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This corrects the article DOI: 10.1038/onc.2016.209.
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Canonical Wnt signaling induces the stabilization of ß-catenin, its translocation to the nucleus and the activation of target promoters. This pathway is initiated by the binding of Wnt ligands to the Frizzled receptor, the association of the LRP5/6 co-receptor and the formation of a complex comprising Dvl-2, Axin and protein kinases CK1α, É, γ and GSK3. Among these, activation of CK1É, constitutively bound to LRP5/6 through p120-catenin, is required for the association of the rest of the components. We describe here that CK1É is activated by the PP2A/PR61É phosphatase. Binding of Wnt ligands promotes the interaction of LRP5/6-associated CK1É with Frizzled-bound PR61É regulatory subunit, facilitating the access of PP2A catalytic subunit to CK1É and its activation, what enables the recruitment of Dvl-2 to the receptor complex and the initiation of the Wnt pathway. Our results uncover the mechanism of activation of the canonical Wnt pathway by its ligands.
Assuntos
Caseína Quinase Idelta/metabolismo , Proteína Fosfatase 2/metabolismo , Receptores Frizzled/metabolismo , Células HEK293 , Células HT29 , Humanos , Via de Sinalização WntRESUMO
Snail1 is a master regulator of the epithelial-mesenchymal transition (EMT) and has been implicated in key tumor biological processes such as invasion and metastasis. It has been previously shown that poly(ADP-ribose) polymerase-1 (PARP-1) knockdown, but not PARP inhibition, downregulates the expression of Snail1. In this study we have characterized a novel regulatory mechanism controlling Snail1 protein expression through poly(ADP-ribosyl)ation. The effect is not only limited to repression of Snail1 transcription but also to downregulated Snail1 protein stability. PARP-1 (but not PARP-2) poly(ADP) ribosylates Snail1, both in vivo and in vitro, and interacts with Snail1, an association that is sensitive to PARP inhibitors. PARP inhibition has also clear effects on EMT phenotype of different tumor cells, including Snail1 downregulation, E-cadherin upregulation, decreased cell elongation and invasiveness. Therefore, this study reveals a new regulatory mechanism of Snail1 activation through poly(ADP-ribosyl)ation with consequences in malignant transformation through EMT.
Assuntos
Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Fatores de Transcrição/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fluorbenzenos/farmacologia , Humanos , Melanoma/metabolismo , Invasividade Neoplásica/patologia , Fenantrenos/farmacologia , Ftalazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1 , Poli Adenosina Difosfato Ribose/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases , Estabilidade Proteica , Neoplasias Cutâneas/metabolismo , Fatores de Transcrição da Família SnailRESUMO
SNAI1, ZEB1, E-cadherin (CDH1), and vitamin D receptor (VDR) genes regulate the epithelial-mesenchymal transition (EMT) that initiates the invasion process of many tumor cells. We hypothesized that this process could also affect the behavior of normal cells adjacent to the tumor. To verify this hypothesis, the expression level of these genes was determined by quantitative RT-PCR in tumor, normal adjacent, and normal distant tissues from 32 colorectal cancer (CC) patients. In addition, we extended the study to human HaCaT normal keratinocytes and SW480-ADH colon cancer cells co-cultured with SW480-ADH cells overexpressing the mouse Snai1 gene. Of 18 CC cases with SNAI1 expression in tumor tissue, five also had SNAI1 in normal adjacent tissue (NAT). Expression of SNAI1 in tumor tissue correlated with downregulation of CDH1 and VDR genes in both tumor (P=0.047 and P=0.014, respectively) and NAT lacking SNAI1 expression (P=0.054 and P=0.003). ZEB1 expression was directly related to VDR expression in tumor tissue (r=0.39; P=0.027) and inversely to CDH1 in NAT (r=-0.46; P=0.010). CDH1 and VDR were also downregulated in SW480-ADH and MaCaT cells, respectively, when they were co-cultured with Snai1-expressing cells. Furthermore, cytokine analysis showed differences in the conditioned media obtained from the two cell types. These results indicate that histologically normal tissue adjacent to tumor tissue expressing the EMT-inducing gene SNAI1 shows alterations in the expression of epithelial differentiation genes such as CDH1 and VDR.
Assuntos
Caderinas/genética , Carcinoma/genética , Colo/metabolismo , Neoplasias do Colo/genética , Receptores de Calcitriol/genética , Fatores de Transcrição/genética , Animais , Antígenos CD , Caderinas/metabolismo , Carcinoma/patologia , Neoplasias do Colo/patologia , Regulação para Baixo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/fisiologia , Genes ras/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Calcitriol/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Transfecção , Células Tumorais Cultivadas , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
The beta-catenin signaling pathway is deregulated in nearly all colon cancers. Nonhypercalcemic vitamin D3 (1alpha,25-dehydroxyvitamin D(3)) analogues are candidate drugs to treat this neoplasia. We show that these compounds promote the differentiation of human colon carcinoma SW480 cells expressing vitamin D receptors (VDRs) (SW480-ADH) but not that of a malignant subline (SW480-R) or metastasic derivative (SW620) cells lacking VDR. 1alpha,25(OH)2D(3) induced the expression of E-cadherin and other adhesion proteins (occludin, Zonula occludens [ZO]-1, ZO-2, vinculin) and promoted the translocation of beta-catenin, plakoglobin, and ZO-1 from the nucleus to the plasma membrane. Ligand-activated VDR competed with T cell transcription factor (TCF)-4 for beta-catenin binding. Accordingly, 1alpha,25(OH)2D(3) repressed beta-catenin-TCF-4 transcriptional activity. Moreover, VDR activity was enhanced by ectopic beta-catenin and reduced by TCF-4. Also, 1alpha,25(OH)2D(3) inhibited expression of beta-catenin-TCF-4-responsive genes, c-myc, peroxisome proliferator-activated receptor delta, Tcf-1, and CD44, whereas it induced expression of ZO-1. Our results show that 1alpha,25(OH)2D(3) induces E-cadherin and modulates beta-catenin-TCF-4 target genes in a manner opposite to that of beta-catenin, promoting the differentiation of colon carcinoma cells.
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Adenocarcinoma/metabolismo , Caderinas/biossíntese , Calcitriol/análogos & derivados , Diferenciação Celular/efeitos dos fármacos , Colecalciferol/farmacologia , Neoplasias do Colo/metabolismo , Proteínas do Citoesqueleto/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transativadores , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Adenocarcinoma/patologia , Antineoplásicos , Calcitriol/farmacologia , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Neoplasias do Colo/patologia , Proteínas do Citoesqueleto/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Substâncias Macromoleculares , Fenótipo , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores de Calcitriol/metabolismo , Fatores de Transcrição TCF , Proteína 2 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Células Tumorais Cultivadas , Vitamina D/análogos & derivados , Vitamina D/farmacologia , beta CateninaRESUMO
Malignant transformation of melanocytes frequently coincides with loss of E-cadherin expression. Here we show that loss of E-cadherin in melanoma cell lines does not involve mutations in the E-cadherin gene, promoter methylation, or alterations in expression of AP-2 transcription factors as suggested previously. In a panel of different melanoma cell lines, E-cadherin expression was negatively regulated by up-regulation of the transcription factor Snail. In comparison with primary human melanocytes, where Snail expression was not detected by reverse transcription-polymerase chain reaction, significant expression was found in all eight melanoma cell lines. In parallel, Western blot and reverse transcription-polymerase chain reaction analysis revealed strong reduction of E-cadherin expression in the melanoma cells. Consistently, transient transfection of a Snail expression plasmid into human primary melanocytes led to significant down-regulation of E-cadherin, whereas transient and stable transfection of an antisense Snail construct induced reexpression of E-cadherin in Mel Ju and Mel Im melanomas. In summary, we conclude that activation of Snail expression plays an important role in down-regulation of E-cadherin and tumorigenesis of malignant melanomas.
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Caderinas/biossíntese , Transformação Celular Neoplásica , Proteínas de Ligação a DNA/metabolismo , Melanoma/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima , Western Blotting , Humanos , Metilação , Oligonucleotídeos Antissenso/farmacologia , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Fator de Transcrição AP-2 , Transfecção , Células Tumorais CultivadasRESUMO
Loss of functional adenomatous polyposis coli (APC) protein results in the stabilization of cytosolic beta-catenin and activation of genes that are responsive to Lef/Tcf family transcription factors. We have recently shown that an independent cell adhesion and integrin linked kinase (ILK)-dependent pathway can also activate beta-catenin/LEF mediated gene transcription and downregulate E-cadherin expression. In addition, ILK activity and expression are elevated in adenomatous polyposis and colon carcinomas. To examine the role of this pathway in the background of APC mutations we inhibited ILK activity in APC-/- human colon carcinoma cell lines. In all cases, inhibition of ILK resulted in substantial inhibition of TCF mediated gene transcription and inhibition of transcription and expression of the TCF regulated gene, cyclin D1. Inhibition of ILK resulted in decreased nuclear beta-catenin expression, and in the inhibition of phosphorylation of GSK-3 and stimulation of its activity, leading to accelerated degradation of beta-catenin. In addition, inhibition of ILK suppressed cell growth in culture as well as growth of human colon carcinoma cells in SCID mice. Strikingly, inhibition of ILK also resulted in the transcriptional stimulation of E-cadherin expression and correlated with the inhibition of gene transcription of snail, a repressor of E-cadherin gene expression. Overexpression of ILK caused a stimulation of expression of snail, but snail expression was found not to be regulated by beta-catenin/Tcf. These data demonstrate that ILK can regulate beta-catenin/TCF and snail transcription factors by distinct pathways. We propose that inhibition of ILK may be a useful strategy in the control of progression of colon as well as other carcinomas. Oncogene (2001) 20, 133 - 140.
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Caderinas/fisiologia , Neoplasias do Colo/metabolismo , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transativadores , Fatores de Transcrição/antagonistas & inibidores , Transcrição Gênica , Polipose Adenomatosa do Colo/enzimologia , Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo , Caderinas/genética , Caderinas/metabolismo , Neoplasias do Colo/genética , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Genes APC , Humanos , Integrinas/fisiologia , Fator 1 de Ligação ao Facilitador Linfoide , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Células Tumorais Cultivadas , beta CateninaRESUMO
beta-Catenin plays a dual role as a key effector in the regulation of adherens junctions and as a transcriptional coactivator. Phosphorylation of Tyr-654, a residue placed in the last armadillo repeat of beta-catenin, decreases its binding to E-cadherin. We show here that phosphorylation of Tyr-654 also stimulates the association of beta-catenin to the basal transcription factor TATA-binding protein. The structural bases of these different affinities were investigated. Our results indicate that the beta-catenin C-terminal tail interacts with the armadillo repeat domain, hindering the association of the armadillo region to the TATA-binding protein or to E-cadherin. Phosphorylation of beta-catenin Tyr-654 decreases armadillo-C-terminal tail association, uncovering the last armadillo repeats. In a C-terminal-depleted beta-catenin, the presence of a negative charge at Tyr-654 does not affect the interaction of the TATA-binding protein to the armadillo domain. However, in the case of E-cadherin, the establishment of ion pairs dominates its association with beta-catenin, and its binding is greatly dependent on the absence of a negative charge at Tyr-654. Thus, phosphorylation of Tyr-654 blocks the Ecadherin-beta-catenin interaction, even though the steric hindrance of the C-tail is no longer present. These results explain how phosphorylation of beta-catenin in Tyr-654 modifies the tertiary structure of this protein and the interaction with its different partners.
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Proteínas do Citoesqueleto/metabolismo , Transativadores , Tirosina/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Proteína de Ligação a TATA-Box , Fatores de Transcrição/metabolismo , beta CateninaRESUMO
The phorbol ester phorbol 12-myristate 13-acetate induces remarkable phenotypic changes in intestinal HT-29 M6 cells; these changes consist of loss of homotypic adhesion and inactivation of E-cadherin. In parallel, cell growth is retarded. We have transfected HT-29 M6 cells with an activated form of the conventional protein kinase Calpha (cPK-Calpha). Expression of this isoform induced the acquisition of a scattered phenotype, similar to that adopted by cells after addition of phorbol 12-myristate 13-acetate, with very low cell-to-cell aggregation and undetectable levels of functional E-cadherin. These cell clones were highly motile and rapidly invaded embryonic chick heart fragments. Furthermore, cells expressing activated-cPK-Calpha showed decreased proliferation in comparison to control clones. We have also studied how these two apparently antagonistic changes affect the tumorigenic ability of HT-29 M6 cells. When the different cell clones were xenografted into athymic mice, the effect on cell growth seemed to predominate. Expression of activated-cPK-Calpha significantly reduced the size of the tumors; the cells with the highest level of expression did not even form subcutaneous tumors. Besides their smaller size, the morphology of these tumors was clearly different from those originated by HT-29 M6 cells, and they could be defined as infiltrative on anatomo-pathological basis. These results indicate that cPK-Calpha controls both cell-to-cell adhesion and proliferation of intestinal cells.
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Adesão Celular/genética , Movimento Celular/genética , Invasividade Neoplásica/fisiopatologia , Proteína Quinase C/fisiologia , Animais , Caderinas/análise , Carbazóis/farmacologia , Tamanho Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Indóis/farmacologia , Maleimidas/farmacologia , Camundongos , Camundongos Nus , Mutagênese Sítio-Dirigida/genética , Neoplasias Experimentais/patologia , Proteína Quinase C/genética , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologia , Transfecção/genética , Transplante Heterólogo/patologia , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/análiseRESUMO
We have previously reported that addition of Ca2+ and phospholipid (PL) inhibits translation in hemin-containing reticulocyte lysates through activation of a eukaryotic protein synthesis initiation factor (eIF-2) kinase. The possibility that this activation was mediated by a Ca2+-PL-dependent protein kinase (protein kinase C, PKC) appeared unlikely by the observation that it was prevented or reversed by NADPH-generating systems. Nevertheless, reticulocyte lysates contain a potent PKC activity and we deemed it desirable to isolate this enzyme to answer unequivocally the question whether it does or does not activate eIF-2 alpha kinase. We have purified reticulocyte PKC to near homogeneity with Mr 95,500 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme absolutely depended upon both Ca2+ and phosphatidylserine for activity on histone H1 or the beta-subunit of initiation factor eIF-2 and underwent autophosphorylation in a Ca2+- and PL-dependent manner. Mild treatment with trypsin yielded an Mr 82,000 polypeptide that still required Ca2+ and PL for activity. This Mr agrees with that reported for other PKCs, suggesting that these enzymes may undergo limited degradation during isolation. Further proteolytic treatment converted the reticulocyte enzyme into a Ca2+- and PL-dependent form, as is known for PKCs from other sources. The highly purified PKC had no effect on translation in hemin-supplemented reticulocyte lysates.
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Proteína Quinase C/sangue , Reticulócitos/enzimologia , Animais , Cálcio/farmacologia , Centrifugação com Gradiente de Concentração/métodos , Cromatografia/métodos , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Durapatita , Eletroforese em Gel de Poliacrilamida/métodos , Hidroxiapatitas , Cinética , Peso Molecular , Proteína Quinase C/isolamento & purificação , CoelhosRESUMO
The polypeptide chain initiation factor 2 (eIF-2) binds phospholipid (PL) and becomes a potent inhibitor of translation in hemin-supplemented reticulocyte lysates. This binding is markedly reduced by prior treatment of eIF-2 with N-ethylmaleimide. Although PL is probably bound by all three eIF-2 subunits, our results suggest that the inhibitory molecule is produced by binding to the alpha subunit because, functionally, PL binding has the same effect on eIF-2 as alpha-subunit phosphorylation. This is suggested by the following findings. (i) Like translational inhibition due to heme deficiency, inhibition by small amounts of the eIF-2 X PL complex is prevented by small amounts of the GDP exchange factor (GEF). (ii) In the presence of Mg2+, the GEF-catalyzed formation of a ternary complex (eIF-2 X GTP X Met-tRNAi in which Met-tRNAi is the eukaryotic initiator methionyl tRNA) is inhibited by eIF-2 X PL just as well as by eIF-2 alpha-subunit phosphorylation. (iii) Also in the presence of Mg2+, GEF is unable to catalyze the exchange of free GDP with eIF-2 X PL-bound GDP, as it fails to catalyze the exchange of free GDP with GDP that is bound to alpha-subunit-phosphorylated eIF-2. These observations suggest that, like alpha-subunit-phosphorylated eIF-2, eIF-2 X PL traps GEF in a nondissociable eIF-2 X PL X GEF complex, whereby GEF is no longer able to catalyze ternary complex formation and initiation is inhibited.
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Fatores de Iniciação de Peptídeos/metabolismo , Fosfolipídeos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas/metabolismo , Animais , Fator de Iniciação 2 em Eucariotos , Guanosina Difosfato/metabolismo , Fatores de Iniciação de Peptídeos/farmacologia , Fosforilação , Proteínas/farmacologia , Coelhos , TrítioRESUMO
We have reported previously that calcium ions and phospholipid activate the heme-stabilized proinhibitor form (pro-HCI) of the heme-controlled translational inhibitor (HCI) in reticulocyte lysates and promote the first step of the reaction pro-HCI in equilibrium reversible HCI----irreversible HCI. This suggested the possible involvement of a Ca2+/phospholipid-dependent protein kinase (protein kinase C) in the activation. However, further investigation revealed, among other things, that polyunsaturated fatty acids (e.g., arachidonic acid) were as effective as Ca2+/phospholipid in promoting translational inhibition and phosphorylation of the alpha subunit of the chain-initiation factor eIF-2 and, moreover, HCI activation could be prevented or reversed in either case by NADPH-generating systems or by dithiols. Our results suggest that pro-HCI is activated by lipoperoxides produced in reticulocyte lysates from either phospholipid or polyunsaturated fatty acids; the presence of Ca2+ is required in the former but not in the latter case. The reversible activation of HCI by Ca2+ and phospholipid might suggest a possible modulatory role of Ca2+ in translational control.
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Cálcio/fisiologia , Iniciação Traducional da Cadeia Peptídica , Fosfolipídeos/farmacologia , Proteínas Quinases/metabolismo , Reticulócitos/fisiologia , Ditiotreitol/farmacologia , Ácido Egtázico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos Insaturados/farmacologia , Peróxidos Lipídicos/farmacologia , NADP/farmacologia , Oxirredução , Fatores de Iniciação de Peptídeos/metabolismo , Proteína Quinase C , eIF-2 QuinaseRESUMO
The synthesis of globin, the major protein synthesized by reticulocytes, requires the presence of heme, the prosthetic group of hemoglobin. The absence of heme leads to the activation of a nucleotide-independent protein kinase that phosphorylates the alpha subunit of the chain initiation factor eIF-2. This modification interferes with the catalytic function of eIF-2 in protein synthesis initiation. Recent progress in our understanding of the molecular mechanism of this inhibition is briefly reviewed. The same phosphorylation is catalyzed by a different enzyme (DAI) which, while constitutive in reticulocytes, is induced by interferon in other cells. This enzyme is activated by low concentrations of double-stranded RNA in conjunction with ATP. The mechanisms of activation of these enzymes are still poorly understood. HCI is believed to form an inactive complex with heme and become active when the heme is removed by hemoglobin formation. The proinhibitor form of HCI (proHCI) is unstable in vitro and, even in the presence of heme, is irreversibly inactivated by SH-binding reagents, alkaline pH, slightly elevated temperatures, or high hydrostatic pressure. In hemin-supplemented reticulocyte lysates proHCI can also be reversibly activated by oxidized glutathione (GSSG) or NADPH depletion as well as by polyunsaturated fatty acids and by Ca2+-phospholipid. The mechanism of activation of HCI by GSSG has not been clarified although it appears to involve oxidation of proHCI SH groups to disulfides. Like activation by GSSG, the activation of HCI by polyunsaturated fatty acids and by Ca2+-phospholipid also appears to be largely due to oxidation of some of the enzyme's SH groups. There thus appear to be two fully independent mechanisms of HCI activation in reticulocyte lysates, one involving heme deficiency, the other involving oxidation of proHCI SH groups. The latter, but not the former, can be prevented or reversed by NADPH generators or dithiols. ProHCI appears to be maintained in the reduced, inactive state by a system involving NADPH, thioredoxin, and thioredoxin reductase.
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Proteínas Sanguíneas/metabolismo , Cálcio/farmacologia , Heme/farmacologia , Fosfolipídeos/farmacologia , Biossíntese de Proteínas , Reticulócitos/metabolismo , Animais , Ativação Enzimática , Fator de Iniciação 2 em Eucariotos , Heme/fisiologia , Homeostase , Cinética , Fatores de Iniciação de Peptídeos/sangue , Fosforilação , Proteínas Quinases/sangue , eIF-2 QuinaseRESUMO
Hemin-supplemented reticulocyte lysates can be activated for translational inhibition by addition of Ca2+ or phospholipid. The fact that this inhibition is prevented or decreased in both cases either by the Ca2+ chelator EGTA or by polymyxin B, an inhibitor of the recently described Ca2+- and phospholipid-dependent protein kinases, suggests the involvement of both Ca2+ and phospholipid in this activation. The inhibition by Ca2+ or phospholipid is accompanied by phosphorylation of the 38-kilodalton subunit of the eukaryotic initiation factor 2 (eIF-2) and the 90-kilodalton band of the heme-controlled translational inhibitor (HCI) and can be reversed by high concentrations of eIF-2 or GTP. When incubation is conducted at 30 degrees C, the inhibition produced by Ca2+ is not reversed by EGTA after 15 min. However, at 20 degrees C, Ca2+ inhibition can be fully reversed as late as 90 min from the start of incubation and phosphorylation of the eIF-2 alpha-subunit is correspondingly decreased. These results are consistent with the idea that, like heme deprivation, the activation by Ca2+ and phospholipid promotes the first step of the reaction pro-inhibitor in equilibrium reversible inhibitor leads to irreversible inhibitor and suggest that, in the presence of hemin albeit by a different mechanism, this activation affects the same inhibitor that is activated in the absence of heme--namely, HCI. Whether this activation is direct or indirect--e.g., via a separate Ca2+- and phospholipid-dependent protein kinase--remains to be determined.
Assuntos
Cálcio/farmacologia , Heme/farmacologia , Fosfolipídeos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Reticulócitos/metabolismo , Animais , Calmodulina/farmacologia , Ácido Egtázico/farmacologia , Fator de Iniciação 2 em Eucariotos , Cinética , Peso Molecular , Fatores de Iniciação de Peptídeos/metabolismo , Fosforilação , Polimixina B/farmacologia , Proteínas/metabolismo , Reticulócitos/efeitos dos fármacos , TemperaturaRESUMO
Inhibition of translation in hemin-containing reticulocyte lysates by catalytic subunit (cS) preparations of cAMP-dependent protein kinase from bovine heart, reported earlier by our group, is due to a highly active heat-stable protein contaminant (HS). The specific activity for translational inhibition goes up by a factor of 10 when cS is heated for 10 min at 80 degrees C, which completely destroys histone phosphorylation activity. HS has been purified to homogeneity from bovine heart. It consists of a single polypeptide chain (Mr approximately 68,000). HS inhibits translation with biphasic kinetics similar to those of hemin deficiency and induces pronounced phosphorylation of the alpha subunit of the eukaryotic initiation factor eIF-2. The inhibition is relieved by eIF-2 or GTP but not by high concentrations of double-stranded RNA, thus ruling out involvement of the double-stranded RNA-activated inhibitor. Judged by poly(U) translation, HS has no effect on chain elongation. When added to crude preparations of the proinhibitor form (proHCI) of the heme-controlled translational inhibitor (HCI), HS appears to produce an increase of the HCI-to proHCI ratio. The mode of action of HS is as yet unknown.