Deubiquitinating enzyme CYLD negatively regulates the ubiquitin-dependent kinase Tak1 and prevents abnormal T cell responses

Gram-positive bacterium Streptococcus pneumoniae is an important human pathogen that colonizes the upper respiratory tract and is also the major cause of morbidity and mortality worldwide. S. pneumoniae causes invasive diseases such as pneumonia, meningitis, and otitis media. Despite the importance of pneumococcal diseases, little is known about the molecular mechanisms by which S. pneumoniae-induced inflammation is regulated, especially the negative regulatory mechanisms. Here we show that S. pneumoniae activates nuclear factor of activated T cells (NFAT) signaling pathway and the subsequent up-regulation of inflammatory mediators via a key pneumococcal virulence factor, pneumolysin. We also demonstrate that S. pneumoniae activates NFAT transcription factor independently of Toll-like receptors 2 and 4. Moreover, S. pneumoniae induces NFAT activation via both Ca 2؉ -calcineurin and transforming growth factor-␤-activated kinase 1 (TAK1)-mitogen-activated protein kinase kinase (MKK) 3/6-p38␣/␤-dependent signaling pathways. Interestingly, we found for the first time that tumor suppressor cylindromatosis (CYLD) acts as a negative regulator for S. pneumoniae-induced NFAT signaling pathway via a deubiquitination-dependent mechanism. Finally, we showed that CYLD interacts with and deubiquitinates TAK1 to negatively regulate the activation of the downstream MKK3/6-p38␣/␤ pathway. Our studies thus bring new insights into the molecular pathogenesis of S. pneumoniae infections through the NFAT-dependent mechanism and further identify CYLD as a negative regulator for NFAT signaling, thereby opening up new therapeutic targets for these diseases.

Section: Discussionsupporting

confidence: 81%

Section: S Pneumoniae Activates Nfat Through Camentioning

confidence: 99%

Tumor Suppressor Cylindromatosis Acts as a Negative Regulator for Streptococcus pneumoniae-induced NFAT Signaling

Koga

Lim

Jono

et al. 2008

“…6, B and D). These include transforming growth factor ␤-activated kinase 1 (TAK1), TAK1-binding proteins 1-3 (TAB1-3), NF-B essential modulator (NEMO), and deubiquitinating enzymes such as A20 and cylindromatosis tumor suppressor protein (CYLD) (27,(52)(53)(54)(55)(56). Among these, Li et al reported that AMPK␣2 associates with TAB1 and activates p38 MAPK in mouse heart (57).…”

Section: Discussionmentioning

confidence: 99%

“…6E). As IB␣ is degraded by the ubiquitin process after phosphorylation (24 -26), these findings suggest that IB␣ might undergo chronic degradation and resynthesis in AMPK␣ Ϫ/Ϫ ⌴⌭Fs and that the AMPK␣ deletion causes constitutive activation of the NF-〉 pathway in MEFs (27).…”

Section: -Foldmentioning

confidence: 99%

AMP-activated Protein Kinase Suppresses Matrix Metalloproteinase-9 Expression in Mouse Embryonic Fibroblasts

Morizane

Thanos

Takeuchi

et al. 2011

Matrix metalloproteinase-9 (MMP-9) plays a critical role in tissue remodeling under both physiological and pathological conditions. Although MMP-9 expression is low in most cells and is tightly controlled, the mechanism of its regulation is poorly understood. We utilized mouse embryonic fibroblasts (MEFs) that were nullizygous for the catalytic ␣ subunit of AMP-activated protein kinase (AMPK), which is a key regulator of energy homeostasis, to identify AMPK as a suppressor of MMP-9 expression. Total AMPK␣ deletion significantly elevated MMP-9 expression compared with wild-type (WT) MEFs, whereas single knock-out of the isoforms AMPK␣1 and AMPK␣2 caused minimal change in the level of MMP-9 expression. The suppressive role of AMPK on MMP-9 expression was mediated through both its activity and presence. Matrix metalloproteinase-9 (MMP-9, 2 gelatinase B) degrades denatured collagens and native collagen type IV, which is a major component of the extracellular matrix (ECM) and basement membranes (1). Under normal circumstances, the degradation of the ECM by MMP-9 is a tightly controlled process involved in physiological wound healing and embryo development (1, 2). Conversely, aberrant degradation of ECM by excess MMP-9 expression results in the pathologic destruction of connective tissue seen in cancer, arterial sclerosis, and rheumatoid arthritis (1, 3). Therefore, under physiological conditions, regulated MMP-9 expression is low (1), but the mechanisms behind this are obscure.AMP-activated protein kinase (AMPK) is a serine/threonine kinase, which regulates energy homeostasis and metabolic stress (4). AMPK acts as a sensor of cellular energy status and maintains the balance between ATP production and consumption. In mammals, AMPK exists as a heterotrimer with ␣, ␤, and ␥ subunits, each of which is encoded by two or three genes (␣1, ␣2, ␤1, ␤2, ␥1, ␥2, and ␥3). The ␣ subunit possesses catalytic activity, whereas the ␤ and ␥ subunits are regulatory and maintain the stability of the heterotrimer complex. The importance of AMPK␣ is illustrated by the fact that dual deficiency of AMPK␣1 and AMPK␣2 is embryonic lethal (5).Recent evidence suggests that AMPK has a much wider range of functions, including the regulation of cell growth, cell proliferation, cell polarity, and autophagy (6, 7). Because these functions are closely linked to the pathology of MMP-9-related diseases, including cancer, arterial sclerosis, and rheumatoid arthritis, we hypothesized that AMPK regulates MMP-9 expression. To address this, in the present study, we utilized AMPK␣-deficient mouse embryonic fibroblasts (MEFs) to investigate the effect of the genetic deletion and activation of AMPK on MMP-9 expression. EXPERIMENTAL PROCEDURESAntibodies, Recombinant Proteins, and Reagents-All antibodies, except for MMP-9 (Abcam, Cambridge, MA) and AMPK␣2 (Santa Cruz Biotechnology, Santa Cruz, CA), were purchased from Cell Signaling (Beverly, MA). Recombinant mouse TNF-␣, MMP-9, and MMP-2 proteins were obtained from R&D Systems (

“…and Cyld ϩ/ϩ littermates, and genotyping was performed by PCR using tail DNA (30). All mice were housed in specific pathogen-free cages and monitored periodically (every 3 months) for the lack of common pathogens.…”

Section: Methodsmentioning

confidence: 99%

Regulation of IκB Kinase-related Kinases and Antiviral Responses by Tumor Suppressor CYLD

Zhang

Lee

et al. 2008

Self Cite

115

101

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...