cylindromatosis ortholog (H. sapiens): Biological Overview | References
Gene name - cylindromatosis ortholog (H. sapiens)
Synonyms - CG5603, CYLD
Cytological map position - 31C7-31D1
Function - enzyme
Symbol - CYLD
FlyBase ID: FBgn0032210
Genetic map position - 2L:10,295,856..10,299,827 [-]
Classification - Peptidase, CAP-Gly domain
Cellular location - cytoplasmic
CYLD encodes a tumor suppressor that is mutated in familial cylindromatosis. Despite biochemical and cell culture studies, the physiological functions of CYLD in animal development and tumorigenesis remain poorly understood. To address these questions, Drosophila CYLD (dCYLD) mutant and transgenic flies were generated expressing wild-type and mutant dCYLD proteins. dCYLD is essential for JNK-dependent oxidative stress resistance and normal lifespan. Furthermore, dCYLD regulates TNF-induced JNK activation and cell death through dTRAF2, which acts downstream of the TNF receptor Wengen and upstream of the JNKK kinase dTAK1. dCYLD encodes a deubiquitinating enzyme that deubiquitinates dTRAF2 and prevents dTRAF2 from ubiquitin-mediated proteolytic degradation. These data provide a molecular mechanism for the tumor suppressor function of this evolutionary conserved molecule by indicating that dCYLD plays a critical role in modulating TNF-JNK-mediated cell death (Xue, 2007).
Mutations in the tumor suppressor CYLD have been associated with familial cylindromatosis (Brooke-Spiegler syndrome), an autosomal dominant inherited disease characterized by the development of multiple skin appendage tumors such as cylindroma, trichoepithelioma, and spiradenoma (Bignell, 2000; Lee, 2005). The physiological functions of CYLD in animal development and tumorigenesis remain largely unclear. Recent studies have indicated that CYLD encodes a deubiquitinating enzyme that, by removing K46- or K63-linked polyubiquitin chains (or both) from its target, modulates multiple signaling pathways in immune and epithelial cells. In vitro studies have suggested that CYLD negatively regulates the p65/p50 NF-κB pathway by removing K63-linked polyubiquitin chains from TRAF2/6 (Brummelkamp, 2003; Kovalenko, 2003; Massoumi, 2006; Trompouki, 2003). Hence, it was proposed that loss of CYLD would result in hyperactivation of p65/p50 NF-κB and survival of tumor cells. However, in contrast to this hypothesis, ectopic activation of this signaling in mouse epithelial tissue results in epidermal hypoplasia and growth inhibition rather than epidermal hyperplasia (Seitz, 1998). Another study has suggested that CYLD deubiquitinates K63-linked polyubiquitination from Bcl-3 and inhibits p50/Bcl-3- or p52/Bcl-3-depedent proliferation in mouse keratinocytes (Massoumi, 2006). Recently, it has been shown that CYLD deubiquitinates both K48- and K63-linked polyubiquitin chains from Lck and regulates TCR signaling in thymocytes (Reiley, 2006). Thus, depending on the specific cellular context, CYLD may execute its functions via two different molecular mechanisms: the K48-linked, polyubiquitination-mediated, proteasome-dependent protein degradation and K63-linked, polyubiquitination-mediated endocytosis and signal transduction (Xue, 2007).
The c-Jun N-terminal kinase (JNK) signaling pathway has been conserved from flies to humans and plays a wide range of roles from stress response to apoptosis. Interestingly, mice deficient for jnk1 show enhanced skin tumor development, a phenotype similar to cyld mutants. However, the role of CYLD in JNK signaling has remained controversial, and the mechanism unknown (Reiley, 2004, Reiley, 2006; Zhang, 2006). In Drosophila, the Tumor Necrosis Factor (TNF) orthlog Eiger (Egr) triggers the cell death pathway through its receptor Wengen (Wgn), which in turn activates the conserved JNK cascade, the JNKK kinase dTAK1, the JNK kinase Hep, and Drosophila JNK Bsk (Xue, 2007).
Drosophila has been used as a powerful model organism for the genetic dissection of conserved signaling pathways in development. Given that CYLD is conserved from invertebrates to mammals (Bignell, 2000), the in vivo functions of CYLD were characterized in Drosophila by generating dCYLD mutants and transgenic animals expressing wild-type or mutant dCYLD proteins. This genetic and biochemical analyses indicated that a major function of dCYLD is regulating JNK-mediated cell death through deubiquitinating dTRAF2 (Xue, 2007).
The Drosophila genome encodes a single CYLD ortholog, dCYLD (CG5603). To obtain the dCYLD mutant, a 5 kb genomic deletion was generated by male recombination. This deficiency, termed dCYLDmr4, deletes the entire upstream region and the first 57 amino acids of dCYLD, as well as the coding regions of two adjacent genes, CG5395 and CG13138, and thus represents null mutations for these three genes. To obtain flies only mutant for dCYLD, a transgene, pLX107, containing the genomic region for CG5395 and CG13138, was introduced into the fly genome and was able to rescue the lethality of homozygous dCYLDmr4 flies. The rescued flies, which represent null mutants only for dCYLD, were viable and fertile, with no obvious morphological abnormalities (data not shown), suggesting that dCYLD is dispensable for viability and fertility. RT-PCR experiments confirmed that no dCYLD transcripts were produced in homozygous dCYLDmr4 pLX107 flies, as compared with wild-type. However, it was found that dCYLDmr4 pLX107 mutant animals were shorter lived than wild-type or heterozygous dCYLD flies. This phenotype could be rescued by adding one copy of dCYLDRes, a genomic rescue transgene for dCYLD, which confirms that loss of dCYLD is the cause of this defect. Similarly, other defects associated with dCYLDmr4 pLX107 are also rescued by the dCYLDRes transgene. Thus, dCYLDmr4 pLX107 is referred to as dCYLD mutants (Xue, 2007).
Shortened animal lifespan may result from compromised oxidative stress tolerance. To examine the oxidative stress resistance, 3-day-old flies were challenged with paraquat for a prolonged period of time and their survival rates were measured. It was found that dCYLD mutants exhibited a significant reduction in survival rate as compared with wild-type or heterozygous dCYLD flies after 24 hr or 36 hr of exposure to paraquat, suggesting dCYLD plays a pivotal role in regulating oxidative stress resistance (Xue, 2007).
JNK signaling has been reported to play an important role in regulating oxidative stress resistance and lifespan in Drosophila (Wang, 2003). Ubiquitous expression of Bsk, the Drosophila JNK ortholog, under the control of tubulin promoter, rescues both lifespan and oxidative stress resistance defects in dCYLD mutants, suggesting that dCYLD regulates these two physiological effects through the JNK signaling pathway (Xue, 2007).
This study was extended to other stress conditions and it was found that dCYLD mutants are less resistant to dry starvation (no food and water), a phenotype that has been associated with reduced JNK activity (Wang, 2003). In contrast, dCYLD mutants do not affect animal survival at high and low temperature conditions (Xue, 2007).
To further examine the role of dCYLD in regulating JNK signaling in animal development, the genetic interactions between dCYLD and Eiger (Egr), the Drosophila ortholog of TNF that triggers the JNK pathway, was tested. Ectopic expression of Egr, under the control of the GMR promoter (GMR > Egr) and using the Gal4/UAS binary system, induces JNK activation and cell death in the developing eye that results in vastly reduced adult eye size (Igaki, 2002; Moreno, 2002). The Egr-induced JNK activation and small-eye phenotype was suppressed modestly by deleting one copy of dCYLD and suppressed soundly by removing both copies. The strong suppression of the Egr eye phenotype in homozygous dCYLD mutants was partially reverted by adding one copy of dCYLDRes. These results indicate that dCYLD is required for Egr-triggered JNK activation and cell death (Xue, 2007).
dCYLD encodes a protein of 640 amino acids, containing in its N terminal portion a cytoskeleton-associated protein (CAP) domain that is present in proteins associated with microtubules and the cytoskeletal network, two ubiquitin carboxyl-terminal hydrolases (UCH) domains that are commonly associated with deubiquitinating enzyme activity, and three CXXC zinc-finger (ZF) motifs with potential protein-protein interaction ability. To functionally characterize these motifs, UAS transgenes were generated expressing the wild-type or three mutant versions of dCYLD that delete the CAP domain, the two UCH domains, or the three ZF motifs. When expressed under the control of the GMR promoter, neither the full-length nor the dCYLD mutants displayed any detectable phenotype. When introduced into the GMR > Egr; dCYLD−/− background, wild-type dCYLD released the suppression of the Egr eye phenotype, confirming that the suppressive effect was due to the loss of dCYLD functions. In contrast, dCYLDΔUCH had no effect on the suppression of the Egr eye phenotype, and dCYLDΔCAP could only partially relieve this suppression, implying that the UCH domains are necessary for dCYLD functions and that the CAP domain is essential for dCYLD to execute its full activity in vivo. Interestingly, expression of dCYLDΔZF completely abolished the suppression effect, suggesting that the ZF motifs are dispensable in dCYLD regulation of Egr-induced cell death (Xue, 2007).
Ubiquitous expression of the full-length dCYLD, but not dCYLDΔUCH, rescues both shortened lifespan and hypersensitivity to paraquat in dCYLD mutants, suggesting that the deubiquitinating activity is indispensable for dCYLD to regulate JNK-dependent oxidative stress resistance and lifespan (Xue, 2007).
TNF receptor-associated factors (TRAFs) are important adaptor proteins that bind to TNF receptors and relay TNF signals to the JNK and NF-κB pathways in mammals. In Drosophila, Egr signal is mediated exclusively by the JNK pathway (Igaki, 2002; Moreno, 2002). However, the role of Drosophila TRAF proteins in Egr-JNK signaling remains unclear. The Drosophila genome encodes two TRAFs: dTRAF1, the TRAF2 ortholog; and dTRAF2, the TRAF6 ortholog. To determine the role of dTRAF1 and dTRAF2 in Egr-JNK signaling, the effects were examined of loss-of-function mutations and RNAi-mediated downregulation of dTRAF1 or dTRAF2 on the Egr eye phenotype (Cha, 2003; Kuranaga, 2002). The Egr-induced small-eye phenotype was not suppressed by either deletion of one copy of the dTRAF1 gene or coexpression of a dTRAF1 RNAi. In contrast, the Egr eye phenotype was suppressed strongly by removing half of the dosage of dTRAF2 and suppressed completely by deleting the dTRAF2 gene. Consistently, coexpression of a dTRAF2 RNAi significantly suppressed the Egr eye phenotype. In agreement with genetic data, dTRAF2 exhibited a much stronger physical interaction with Wgn and dTAK1 than did dTRAF1 (Geuking, 2005 ; Kauppila, 2003). Together, the above results point to dTRAF2, but not dTRAF1, as the adaptor protein that mediates Egr signaling in Drosophila (Xue, 2007).
To investigate the physiological functions of dCYLD and dTRAF2 in JNK activation, the expression pattern of puckered (puc) was checked in dCYLD or dTRAF2 mutants. puc encodes a JNK phosphatase whose expression is positively regulated by the JNK pathway, and thus, the puc-LacZ expression of the pucE69 enhancer-trap allele can be used as a readout of JNK activity in vivo. puc is weakly expressed in wild-type third-instar eye discs, and can be detected by prolonged staining. It has been previously shown that puc expression posterior to the morphogenetic furrow (MF) depends on endogenous Egr signaling (Igaki, 2002). This study found that such expression patterns are reduced dramatically in dCYLD mutants and dTRAF2 RNAi animals. In contrast, puc expression in the disc margin, which is independent of Egr signaling (Igaki, 2002), was not affected. GMR > Egr strongly activated puc transcription posterior to the MF (Igaki, 2002). This ectopic Egr-induced puc expression was largely blocked by loss of dCYLD or by expression of dTRAF2 RNAi. Taken together, these observations indicate that both dCYLD and dTRAF2 are physiologically required by the endogenous JNK pathway (Xue, 2007).
The role of CYLD in modulating JNK signaling in mammalian cells has remained controversial. Consistent with the current observation, it was reported that JNK activity diminished in Cyld−/− thymocytes (Reiley, 2006), which implies that CYLD is physiologically required for JNK activation. However, CYLD was also reported to negatively regulate JNK signaling in culture cells (Reiley, 2004) and macrophages (Zhang, 2006). Thus, CYLD could positively or negatively regulate JNK signaling in a cell-type-specific manner (Xue, 2007).
To genetically map the epistasis of dCYLD and dTRAF2 in the Egr-JNK pathway, the genetic interaction between dTAK1 (JNKKK) and dCYLD or dTRAF2 was examined in the developing eyes. Expression of dTAK1 under the control of the GMR promoter resulted in pupa lethality, while dTAK1 expression under the control of the sevenless (sev) promoter (sev > dTAK1) induced extensive cell death in larval eye discs and gave rise to rough eyes with a reduced size. Loss of dCYLD or dTRAF2, or coexpression of a dTRAF2 RNAi, had no effect on the sev > dTAK1 phenotype, while removal of one copy of hep (JNKK) or bsk (JNK) partially suppressed the sev > dTAK1 phenotype, suggesting that dCYLD and dTRAF2 operate upstream of dTAK1 in the Egr-JNK pathway (Xue, 2007).
Ectopic Egr expression in the dorsal thorax driven by the potent pannier-GAL4 driver resulted in pupa lethality. However, when reared at 18°C, these animals survived to adulthood, presumably due to lessened Egr expression caused by reduced Gal4 activity, and produced a small-scutellum phenotype. This phenotype could be suppressed by RNAi inactivation of JNK signaling components, e.g., wgn, dTRAF2, dTAK1, hep, or bsk, suggesting that the phenotype is caused by activation of JNK signaling. Ectopic expression of dCYLD driven by pannier-GAL4 produced a similar but weaker phenotype, which could be fully suppressed by the coexpression of an RNAi of dTRAF2 and dTAK1, but not that of wgn. These results indicate that dCYLD functions downstream of Wgn, but upstream of dTRAF2 and dTAK1, in modulating JNK signaling (Xue, 2007).
RNAi-mediated downregulation of dTRAF2, but not dTRAF1, resulted in compromised oxidative stress resistance and shortened lifespan, suggesting that the role of dTRAF2 in dCYLD-JNK signaling has been conserved in different physiological contexts (Xue, 2007).
Previous studies have reported that the Shark tyrosine kinase and Src42A regulate JNK signaling in epidermal closure during embryogenesis and metamorphosis. However, null mutants for egr and dCYLD are fully viable and do not display the epidermal closure defect, implying that Shark and Src42A act in parallel to Egr and dCYLD in modulating JNK signaling. Consistent with this interpretation, loss of shark or src42A failed to suppress the GMR > Egr or pnr > dCYLD phenotype. In addition, it was found that loss of the transcription factor dFOXO could suppress both GMR > Egr and pnr > dCYLD phenotypes, suggesting that dFOXO acts downstream of dCYLD in JNK signaling. Consistent with this observation, dFOXO is required downstream of JNK in modulating cell death, oxidative stress resistance, and lifespan (Xue, 2007).
Overexpression of CYLD in mammalian tissue culture cells negatively regulates NF-κB signaling by deubiquitinating TRAF2/6 (Brummelkamp, 2003; Kovalenko, 2003; Trompouki, 2003). dCYLD, like its mammalian counterpart, contains two UCH deubiquitinating domains (Bignell, 2000). Indeed, genetic analysis revealed that the UCH deubiquitinating domains are crucial for the in vivo functions of dCYLD. Furthermore, genetic epistasis data show that dCYLD acts upstream of dTRAF2 in the JNK pathway. Thus, it was hypothesized that dCYLD might act in the JNK pathway to deubiquitinate and subsequently stabilize dTRAF2 by preventing its ubiquitination-mediated proteolytic degradation. To examine this hypothesis in vivo, a FLAG-tagged dTRAF2 transgene (GMR > FLAG-dTRAF2) was introduced into dCYLD mutants and transgenic flies. Proteins were extracted from the heads of these flies for biochemical analyses. It was found that loss of dCYLD resulted in a significant reduction in dTRAF2 protein level, while the ubiquitination of dTRAF2 was markedly enhanced. Both changes were suppressed by dCYLDRes. Consistently, overexpression of dCYLD, but not dCYLDΔUCH, increased dTRAF2 protein level and decreased its ubiquitination. These results show that dCYLD functions as a deubiquitinating enzyme that deubiquitinates dTRAF2 and promotes dTRAF2 accumulation in vivo (Xue, 2007).
Polyubiquitination chains are usually formed on two lysine residues, K48 and K63. It is generally believed that the K48-linked polyubiquitination mediates proteasome-dependent protein degradation, while the K63-linked polyubiquitination mediates endocytosis and signal transduction (Mukhopadhyay, 2007). Previous work in mammalian culture cells has implicated that CYLD encodes a deubiquitinating enzyme that preferentially cleaves K63-linked polyubiquitin chain from its target proteins for NF-κB signaling. However, a recent in vivo study in Cyld−/− mice has reported that CYLD could regulate the stability of its target protein by effectively removing K48-linked polyubiquitin chain in thymocytes (Reiley, 2006). Interestingly, JNK activity also diminished in Cyld−/− thymocytes (Reiley, 2006). Thus, the role of CYLD in regulating protein stability and positively modulating JNK signaling could be conserved in mammals (Xue, 2007).
CYLD mutations in human patients cause dramatic skin tumors. However, the physiological function of CYLD and the mechanism underlying CYLD deficiency-induced tumorigenesis remain largely unknown. By generating the null dCYLD mutation and dCYLD transgenic animals and performing genetic analysis, this study has shown that dCYLD is a critical regulatory component of the JNK signaling pathway. Genetic epistasis and biochemical analysis further reveal that dCYLD modulates JNK signaling by deubiquitinating dTRAF2 and thus preventing dTRAF2 from ubiquitination-mediated proteolytic degradation. Loss of dCYLD results in augmented ubiquitination and degradation of dTRAF2, which renders cells resistant to apoptosis triggered by JNK signaling. Deregulation of apoptosis has been implicated as a major cause of tumorigenesis. Consistently, mice deficient for both jnk1 and jnk2 were resistant to apoptosis induced by UV irradiation, anisomycin, and MMS (Tournier, 2000), and jnk1−/− mice exhibited enhanced skin tumor development (She, 2002), a phenotype that is pathogenically similar to cylindromatosis in CYLD human patients. Together these data argue that modulation of JNK signaling could be a conserved mechanism underlying familial cylindromatosis in CYLD patients (Xue, 2007).
Search PubMed for articles about Drosophila CYLD
Bignell, G., et al. (2000). Identification of the familial cylindromatosis tumour-suppressor gene, Nat. Genet. 25: 160-165. PubMed ID: 10835629
Brummelkamp, T., Nijman, S., Dirac, A. and Bernards, R. (2003). Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-kappaB. Nature 424: 797-801. PubMed ID: 12917690
Cha, G. H., Cho, K. S., Lee, J. H., Kim, M., Kim, E., Park, J., Lee, S. B. and Chung. J. (2003). Discrete functions of TRAF1 and TRAF2 in Drosophila melanogaster mediated by c-Jun N-terminal kinase and NF-kappaB-dependent signaling pathways. Mol. Cell. Biol. 23(22): 7982-91. PubMed ID: 14585960
Geuking, P., Narasimamurthy, R. and Basler, K. (2005). A genetic screen targeting the tumor necrosis factor/Eiger signaling pathway: identification of Drosophila TAB2 as a functionally conserved component. Genetics 171: 1683-1694. PubMed ID: 16079232
Igaki, T., et al. (2002). Eiger, a TNF superfamily ligand that triggers the Drosophila JNK pathway. EMBO J. 21: 3009-3018. PubMed ID: 12065414
Kauppila, S., et al. (2003). Eiger and its receptor, Wengen, comprise a TNF-like system in Drosophila, Oncogene 22: 4860-4867. PubMed ID: 12894227
Kovalenko, A., et al. (2003). The tumour suppressor CYLD negatively regulates NF-kappaB signalling by deubiquitination. Nature 424: 801-805. PubMed ID: 12917691
Kuranaga, E., et al. (2002). Reaper-mediated inhibition of DIAP1-induced DTRAF1 degradation results in activation of JNK in Drosophila. Nat. Cell Biol. 4(9): 705-10. PubMed ID: 12198495
Lee, D., Grossman, M., Schneiderman, P. and Celebi, J. (2005). Genetics of skin appendage neoplasms and related syndromes. J. Med. Genet. 42: 811-819. PubMed ID: 16272260
Massoumi, R., et al. (2006). Cyld inhibits tumor cell proliferation by blocking Bcl-3-dependent NF-kappaB signaling. Cell 125: 665-677. PubMed ID: 16713561
Moreno, E., Yan, M. and Basler, K. (2002). Evolution of TNF signaling mechanisms: JNK-dependent apoptosis triggered by Eiger, the Drosophila homolog of the TNF superfamily. Curr. Biol. 12: 1263-1268. PubMed ID: 12176339
Mukhopadhyay, D. and Riezman, H. (2007). Proteasome-independent functions of ubiquitin in endocytosis and signaling, Science 315: 201-205. PubMed ID: 17218518
Reiley, W., Zhang, M. and Sun, W. (2004). Negative regulation of JNK signaling by the tumor suppressor CYLD. J. Biol. Chem. 279: 55161-55167. PubMed ID: 15496400
Reiley, W., et al. (2006). Regulation of T cell development by the deubiquitinating enzyme CYLD. Nat. Immunol. 7: 411-417. PubMed ID: 16501569
Seitz, C., Lin, Q., Deng, H. and Khavari, P. (1998). Alterations in NF-kappaB function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-kappaB. Proc. Natl. Acad. Sci. 95: 2307-2312. PubMed ID: 9482881
She, Q., et al. (2002). Deficiency of c-Jun-NH(2)-terminal kinase-1 in mice enhances skin tumor development by 12-O-tetradecanoylphorbol-13-acetate. Cancer Res. 62: 1343-1348. PubMed ID: 11888903
Tournier, C., Tournier, C., Hess, P. and Yang, D. (2000). Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science 288: 870-874. PubMed ID: 10797012
Trompouki, E., et al. (2003). CYLD is a deubiquitinating enzyme that negatively regulates NF-kappaB activation by TNFR family members. Nature 424: 793-796. PubMed ID: 12917689
Wang, M., Bohmann, D. and Jasper, H. (2005). JNK extends life span and limits growth by antagonizing cellular and organism-wide responses to insulin signaling. Cell 121: 115-125. PubMed ID: 15820683
Xue, L., et al.(2007). Tumor suppressor CYLD regulates JNK-induced cell death in Drosophila. Dev. Cell 13: 446-454. PubMed ID: 17765686
Zhang, J., et al. (2006). Impaired regulation of NF-kappaB and increased susceptibility to colitis-associated tumorigenesis in CYLD-deficient mice. J. Clin. Invest. 116: 3042-3049. PubMed ID: 17053834
date revised: 15 May 2008
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