RESUMEN
RAF family protein kinases are a key node in the RAS/RAF/MAP kinase pathway, the signaling cascade that controls cellular proliferation, differentiation, and survival in response to engagement of growth factor receptors on the cell surface. Over the past few years, structural and biochemical studies have provided new understanding of RAF autoregulation, RAF activation by RAS and the SHOC2 phosphatase complex, and RAF engagement with HSP90-CDC37 chaperone complexes. These studies have important implications for pharmacologic targeting of the pathway. They reveal RAF in distinct regulatory states and show that the functional RAF switch is an integrated complex of RAF with its substrate (MEK) and a 14-3-3 dimer. Here we review these advances, placing them in the context of decades of investigation of RAF regulation. We explore the insights they provide into aberrant activation of the pathway in cancer and RASopathies (developmental syndromes caused by germline mutations in components of the pathway).
Asunto(s)
Transducción de Señal , Quinasas raf , Proteínas ras , Humanos , Proteínas ras/metabolismo , Proteínas ras/genética , Proteínas ras/química , Quinasas raf/metabolismo , Quinasas raf/genética , Animales , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patología , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genéticaRESUMEN
The nicotinic acetylcholine receptor has served, since its biochemical identification in the 1970s, as a model of an allosteric ligand-gated ion channel mediating signal transition at the synapse. In recent years, the application of X-ray crystallography and high-resolution cryo-electron microscopy, together with molecular dynamic simulations of nicotinic receptors and homologs, have opened a new era in the understanding of channel gating by the neurotransmitter. They reveal, at atomic resolution, the diversity and flexibility of the multiple ligand-binding sites, including recently discovered allosteric modulatory sites distinct from the neurotransmitter orthosteric site, and the conformational dynamics of the activation process as a molecular switch linking these multiple sites. The model emerging from these studies paves the way for a new pharmacology based, first, upon the occurrence of an original mode of indirect allosteric modulation, distinct from a steric competition for a single and rigid binding site, and second, the design of drugs that specifically interact with privileged conformations of the receptor such as agonists, antagonists, and desensitizers. Research on nicotinic receptors is still at the forefront of understanding the mode of action of drugs on the nervous system.
Asunto(s)
Sitio Alostérico , Microscopía por Crioelectrón , Simulación de Dinámica Molecular , Receptores Nicotínicos , Transducción de Señal , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Regulación Alostérica , Humanos , Animales , Cristalografía por Rayos X , Sitios de Unión , Conformación Proteica , Ligandos , Modelos Moleculares , Multimerización de Proteína , Agonistas Nicotínicos/química , Agonistas Nicotínicos/farmacología , Agonistas Nicotínicos/metabolismoRESUMEN
During development, morphogens pattern tissues by instructing cell fate across long distances. Directly visualizing morphogen transport in situ has been inaccessible, so the molecular mechanisms ensuring successful morphogen delivery remain unclear. To tackle this longstanding problem, we developed a mouse model for compromised sonic hedgehog (SHH) morphogen delivery and discovered that endocytic recycling promotes SHH loading into signaling filopodia called cytonemes. We optimized methods to preserve in vivo cytonemes for advanced microscopy and show endogenous SHH localized to cytonemes in developing mouse neural tubes. Depletion of SHH from neural tube cytonemes alters neuronal cell fates and compromises neurodevelopment. Mutation of the filopodial motor myosin 10 (MYO10) reduces cytoneme length and density, which corrupts neuronal signaling activity of both SHH and WNT. Combined, these results demonstrate that cytoneme-based signal transport provides essential contributions to morphogen dispersion during mammalian tissue development and suggest MYO10 is a key regulator of cytoneme function.
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Estructuras de la Membrana Celular , Miosinas , Tubo Neural , Transducción de Señal , Animales , Ratones , Transporte Biológico , Estructuras de la Membrana Celular/metabolismo , Proteínas Hedgehog/metabolismo , Miosinas/metabolismo , Seudópodos/metabolismo , Tubo Neural/citología , Tubo Neural/metabolismoRESUMEN
Many of the world's most devastating crop diseases are caused by fungal pathogens that elaborate specialized infection structures to invade plant tissue. Here, we present a quantitative mass-spectrometry-based phosphoproteomic analysis of infection-related development by the rice blast fungus Magnaporthe oryzae, which threatens global food security. We mapped 8,005 phosphosites on 2,062 fungal proteins following germination on a hydrophobic surface, revealing major re-wiring of phosphorylation-based signaling cascades during appressorium development. Comparing phosphosite conservation across 41 fungal species reveals phosphorylation signatures specifically associated with biotrophic and hemibiotrophic fungal infection. We then used parallel reaction monitoring (PRM) to identify phosphoproteins regulated by the fungal Pmk1 MAPK that controls plant infection by M. oryzae. We define 32 substrates of Pmk1 and show that Pmk1-dependent phosphorylation of regulator Vts1 is required for rice blast disease. Defining the phosphorylation landscape of infection therefore identifies potential therapeutic interventions for the control of plant diseases.
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Proteínas Fúngicas , Oryza , Enfermedades de las Plantas , Fosforilación , Oryza/microbiología , Oryza/metabolismo , Enfermedades de las Plantas/microbiología , Proteínas Fúngicas/metabolismo , Fosfoproteínas/metabolismo , Ascomicetos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteómica , Transducción de SeñalRESUMEN
It is currently not known whether mRNAs fulfill structural roles in the cytoplasm. Here, we report the fragile X-related protein 1 (FXR1) network, an mRNA-protein (mRNP) network present throughout the cytoplasm, formed by FXR1-mediated packaging of exceptionally long mRNAs. These mRNAs serve as an underlying condensate scaffold and concentrate FXR1 molecules. The FXR1 network contains multiple protein binding sites and functions as a signaling scaffold for interacting proteins. We show that it is necessary for RhoA signaling-induced actomyosin reorganization to provide spatial proximity between kinases and their substrates. Point mutations in FXR1, found in its homolog FMR1, where they cause fragile X syndrome, disrupt the network. FXR1 network disruption prevents actomyosin remodeling-an essential and ubiquitous process for the regulation of cell shape, migration, and synaptic function. Our findings uncover a structural role for cytoplasmic mRNA and show how the FXR1 RNA-binding protein as part of the FXR1 network acts as an organizer of signaling reactions.
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Actomiosina , ARN Mensajero , Proteínas de Unión al ARN , Transducción de Señal , Proteína de Unión al GTP rhoA , Proteínas de Unión al ARN/metabolismo , Humanos , Actomiosina/metabolismo , Animales , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteína de Unión al GTP rhoA/metabolismo , Ratones , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Citoplasma/metabolismo , Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/genéticaRESUMEN
The protein kinase C (PKC) family, discovered in the late 1970s, is composed of at least 10 serine/threonine kinases, divided into three groups based on their molecular architecture and cofactor requirements. PKC enzymes have been conserved throughout evolution and are expressed in virtually all cell types; they represent critical signal transducers regulating cell activation, differentiation, proliferation, death, and effector functions. PKC family members play important roles in a diverse array of hematopoietic and immune responses. This review covers the discovery and history of this enzyme family, discusses the roles of PKC enzymes in the development and effector functions of major hematopoietic and immune cell types, and points out gaps in our knowledge, which should ignite interest and further exploration, ultimately leading to better understanding of this enzyme family and, above all, its role in the many facets of the immune system.
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Hematopoyesis , Sistema Inmunológico , Proteína Quinasa C/metabolismo , Animales , Coenzimas/metabolismo , Activación Enzimática/inmunología , Humanos , Proteína Quinasa C/inmunología , Transducción de SeñalRESUMEN
Although Ca2+ has long been recognized as an obligatory intermediate in visual transduction, its role in plant phototransduction remains elusive. Here, we report a Ca2+ signaling that controls photoreceptor phyB nuclear translocation in etiolated seedlings during dark-to-light transition. Red light stimulates acute cytosolic Ca2+ increases via phyB, which are sensed by Ca2+-binding protein kinases, CPK6 and CPK12 (CPK6/12). Upon Ca2+ activation, CPK6/12 in turn directly interact with and phosphorylate photo-activated phyB at Ser80/Ser106 to initiate phyB nuclear import. Non-phosphorylatable mutation, phyBS80A/S106A, abolishes nuclear translocation and fails to complement phyB mutant, which is fully restored by combining phyBS80A/S106A with a nuclear localization signal. We further show that CPK6/12 function specifically in the early phyB-mediated cotyledon expansion, while Ser80/Ser106 phosphorylation generally governs phyB nuclear translocation. Our results uncover a biochemical regulatory loop centered in phyB phototransduction and provide a paradigm for linking ubiquitous Ca2+ increases to specific responses in sensory stimulus processing.
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Proteínas de Arabidopsis , Arabidopsis , Fitocromo , Fitocromo B/genética , Fitocromo B/metabolismo , Fitocromo/genética , Fitocromo/metabolismo , Calcio/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Luz , Fototransducción , MutaciónRESUMEN
Molecular signals interact in networks to mediate biological processes. To analyze these networks, it would be useful to image many signals at once, in the same living cell, using standard microscopes and genetically encoded fluorescent reporters. Here, we report temporally multiplexed imaging (TMI), which uses genetically encoded fluorescent proteins with different clocklike properties-such as reversibly photoswitchable fluorescent proteins with different switching kinetics-to represent different cellular signals. We linearly decompose a brief (few-second-long) trace of the fluorescence fluctuations, at each point in a cell, into a weighted sum of the traces exhibited by each fluorophore expressed in the cell. The weights then represent the signal amplitudes. We use TMI to analyze relationships between different kinase activities in individual cells, as well as between different cell-cycle signals, pointing toward broad utility throughout biology in the analysis of signal transduction cascades in living systems.
Asunto(s)
Proteínas , Transducción de Señal , Animales , Humanos , Ratones , Línea Celular , Colorantes Fluorescentes , Microscopía Fluorescente/métodos , Fosforilación , Supervivencia CelularRESUMEN
The Wnt pathway is central to a host of developmental and disease-related processes. The remarkable conservation of this intercellular signaling cascade throughout metazoan lineages indicates that it coevolved with multicellularity to regulate the generation and spatial arrangement of distinct cell types. By regulating cell fate specification, mitotic activity, and cell polarity, Wnt signaling orchestrates development and tissue homeostasis, and its dysregulation is implicated in developmental defects, cancer, and degenerative disorders. We review advances in our understanding of this key pathway, from Wnt protein production and secretion to relay of the signal in the cytoplasm of the receiving cell. We discuss the evolutionary history of this pathway as well as endogenous and synthetic modulators of its activity. Finally, we highlight remaining gaps in our knowledge of Wnt signal transduction and avenues for future research.
Asunto(s)
Neoplasias , Vía de Señalización Wnt , Animales , Diferenciación Celular , Neoplasias/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismoRESUMEN
T cells carry out the formidable task of identifying small numbers of foreign antigenic peptides rapidly and specifically against a very noisy environmental background of endogenous self-peptides. Early steps in T cell activation have thus fascinated biologists and are among the best-studied models of cell stimulation. This remarkable process, critical in adaptive immune responses, approaches and even seems to exceed the limitations set by the physical laws ruling molecular behavior. Despite the enormous amount of information concerning the nature of molecules involved in the T cell antigen receptor (TCR) signal transduction network, and the description of the nanoscale organization and real-time analysis of T cell responses, the general principles of information gathering and processing remain incompletely understood. Here we review currently accepted key data on TCR function, discuss the limitations of current research strategies, and suggest a novel model of TCR triggering and a few promising ways of going further into the integration of available data.
Asunto(s)
Activación de Linfocitos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Humanos , Modelos Inmunológicos , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de SeñalRESUMEN
Optobiochemical control of protein activities allows the investigation of protein functions in living cells with high spatiotemporal resolution. Over the last two decades, numerous natural photosensory domains have been characterized and synthetic domains engineered and assembled into photoregulatory systems to control protein function with light. Here, we review the field of optobiochemistry, categorizing photosensory domains by chromophore, describing photoregulatory systems by mechanism of action, and discussing protein classes frequently investigated using optical methods. We also present examples of how spatial or temporal control of proteins in living cells has provided new insights not possible with traditional biochemical or cell biological techniques.
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Bioquímica/métodos , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Criptocromos/química , Criptocromos/metabolismo , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/metabolismo , Luz , Optogenética/métodos , Procesos Fotoquímicos , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Fitocromo/química , Fitocromo/metabolismo , Dominios Proteicos , Ingeniería de Proteínas/métodos , Vitamina B 12/metabolismoRESUMEN
Hedgehog pathway components and select G protein-coupled receptors (GPCRs) localize to the primary cilium, an organelle specialized for signal transduction. We investigated whether cells distinguish between ciliary and extraciliary GPCR signaling. To test whether ciliary and extraciliary cyclic AMP (cAMP) convey different information, we engineered optogenetic and chemogenetic tools to control the subcellular site of cAMP generation. Generating equal amounts of ciliary and cytoplasmic cAMP in zebrafish and mammalian cells revealed that ciliary cAMP, but not cytoplasmic cAMP, inhibited Hedgehog signaling. Modeling suggested that the distinct geometries of the cilium and cell body differentially activate local effectors. The search for effectors identified a ciliary pool of protein kinase A (PKA). Blocking the function of ciliary PKA, but not extraciliary PKA, activated Hedgehog signal transduction and reversed the effects of ciliary cAMP. Therefore, cells distinguish ciliary and extraciliary cAMP using functionally and spatially distinct pools of PKA, and different subcellular pools of cAMP convey different information.
Asunto(s)
Cilios/metabolismo , AMP Cíclico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/metabolismo , Neuronas/metabolismo , Optogenética/métodos , Transducción de Señal/fisiología , Pez Cebra/metabolismoRESUMEN
During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a "trimer-of-trimers" super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas Ligadas a GPI/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Oncogénicas/metabolismo , Animales , Moléculas de Adhesión Celular Neuronal/química , Movimiento Celular , Receptor DCC/deficiencia , Receptor DCC/genética , Proteínas Ligadas a GPI/química , Conos de Crecimiento/fisiología , Humanos , Ventrículos Laterales/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/química , Neuronas/citología , Neuronas/metabolismo , Proteínas Oncogénicas/química , Proteínas Oncogénicas/genética , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
Animals display wide-ranging evolutionary adaptations based on their ecological niche. Octopuses explore the seafloor with their flexible arms using a specialized "taste by touch" system to locally sense and respond to prey-derived chemicals and movement. How the peripherally distributed octopus nervous system mediates relatively autonomous arm behavior is unknown. Here, we report that octopus arms use a family of cephalopod-specific chemotactile receptors (CRs) to detect poorly soluble natural products, thereby defining a form of contact-dependent, aquatic chemosensation. CRs form discrete ion channel complexes that mediate the detection of diverse stimuli and transduction of specific ionic signals. Furthermore, distinct chemo- and mechanosensory cells exhibit specific receptor expression and electrical activities to support peripheral information coding and complex chemotactile behaviors. These findings demonstrate that the peripherally distributed octopus nervous system is a key site for signal processing and highlight how molecular and anatomical features synergistically evolve to suit an animal's environmental context.
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Células Quimiorreceptoras/metabolismo , Octopodiformes/fisiología , Tacto/fisiología , Acetilcolina/farmacología , Secuencia de Aminoácidos , Animales , Conducta Animal , Femenino , Células HEK293 , Humanos , Octopodiformes/anatomía & histología , Octopodiformes/genética , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Receptores Colinérgicos/metabolismo , Transducción de SeñalRESUMEN
Hallucinogens like lysergic acid diethylamide (LSD), psilocybin, and substituted N-benzyl phenylalkylamines are widely used recreationally with psilocybin being considered as a therapeutic for many neuropsychiatric disorders including depression, anxiety, and substance abuse. How psychedelics mediate their actions-both therapeutic and hallucinogenic-are not understood, although activation of the 5-HT2A serotonin receptor (HTR2A) is key. To gain molecular insights into psychedelic actions, we determined the active-state structure of HTR2A bound to 25-CN-NBOH-a prototypical hallucinogen-in complex with an engineered Gαq heterotrimer by cryoelectron microscopy (cryo-EM). We also obtained the X-ray crystal structures of HTR2A complexed with the arrestin-biased ligand LSD or the inverse agonist methiothepin. Comparisons of these structures reveal determinants responsible for HTR2A-Gαq protein interactions as well as the conformational rearrangements involved in active-state transitions. Given the potential therapeutic actions of hallucinogens, these findings could accelerate the discovery of more selective drugs for the treatment of a variety of neuropsychiatric disorders.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Alucinógenos/química , Receptor de Serotonina 5-HT2A/química , Receptor de Serotonina 5-HT2A/metabolismo , Animales , Microscopía por Crioelectrón , Cristalografía por Rayos X , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Expresión Génica , Células HEK293 , Alucinógenos/farmacología , Alucinógenos/uso terapéutico , Humanos , Ligandos , Dietilamida del Ácido Lisérgico/química , Dietilamida del Ácido Lisérgico/farmacología , Metiotepina/química , Metiotepina/metabolismo , Modelos Químicos , Mutación , Conformación Proteica en Hélice alfa , Receptor de Serotonina 5-HT2A/genética , Proteínas Recombinantes , Serotonina/metabolismo , SpodopteraRESUMEN
The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.
Asunto(s)
Carcinogénesis/metabolismo , Carcinoma Hepatocelular/genética , Compartimento Celular/genética , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas del Choque Térmico HSP40/genética , Neoplasias Hepáticas/genética , Transducción de Señal , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinoma Hepatocelular/metabolismo , Compartimento Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , AMP Cíclico/farmacología , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Ratones , Oncogenes/genética , Dominios Proteicos , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión , Espectroscopía Infrarroja por Transformada de Fourier , Imagen de Lapso de Tiempo/métodosRESUMEN
In order to analyze how a signal transduction network converts cellular inputs into cellular outputs, ideally one would measure the dynamics of many signals within the network simultaneously. We found that, by fusing a fluorescent reporter to a pair of self-assembling peptides, it could be stably clustered within cells at random points, distant enough to be resolved by a microscope but close enough to spatially sample the relevant biology. Because such clusters, which we call signaling reporter islands (SiRIs), can be modularly designed, they permit a set of fluorescent reporters to be efficiently adapted for simultaneous measurement of multiple nodes of a signal transduction network within single cells. We created SiRIs for indicators of second messengers and kinases and used them, in hippocampal neurons in culture and intact brain slices, to discover relationships between the speed of calcium signaling, and the amplitude of PKA signaling, upon receiving a cAMP-driving stimulus.
Asunto(s)
Colorantes Fluorescentes/metabolismo , Genes Reporteros , Imagen Óptica , Transducción de Señal , Animales , Calcio/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Hipocampo/metabolismo , Humanos , Ratones , Neuronas/metabolismo , Péptidos/metabolismo , Proteínas/metabolismo , Células Piramidales/metabolismoRESUMEN
The Hippo pathway was initially discovered in Drosophila melanogaster as a key regulator of tissue growth. It is an evolutionarily conserved signaling cascade regulating numerous biological processes, including cell growth and fate decision, organ size control, and regeneration. The core of the Hippo pathway in mammals consists of a kinase cascade, MST1/2 and LATS1/2, as well as downstream effectors, transcriptional coactivators YAP and TAZ. These core components of the Hippo pathway control transcriptional programs involved in cell proliferation, survival, mobility, stemness, and differentiation. The Hippo pathway is tightly regulated by both intrinsic and extrinsic signals, such as mechanical force, cell-cell contact, polarity, energy status, stress, and many diffusible hormonal factors, the majority of which act through G protein-coupled receptors. Here, we review the current understanding of molecular mechanisms by which signals regulate the Hippo pathway with an emphasis on mechanotransduction and the effects of this pathway on basic biology and human diseases.
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Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Animales , Vía de Señalización Hippo , Humanos , Mecanotransducción Celular , Proteínas Serina-Treonina Quinasas/fisiología , Serina-Treonina Quinasa 3 , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Varying pH of luminal fluid along the female reproductive tract is a physiological cue that modulates sperm motility. CatSper is a sperm-specific, pH-sensitive calcium channel essential for hyperactivated motility and male fertility. Multi-subunit CatSper channel complexes organize linear Ca2+ signaling nanodomains along the sperm tail. Here, we identify EF-hand calcium-binding domain-containing protein 9 (EFCAB9) as a bifunctional, cytoplasmic machine modulating the channel activity and the domain organization of CatSper. Knockout mice studies demonstrate that EFCAB9, in complex with the CatSper subunit, CATSPERζ, is essential for pH-dependent and Ca2+-sensitive activation of the CatSper channel. In the absence of EFCAB9, sperm motility and fertility is compromised, and the linear arrangement of the Ca2+ signaling domains is disrupted. EFCAB9 interacts directly with CATSPERζ in a Ca2+-dependent manner and dissociates at elevated pH. These observations suggest that EFCAB9 is a long-sought, intracellular, pH-dependent Ca2+ sensor that triggers changes in sperm motility.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Motilidad Espermática/fisiología , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Proteínas de Unión al Calcio/fisiología , Línea Celular , Membrana Celular/metabolismo , Fertilidad , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Espermatozoides/metabolismoRESUMEN
The ubiquitin-binding endoribonuclease N4BP1 potently suppresses cytokine production by Toll-like receptors (TLRs) that signal through the adaptor MyD88 but is inactivated via caspase-8-mediated cleavage downstream of death receptors, TLR3, or TLR4. Here, we examined the mechanism whereby N4BP1 limits inflammatory responses. In macrophages, deletion of N4BP1 prolonged activation of inflammatory gene transcription at late time points after TRIF-independent TLR activation. Optimal suppression of inflammatory cytokines by N4BP1 depended on its ability to bind polyubiquitin chains, as macrophages and mice-bearing inactivating mutations in a ubiquitin-binding motif in N4BP1 displayed increased TLR-induced cytokine production. Deletion of the noncanonical IκB kinases (ncIKKs), Tbk1 and Ikke, or their adaptor Tank phenocopied N4bp1 deficiency and enhanced macrophage responses to TLR1/2, TLR7, or TLR9 stimulation. Mechanistically, N4BP1 acted in concert with the ncIKKs to limit the duration of canonical IκB kinase (IKKα/ß) signaling. Thus, N4BP1 and the ncIKKs serve as an important checkpoint against over-exuberant innate immune responses.