer. Canonical Wnt/b-catenin signaling leads to the activation of b-catenin and involves recruitment of Dvl and Axin to the Frizzled/LRP6 coreceptor complex (MacDonald et al., 2009). The adaptor molecule Dvl polymerizes, recruiting the b-catenin inhibitory complex consisting of Axin, adenomatous polyposis coli (APC), casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3) to the plasma membrane, which results in inhibition of GSK3. This leads to accumulation of b-catenin because the GSK3-mediated constitutive degradation of b-catenin is now switched off, and b-catenin can act as a transcriptional coactivator (MacDonald et al., 2009). Lys63 Polyubiquitin Chains Activate Wnt/b-Catenin Signaling Tauriello et al. identify the deubiquitinating enzyme CYLD as a new negative regulator of Dvl-mediated signaling (Tauriello et al., 2010). A siRNA screen against ubiquitin-specific protease (USP) family deubiquitinases indicated that CYLD depletion leads to elevated b-catenin accumulation and Wnt target gene transcription. Subsequent epistasis analysis
to identify the point of CYLD interference with Wnt signaling suggested a role for the deubiquitinase upstream of b-catenin activation, at the level of Dvl. Knockdown of CYLD led to hyperubiquitination of overexpressed and endogenous Dvl, and by using ubiquitin mutants and mass spectroscopy, these ubiquitin chains were found to be primarily Lys63 linked. CYLD is best known for inhibiting NFkB activation; however, the authors find no evidence for NFkB-mediated effects on Wnt signaling. Interestingly, CYLD seems to act on upstream components of the signaling cascades in both pathways. Lys63-linked ubiquitin chains have prominent upstream roles in NFkB activation (Sun, 2010). The CYLD deubiquitinase domain is endowed with Lys63 specificity and mediates removal of these chains. CYLD is constitutively active and may function as a general downregulator of Lys63 ubiquitination events; however, its activity can be regulated. For example, phosphorylation can inhibit CYLD, and as a result, Lys63 chains accumulate on CYLD targets (Sun, 2010). The identification of CYLD leads to the question of how Lys63-linked ubiquitination positively modulates Dvl signaling. Tauriello et al. mapped ubiquitination sites on Dvl, and it was found that seven conserved Lys residues in the DIX domain are ubiquitinated. The DIX domain of Dvl undergoes reversible head-to-tail polymerization in vitro, and overexpressed polymerization-competent Dvl forms highly dynamic puncta in cells (SchwarzRomond et al., 2005, 2007). Polymerization-deficient Dvl mutants are dominant negative and interfere with Wnt signaling (Bilic et al., 2007). Tauriello et al. find that ubiquitination of these mutants is
decreased. Mutation of seven Lys residues in the DIX domain leads to a 2-fold reduction in Dvl-mediated signaling and a slight reduction in observed ubiquitination of Dvl. DIX domain ubiquitination does not seem to be required for DIXmediated puncta formation because Dvl still forms puncta in cells when DIX domain Lys residues are mutated. DIX domains adopt a ubiquitin-like fold with resemblance to PB1 domains (Schwarz-Romond et al., 2007), and therefore ubiquitination of DIX polymers would lead to a branched, heterogeneous meshwork of DIX and ubiquitin chains that might stabilize Dvl protein assemblies. A signalosome-stabilizing role for polyubiquitination has recently also been suggested for TNF receptor complexes (Haas et al., 2009). It will be interesting to analyze whether ubiquitination affects the dynamic behavior of Dvl selfassembly. In order to achieve its signaling function, Dvl binds to and inhibits the b-catenin inhibitory complex (Axin, APC, CK1, GSK3), yet how this is performed is still unclear (MacDonald et al., 2009). The new data suggest that ubiquitinated Dvl is more efficient in inhibiting this complex, and it is tempting to speculate that Lys63linked ubiquitin chains attract the b-catenin inhibitory complex. Yet, none of the mentioned proteins have discernible ubiquitin-binding domains, and whether they bind to ubiquitin has not been tested. Wnt Signaling as a Contributor to Cylindromatosis? Functional loss of the tumor suppressor gene CYLD gives rise to familial cylindromatosis, a rare skin condition in which patients develop benign tumors in hair
Molecular Cell 37, March 12, 2010 ª2010 Elsevier Inc. 589
Molecular Cell
Previews follicles and sweat glands of head and neck (Bignell et al., 2000). Tauriello et al. show that b-catenin levels are increased in some cells of cylindroma tumor samples, indicative of Wnt pathway activation and consistent with a role of CYLD in Wnt/ b-catenin signaling. The tumor suppressor function of CYLD has been linked to its role in NFkB signaling, but it is possible that Wnt/b-catenin signaling contributes to cylindromatosis. In addition to roles in NFkB activation, recent data have implicated CYLD also in cell-cycle regulation and a number of cell-type-specific pathways (Sun, 2010). It is, however, surprising that, despite the important pathways that CYLD was suggested to affect, knockout mouse models of CYLD have resulted in relatively mild phenotypes, and these mice do not develop tumors spontaneously (Sun, 2010). However, cylindromatosis arises from loss of heterozygosity and truncation of the second allele, suggesting potential dominant-negative roles of CYLD fragments. Mouse models with patientderived mutations would be informative to shed further light on this disease. Activating versus Inhibitory Roles of Lys63-Linked Polyubiquitin in Wnt Signaling Lys63-linked ubiquitin chains seem to upregulate b-catenin activity due to modulating upstream signaling events. This is intriguing because, further downstream, the same chain type was reported to dampen Wnt/b-catenin signaling (Tran et al., 2008). Subsequent to b-catenin activation, Lys63-linked ubiquitin chains on APC have been suggested to inhibit the TCF/b-catenin transcription complex, and here, another deubiquitinase with
Lys63 specificity, TRABID, was suggested to act as a positive regulator of Wnt signaling (Tran et al., 2008). Another study has recently found that loss of the E2 ubiquitin-conjugating enzyme Ubc13, which is Lys63 specific and attributed to most functions of this chain type, leads to accumulation of b-catenin and increased Wnt target gene transcription, consistent with an inhibitory role for Lys63-linked ubiquitin chains in b-catenin activation (Wu et al., 2009). Indeed, the positive modulation further upstream, mediated by Dvl ubiquitination, is also dependent on Ubc13 (Tauriello et al., 2010), leading to an apparent disagreement between these recent reports. It appears that the same type of signaling ubiquitin chain mediates positive as well as negative effects. However, because the negative effects of increased Lys63linked chains are further downstream, they are epistatic and overrule their positive modulation of the pathway upstream in the cascade. How do the deubiquitinases CYLD and TRABID achieve their specificity for different stages of signaling? The answer may lie in the subcellular localization of the deubiquitinases. CYLD is excluded from the nucleus, whereas TRABID is found throughout the cell, including in the nucleus, where it might modulate b-catenin activity. Alternatively, selective substrate interaction may provide the required specificity. What Is the Ligase? Three independent reports have now identified Lys63-linked ubiquitin chains as modulators of Wnt/b-catenin signaling, and the Lys63-specific enzymes CYLD, TRABID, and Ubc13 have been implicated in this pathway. What is not known
590 Molecular Cell 37, March 12, 2010 ª2010 Elsevier Inc.
is the identity of the E3 ligase(s) that mediate(s) Lys63-linked ubiquitination. So far, the focus has been on the degradative roles of ubiquitination in Wnt/b-catenin signaling, yet in order to understand this intricate regulatory network, nondegradative ubiquitination events also need to be taken into account. ACKNOWLEDGMENTS I would like to thank Mariann Bienz for valuable comments and discussions. REFERENCES Bignell, G.R., Warren, W., Seal, S., Takahashi, M., Rapley, E., Barfoot, R., Green, H., Brown, C., Biggs, P.J., Lakhani, S.R., et al. (2000). Nat. Genet. 25, 160–165. Bilic, J., Huang, Y.L., Davidson, G., Zimmermann, T., Cruciat, C.M., Bienz, M., and Niehrs, C. (2007). Science 316, 1619–1622. Haas, T.L., Emmerich, C.H., Gerlach, B., Schmukle, A.C., Cordier, S.M., Rieser, E., Feltham, R., Vince, J., Warnken, U., Wenger, T., et al. (2009). Mol. Cell 36, 831–844. MacDonald, B.T., Tamai, K., and He, X. (2009). Dev. Cell 17, 9–26. Schwarz-Romond, T., Merrifield, C., Nichols, B.J., and Bienz, M. (2005). J. Cell Sci. 118, 5269–5277. Schwarz-Romond, T., Fiedler, M., Shibata, N., Butler, P.J.G., Kikuchi, A., Higuchi, Y., and Bienz, M. (2007). Nat. Struct. Mol. Biol. 14, 484–492. Sun, S.C. (2010). Cell Death Differ. 17, 25–34. Tauriello, D.V.F., Haegebarth, A., Kuper, I., Edelmann, M.J., Henraat, M., Canninga-van Dijk, M.R., Kessler, B.M., Clevers, H., and Maurice, M.M. (2010). Mol. Cell 37, this issue, 607–619. Tran, H., Hamada, F., Schwarz-Romond, T., and Bienz, M. (2008). Genes Dev. 22, 528–542. Wu, X., Yamamoto, M., Akira, S., and Sun, S.C. (2009). Proc Natl. Acad. Sci. USA, in press. Published online November 19, 2009. 10.1073/pnas. 0906547106.