RESUMO
Phosphorylation of gap junction proteins, connexins, plays a role in global signaling events involving kinases. Connexin43 (Cx43), a ubiquitous and important connexin, has several phosphorylation sites for specific kinases. We appended an imaging reporter tag for the activity of the δ isoform of protein kinase C (PKCδ) to the carboxyl terminus of Cx43. The FRET signal of this reporter is inversely related to the phosphorylation of serine 368 of Cx43. By activating PKC with the phorbol ester phorbol 12,13-dibutyrate (PDBu) or a natural stimulant, UTP, time lapse live cell imaging movies indicated phosphorylated Ser-368 Cx43 separated into discrete domains within gap junctions and was internalized in small vesicles, after which it was degraded by lysosomes and proteasomes. Mutation of Ser-368 to an Ala eliminated the response to PDBu and changes in phosphorylation of the reporter. A phosphatase inhibitor, calyculin A, does not change this pattern, indicating PKC phosphorylation causes degradation of Cx43 without dephosphorylation, which is in accordance with current hypotheses that cells control their intercellular communication by a fast and constant turnover of connexins, using phosphorylation as part of this mechanism.
Assuntos
Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Movimento , Proteína Quinase C-delta/metabolismo , Proteólise , Animais , Células COS , Chlorocebus aethiops , Conexina 43/química , Junções Comunicantes/efeitos dos fármacos , Células HeLa , Humanos , Movimento/efeitos dos fármacos , Ésteres de Forbol/farmacologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Proteína Quinase C-delta/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Serina/metabolismoRESUMO
Connexin proteins form hexameric assemblies known as hemichannels. When docked to form gap junction (GJ) channels, hemichannels play a critical role in cell-cell communication and cellular homeostasis, but often are functional entities on their own in unapposed cell membranes. Defects in the Connexin26 (Cx26) gene are the major cause of hereditary deafness arising from dysfunctional hemichannels in the cochlea. Structural studies of Cx26 hemichannels properly trafficked and inserted in plasma membranes, including their clustering that forms a plaque-like feature in whole gap junctions, are limited. We used atomic force microscopy (AFM) to study the surface topography of Cx26 hemichannels using two different membrane preparations. Rat Cx26 containing appended carboxy terminal V5 and hexahistidine tags were expressed in baculovirus/Sf9 cell systems. The expressed Cx26 proteins form hemichannels in situ in Sf9 cells that were then purified either as (1) Sf9 membrane fragments containing Cx26 hemichannels or (2) solubilized hemichannels. The latter were subsequently reconstituted in liposomes. AFM images of purified membrane fragments showed clusters of protein macromolecular structures in the membrane that at higher magnification corresponded to Cx26 hemichannels. Hemichannels reconstituted into DOPC bilayers displayed two populations of channel heights likely resulting from differences in orientations of inserted hemichannels. Hemichannels in the protein rich portions of purified membranes also showed a reduced channel height above the bilayer compared to membranes with reconstituted hemichannels perhaps due to reduced AFM probe access to the lipid bilayer. These preparations of purified membranes enriched for connexin hemichannels that have been properly trafficked and inserted in membranes provide a platform for high-resolution AFM imaging of the structure, interconnexon interactions, and cooperativity of properly trafficked and inserted noncrystalline connexin hemichannels.
Assuntos
Membrana Celular/metabolismo , Conexinas/química , Microscopia de Força Atômica , Fragmentos de Peptídeos/química , Animais , Conexina 26 , Conexinas/metabolismo , Detergentes/farmacologia , Bicamadas Lipídicas/metabolismo , Fragmentos de Peptídeos/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Ratos , Células Sf9 , SpodopteraRESUMO
Fibrosis following myocardial infarction is associated with increases in arrhythmias and sudden cardiac death. Initial steps in the development of fibrosis are not clear; however, it is likely that cardiac fibroblasts play an important role. In immune cells, ATP release from pannexin 1 (Panx1) channels acts as a paracrine signal initiating activation of innate immunity. ATP has been shown in noncardiac systems to initiate fibroblast activation. Therefore, we propose that ATP release through Panx1 channels and subsequent fibroblast activation in the heart drives the development of fibrosis in the heart following myocardial infarction. We identified for the first time that Panx1 is localized within sarcolemmal membranes of canine cardiac myocytes where it directly interacts with the postsynaptic density 95/Drosophila disk large/zonula occludens-1-containing scaffolding protein synapse-associated protein 97 via its carboxyl terminal domain (amino acids 300-357). Induced ischemia rapidly increased glycosylation of Panx1, resulting in increased trafficking to the plasma membrane as well as increased interaction with synapse-associated protein 97. Cellular stress enhanced ATP release from myocyte Panx1 channels, which, in turn, causes fibroblast transformation to the activated myofibroblast phenotype via activation of the MAPK and p53 pathways, both of which are involved in the development of cardiac fibrosis. ATP release through Panx1 channels in cardiac myocytes during ischemia may be an early paracrine event leading to profibrotic responses to ischemic cardiac injury.
Assuntos
Trifosfato de Adenosina/metabolismo , Conexinas/metabolismo , Fibroblastos/metabolismo , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Comunicação Parácrina , Animais , Membrana Celular/metabolismo , Técnicas de Cocultura , Conexinas/genética , Modelos Animais de Doenças , Cães , Fibroblastos/patologia , Fibrose , Glicosilação , Células Madin Darby de Rim Canino , Camundongos , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miócitos Cardíacos/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Proteínas do Tecido Nervoso/genética , Fenótipo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Transporte Proteico , Sarcolema/metabolismo , Transdução de Sinais , Fatores de Tempo , Regulação para CimaRESUMO
Pannexins are homologous to innexins, the invertebrate gap junction family. However, mammalian pannexin1 does not form canonical gap junctions, instead forming hexameric oligomers in single plasma membranes and intracellularly. Pannexin1 acts as an ATP release channel, whereas less is known about the function of Pannexin2. We purified cellular membranes isolated from MDCK cells stably expressing rat Pannexin1 or Pannexin2 and identified pannexin channels (pannexons) in single membranes by negative stain and immunogold labeling. Protein gel and Western blot analysis confirmed Pannexin1 (Panx1) or Pannexin2 (Panx2) as the channel-forming proteins. We expressed and purified Panx1 and Panx2 using a baculovirus Sf9 expression system and obtained doughnut-like structures similar to those seen previously in purified connexin hemichannels (connexons) and mammalian membranes. Purified pannexons were comparable in size and overall appearance to Connexin46 and Connexin50 connexons. Pannexons and connexons were further analyzed by single-particle averaging for oligomer and pore diameters. The oligomer diameter increased with increasing monomer molecular mass, and we found that the measured oligomeric pore diameter for Panxs was larger than for Connexin26. Panx1 and Panx2 formed active homomeric channels in Xenopus oocytes and in vitro vesicle assays. Cross-linking and native gels of purified homomeric full-length and a C-terminal Panx2 truncation mutant showed a banding pattern more consistent with an octamer. We purified Panx1/Panx2 heteromeric channels and found that they were unstable over time, possibly because Panx1 and Panx2 homomeric pannexons have different monomer sizes and oligomeric symmetry from each other.
Assuntos
Conexinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Conexina 26 , Citocromos c/química , Dimerização , Cães , Junções Comunicantes/metabolismo , Células HeLa , Humanos , Imuno-Histoquímica , Microscopia Eletrônica/métodos , Oócitos/metabolismo , Isoformas de Proteínas , Ratos , Xenopus/metabolismoRESUMO
Connexin26 is a ubiquitous gap junction protein that serves critical homeostatic functions. Four single-site mutations found in the transmembrane helices (M1-M4) cause different types of dysfunctional channels: 1), Cx26T135A in M3 produces a closed channel; 2), Cx26M34A in M1 severely decreases channel activity; 3), Cx26P87L in M2 has been implicated in defective channel gating; and 4), Cx26V84L in M2, a nonsyndromic deafness mutant, retains normal dye coupling and electrophysiological properties but is deficient in IP(3) transfer. These mutations do not affect Cx26 trafficking in mammalian cells, and make normal-appearing channels in baculovirus-infected Sf9 membranes when imaged by negative stain electron microscopy. Upon dodecylmaltoside solubilization of the membrane fraction, Cx26M34A and Cx26V84L are stable as hexamers or dodecamers, but Cx26T135A and Cx26P87L oligomers are not. This instability is also found in Cx26T135A and Cx26P87L hemichannels isolated from mammalian cells. In this work, coexpression of both wild-type Cx26 and Cx26P87L in Sf9 cells rescued P87L hexamer stability. Similarly, in paired Xenopus oocytes, coexpression with wild-type restored function. In contrast, the stability of Cx26T135A hemichannels could not be rescued by coexpression with WT. Thus, T135 and P87 residues are in positions that are important for oligomer stability and can affect gap junction gating.
Assuntos
Conexinas/química , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Multimerização Proteica , Sequência de Aminoácidos , Animais , Conexina 26 , Conexinas/genética , Conexinas/isolamento & purificação , Detergentes/química , Células HeLa , Humanos , Hidróxidos/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/isolamento & purificação , Octoxinol/química , Oócitos/metabolismo , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Ratos , Solubilidade , Treonina/metabolismo , XenopusRESUMO
Connexin26 (Cx26) is a member of the connexin family, the building blocks for gap junction intercellular channels. These dodecameric assemblies are involved in gap junction-mediated cell-cell communication allowing the passage of ions and small molecules between two neighboring cells. Mutations in Cx26 lead to the disruption of gap junction-mediated intercellular communication with consequences such as hearing loss and skin disorders. We show here that a mutant of Cx26, M34A, forms an active hemichannel in lipid bilayer experiments. A comparison with the Cx26 wild-type is presented. Two different techniques using micro/nano-structured substrates for the formation of pore-suspending lipid membranes are used. We reconstituted the Cx26 wild-type and Cx26M34A into artificial lipid bilayers and observed single channel activity for each technique, with conductance levels of around 35, 70 and 165 pS for the wild-type. The conductance levels of Cx26M34A were found at around 45 and 70 pS.
Assuntos
Conexinas/química , Conexinas/metabolismo , Junções Comunicantes/química , Junções Comunicantes/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Animais , Linhagem Celular , Conexina 26 , Conexinas/genética , Humanos , Mutação , RatosRESUMO
High mobility group A1 (HMGA1) is an architectural transcription factor and a putative protoncogene. Deregulation of its expression has been shown in most human cancers. We have previously shown that the expression of the HMGA family members is deregulated in neuroblastoma cell lines and primary tumors. On retinoic acid (RA) treatment of MYCN-amplified neuroblastoma cell lines, HMGA1 decreases with a kinetics that strictly follows MYCN repression. In addition, MYCN constitutive expression abolishes HMGA1 repression by RA. Here we explored the possibility that HMGA1 expression might be sustained by MYCN in amplified cells. Indeed, MYCN transfection induced HMGA1 expression in several neuroblastoma cell lines. HMGA1 expression increased in a transgene dose-dependent fashion in neuroblastoma-like tumors of MYCN transgenic mice. In addition, it was significantly more expressed in MYCN-amplified compared with MYCN single-copy primary human neuroblastomas. MYCN cotransfection activated a promoter/luciferase reporter containing a 1,600 bp region surrounding the first three transcription start sites of the human HMGA1 and eight imperfect E-boxes. By heterodimerizing with its partner MAX, MYCN could bind to multiple DNA fragments within the 1,600 bp. Either 5' or 3' deletion variants of the 1,600 bp promoter/luciferase reporter strongly decreased luciferase activity, suggesting that, more than a single site, the cooperative function of multiple cis-acting elements mediates direct HMGA1 transactivation by MYCN. Finally, HMGA1 repression by RNA interference reduced neuroblastoma cell proliferation, indicating that HMGA1 is a novel MYCN target gene relevant for neuroblastoma tumorigenesis.
Assuntos
Proteínas HMGA/genética , Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Animais , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Criança , Amplificação de Genes , Genes Reporter , Proteínas HMGA/biossíntese , Humanos , Luciferases/biossíntese , Luciferases/genética , Camundongos , Camundongos Transgênicos , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/metabolismo , Regiões Promotoras Genéticas , Interferência de RNA , Ativação Transcricional , TransfecçãoRESUMO
Gap junctions are membrane specialization domains identified in most tissue types where cells abut each other. The connexin channels found in these membrane domains are conduits for direct cell-to-cell transfer of ions and molecules. Connexin43 (Cx43) is the most ubiquitous connexin, with critical roles in heart, skin, and brain. Several studies described the interaction between Cx43 and the cytoskeleton involving the actin binding proteins Zonula occludens (ZO-1) and drebrin, as well as with tubulin. However, a direct interaction has not been identified between drebrin and Cx43. In this study, co-IP and NMR experiments were used to demonstrate that the Cx43-CT directly interacts with the highly conserved N-terminus region of drebrin. Three Cx43-CT areas were found to be involved in drebrin binding, with residues 264-275 being critical for the interaction. Mimicking Src phosphorylation within this region (Y265) significantly disrupted the interaction between the Cx43-CT and drebrin. Immunofluorescence showed colocalization of Cx43, drebrin, and F-actin in astrocytes and Vero cells membrane, indicating that Cx43 forms a submembrane protein complex with cytoskeletal and scaffolding proteins. The co-IP data suggest that Cx43 indirectly interacts with F-actin through drebrin. Along with the known interaction of the Cx43-CT with ZO-1 and tubulin, the data presented here for drebrin indicate non-overlapping and separated binding sites for all three proteins for which simultaneous binding could be important in regulating cytoskeleton rearrangements, especially for neuronal migration during brain development.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Conexina 43/metabolismo , Complexos Multiproteicos/metabolismo , Neuropeptídeos/metabolismo , Tubulina (Proteína)/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Citoesqueleto de Actina , Animais , Astrócitos/citologia , Sítios de Ligação , Encéfalo/citologia , Movimento Celular , Células Cultivadas , Chlorocebus aethiops , Feminino , Junções Comunicantes , Humanos , Domínios PDZ , Fosforilação , Ligação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
Very soon after their original identification in HeLa cells in 1983, HMGA proteins appeared as interesting cancer-related molecules. Indeed, they were immediately noted as a sub-class of High Mobility Group proteins induced in fibroblast or epithelial cells transformed with sarcoma viruses. After more than 20 years, the association between HMGA protein expressions and cellular transformation has been largely confirmed and HMGA are among the most widely expressed cancer-associated proteins. Nevertheless, their functional contribution to tumour development and progression is far from being completely understood. Furthermore, although HMGA1 expression has been reported to be inducible by a number of factors and circumstances, the question of how their expression is deregulated in cancer is even less clear and somehow has been ignored from most researchers. An active AP1 site is the only characterized element of the HMGA1 human promoter, that remains a rather complicated and unexplored source of information to answer this question. Following the indication that c-Myc might bind and activate the mouse HMGA1 gene promoter, we have demonstrated that HMGA1 is a new target for MYCN in human neuroblastomas. In this report, we overview part of the current information on HMGA1 and focus our attention on the analysis of its human promoter.
Assuntos
Proteína HMGA1a/metabolismo , Neuroblastoma/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , Divisão Celular , Regulação Neoplásica da Expressão Gênica , Proteína HMGA1a/química , Proteína HMGA1a/genética , Proteína HMGA1a/fisiologia , Humanos , Camundongos , Dados de Sequência Molecular , Neuroblastoma/patologia , Regiões Promotoras Genéticas , Homologia de Sequência de Aminoácidos , Transcrição Gênica/fisiologiaRESUMO
Pannexin1 (Panx1) participates in several signaling events that involve adenosine triphosphate (ATP) release, including the innate immune response, ciliary beat in airway epithelia, and oxygen supply in the vasculature. The view that Panx1 forms a large ATP release channel has been challenged by the association of a low-conductance, small anion-selective channel with the presence of Panx1. We showed that Panx1 membrane channels can function in two distinct modes with different conductances and permeabilities when heterologously expressed in Xenopus oocytes. When stimulated by potassium ions (K(+)), Panx1 formed a high-conductance channel of ~500 pS that was permeable to ATP. Various physiological stimuli can induce this ATP-permeable conformation of the channel in several cell types. In contrast, the channel had a low conductance (~50 pS) with no detectable ATP permeability when activated by voltage in the absence of K(+). The two channel states were associated with different reactivities of the terminal cysteine of Panx1 to thiol reagents, suggesting different conformations. Single-particle electron microscopic analysis revealed that K(+) stimulated the formation of channels with a larger pore diameter than those formed in the absence of K(+). These data suggest that different stimuli lead to distinct channel structures with distinct biophysical properties.
Assuntos
Trifosfato de Adenosina/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Conexinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/fisiologia , Trifosfato de Adenosina/genética , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/fisiologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Conexinas/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Potássio/farmacologia , Transdução de Sinais/efeitos dos fármacos , Xenopus laevisRESUMO
Pannexin1 (Panx1) channels release cytosolic ATP in response to signaling pathways. Panx1 is highly expressed in the central nervous system. We used four antibodies with different Panx1 anti-peptide epitopes to analyze four regions of rat brain. These antibodies labeled the same bands in Western blots and had highly similar patterns of immunofluorescence in tissue culture cells expressing Panx1, but Western blots of brain lysates from Panx1 knockout and control mice showed different banding patterns. Localizations of Panx1 in brain slices were generated using automated wide field mosaic confocal microscopy for imaging large regions of interest while retaining maximum resolution for examining cell populations and compartments. We compared Panx1 expression over the cerebellum, hippocampus with adjacent cortex, thalamus, and olfactory bulb. While Panx1 localizes to the same neuronal cell types, subcellular localizations differ. Two antibodies with epitopes against the intracellular loop and one against the carboxy terminus preferentially labeled cell bodies, while an antibody raised against an N-terminal peptide highlighted neuronal processes more than cell bodies. These labeling patterns may be a reflection of different cellular and subcellular localizations of full-length and/or modified Panx1 channels where each antibody is highlighting unique or differentially accessible Panx1 populations. However, we cannot rule out that one or more of these antibodies have specificity issues. All data associated with experiments from these four antibodies are presented in a manner that allows them to be compared and our claims thoroughly evaluated, rather than eliminating results that were questionable. Each antibody is given a unique identifier through the NIF Antibody Registry that can be used to track usage of individual antibodies across papers and all image and metadata are made available in the public repository, the Cell Centered Database, for on-line viewing, and download.
RESUMO
Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure.
Assuntos
Conexinas/genética , Conexinas/metabolismo , Surdez/genética , Surdez/metabolismo , Mutação , Animais , Linhagem Celular , Membrana Celular/metabolismo , Conexina 26 , Conexinas/química , Junções Comunicantes/metabolismo , Humanos , Modelos Moleculares , Permeabilidade , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Transporte Proteico , Células Sf9RESUMO
Pannexins are newly discovered channel proteins expressed in many different tissues and abundantly in the vertebrate central nervous system. Based on membrane topology, folding and secondary structure prediction, pannexins are proposed to form gap junction-like structures. We show here that Pannexin1 forms a hexameric channel and reaches the cell surface but, unlike connexins, is N-glycosylated. Using site-directed mutagenesis we analyzed three putative N-linked glycosylation sites and examined the effects of each mutation on channel expression. We show for the first time that Pannexin1 is glycosylated at Asn-254 and that this residue is important for plasma membrane targeting. The glycosylation of Pannexin1 at its extracellular surface makes it unlikely that two oligomers could dock to form an intercellular channel. Ultrastructural analysis by electron microscopy confirmed that Pannexin1 junctional areas do not appear as canonical gap junctions. Rather, Pannexin1 channels are distributed throughout the plasma membrane. We propose that N-glycosylation of Pannexin1 could be a significant mechanism for regulating the trafficking of these membrane proteins to the cell surface in different tissues.