RESUMO
Atypical PKC (ι/λ and ζ; hereafter referred to as aPKC) is a key player in the acquisition of epithelial polarity and participates in other signaling cascades including the control of NF-κB signaling. This kinase is post-translationally regulated through Hsp70-mediated refolding. Previous work has shown that such a chaperoning activity is specifically localized to keratin intermediate filaments. Our work was performed with the goal of identifying the molecule(s) that block Hsp70 activity on keratin filaments during inflammation. A transcriptional screen allowed us to focus on BAG-1, a multi-functional protein that assists Hsp70 in nucleotide exchange but also blocks its activity at higher concentrations. We found the BAG-1 isoform BAG-1M upregulated threefold in human Caco-2 cells following stimulation with tumor necrosis factor receptor α (TNFα) to induce a pro-inflammatory response, and up to sixfold in mouse enterocytes following treatment with dextran sodium sulfate (DSS) to induce colitis. BAG-1M, but no other isoform, was found to co-purify with intermediate filaments and block Hsp70 activity in the keratin fraction but not in the soluble fraction within the range of concentrations found in epithelial cells cultured under control and inflammation conditions. Constitutive expression of BAG-1M decreased levels of phosphorylated aPKC. By contrast, knockdown of BAG-1, blocked the TNFα-induced decrease of phosphorylated aPKC. We conclude that BAG-1M mediates Hsp70 inhibition downstream of NF-κB.
Assuntos
Colite/enzimologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Intestino Delgado/metabolismo , Queratinas/metabolismo , Proteína Quinase C/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células CACO-2 , Colite/genética , Colite/imunologia , Colite/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Choque Térmico HSP70/genética , Humanos , Intestino Delgado/enzimologia , Intestino Delgado/imunologia , Queratinas/genética , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação , Proteína Quinase C/genética , Fatores de Transcrição/genéticaRESUMO
Microvillus inclusion disease (MVID) is an autosomal recessive condition resulting in intractable secretory diarrhea in newborns due to loss-of-function mutations in myosin Vb (Myo5b). Previous work suggested that the apical recycling endosomal (ARE) compartment is the primary location for phosphoinositide-dependent protein kinase 1 (PDK1) signaling. Because the ARE is disrupted in MVID, we tested the hypothesis that polarized signaling is affected by Myo5b dysfunction. Subcellular distribution of PDK1 was analyzed in human enterocytes from MVID/control patients by immunocytochemistry. Using Myo5b knockdown (kd) in Caco-2BBe cells, we studied phosphorylated kinases downstream of PDK1, electrophysiological parameters, and net water flux. PDK1 was aberrantly localized in human MVID enterocytes and Myo5b-deficient Caco-2BBe cells. Two PDK1 target kinases were differentially affected: phosphorylated atypical protein kinase C (aPKC) increased fivefold and phosohoprotein kinase B slightly decreased compared with control. PDK1 redistributed to a soluble (cytosolic) fraction and copurified with basolateral endosomes in Myo5b kd. Myo5b kd cells showed a decrease in net water absorption that could be reverted with PDK1 inhibitors. We conclude that, in addition to altered apical expression of ion transporters, depolarization of PDK1 in MVID enterocytes may lead to aberrant activation of downstream kinases such as aPKC. The findings in this work suggest that PDK1-dependent signaling may provide a therapeutic target for treating MVID.
Assuntos
Polaridade Celular , Enterócitos/metabolismo , Síndromes de Malabsorção/metabolismo , Microvilosidades/patologia , Mucolipidoses/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Transdução de Sinais , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/antagonistas & inibidores , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Células CACO-2 , Estudos de Casos e Controles , Regulação para Baixo , Endossomos/metabolismo , Enterócitos/efeitos dos fármacos , Humanos , Síndromes de Malabsorção/tratamento farmacológico , Síndromes de Malabsorção/genética , Microvilosidades/genética , Microvilosidades/metabolismo , Terapia de Alvo Molecular , Mucolipidoses/tratamento farmacológico , Mucolipidoses/genética , Mutação , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Fosforilação , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Transfecção , Água/metabolismoRESUMO
BACKGROUND: Extracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation. RESULTS: Here we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells. CONCLUSION: Our results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.
Assuntos
Proteína de Ligação a CREB/metabolismo , Odorantes , Mucosa Olfatória/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Antagonistas Purinérgicos/farmacologia , Receptores Odorantes/metabolismo , Olfato/fisiologia , Animais , Células Cultivadas , Camundongos , Mucosa Olfatória/citologia , Mucosa Olfatória/efeitos dos fármacos , Neurônios Receptores Olfatórios/efeitos dos fármacos , Fosforilação , Olfato/efeitos dos fármacosRESUMO
Par1b/MARK2 is a Ser/Thr kinase with pleiotropic effects that participates in the generation of apico-basal polarity in Caenorhabditis elegans. It is phosphorylated by atypical PKC(ι/λ) in Thr595 and inhibited. Because previous work showed a decrease in atypical protein kinase C (aPKC) activity under proinflammatory conditions, we analyzed the hypothesis that the resulting decrease in Thr595-MARK2 with increased kinase activity may also participate in innate immunity. We confirmed that pT595-MARK2 was decreased under inflammatory stimulation. The increase in MARK2 activity was verified by Par3 delocalization and rescue with a specific inhibitor. MARK2 overexpression significantly enhanced the transcriptional activity of NF-kB for a subset of transcripts. It also resulted in phosphorylation of a single band (â¼Mr 80,000) coimmunoprecipitating with RelA, identified as Med17. In vitro phosphorylation showed direct phosphorylation of Med17 in Ser152 by recombinant MARK2. Expression of S152D-Med17 mimicked the effect of MARK2 activation on downstream transcriptional regulation, which was antagonized by S152A-Med17. The decrease in pThr595 phosphorylation was validated in aPKC-deficient mouse jejunal mucosae. The transcriptional effects were confirmed in transcriptome analysis and transcript enrichment determinations in cells expressing S152D-Med17. We conclude that theMARK2-Med17 axis represents a novel form of cross-talk between polarity signaling and transcriptional regulation including, but not restricted to, innate immunity responses.
Assuntos
Polaridade Celular/fisiologia , Complexo Mediador/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Humanos , Imunidade Inata/fisiologia , Complexo Mediador/fisiologia , Camundongos , NF-kappa B/metabolismo , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de SinaisRESUMO
Odorant receptors comprise the biggest subfamily of G-protein-coupled receptors. Although the endocytic mechanisms of other G-protein-coupled receptors have been characterized extensively, almost nothing is known about the intracellular trafficking of odorant receptors. The present study describes the endocytic pathway of mammalian odorant receptors, which bind beta-arrestin2 with high affinity and are internalized via a clathrin-dependent mechanism. After prolonged odorant exposure, receptors are not targeted to lysosomal degradation but accumulate in recycling endosomes. Odorant-induced odorant receptor desensitization is promoted by cAMP-dependent protein kinase A phosphorylation and is dependent on serine and threonine residues within the third intracellular loop of the receptor. Moreover, beta-arrestin2 is redistributed into the dendritic knobs of mouse olfactory receptor neurons after treatment with a complex odorant mixture. Prolonged odorant exposure resulted in accumulation of beta-arrestin2 in intracellular vesicles. Adaptation of olfactory receptor neurons to odorants can be abolished by the inhibition of clathrin-mediated endocytosis, showing the physiological relevance of the here described mechanism of odorant receptor desensitization. A better understanding of odorant receptor trafficking and additional insight into the molecular determinants underlying the interactions of odorant receptors with beta-arrestin2 and other trafficking proteins will therefore be important to fully understand the mechanisms of adaptation and sensitization in the olfactory epithelium.
Assuntos
Arrestinas/fisiologia , Butiratos/farmacologia , Endocitose/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Odorantes , Receptores Odorantes/metabolismo , Motivos de Aminoácidos , Animais , Arrestinas/genética , Sinalização do Cálcio , Linhagem Celular , Membrana Celular/metabolismo , Vesículas Revestidas por Clatrina/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Regulação para Baixo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Genes Reporter , Habituação Psicofisiológica , Humanos , Isoquinolinas/farmacologia , Rim/citologia , Rim/embriologia , Ligantes , Masculino , Camundongos , Mutagênese Sítio-Dirigida , Fosforilação , Processamento de Proteína Pós-Traducional , Transporte Proteico , Receptores Odorantes/genética , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/farmacologia , Fatores de Tempo , Transfecção , Transferrina/metabolismo , beta-ArrestinasRESUMO
A growing body of evidence from several laboratories points at nonmechanical functions of keratin intermediate filaments (IF), such as control of apoptosis, modulation of signaling, or regulation of innate immunity, among others. While these functions are generally assigned to the ability of IF to scaffold other proteins, direct mechanistic causal relationships between filamentous keratins and the observed effects of keratin knockout or mutations are still missing. We have proposed that the scaffolding of chaperones such as Hsp70/40 may be key to understand some IF nonmechanical functions if unique features or specificity of the chaperoning activity in the IF scaffold can be demonstrated. The same criteria of uniqueness could be applied to other biochemical functions of the IF scaffold. Here, we describe a subcellular fractionation technique based on established methods of keratin purification. The resulting keratin-enriched fraction contains several proteins tightly associated with the IF scaffold, including Hsp70/40 chaperones. Being nondenaturing, this fractionation method enables direct testing of chaperoning and other enzymatic activities associated with IF, as well as supplementation experiments to determine the need for soluble (cytosolic) proteins. This method also permits to analyze inhibitory activity of cytosolic proteins at independently characterized physiological concentrations. When used as complementary approaches to knockout, knockdown, or site-directed mutagenesis, these techniques are expected to shed light on molecular mechanisms involved in the effects of IF loss of function.
Assuntos
Proteínas de Choque Térmico/química , Queratinas Específicas do Cabelo/química , Proteína Quinase C/química , Animais , Células CACO-2 , Fracionamento Celular , Humanos , Filamentos Intermediários/enzimologia , Mucosa Intestinal/citologia , Camundongos , Fosforilação , Dobramento de Proteína , Processamento de Proteína Pós-TraducionalRESUMO
As multicellular organisms evolved a family of cytoskeletal proteins, the keratins (types I and II) expressed in epithelial cells diversified in more than 20 genes in vertebrates. There is no question that keratin filaments confer mechanical stiffness to cells. However, such a number of genes can hardly be explained by evolutionary advantages in mechanical features. The use of transgenic mouse models has revealed unexpected functional relationships between keratin intermediate filaments and intracellular signaling. Accordingly, loss of keratins or mutations in keratins that cause or predispose to human diseases, result in increased sensitivity to apoptosis, regulation of innate immunity, permeabilization of tight junctions, and mistargeting of apical proteins in different epithelia. Precise mechanistic explanations for these phenomena are still lacking. However, immobilization of membrane or cytoplasmic proteins, including chaperones, on intermediate filaments ("scaffolding") appear as common molecular mechanisms and may explain the need for so many different keratin genes in vertebrates.
Assuntos
Polaridade Celular , Células Epiteliais/metabolismo , Queratinas/metabolismo , Animais , Apoptose , Células Epiteliais/citologia , Células Epiteliais/imunologia , Humanos , Imunidade Inata , Queratinas/química , Queratinas/genética , Transdução de SinaisRESUMO
The conserved proteins of the polarity complex made up of atypical PKC (aPKC, isoforms ι and ζ), Par6, and Par3 determine asymmetry in several cell types, from Caenorhabditis elegans oocytes to vertebrate epithelia and neurons. We previously showed that aPKC is down-regulated in intestinal epithelia under inflammatory stimulation. Further, expression of constitutively active PKCι decreases NF-κB activity in an epithelial cell line, the opposite of the effect reported in other cells. Here we tested the hypothesis that aPKC has a dual function in epithelia, inhibiting the NF-κB pathway in addition to having a role in apicobasal polarity. We achieved full aPKC down-regulation in small intestine villi and colon surface epithelium using a conditional epithelium-specific knockout mouse. The results show that aPKC is dispensable for polarity after cell differentiation, except for known targets, including ROCK and ezrin, claudin-4 expression, and barrier permeability. The aPKC defect resulted in increased NF-κB activity, which could be rescued by IKK and ROCK inhibitors. It also increased expression of proinflammatory cytokines. In contrast, expression of anti-inflammatory IL-10 decreased. We conclude that epithelial aPKC acts upstream of multiple mechanisms that participate in the inflammatory response in the intestine, including, but not restricted to, NF-κB.
Assuntos
Isoenzimas/metabolismo , NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Animais , Diferenciação Celular/fisiologia , Polaridade Celular/fisiologia , Regulação para Baixo , Células Epiteliais/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Isoenzimas/deficiência , Isoenzimas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase C/deficiência , Proteína Quinase C/genética , Transdução de SinaisRESUMO
Components of the Par-complex, atypical PKC and Par3, have been found to be downregulated upon activation of NF-κB in intestinal epithelial cells. To determine their possible role in pro-inflammatory responses we transduced Caco-2 human colon carcinoma cells with constitutively active (ca) PKCι or anti-Par3 shRNA-expressing lentiviral particles. Contrary to previous reports in other cell types, ca-PKCι did not activate, but rather decreased, baseline NF-κB activity in a luminiscence reporter assay. An identical observation applied to a PB1 domain deletion PKCι, which fails to localize to the tight-junction. Conversely, as expected, the same ca-PKCι activated NF-κB in non-polarized HEK293 cells. Likewise, knockdown of Par3 increased NF-κB activity and, surprisingly, greatly enhanced its response to TNFα, as shown by transcription of IL-8, GRO-1, GRO-2 and GRO-3. We conclude that aPKC and Par3 are inhibitors of the canonical NF-κB activation pathway, although perhaps acting through independent pathways, and may be involved in pro-inflammatory responses.
RESUMO
Phosphorylation of the activation domain of protein kinase C (PKC) isoforms is essential to start a conformational change that results in an active catalytic domain. This activation is necessary not only for newly synthesized molecules, but also for kinase molecules that become dephosphorylated and need to be refolded and rephosphorylated. This "rescue" mechanism is responsible for the maintenance of the steady-state levels of atypical PKC (aPKC [PKCι/λ and ζ]) and is blocked in inflammation. Although there is consensus that phosphoinositide-dependent protein kinase 1 (PDK1) is the activating kinase for newly synthesized molecules, it is unclear what kinase performs that function during the rescue and where the rescue takes place. To identify the activating kinase during the rescue mechanism, we inhibited protein synthesis and analyzed the stability of the remaining aPKC pool. PDK1 knockdown and two different PDK1 inhibitors-BX-912 and a specific pseudosubstrate peptide-destabilized PKCι. PDK1 coimmunoprecipitated with PKCι in cells without protein synthesis, confirming that the interaction is direct. In addition, we showed that PDK1 aids the rescue of aPKC in in vitro rephosphorylation assays using immunodepletion and rescue with recombinant protein. Surprisingly, we found that in Caco-2 epithelial cells and intestinal crypt enterocytes PDK1 distributes to an apical membrane compartment comprising plasma membrane and apical endosomes, which, in turn, are in close contact with intermediate filaments. PDK1 comigrated with the Rab11 compartment and, to some extent, with the transferrin compartment in sucrose gradients. PDK1, pT555-aPKC, and pAkt were dependent on dynamin activity. These results highlight a novel signaling function of apical endosomes in polarized cells.
Assuntos
Endossomos/enzimologia , Enterócitos/enzimologia , Filamentos Intermediários/enzimologia , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Células CACO-2 , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Enterócitos/citologia , Técnicas de Silenciamento de Genes , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Transdução de SinaisRESUMO
Inflammatory processes disrupt the barrier function in epithelia. Increased permeability often leads to chronic of inflammation. Important among other cytokines, tumor necrosis factor alpha (TNF-α) initiates an NF-κB-mediated response that leads to upregulation of myosin light chain kinase (MLCK), a hallmark of the pathogenesis of inflammatory bowel disease. Here, we found that two components of the evolutionarily conserved organizer of tight junctions and polarity, the polarity complex (atypical protein kinase C [aPKC]-PAR6-PAR3) were downregulated by TNF-α signaling in intestinal epithelial cells and also in vivo during intestinal inflammation. Decreases in aPKC levels were due to decreased chaperoning activity of Hsp70 proteins, with failure of the aPKC rescue machinery, and these effects were rescued by NF-κB inhibition. Comparable downregulation of aPKC shRNA phenocopied effects of TNF-α signaling, including apical nonmuscle myosin II accumulation and myosin light chain phosphorylation. These effects, including ZO-1 downregulation, were rescued by overexpression of constitutively active aPKC. We conclude that this novel mechanism is a complementary effector pathway for TNF-α signaling.
Assuntos
Inflamação/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sequência de Bases , Células CACO-2 , Proteínas de Ciclo Celular/metabolismo , Polaridade Celular , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP70/deficiência , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Inflamação/genética , Doenças Inflamatórias Intestinais/etiologia , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Complexos Multiproteicos , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/genética , Transdução de Sinais , Junções Íntimas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
Epithelial barrier function is contingent on appropriate polarization of key protein components. Work in intestinal epithelial cell cultures and animal models of bowel inflammation suggested that atypical PKC (aPKC), the kinase component of the Par3-Par6 polarity complex, is downregulated by pro-inflammatory signaling. Data from other laboratories showed the participation of myosin light chain kinase in intestinal inflammation, but there is paucity of evidence for assembly of its major target, non-muscle myosin II, in inflammatory bowel disease (IBD). In addition, we showed before that non-muscle myosin IIA (nmMyoIIA) is upregulated in intestinal inflammation in mice and TNFα-treated Caco-2 cells. Thus far, it is unknown if a similar phenomena occur in patients with IBD. Moreover, it is unclear whether aPKC downregulation is directly correlated with local mucosal inflammation or occurs in uninvolved areas. Frozen sections from colonoscopy material were stained for immunofluorescence with extensively validated specific antibodies against phosphorylated aPKC turn motif (active form) and nmMyoIIA. Inflammation was scored for the local area from where the material was obtained. We found a significant negative correlation between the expression of active aPKC and local inflammation, and a significant increase in the apical expression of nmMyoIIA in surface colon epithelia in inflamed areas, but not in non-inflamed mucosa even in the same patients. Changes in aPKC and nmMyoIIA expression are likely to participate in the pathogenesis of epithelial barrier function in response to local pro-inflammatory signals. These results provide a rationale for pursuing mechanistic studies on the regulation of these proteins.
Assuntos
Doenças Inflamatórias Intestinais/fisiopatologia , Isoenzimas/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Proteína Quinase C/metabolismo , Membrana Celular/metabolismo , Polaridade Celular , Estudos de Coortes , Colite/metabolismo , Colo/metabolismo , Regulação para Baixo , Feminino , Imunofluorescência , Humanos , Mucosa Intestinal/metabolismo , Masculino , Microscopia Confocal , Permeabilidade , Fosforilação , Coloração e Rotulagem , Distribuição Tecidual , Regulação para CimaRESUMO
Atypical PKC (PKC iota) is a key organizer of cellular asymmetry. Sequential extractions of intestinal cells showed a pool of enzymatically active PKC iota and the chaperone Hsp70.1 attached to the apical cytoskeleton. Pull-down experiments using purified and recombinant proteins showed a complex of Hsp70 and atypical PKC on filamentous keratins. Transgenic animals overexpressing keratin 8 displayed delocalization of Hsp70 and atypical PKC. Two different keratin-null mouse models, as well as keratin-8 knockdown cells in tissue culture, also showed redistribution of Hsp70 and a sharp decrease in the active form of atypical PKC, which was also reduced by Hsp70 knockdown. An in-vitro turn motif rephosphorylation assay indicated that PKC iota is dephosphorylated by prolonged activity. The Triton-soluble fraction could rephosphorylate PKC iota only when supplemented with the cytoskeletal pellet or filamentous highly purified keratins, a function abolished by immunodepletion of Hsp70 but rescued by recombinant Hsp70. We conclude that both filamentous keratins and Hsp70 are required for the rescue rephosphorylation of mature atypical PKC, regulating the subcellular distribution and steady-state levels of active PKC iota.
Assuntos
Enterócitos/enzimologia , Proteínas de Choque Térmico HSP70/metabolismo , Filamentos Intermediários/enzimologia , Isoenzimas/metabolismo , Queratinas/metabolismo , Proteína Quinase C/metabolismo , Animais , Células CACO-2 , Proteínas de Choque Térmico HSP70/genética , Humanos , Isoenzimas/genética , Queratina-18/metabolismo , Queratina-19/metabolismo , Queratina-8/metabolismo , Queratinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fosforilação , Proteína Quinase C/genética , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNARESUMO
Atypical protein kinase iota (PKCiota) is a key organizer of the apical domain in epithelial cells. Ezrin is a cytosolic protein that, upon activation by phosphorylation of T567, is localized under the apical membrane where it connects actin filaments to membrane proteins and recruits protein kinase A (PKA). To identify the kinase that phosphorylates ezrin T567 in simple epithelia, we analyzed the expression of active PKC and the appearance of T567-P during enterocyte differentiation in vivo. PKCiota phosphorylated ezrin on T567 in vitro, and in Sf9 cells that do not activate human ezrin. In CACO-2 human intestinal cells in culture, PKCiota co-immunoprecipitated with ezrin and was knocked down by shRNA expression. The resulting phenotype showed a modest decrease in total ezrin, but a steep decrease in T567 phosphorylation. The PKCiota-depleted cells showed fewer and shorter microvilli and redistribution of the PKA regulatory subunit. Expression of a dominant-negative form of PKCiota also decreased T567-P signal, and expression of a constitutively active PKCiota mutant showed depolarized distribution of T567-P. We conclude that, although other molecular mechanisms contribute to ezrin activation, apically localized phosphorylation by PKCiota is essential for the activation and normal distribution of ezrin at the early stages of intestinal epithelial cell differentiation.
Assuntos
Membrana Celular/enzimologia , Proteínas do Citoesqueleto/metabolismo , Mucosa Intestinal/enzimologia , Isoenzimas/metabolismo , Microdomínios da Membrana/enzimologia , Proteína Quinase C/metabolismo , Sequência de Aminoácidos/fisiologia , Animais , Sítios de Ligação/fisiologia , Células CACO-2 , Diferenciação Celular/fisiologia , Membrana Celular/ultraestrutura , Polaridade Celular/fisiologia , Proteínas do Citoesqueleto/química , Regulação para Baixo/fisiologia , Ativação Enzimática/fisiologia , Humanos , Insetos , Mucosa Intestinal/citologia , Isoenzimas/genética , Microdomínios da Membrana/ultraestrutura , Camundongos , Microvilosidades/enzimologia , Microvilosidades/ultraestrutura , Fosforilação , Proteína Quinase C/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/genética , Tirosina/metabolismoRESUMO
The olfactory system is exposed to a plethora of chemical compounds throughout an organism's lifespan. Anticipation of stimuli and construction of appropriate neural filters present a significant challenge. This may be addressed via modulation of the protein composition of the sensory epithelium in response to environmental conditions. To reveal the mechanisms governing these changes, we employed a comprehensive quantitative proteomics strategy. Two groups of juvenile mice were treated with either pulsed or continuous application of octanal. After 20 days of treatment, we performed a behavioral study and conducted electrophysiological recordings from the olfactory epithelium (OE). Both treated groups demonstrated peripheral desensitization to octanal; however, only the 'continuous' group exhibited habituation. To obtain novel insight into the molecular mechanisms underpinning the peripheral desensitization to octanal, the OE proteomes of octanal-treated mice versus control were quantitatively analyzed using two-dimensional difference gel electrophoresis. We identified several significantly regulated proteins that were functionally classified as calcium-binding proteins, cytoskeletal proteins, and lipocalins. The calcium-binding proteins and cytoskeletal proteins were up-regulated in the 'pulsed' group, whereas in the 'continuous' group, four lipocalins were significantly down-regulated. Uniquely, the lipocalin odorant-binding protein Ia was drastically down-regulated in both groups. The identified proteins reflect changes throughout the entire OE, corresponding to changes in neuronal, non-neuronal, and pericellular processes. We report the regulation of several promising candidates for the investigation of odorant-induced changes of the OE. Among these proteins are different lipocalins, which seem to play a crucial role in the regulation of the sensitivity of the olfactory system.
Assuntos
Plasticidade Neuronal/fisiologia , Mucosa Olfatória/metabolismo , Proteômica/métodos , Aldeídos/química , Animais , Calbindina 2 , Proteínas de Ligação ao Cálcio/análise , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/metabolismo , Regulação para Baixo , Eletroforese em Gel Bidimensional , Eletrofisiologia , Feminino , Habituação Psicofisiológica , Lipocalinas/análise , Lipocalinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucosa Olfatória/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Gravidez , Proteínas/análise , Proteínas/metabolismo , Receptores Odorantes/análise , Receptores Odorantes/metabolismo , Proteína G de Ligação ao Cálcio S100/análise , Proteína G de Ligação ao Cálcio S100/metabolismo , Olfato/fisiologia , Espectrometria de Massas em Tandem , Regulação para CimaRESUMO
Multiple trials failed to express significant amounts of olfactory receptors in heterologous cells as they are typically retained in the endoplasmic reticulum (ER). Evidence is accumulating that cell-type-specific accessory proteins regulate the folding of olfactory receptors, their exit from the ER, and the trafficking to the plasma membrane of the olfactory cilia where the receptors gain access to odorants. We found Hsc70t, a testis-enriched variant of the Hsp70 family of heat shock proteins which is specifically expressed in post-meiotic germ cells, in the olfactory epithelium of mouse and human. Cotransfected HEK293 cells with Hsc70t and different green fluorescent protein-tagged odorant receptors (ORs) from mouse and man showed a significantly enhanced OR expression. Hsc70t expression also changed the amount of cells functionally expressing olfactory receptors at the cell surface as the number of cells responding to odorants in Ca2+-imaging experiments significantly increased. Our results show that Hsc70t helps expression of ORs in heterologous cell systems and helped the characterization of an "orphan" human olfactory receptor.
Assuntos
Proteínas de Choque Térmico HSC70/farmacologia , Receptores Odorantes/efeitos dos fármacos , Receptores Odorantes/genética , Animais , Linhagem Celular , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Proteínas de Choque Térmico HSC70/genética , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Mucosa Olfatória/química , Mucosa Olfatória/citologia , Mucosa Olfatória/metabolismo , Especificidade de Órgãos , Receptores Odorantes/fisiologia , Testículo/química , Testículo/citologia , Testículo/metabolismo , Fatores de TempoRESUMO
A growing number of proteins originally found in endocytic structures of the plasma membrane appear to be able to traffic into the nucleus, but the cellular function of this translocation remains unclear. We have found that beta-arrestin2, which typically shows a cytoplasmic localization owing to constitutive nuclear export, appears in the nucleus after stimulation of the G-protein-coupled odorant receptor hOR17-4. In the nucleus, beta-arrestin2 was involved in transcriptional regulation as shown by a Gal4-based transactivation assay. Moreover, we discovered that beta-arrestin2 and hOR17-4, a receptor known to have a role in sperm-egg communication, colocalize in the midpiece of mature human spermatozoa. Stimulation of hOR17-4 in spermatozoa induced PKA-dependent translocation of beta-arrestin2 to the nucleus and nuclear accumulation of phosphorylated MAPKs. Analysis of the interaction partners of beta-arrestin2 indicates that odorant receptor signaling in spermatozoa may be important for the regulation of gene expression during the early processes of fertilization.