The deubiquitinating enzyme CYLD has recently been implicated in the regulation of signal transduction, but its physiological function and mechanism of action are still elusive. In this study, we show that CYLD plays a pivotal role in regulating T cell activation and homeostasis. T cells derived from Cyld knockout mice display a hyperresponsive phenotype and mediate the spontaneous development of intestinal inflammation. Interestingly, CYLD targets a ubiquitin-dependent kinase, transforming growth factor–β-activated kinase 1 (Tak1), and inhibits its ubiquitination and autoactivation. Cyld-deficient T cells exhibit constitutively active Tak1 and its downstream kinases c-Jun N-terminal kinase and IκB kinase β. These results emphasize a critical role for CYLD in preventing spontaneous activation of the Tak1 axis of T cell signaling and, thereby, maintaining normal T cell function.

Spermatogenesis involves an early wave of germ cell apoptosis, which is required for maintaining the balance between germ cells and supporting Sertoli cells. However, the signaling mechanism regulating this apoptotic event is poorly defined. Here we show that genetic deficiency of Cyld, a recently identified deubiquitinating enzyme, attenuates the early wave of germ cell apoptosis and causes impaired spermatogenesis in mice. Interestingly, the loss of CYLD in testicular cells leads to activation of the transcription factor NF-kappaB and aberrant expression of antiapoptotic genes. We further show that CYLD negatively regulates a ubiquitin-dependent NF-kappaB activator, RIP1. CYLD binds to RIP1 and inhibits its ubiquitination and signaling function. These findings establish CYLD as a pivotal deubiquitinating enzyme (DUB) that regulates germ cell apoptosis and spermatogenesis and suggest an essential role for CYLD in controlling the RIP1/NF-kappaB signaling axis in testis.

Osteoclastogenesis is a tightly regulated biological process, and deregulation can lead to severe bone disorders such as osteoporosis. The regulation of osteoclastic signaling is incompletely understood, but ubiquitination of TNF receptor-associated factor 6 (TRAF6) has recently been shown to be important in mediating this process. We therefore investigated the role of the recently identified deubiquitinating enzyme CYLD in osteoclastogenesis and found that mice with a genetic deficiency of CYLD had aberrant osteoclast differentiation and developed severe osteoporosis. Cultured osteoclast precursors derived from CYLD-deficient mice were hyperresponsive to RANKL-induced differentiation and produced more and larger osteoclasts than did controls upon stimulation. We assessed the expression pattern of CYLD and found that it was drastically upregulated during RANKL-induced differentiation of preosteoclasts. Furthermore, CYLD negatively regulated RANK signaling by inhibiting TRAF6 ubiquitination and activation of downstream signaling events. Interestingly, we found that CYLD interacted physically with the signaling adaptor p62 and thereby was recruited to TRAF6. These findings establish CYLD as a crucial negative regulator of osteoclastogenesis and suggest its involvement in the p62/TRAF6 signaling axis.

The IB kinase (IKK)-related kinases, IKK⑀ and TBK1, participate in the induction of type I interferons (IFNs) during viral infections. Deregulated activation of IKK⑀ and TBK1 also contributes to the abnormal cell survival and transformation. However, how these kinases are negatively regulated remains unclear. We show here that the tumor suppressor CYLD has an essential role in preventing aberrant activation of IKK⑀/TBK1. CYLD deficiency causes constitutive activation of IKK⑀/TBK1, which is associated with hyper-induction of IFNs in virus-infected cells. We further show that CYLD targets a cytoplasmic RNA sensor, RIG-I, and inhibits the ubiquitination of this IKK⑀/ TBK1 stimulator. Consistent with the requirement of ubiquitination in RIG-I function, CYLD potently inhibits RIG-I-mediated activation of the IFN-␤ promoter. These findings establish CYLD as a key negative regulator of IKK⑀/TBK1 and suggest a role for CYLD in the control of RIG-I ubiquitination.Innate immunity serves as the first line of host defense against microbial infections. A central step in antiviral innate immunity involves the production of type I interferons (IFNs), 3 which is triggered when host pattern recognition receptors (PRRs) detect viral products (1, 2). Two recently identified cytoplasmic PRRs, retinoic acid-induced gene I (RIG-I) and melanoma differentiation-associated gene 5, play critical roles in mediating antiviral innate immune responses (3). These PRRs signal for IFN production through common downstream signaling pathways that involve activation of two homologous protein kinases, IKK⑀ and TBK1, as well as the typical IB kinase (IKK) (4 -9).IKK⑀ (also named IKKi) and TBK1 (also named T2K and NAK) are known as IKK-related kinases because of their structural homology to IKK (10 -14), a central component of the NF-B signaling pathway (15,16). Unlike the typical IKK, which phosphorylates NF-B inhibitors (IBs) and mediates NF-B activation, IKK⑀ and TBK1 phosphorylate IRF3 and IRF7 to trigger their dimerization and nuclear translocation, thereby mediating induction of IFN gene expression (9, 16). In addition, emerging evidence suggests that IKK⑀ and TBK1 also regulate cell survival and oncogenesis via an IFN-independent mechanism (17-20). However, how IKK⑀/TBK1 is negatively regulated is currently not well understood.An emerging mechanism that regulates signal transduction in various biological processes is protein ubiquitination (21). Analogous to protein phosphorylation, which is reversibly controlled by protein kinases and phosphatases, protein ubiquitination is a reversible event mediated by the counteractive actions of ubiquitin-conjugating enzymes and deubiquitinating enzymes (DUBs) (22). Although ubiquitination is traditionally known as a process that mediates protein degradation by the proteasome, it is now evident that specific ubiquitin chains facilitate protein/protein interactions that lead to the activation of signaling molecules (23). Notably, ubiquitination of RIG-I, a PRR that recognizes double-stranded and 5Ј-phospho...