RESUMO
CREB (cAMP-response-element-binding protein) is an important transcription factor for the activation of a number of immediate early genes. CREB is phosphorylated on Ser133 by PKA (protein kinase A), promoting the recruitment of the co-activator proteins CBP (CREB-binding protein) and p300; this has been proposed to increase the transcription of CREB-dependent genes. CREB is also phosphorylated on Ser133 by MSK1/2 (mitogen- and stress-activated kinase 1/2) in cells in response to the activation of MAPK (mitogen-activated protein kinase) signalling; however, the relevance of this to gene transcription has been controversial. To resolve this problem, we created a mouse with a Ser133 to alanine residue mutation in the endogenous Creb gene. Unlike the total CREB knockout, which is perinatally lethal, these mice were viable, but born at less than the expected Mendelian frequency on a C57Bl/6 background. Using embryonic fibroblasts from the S133A-knockin mice we show in the present study that Ser133 phosphorylation downstream of PKA is required for CBP/p300 recruitment. The requirement of Ser133 phosphorylation for the PKA-mediated induction of CREB-dependent genes was, however, promoter-specific. Furthermore, we show that in cells the phosphorylation of CREB on Ser133 by MSKs does not promote strong recruitment of CBP or p300. Despite this, MSK-mediated CREB phosphorylation is critical for the induction of CREB-dependent genes downstream of MAPK signalling.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Transdução de Sinais , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Técnicas de Introdução de Genes , Genes Precoces , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Mutação , Fosforilação , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Serina/genética , Transcrição GênicaRESUMO
Four evolutionarily conserved proteins are required for mammalian regulated exocytosis: three SNARE proteins, syntaxin, SNAP-25, and synaptobrevin, and the SM protein, Munc18-1. Here, using single-molecule imaging, we measured the spatial distribution of large cohorts of single Munc18-1 molecules correlated with the positions of single secretory vesicles in a functionally rescued Munc18-1-null cellular model. Munc18-1 molecules were nonrandomly distributed across the plasma membrane in a manner not directed by mode of interaction with syntaxin1, with a small mean number of molecules observed to reside under membrane resident vesicles. Surprisingly, we found that the majority of vesicles in fully secretion-competent cells had no Munc18-1 associated within distances relevant to plasma membrane-vesicle SNARE interactions. Live cell imaging of Munc18-1 molecule dynamics revealed that the density of Munc18-1 molecules at the plasma membrane anticorrelated with molecular speed, with single Munc18-1 molecules displaying directed motion between membrane hotspots enriched in syntaxin1a. Our findings demonstrate that Munc18-1 molecules move between membrane depots distinct from vesicle morphological docking sites.
Assuntos
Proteínas Munc18/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , Biofísica/métodos , Linhagem Celular , Membrana Celular/metabolismo , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Células PC12 , Ligação Proteica , Ratos , Proteínas SNARE/metabolismoRESUMO
The ability of neurons to modulate synaptic strength underpins synaptic plasticity, learning and memory, and adaptation to sensory experience. Despite the importance of synaptic adaptation in directing, reinforcing, and revising the behavioral response to environmental influences, the cellular and molecular mechanisms underlying synaptic adaptation are far from clear. Brain-derived neurotrophic factor (BDNF) is a prime initiator of structural and functional synaptic adaptation. However, the signaling cascade activated by BDNF to initiate these adaptive changes has not been elucidated. We have previously shown that BDNF activates mitogen- and stress-activated kinase 1 (MSK1), which regulates gene transcription via the phosphorylation of both CREB and histone H3. Using mice with a kinase-dead knock-in mutation of MSK1, we now show that MSK1 is necessary for the upregulation of synaptic strength in response to environmental enrichment in vivo. Furthermore, neurons from MSK1 kinase-dead mice failed to show scaling of synaptic transmission in response to activity deprivation in vitro, a deficit that could be rescued by reintroduction of wild-type MSK1. We also show that MSK1 forms part of a BDNF- and MAPK-dependent signaling cascade required for homeostatic synaptic scaling, which likely resides in the ability of MSK1 to regulate cell surface GluA1 expression via the induction of Arc/Arg3.1. These results demonstrate that MSK1 is an integral part of a signaling pathway that underlies the adaptive response to synaptic and environmental experience. MSK1 may thus act as a key homeostat in the activity- and experience-dependent regulation of synaptic strength.
Assuntos
Homeostase/genética , Plasticidade Neuronal/genética , Neurônios/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Sinapses/fisiologia , Análise de Variância , Animais , Animais Recém-Nascidos , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Espinhas Dendríticas/fisiologia , Meio Ambiente , Inibidores Enzimáticos/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Homeostase/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/ultraestrutura , Técnicas de Patch-Clamp , Mutação Puntual/genética , Receptores de AMPA/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Bloqueadores dos Canais de Sódio/farmacologia , Sinapses/genética , Tetrodotoxina/farmacologia , Fatores de TempoRESUMO
Neurotrophins are growth factors that are important in neuronal development and survival as well as synapse formation and plasticity. Many of the effects of neurotrophins are mediated by changes in protein expression as a result of altered transcription or translation. To determine whether neurotrophins regulate the production of microRNAs (miRNAs), small RNA species that modulate protein translation or mRNA stability, we used deep sequencing to identify BDNF (brain-derived neurotrophic factor)-induced miRNAs in cultured primary cortical mouse neurons. This revealed that the miR-212/132 cluster contained the miRNAs most responsive to BDNF treatment. This cluster was found to produce four miRNAs: miR-132, miR-132*, miR-212 and miR-212*. Using specific inhibitors, mouse models and promoter analysis we have shown that the regulation of the transcription of the miR-212/132 miRNA cluster and the miRNAs derived from it are regulated by the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway, via both MSK (mitogen and stress-activated kinase)-dependent and -independent mechanisms.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , MicroRNAs/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/fisiologia , Animais , Sequência de Bases , Benzamidas/farmacologia , Northern Blotting , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Dados de Sequência Molecular , Neurônios/metabolismo , Neurônios/microbiologia , Técnicas de Amplificação de Ácido Nucleico , Fosforilação/genética , Fosforilação/fisiologia , Reação em Cadeia da Polimerase , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Homologia de Sequência do Ácido NucleicoRESUMO
Phosphorylation of Ser133 in the transcription factor CREB is an important mechanism for regulating its transcriptional activity, however recent work has suggested significant roles for other regulatory inputs into CREB. To allow study of this in vivo, we have generated a Ser133 to alanine knockin mutation in the mouse CREB locus. As CREB knockout is perinatal lethal, a minigene strategy was used to allow conditional knockin of the Ser133Ala mutation in adult mice using Cre recombinase. While some expression of the mutated protein was observed prior to Cre expression, following Cre expression in either T cells or neurons only the mutated CREB protein was detected.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Técnicas de Introdução de Genes/métodos , Mutação , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Integrases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Pancreatic ductal adenocarcinoma is one of the most invasive and metastatic cancers and has a dismal 5-year survival rate. We show that N-WASP drives pancreatic cancer metastasis, with roles in both chemotaxis and matrix remodeling. lysophosphatidic acid, a signaling lipid abundant in blood and ascites fluid, is both a mitogen and chemoattractant for cancer cells. Pancreatic cancer cells break lysophosphatidic acid down as they respond to it, setting up a self-generated gradient driving tumor egress. N-WASP-depleted cells do not recognize lysophosphatidic acid gradients, leading to altered RhoA activation, decreased contractility and traction forces, and reduced metastasis. We describe a signaling loop whereby N-WASP and the endocytic adapter SNX18 promote lysophosphatidic acid-induced RhoA-mediated contractility and force generation by controlling lysophosphatidic acid receptor recycling and preventing degradation. This chemotactic loop drives collagen remodeling, tumor invasion, and metastasis and could be an important target against pancreatic cancer spread.
Assuntos
Lisofosfolipídeos/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Quimiotaxia , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Transporte Proteico , Ratos , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/isolamento & purificação , Transdução de Sinais , Nexinas de Classificação/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
FRET biosensors have proven very useful tools for studying the activation of specific signalling pathways in living cells. Most biosensors designed to date have been predicated on fluorescent protein pairs that were identified by, and for use in, intensity based measurements, however fluorescence lifetime provides a more reliable measurement of FRET. Both the technology and fluorescent proteins available for FRET have moved on dramatically in the last decade. Lifetime imaging systems have become increasingly accessible and user-friendly, and there is an entire field of biology dedicated to refining and adapting different characteristics of existing and novel fluorescent proteins. This growing pool of fluorescent proteins includes the long-lifetime green and cyan fluorescent proteins Clover and mTurquoise2, the red variant mRuby2, and the dark acceptor sREACh. Here, we have tested these donors and acceptors in appropriate combinations against the standard or recommended norms (EGFP and mTFP as donors, mCherry and either Ypet or Venus as acceptors) to determine if they could provide more reliable, reproducible and quantifiable FLIM-FRET data to improve on the dynamic range compared to other donors and breadth of application of biosensor technologies. These tests were performed for comparison on both a wide-field, frequency domain system and a multiphoton, TCSPC time domain FLIM system. Clover proved to be an excellent donor with extended dynamic range in combination with mCherry on both platforms, while mRuby2 showed a high degree of variability and poor FRET efficiencies in all cases. mTFP-Venus was the most consistent cyan-yellow pair between the two FLIM methodologies, but mTurquoise2 has better dynamic range and transfers energy consistently over time to the dark acceptor sRCh. Combination of mTFP-sRCh with Clover-mCherry would allow the simultaneous use of two FLIM-FRET biosensors within one sample by eliminating the crosstalk between the yellow acceptor and green donor emissions.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Luminescentes/metabolismo , Técnicas Biossensoriais , Fluorescência , Ligação ProteicaRESUMO
Actin-based protrusions are reinforced through positive feedback, but it is unclear what restricts their size, or limits positive signals when they retract or split. We identify an evolutionarily conserved regulator of actin-based protrusion: CYRI (CYFIP-related Rac interactor) also known as Fam49 (family of unknown function 49). CYRI binds activated Rac1 via a domain of unknown function (DUF1394) shared with CYFIP, defining DUF1394 as a Rac1-binding module. CYRI-depleted cells have broad lamellipodia enriched in Scar/WAVE, but reduced protrusion-retraction dynamics. Pseudopods induced by optogenetic Rac1 activation in CYRI-depleted cells are larger and longer lived. Conversely, CYRI overexpression suppresses recruitment of active Scar/WAVE to the cell edge, resulting in short-lived, unproductive protrusions. CYRI thus focuses protrusion signals and regulates pseudopod complexity by inhibiting Scar/WAVE-induced actin polymerization. It thus behaves like a 'local inhibitor' as predicted in widely accepted mathematical models, but not previously identified in cells. CYRI therefore regulates chemotaxis, cell migration and epithelial polarization by controlling the polarity and plasticity of protrusions.
Assuntos
Movimento Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pseudópodes/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Quimiotaxia/genética , Chlorocebus aethiops , Cães , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Madin Darby de Rim Canino , Polimerização , Ligação Proteica , Pseudópodes/genética , Transdução de Sinais/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
The immediate early gene activity-regulated cytoskeletal protein (Arc)/Arg3.1 and the neurotrophin brain-derived neurotrophic factor (BDNF) play important roles in synaptic plasticity and learning and memory in the mammalian brain. However, the mechanisms by which BDNF regulates the expression of Arc/Arg3.1 are unclear. In this study, we show that BDNF acts via the ERK1/2 pathway to activate the nuclear kinase mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 then induces Arc/Arg3.1 expression via the phosphorylation of histone H3 at the Arc/Arg3.1 promoter. MSK1 can also phosphorylate the transcription factor cyclic-AMP response element-binding protein (CREB) on Ser133. However, this is not required for BDNF-induced Arc.Arg3.1 transcription as a Ser133Ala knockin mutation had no effect on Arc/Arg3.1 induction. In parallel, ERK1/2 directly activates Arc/Arg3.1 mRNA transcription via at least one serum response element on the promoter, which bind a complex of the Serum Response Factor (SRF) and a Ternary Complex Factor (TCF).
RESUMO
Neuronal synapses are among the most scrutinized of cellular systems, serving as a model for all membrane trafficking studies. Despite this, synaptic biology has proven difficult to interrogate directly in situ due to the small size and dynamic nature of central synapses and the molecules within them. Here we determine the spatial and temporal interaction status of presynaptic proteins, imaging large cohorts of single molecules inside active synapses. Measuring rapid interaction dynamics during synaptic depolarization identified the small number of syntaxin1a and munc18-1 protein molecules required to support synaptic vesicle exocytosis. After vesicle fusion and subsequent SNARE complex disassembly, a prompt switch in syntaxin1a and munc18-1-binding mode, regulated by charge alteration on the syntaxin1a N-terminal, sequesters monomeric syntaxin1a from other disassembled fusion complex components, preventing ectopic SNARE complex formation, readying the synapse for subsequent rounds of neurotransmission.
Assuntos
Exocitose/genética , Proteínas Munc18/metabolismo , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Sintaxina 1/metabolismo , Animais , Toxinas Botulínicas/farmacologia , Toxinas Botulínicas Tipo A/farmacologia , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Embrião de Mamíferos , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fusão de Membrana , Imagem Molecular , Proteínas Munc18/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Cultura Primária de Células , Ligação Proteica , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/ultraestrutura , Transmissão Sináptica , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/ultraestrutura , Sintaxina 1/genética , Proteína Vermelha FluorescenteRESUMO
Signal transduction cascades, including the MAPK, PI3 kinase, Ca(2+) and PKC pathways, play important roles in neurons downstream of multiple signals including neurotrophins and neurotransmitters. Small molecule kinase inhibitors that block these pathways provide a powerful way of studying the in vivo or cellular roles of these signaling systems. Over the last 15 years there has been a major effort by the pharmaceutical industry to develop kinase inhibitors as potential drugs for a variety of diseases including cancer and auto-immunity. As a result of this there are now many compounds available that can be used as research tools. One major drawback is however that many of these compounds are not truly selective for a single kinase, and therefore the possibility that their cellular effects may be due to an off target activity must be considered. This problem has been brought into sharp relief by modern in vitro screening methods that allow an inhibitor to be screened against a significant proportion of the kinome. In this review we discuss the advantages and problems with the use of kinase inhibitors as research tools and describe some of the available compounds that target pathways important to neurons.
Assuntos
Neurociências , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , PesquisaRESUMO
Intercellular communication is commonly mediated by the regulated fusion, or exocytosis, of vesicles with the cell surface. SNARE (soluble N-ethymaleimide sensitive factor attachment protein receptor) proteins are the catalytic core of the secretory machinery, driving vesicle and plasma membrane merger. Plasma membrane SNAREs (tSNAREs) are proposed to reside in dense clusters containing many molecules, thus providing a concentrated reservoir to promote membrane fusion. However, biophysical experiments suggest that a small number of SNAREs are sufficient to drive a single fusion event. Here we show, using molecular imaging, that the majority of tSNARE molecules are spatially separated from secretory vesicles. Furthermore, the motilities of the individual tSNAREs are constrained in membrane micro-domains, maintaining a non-random molecular distribution and limiting the maximum number of molecules encountered by secretory vesicles. Together our results provide a new model for the molecular mechanism of regulated exocytosis and demonstrate the exquisite organization of the plasma membrane at the level of individual molecular machines.
Assuntos
Proteínas SNARE/metabolismo , Vesículas Secretórias/metabolismo , Animais , Comunicação Celular , Linhagem Celular , Membrana Celular/metabolismo , Microscopia de Fluorescência , Imagem Molecular , Células PC12 , Transporte Proteico , RatosRESUMO
Neurotrophins are important mediators of neuronal development, survival and plasticity. They act via binding to Trk receptors, which results in the stimulation of the intracellular tyrosine kinase domain of the receptor leading to autophosphorylation of this domain. This in turn creates a scaffold that recruits various adapter proteins allowing the activation of intracellular signaling cascades including the PLCγ, MAPK and PI3K pathways. Compounds that specifically block the activity of the tyrosine kinase domain of Trk receptors would provide a powerful tool to study the role of these receptors in cells. K252a has previously been used for this purpose, however we show here that it can inhibit many tyrosine and serine/threonine kinases in vitro. Profiling of 3 newer inhibitors, referred to here as SHN-753, SHN-722 and GSK-Trk, demonstrate that they have significantly improved specificity for the kinase activity of TrkA in vitro compared to K252a. In addition these compounds were found to block the TrkB mediated activation of ERK1/2 by BDNF, but did not affect NMDA induced ERK1/2 activation. These compounds, while still not completely specific for Trk receptor kinase activity, do represent a considerable improvement over K252a and should prove valuable in the study of neurotrophin-mediated actions in the nervous system.