RESUMEN
A hallmark of Spemann organizer function is its expression of Wnt antagonists that regulate axial embryonic patterning. Here we identify the tumor suppressor Protein tyrosine phosphatase receptor-type kappa (PTPRK), as a Wnt inhibitor in human cancer cells and in the Spemann organizer of Xenopus embryos. We show that PTPRK acts via the transmembrane E3 ubiquitin ligase ZNRF3, a negative regulator of Wnt signaling promoting Wnt receptor degradation, which is also expressed in the organizer. Deficiency of Xenopus Ptprk increases Wnt signaling, leading to reduced expression of Spemann organizer effector genes and inducing head and axial defects. We identify a '4Y' endocytic signal in ZNRF3, which PTPRK maintains unphosphorylated to promote Wnt receptor depletion. Our discovery of PTPRK as a negative regulator of Wnt receptor turnover provides a rationale for its tumor suppressive function and reveals that in PTPRK-RSPO3 recurrent cancer fusions both fusion partners, in fact, encode ZNRF3 regulators.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Wnt/antagonistas & inhibidores , Animales , Tipificación del Cuerpo/genética , Endocitosis , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Organizadores Embrionarios/metabolismo , Transducción de Señal , Proteínas de Xenopus/metabolismo , Xenopus laevis , beta Catenina/metabolismoRESUMEN
Angiopoietin-like 4 (ANGPTL4) is a secreted signaling protein that is implicated in cardiovascular disease, metabolic disorder, and cancer. Outside of its role in lipid metabolism, ANGPTL4 signaling remains poorly understood. Here, we identify ANGPTL4 as a Wnt signaling antagonist that binds to syndecans and forms a ternary complex with the Wnt co-receptor Lipoprotein receptor-related protein 6 (LRP6). This protein complex is internalized via clathrin-mediated endocytosis and degraded in lysosomes, leading to attenuation of Wnt/ß-catenin signaling. Angptl4 is expressed in the Spemann organizer of Xenopus embryos and acts as a Wnt antagonist to promote notochord formation and prevent muscle differentiation. This unexpected function of ANGPTL4 invites re-interpretation of its diverse physiological effects in light of Wnt signaling and may open therapeutic avenues for human disease.
Asunto(s)
Angiopoyetinas/metabolismo , Endocitosis/fisiología , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Receptores de LDL/metabolismo , Vía de Señalización Wnt/fisiología , Proteínas de Xenopus/metabolismo , beta Catenina/metabolismo , Proteína 4 Similar a la Angiopoyetina , Angiopoyetinas/genética , Animales , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Fosforilación , Transducción de Señal/fisiología , Proteína Wnt3A/metabolismo , Xenopus , Proteínas de Xenopus/genéticaRESUMEN
UNLABELLED: The cellular endosomal sorting complex required for transport (ESCRT) was recently found to mediate important morphogenesis processes at the nuclear envelope (NE). We previously showed that the Epstein-Barr virus (EBV) BFRF1 protein recruits the ESCRT-associated protein Alix to modulate NE structure and promote EBV nuclear egress. Here, we uncover new cellular factors and mechanisms involved in this process. BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. BFRF1 is ubiquitinated, and elimination of possible ubiquitination by either lysine mutations or fusion of a deubiquitinase hampers NE-derived vesicle formation and virus maturation. While it interacts with multiple Nedd4-like ubiquitin ligases, BFRF1 preferentially binds Itch ligase. We show that Itch associates with Alix and BFRF1 and is required for BFRF1-induced NE vesicle formation. Our data demonstrate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE and EBV maturation, uncovering novel regulatory mechanisms of nuclear egress of viral nucleocapsids. IMPORTANCE: The nuclear envelope (NE) of eukaryotic cells not only serves as a transverse scaffold for cellular processes, but also as a natural barrier for most DNA viruses that assemble their nucleocapsids in the nucleus. Previously, we showed that the cellular endosomal sorting complex required for transport (ESCRT) machinery is required for the nuclear egress of EBV. Here, we further report the molecular interplay among viral BFRF1, the ESCRT adaptor Alix, and the ubiquitin ligase Itch. We found that BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. The lysine residues and the ubiquitination of BFRF1 regulate the formation of BFRF1-induced NE-derived vesicles and EBV maturation. During the process, a ubiquitin ligase, Itch, preferably associates with BFRF1 and is required for BFRF1-induced NE vesicle formation. Therefore, our data indicate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE, suggesting novel regulatory mechanisms for ESCRT-mediated NE modulation.
Asunto(s)
Herpesvirus Humano 4/fisiología , Interacciones Huésped-Patógeno , Proteínas de la Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Represoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Virales/metabolismo , Ensamble de Virus , Replicación Viral , Células HeLa , HumanosRESUMEN
Upon virus infection, the host innate immune response is initiated through the activation of IFN regulatory factor 3 (IRF3) and NF-κB signaling pathways to induce IFN production. Previously, we demonstrated EBV BGLF4 kinase suppresses IRF3 function in a kinase activity-dependent manner. The replacement of Ser123, Ser173 and Thr180 into alanines at the proline-rich linker region of IRF3 abolishes BGLF4-mediated suppression. In this study, we show that BGLF4 phosphorylates glutathione-S-transferase (GST)-IRF3(110-202), but not GST-IRF3(110-202)3A mutant (S123/S173/T180A) in vitro. Compared with activation mimicking mutant IRF3(5D), the phosphorylation-defective IRF3(5D)3A shows a higher transactivation activity in reporter assays, whereas the phosphorylation-mimicking IRF3(5D)2D1E, with Ser123 and Ser173 mutated to aspartate and Thr180 to glutamate, has a much lower activity. To explore whether similar cellular regulation also exists in the absence of virus infection, candidate cellular kinases were predicted and the transactivation activity of IRF3 was examined with various kinase inhibitors. Glycogen synthase kinase 3 (GSK3) inhibitor LiCl specifically enhanced both IRF3(5D) and wild type IRF3 activity, even without stimulation. Expression of constitutive active GSK3ß(S9A) represses LiCl-mediated enhancement of IRF3 transactivation activity. In vitro, both GSK3α and GSK3ß phosphorylate IRF3 at the linker region. Collectively, data here suggest GSK3 phosphorylates IRF3 linker region in a way similar to viral kinase BGLF4.
Asunto(s)
Glutatión Transferasa/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Herpesvirus Humano 4/metabolismo , Factor 3 Regulador del Interferón/metabolismo , Virosis/inmunología , Glucógeno Sintasa Quinasa 3/genética , Células HEK293 , Humanos , Factor 3 Regulador del Interferón/genética , Interferón gamma/metabolismo , Mutagénesis Insercional , Mutación/genética , Fosforilación , Activación Transcripcional/efectos de los fármacos , Proteínas Virales de Fusión/metabolismoRESUMEN
Epstein-Barr virus (EBV) BGLF4 is a member of the conserved herpesvirus kinases that regulate multiple cellular and viral substrates and play an important role in the viral lytic cycles. BGLF4 has been found to phosphorylate several cellular and viral transcription factors, modulate their activities, and regulate downstream events. In this study, we identify an NF-κB coactivator, UXT, as a substrate of BGLF4. BGLF4 downregulates not only NF-κB transactivation in reporter assays in response to tumor necrosis factor alpha (TNF-α) and poly(I·C) stimulation, but also NF-κB-regulated cellular gene expression. Furthermore, BGLF4 attenuates NF-κB-mediated repression of the EBV lytic transactivators, Zta and Rta. In EBV-positive NA cells, knockdown of BGLF4 during lytic progression elevates NF-κB activity and downregulates the activity of the EBV oriLyt BHLF1 promoter, which is the first promoter activated upon lytic switch. We show that BGLF4 phosphorylates UXT at the Thr3 residue. This modification interferes with the interaction between UXT and NF-κB. The data also indicate that BGLF4 reduces the interaction between UXT and NF-κB and attenuates NF-κB enhanceosome activity. Upon infection with short hairpin RNA (shRNA) lentivirus to knock down UXT, a spontaneous lytic cycle was observed in NA cells, suggesting UXT is required for maintenance of EBV latency. Overexpression of wild-type, but not phosphorylation-deficient, UXT enhances the expression of lytic proteins both in control and UXT knockdown cells. Taking the data together, transcription involving UXT may also be important for EBV lytic protein expression, whereas BGLF4-mediated phosphorylation of UXT at Thr3 plays a critical role in promoting the lytic cycle.