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1.
Nat Immunol ; 13(12): 1178-86, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23104095

RESUMEN

We report the clinical description and molecular dissection of a new fatal human inherited disorder characterized by chronic autoinflammation, invasive bacterial infections and muscular amylopectinosis. Patients from two kindreds carried biallelic loss-of-expression and loss-of-function mutations in HOIL1 (RBCK1), a component of the linear ubiquitination chain assembly complex (LUBAC). These mutations resulted in impairment of LUBAC stability. NF-κB activation in response to interleukin 1ß (IL-1ß) was compromised in the patients' fibroblasts. By contrast, the patients' mononuclear leukocytes, particularly monocytes, were hyper-responsive to IL-1ß. The consequences of human HOIL-1 and LUBAC deficiencies for IL-1ß responses thus differed between cell types, consistent with the unique association of autoinflammation and immunodeficiency in these patients. These data suggest that LUBAC regulates NF-κB-dependent IL-1ß responses differently in different cell types.


Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo IV/genética , Enfermedades Autoinflamatorias Hereditarias/genética , Síndromes de Inmunodeficiencia/genética , FN-kappa B/metabolismo , Ubiquitina-Proteína Ligasas/genética , Infecciones Bacterianas/genética , Infecciones Bacterianas/inmunología , Proteínas de Ciclo Celular/genética , Línea Celular , Fibroblastos/inmunología , Fibroblastos/metabolismo , Humanos , Síndromes de Inmunodeficiencia/metabolismo , Interleucina-1beta/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Represoras/genética , Factores de Transcripción , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación
2.
Mol Cell ; 45(4): 553-66, 2012 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-22365832

RESUMEN

Plk1 activation is required for progression through mitotic entry to cytokinesis. Here we show that at mitotic entry, Plk1 phosphorylates Optineurin (Optn) at serine 177 and that this dissociates Optn from the Golgi-localized GTPase Rab8, inducing its translocation into the nucleus. Mass spectrometry analysis revealed that Optn is associated with a myosin phosphatase complex (MP), which antagonizes the mitotic function of Plk1. Our data also indicate that Optn functionally connects this complex to Plk1 by promoting phosphorylation of the myosin phosphatase targeting subunit 1 (MYPT1). Accordingly, silencing Optn expression increases Plk1 activity and induces abscission failure and multinucleation, which were rescued upon expression of wild-type (WT) Optn, but not a phospho-deficient mutant (S177A) that cannot translocate into the nucleus during mitosis. Overall, these results highlight an important role of Optn in the spatial and temporal coordination of Plk1 activity.


Asunto(s)
Proteínas de Ciclo Celular/fisiología , Mitosis/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Factor de Transcripción TFIIIA/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Retroalimentación Fisiológica , Células HEK293 , Células HeLa , Humanos , Proteínas de Transporte de Membrana , Fosforilación , Factor de Transcripción TFIIIA/química , Factor de Transcripción TFIIIA/fisiología , Quinasa Tipo Polo 1
3.
J Cell Sci ; 126(Pt 19): 4457-68, 2013 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-23886940

RESUMEN

Notch signaling is a conserved signaling pathway implicated in embryogenesis and adult tissue maintenance. Notch signaling strength is strictly regulated, notably by maintaining a controlled pool of functional receptor at the cell surface. Mammalian non-activated Notch receptor is internalized, ubiquitylated by the Itch E3 ubiquitin ligase and degraded in the lysosomes. Here, we show that ß-arrestins are necessary for Itch-Notch interaction and for Itch-driven ubiquitylation and degradation of Notch. Interestingly, ß-arrestins do not directly bind Itch but heterodimerize with a member of another subfamily of arrestins called ARRDC1 or α-arrestin 1, which harbors PPxY motifs that allow direct interaction with Itch. Cells transfected with ARRDC1 mutated in PPxY motifs show reduced Itch-mediated Notch ubiquitylation and impaired lysosomal degradation of Notch, as observed in ß-arrestin(-/-) or Itch(-/-) cells. Our data show for the first time that ARRDC1 and ß-arrestins heterodimerize and cooperate in the same complex to promote non-activated Notch receptor degradation, thus acting as negative regulators of Notch signaling.


Asunto(s)
Arrestinas/metabolismo , Receptores Notch/metabolismo , Animales , Línea Celular Tumoral , Endocitosis/fisiología , Fibroblastos/metabolismo , Células HEK293 , Humanos , Ratones , Transducción de Señal , Transfección , Ubiquitinación , beta-Arrestinas
4.
FASEB J ; 28(2): 603-14, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24145721

RESUMEN

The Notch signaling pathway is involved in liver development and regeneration. Here, we investigate the role of the 4 mammalian Notch paralogs in the regulation of hepatoblast proliferation and hepatocytic differentiation. Our model is based on bipotential mouse embryonic liver (BMEL) progenitors that can differentiate into hepatocytes or cholangiocytes in vitro and in vivo. BMEL cells were subjected to Notch antagonists or agonists. Blocking Notch activation with a γ-secretase inhibitor, at 50 µM for 48 h, reduced cell growth by 50%. S-phase entry was impaired, but no apoptosis was induced. A systematic paralog-specific strategy was set using lentiviral transduction with constitutively active forms of each Notch receptor along with inhibition of endogenous Notch signaling. This assay demonstrates that proliferation of BMEL cells requires Notch2 and Notch4 activity, resulting in significant down-regulation of p27(Kip1) and p57(Kip2) cyclin-dependent kinase inhibitors. Conversely, Notch3-expressing cells proliferate less and express 3-fold higher levels of p57(Kip2). The Notch3 cells present a hepatocyte-like morphology, enhanced multinucleation, and a ploidy shift. Moreover, Notch3 activity is conducive to hepatocytic differentiation in vitro, while its paralogs impede this fate. Our study provides the first evidence of a functional diversity among the mammalian Notch homologues in the proliferation and hepatocytic-lineage commitment of liver progenitors.


Asunto(s)
Hepatocitos/citología , Hepatocitos/metabolismo , Hígado/citología , Hígado/metabolismo , Receptores Notch/metabolismo , Células Madre/citología , Células Madre/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Proliferación Celular , Técnica del Anticuerpo Fluorescente , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Notch/genética
5.
EMBO J ; 29(3): 619-31, 2010 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-19959994

RESUMEN

In response to stress, cells start transcriptional and transcription-independent programs that can lead to adaptation or death. Here, we show that multiple inducers of autophagy, including nutrient depletion, trigger the activation of the IKK (IkappaB kinase) complex that is best known for its essential role in the activation of the transcription factor NF-kappaB by stress. Constitutively active IKK subunits stimulated autophagy and transduced multiple signals that operate in starvation-induced autophagy, including the phosphorylation of AMPK and JNK1. Genetic inhibition of the nuclear translocation of NF-kappaB or ablation of the p65/RelA NF-kappaB subunit failed to suppress IKK-induced autophagy, indicating that IKK can promote the autophagic pathway in an NF-kappaB-independent manner. In murine and human cells, knockout and/or knockdown of IKK subunits (but not that of p65) prevented the induction of autophagy in response to multiple stimuli. Moreover, the knockout of IKK-beta suppressed the activation of autophagy by food deprivation or rapamycin injections in vivo, in mice. Altogether, these results indicate that IKK has a cardinal role in the stimulation of autophagy by physiological and pharmacological stimuli.


Asunto(s)
Autofagia/fisiología , Quinasa I-kappa B/fisiología , Animales , Autofagia/genética , Células Cultivadas , Células HeLa , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/fisiología , FN-kappa B/genética , FN-kappa B/metabolismo , Células 3T3 NIH , Transducción de Señal/fisiología
6.
J Biol Chem ; 287(35): 29429-41, 2012 Aug 24.
Artículo en Inglés | MEDLINE | ID: mdl-22778262

RESUMEN

Notch signaling is critical for development and adult tissue physiology, controlling cell fate in a context-dependent manner. Upon ligand binding, the transmembrane Notch receptor undergoes two ordered proteolytic cleavages releasing Notch intracellular domain, which regulates the transcription of Notch target genes. The strength of Notch signaling is of crucial importance and depends notably on the quantity of Notch receptor at the cell surface. Using an shRNA library screen monitoring Notch trafficking and degradation in the absence of ligand, we identified mammalian USP12 and its Drosophila melanogaster homolog as novel negative regulators of Notch signaling. USP12 silencing specifically interrupts Notch trafficking to the lysosomes and, as a consequence, leads to an increased amount of receptor at the cell surface and to a higher Notch activity. At the biochemical level, USP12 with its activator UAF1 deubiquitinate the nonactivated form of Notch in cell culture and in vitro. These results characterize a new level of conserved regulation of Notch signaling by the ubiquitin system.


Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas Nucleares/metabolismo , Proteolisis , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Ubiquitina Tiolesterasa/metabolismo , Animales , Línea Celular Tumoral , Proteínas de Drosophila/genética , Drosophila melanogaster , Humanos , Proteínas Nucleares/genética , Transporte de Proteínas/fisiología , Receptores Notch/genética , Ubiquitina Tiolesterasa/genética
7.
EMBO J ; 28(19): 2885-95, 2009 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-19763089

RESUMEN

An important property of NEMO, the core element of the IKK complex involved in NF-kappaB activation, resides in its ability to specifically recognize poly-ubiquitin chains. A small domain called NOA/UBAN has been suggested to be responsible for this property. We recently demonstrated that the C-terminal Zinc Finger (ZF) of NEMO is also able to bind ubiquitin. We show here by ZF swapping and mutagenesis that this represents its only function. While neither NOA nor ZF shows any preference for K63-linked chains, we demonstrate that together they form a bipartite high-affinity K63-specific ubiquitin-binding domain. A similar domain can be found in two other proteins, Optineurin and ABIN2, and can be freely exchanged with that of NEMO without interfering with its activity. This suggests that the main function of the C-terminal half of NEMO is to specifically bind K63-linked poly-ubiquitin chains. We also demonstrate that the recently described binding of NEMO to linear poly-ubiquitin chains is dependent on the NOA alone and does not require the presence of the ZF.


Asunto(s)
Quinasa I-kappa B/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Poliubiquitina/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Regulación hacia Abajo , Humanos , Quinasa I-kappa B/química , Quinasa I-kappa B/genética , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Células Jurkat , Ratones , Datos de Secuencia Molecular , Poliubiquitina/química , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Dedos de Zinc
8.
Blood ; 118(4): 926-35, 2011 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-21622647

RESUMEN

Nuclear factor-κB essential modulator (NEMO), the regulatory subunit of the IκB kinase complex, is a critical component of the NF-κB pathway. Hypomorphic mutations in the X-linked human NEMO gene cause various forms of anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID). All known X-linked EDA-ID-causing mutations impair NEMO protein expression, folding, or both. We describe here 2 EDA-ID-causing missense mutations that affect the same residue in the CC2-LZ domain (D311N and D311G) that do not impair NEMO production or folding. Structural studies based on pull-down experiments showed a defect in noncovalent interaction with K63-linked and linear polyubiquitin chains for these mutant proteins. Functional studies on the patients' cells showed an impairment of the classic NF-κB signaling pathways after activation of 2 NEMO ubiquitin-binding-dependent receptors, the TNF and IL-1ß receptors, and in the CD40-dependent NF-κB pathway. We report the first human NEMO mutations responsible for X-linked EDA-ID found to affect the polyubiquitin binding of NEMO rather than its expression and folding. These experiments demonstrate that the binding of human NEMO to polyubiquitin is essential for NF-κB activation. They also demonstrate that the normal expression and folding of NEMO do not exclude a pathogenic role for NEMO mutations in patients with EDA-ID.


Asunto(s)
Displasia Ectodermal Anhidrótica Tipo 1/genética , Quinasa I-kappa B/genética , Síndromes de Inmunodeficiencia/genética , Ubiquitina/metabolismo , Western Blotting , Displasia Ectodermal Anhidrótica Tipo 1/metabolismo , Activación Enzimática/genética , Femenino , Humanos , Quinasa I-kappa B/metabolismo , Síndromes de Inmunodeficiencia/metabolismo , Masculino , Mutación Missense , FN-kappa B/metabolismo , Linaje , Unión Proteica , Pliegue de Proteína , Transducción de Señal/genética , Adulto Joven
9.
PLoS Biol ; 8(11): e1000545, 2010 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-21124883

RESUMEN

Activation of the mammalian Notch receptor after ligand binding relies on a succession of events including metalloprotease-cleavage, endocytosis, monoubiquitination, and eventually processing by the gamma-secretase, giving rise to a soluble, transcriptionally active molecule. The Notch1 receptor was proposed to be monoubiquitinated before its gamma-secretase cleavage; the targeted lysine has been localized to its submembrane domain. Investigating how this step might be regulated by a deubiquitinase (DUB) activity will provide new insight for understanding Notch receptor activation and downstream signaling. An immunofluorescence-based screening of an shRNA library allowed us to identify eIF3f, previously known as one of the subunits of the translation initiation factor eIF3, as a DUB targeting the activated Notch receptor. We show that eIF3f has an intrinsic DUB activity. Knocking down eIF3f leads to an accumulation of monoubiquitinated forms of activated Notch, an effect counteracted by murine WT eIF3f but not by a catalytically inactive mutant. We also show that eIF3f is recruited to activated Notch on endocytic vesicles by the putative E3 ubiquitin ligase Deltex1, which serves as a bridging factor. Finally, catalytically inactive forms of eIF3f as well as shRNAs targeting eIF3f repress Notch activation in a coculture assay, showing that eIF3f is a new positive regulator of the Notch pathway. Our results support two new and provocative conclusions: (1) The activated form of Notch needs to be deubiquitinated before being processed by the gamma-secretase activity and entering the nucleus, where it fulfills its transcriptional function. (2) The enzyme accounting for this deubiquitinase activity is eIF3f, known so far as a translation initiation factor. These data improve our knowledge of Notch signaling but also open new avenues of research on the Zomes family and the translation initiation factors.


Asunto(s)
Enzimas/metabolismo , Factor 3 de Iniciación Eucariótica/fisiología , Receptores Notch/metabolismo , Ubiquitina/metabolismo , Línea Celular , Factor 3 de Iniciación Eucariótica/genética , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Humanos , Transducción de Señal
10.
J Biol Chem ; 286(21): 18720-30, 2011 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-21464124

RESUMEN

The Notch pathway is involved in cell-cell signaling during development and adulthood from invertebrates to higher eukaryotes. Activation of the Notch receptor by its ligands relies upon a multi-step processing. The extracellular part of the receptor is removed by a metalloprotease of the ADAM family and the remaining fragment is cleaved within its transmembrane domain by a presenilin-dependent γ-secretase activity. γ-Secretase processing of Notch has been shown to depend upon monoubiquitination as well as clathrin-mediated endocytosis (CME). We show here that AAK1, the adaptor-associated kinase 1, directly interacts with the membrane-tethered active form of Notch released by metalloprotease cleavage. Active AAK1 acts upstream of the γ-secretase cleavage by stabilizing both the membrane-tethered activated form of Notch and its monoubiquitinated counterpart. We propose that AAK1 acts as an adaptor for Notch interaction with components of the clathrin-mediated pathway such as Eps15b. Moreover, transfected AAK1 increases the localization of activated Notch to Rab5-positive endocytic vesicles, while AAK1 depletion or overexpression of Numb, an inhibitor of the pathway, interferes with this localization. These results suggest that after ligand-induced activation of Notch, the membrane-tethered form can be directed to different endocytic pathways leading to distinct fates.


Asunto(s)
Endocitosis/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Notch/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Metaloproteasas/genética , Metaloproteasas/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Estabilidad Proteica , Receptores Notch/genética , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismo
11.
J Exp Med ; 203(7): 1745-59, 2006 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-16818673

RESUMEN

Germline mutations in five autosomal genes involved in interleukin (IL)-12-dependent, interferon (IFN)-gamma-mediated immunity cause Mendelian susceptibility to mycobacterial diseases (MSMD). The molecular basis of X-linked recessive (XR)-MSMD remains unknown. We report here mutations in the leucine zipper (LZ) domain of the NF-kappaB essential modulator (NEMO) gene in three unrelated kindreds with XR-MSMD. The mutant proteins were produced in normal amounts in blood and fibroblastic cells. However, the patients' monocytes presented an intrinsic defect in T cell-dependent IL-12 production, resulting in defective IFN-gamma secretion by T cells. IL-12 production was also impaired as the result of a specific defect in NEMO- and NF-kappaB/c-Rel-mediated CD40 signaling after the stimulation of monocytes and dendritic cells by CD40L-expressing T cells and fibroblasts, respectively. However, the CD40-dependent up-regulation of costimulatory molecules of dendritic cells and the proliferation and immunoglobulin class switch of B cells were normal. Moreover, the patients' blood and fibroblastic cells responded to other NF-kappaB activators, such as tumor necrosis factor-alpha, IL-1beta, and lipopolysaccharide. These two mutations in the NEMO LZ domain provide the first genetic etiology of XR-MSMD. They also demonstrate the importance of the T cell- and CD40L-triggered, CD40-, and NEMO/NF-kappaB/c-Rel-mediated induction of IL-12 by monocyte-derived cells for protective immunity to mycobacteria in humans.


Asunto(s)
Antígenos CD40/fisiología , Genes Ligados a X , Predisposición Genética a la Enfermedad , Quinasa I-kappa B/genética , Interleucina-12/biosíntesis , Infecciones por Mycobacterium/genética , Infecciones por Mycobacterium/inmunología , Cromosoma X , Adolescente , Adulto , Animales , Línea Celular Transformada , Células Cultivadas , Niño , Preescolar , Femenino , Humanos , Lactante , Células L , Masculino , Ratones , Linaje
12.
Curr Top Microbiol Immunol ; 349: 73-95, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-20845108

RESUMEN

The IKK kinase complex is the core element of the NF-κB cascade. It is essentially made of two kinases (IKKα and IKKß) and a regulatory subunit, NEMO/IKKγ. Additional components may exist, transiently or permanently, but their characterization is still uncertain. In this review, we will focus on the NEMO molecule, and describe the results which have been obtained, and the hypotheses which have been proposed, to explain how NEMO controls the activation of the IKK complex. NEMO is one of the very few non-redundant components of the NF-κB cascade, and the localization of the gene that encodes it on the X chromosome suggests it is likely to be the target of mutations leading to pathologies: this is indeed the case, and we will also present the current status of our knowledge regarding NEMO-associated pathologies.


Asunto(s)
Quinasa I-kappa B/fisiología , Displasia Ectodérmica/genética , Humanos , Quinasa I-kappa B/química , Quinasa I-kappa B/genética , Incontinencia Pigmentaria/etiología , Incontinencia Pigmentaria/genética , Procesamiento Proteico-Postraduccional
13.
J Allergy Clin Immunol ; 128(3): 610-7.e1-4, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21722947

RESUMEN

BACKGROUND: Children with germline mutations in Toll-like receptor 3 (TLR3), UNC93B1, TNF receptor-associated factor 3, and signal transducer and activator of transcription 1 are prone to herpes simplex virus-1 encephalitis, owing to impaired TLR3-triggered, UNC-93B-dependent, IFN-α/ß, and/or IFN-λ-mediated signal transducer and activator of transcription 1-dependent immunity. OBJECTIVE: We explore here the molecular basis of the pathogenesis of herpes simplex encephalitis in a child with a hypomorphic mutation in nuclear factor-κB (NF-κB) essential modulator, which encodes the regulatory subunit of the inhibitor of the Iκß kinase complex. METHODS: The TLR3 signaling pathway was investigated in the patient's fibroblasts by analyses of IFN-ß, IFN-λ, and IL-6 mRNA and protein levels, by quantitative PCR and ELISA, respectively, upon TLR3 stimulation (TLR3 agonists or TLR3-dependent viruses). NF-κB activation was assessed by electrophoretic mobility shift assay and interferon regulatory factor 3 dimerization on native gels after stimulation with a TLR3 agonist. RESULTS: The patient's fibroblasts displayed impaired responses to TLR3 stimulation in terms of IFN-ß, IFN-λ, and IL-6 production, owing to impaired activation of both NF-κB and IRF-3. Moreover, vesicular stomatitis virus, a potent IFN-inducer in human fibroblasts, and herpes simplex virus-1, induced only low levels of IFN-ß and IFN-λ in the patient's fibroblasts, resulting in enhanced viral replication and cell death, as reported for UNC-93B-deficient fibroblasts. CONCLUSION: Herpes simplex encephalitis may occur in patients carrying NF-κB essential modulator mutations, due to the impairment of NF-κB- and interferon regulatory factor 3-dependent-TLR3-mediated antiviral IFN production.


Asunto(s)
Encefalitis por Herpes Simple/inmunología , Quinasa I-kappa B/metabolismo , Factor 3 Regulador del Interferón/metabolismo , Mutación , FN-kappa B/metabolismo , Receptor Toll-Like 3/inmunología , Preescolar , Encefalitis por Herpes Simple/genética , Encefalitis por Herpes Simple/virología , Fibroblastos/inmunología , Fibroblastos/metabolismo , Humanos , Quinasa I-kappa B/genética , Transducción de Señal , Simplexvirus/inmunología , Receptor Toll-Like 3/metabolismo
14.
PLoS Pathog ; 5(7): e1000521, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19609363

RESUMEN

Nuclear factor (NF)-kappaB is a major survival pathway engaged by the Human T-Lymphotropic Virus type 1 (HTLV-1) Tax protein. Tax1 activation of NF-kappaB occurs predominantly in the cytoplasm, where Tax1 binds NF-kappaB Essential Modulator (NEMO/IKKgamma) and triggers the activation of IkappaB kinases. Several independent studies have shown that Tax1-mediated NF-kappaB activation is dependent on Tax1 ubiquitination. Here, we identify by co-immunoprecipitation assays NEMO-Related Protein (NRP/Optineurin) as a binding partner for Tax1 in HTLV-1 infected and Tax1/NRP co-expressing cells. Immunofluorescence studies reveal that Tax1, NRP and NEMO colocalize in Golgi-associated structures. The interaction between Tax1 and NRP requires the ubiquitin-binding activity of NRP and the ubiquitination sites of Tax1. In addition, we observe that NRP increases the ubiquitination of Tax1 along with Tax1-dependent NF-kappaB signaling. Surprisingly, we find that in addition to Tax1, NRP interacts cooperatively with the Tax1 binding protein TAX1BP1, and that NRP and TAX1BP1 cooperate to modulate Tax1 ubiquitination and NF-kappaB activation. Our data strongly suggest for the first time that NRP is a critical adaptor that regulates the assembly of TAX1BP1 and post-translationally modified forms of Tax1, leading to sustained NF-kappaB activation.


Asunto(s)
Productos del Gen tax/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Factor de Transcripción TFIIIA/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular , Línea Celular Tumoral , Productos del Gen tax/genética , Aparato de Golgi , Células HeLa , Humanos , Inmunoprecipitación , Espacio Intracelular/metabolismo , Proteínas de Transporte de Membrana , Dominios y Motivos de Interacción de Proteínas , Técnicas del Sistema de Dos Híbridos , Ubiquitinación
15.
Proc Natl Acad Sci U S A ; 105(32): 11212-7, 2008 Aug 12.
Artículo en Inglés | MEDLINE | ID: mdl-18676613

RESUMEN

Genetic studies have shown that ubiquitination and endocytosis of the Drosophila ligand Delta in signal-sending cells are required for activation of Notch signaling, but how these events promote Notch activation remains poorly understood. Here, we show that an ubiquitination-defective mutant of the murine Delta-homologue Dll1 is endocytosed but, in contrast to the wild-type Dll1, is unable to subsequently recycle back to the cell surface or to bind Notch1 efficiently. These results demonstrate that ubiquitination, although not required for endocytosis, is essential for Dll1 recycling and that recycling is required to acquire affinity for the receptor. On the other hand, a chimeric molecule encompassing the extracellular domain of Dll1 and the transmembrane/intracellular domain of Dll3, which contains no lysine, is endocytosed, recycled, and interacts with Notch1 but is unable to induce transendocytosis of the extracellular region of Notch1 or to signal. These observations suggest that the chimera uses an ubiquitination-independent signal to recycle, allowing it to acquire affinity for Notch1. Our results support the idea that ligand recycling determines its competence to bind efficiently to the receptor but that this is insufficient to allow it to perform transendocytosis, an event required for activation of Notch signaling. Finally, the present study indicates that Dll1 partially localizes to lipid microdomains, whereas both ubiquitination-defective Dll1 and the Dll1-3 chimera are excluded from these compartments, suggesting that these microdomains provide the environment necessary for Dll1 to activate Notch signaling.


Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal/fisiología , Ubiquitinación/fisiología , Animales , Proteínas de Unión al Calcio , Drosophila , Endocitosis/fisiología , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Ligandos , Microdominios de Membrana/genética , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/genética , Ratones , Transporte de Proteínas/fisiología , Receptor Notch1/genética , Receptor Notch1/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
17.
Nature ; 424(6950): 801-5, 2003 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-12917691

RESUMEN

NF-kappaB transcription factors have key roles in inflammation, immune response, oncogenesis and protection against apoptosis. In most cells, these factors are kept inactive in the cytoplasm through association with IkappaB inhibitors. After stimulation by various reagents, IkappaB is phosphorylated by the IkappaB kinase (IKK) complex and degraded by the proteasome, allowing NF-kappaB to translocate to the nucleus and activate its target genes. Here we report that CYLD, a tumour suppressor that is mutated in familial cylindromatosis, interacts with NEMO, the regulatory subunit of IKK. CYLD also interacts directly with tumour-necrosis factor receptor (TNFR)-associated factor 2 (TRAF2), an adaptor molecule involved in signalling by members of the family of TNF/nerve growth factor receptors. CYLD has deubiquitinating activity that is directed towards non-K48-linked polyubiquitin chains, and negatively modulates TRAF-mediated activation of IKK, strengthening the notion that ubiquitination is involved in IKK activation by TRAFs and suggesting that CYLD functions in this process. Truncations of CYLD found in cylindromatosis result in reduced enzymatic activity, indicating a link between impaired deubiquitination of CYLD substrates and human pathophysiology.


Asunto(s)
FN-kappa B/antagonistas & inhibidores , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Catálisis , Línea Celular , Enzima Desubiquitinante CYLD , Humanos , Quinasa I-kappa B , Datos de Secuencia Molecular , Mutación , FN-kappa B/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Especificidad por Sustrato , Factor 2 Asociado a Receptor de TNF , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética
18.
J Cell Biol ; 166(1): 73-83, 2004 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-15240571

RESUMEN

Activation of mammalian Notch receptor by its ligands induces TNFalpha-converting enzyme-dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent gamma-secretase activity. Here, we demonstrate that a new modification, a monoubiquitination, as well as clathrin-dependent endocytosis, is required for gamma-secretase processing of a constitutively active Notch derivative, DeltaE, which mimics the TNFalpha-converting enzyme-processing product. PS interacts with this modified form of DeltaE, DeltaEu. We identified the lysine residue targeted by the monoubiquitination event and confirmed its importance for activation of Notch receptor by its ligand, Delta-like 1. We propose a new model where monoubiquitination and endocytosis of Notch are a prerequisite for its PS-dependent cleavage, and discuss its relevance for other gamma-secretase substrates.


Asunto(s)
Endocitosis , Endopeptidasas/metabolismo , Proteínas de la Membrana/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas , Línea Celular , Células HeLa , Humanos , Immunoblotting , Ligandos , Lisina/química , Microscopía Confocal , Microscopía Fluorescente , Datos de Secuencia Molecular , Pruebas de Precipitina , Presenilina-1 , Unión Proteica , Estructura Terciaria de Proteína , Receptores Notch , Homología de Secuencia de Aminoácido , Transducción de Señal , Factores de Tiempo , Transfección , Ubiquitina/química
19.
Mol Cell Biol ; 26(8): 2936-46, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16581769

RESUMEN

Synaptic activity-dependent de novo gene transcription is crucial for long-lasting neuronal plasticity and long-term memory. In a forebrain neuronal conditional NF-kappaB-deficient mouse model, we demonstrate here that the transcription factor NF-kappaB regulates spatial memory formation, synaptic transmission, and plasticity. Gene profiling experiments and analysis of regulatory regions identified the alpha catalytic subunit of protein kinase A (PKA), an essential memory regulator, as a new NF-kappaB target gene. Consequently, NF-kappaB inhibition led to a decrease in forskolin-induced CREB phosphorylation. Collectively, these results disclose a novel hierarchical transcriptional network involving NF-kappaB, PKA, and CREB that leads to concerted nuclear transduction of synaptic signals in neurons, accounting for the critical function of NF-kappaB in learning and memory.


Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Memoria/fisiología , FN-kappa B/fisiología , Transmisión Sináptica/fisiología , Animales , Células Cultivadas , Colforsina/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Ensayo de Cambio de Movilidad Electroforética , Electrofisiología , Perfilación de la Expresión Génica , Hipocampo/citología , Hipocampo/fisiología , Inmunohistoquímica , Masculino , Ratones , Ratones Transgénicos , Fosforilación/efectos de los fármacos
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