RESUMEN

Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor whose allele is lost in 50% of liver, breast, lung and 70% of colon cancers. Here, we show that the transcriptional coactivators Megakaryoblastic Leukemia 1 and 2 (MKL1/2) are constitutively localized to the nucleus in hepatocellular and mammary carcinoma cells that lack DLC1. Moreover, DLC1 loss and MKL1 nuclear localization correlate in primary human hepatocellular carcinoma. Nuclear accumulation of MKL1 in DLC1-deficient cancer cells is accomplished by activation of the RhoA/actin signaling pathway and concomitant impairment of MKL1 phosphorylation, which results in constitutive activation of MKL1/2 target genes. We provide evidence that MKL1/2 mediates cancerous transformation in DLC1-deficient hepatocellular and mammary carcinoma cells. Depletion of MKL1/2 suppresses cell migration, cell proliferation and anchorage-independent cell growth induced by DLC1 loss.

Asunto(s)

Neoplasias de la Mama/metabolismo , Carcinoma Hepatocelular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Activadoras de GTPasa/deficiencia , Proteínas Activadoras de GTPasa/genética , Neoplasias Hepáticas/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Neoplasias de la Mama/genética , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Movimiento Celular , Núcleo Celular/metabolismo , Proliferación Celular , Transformación Celular Neoplásica , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Neoplasias Hepáticas/genética , Transducción de Señal , Transactivadores , Proteínas Supresoras de Tumor/metabolismo , Proteína de Unión al GTP rhoA/metabolismo

RESUMEN

Presenilin (PS) proteins facilitate endoproteolysis of selected type I transmembrane proteins such as the Alzheimer's disease (AD) associated beta-Amyloid precursor protein (beta APP) and Notch. beta APP is cleaved within its transmembrane domain by an aspartyl protease activity termed gamma-secretase, which may be identical with PS1 and PS2. Notch also undergoes a PS-dependent intramembraneous proteolysis. A similar gamma-secretase-like cleavage may also occur with IRE1 and ATF6, two signaling molecules of the unfolded protein response (UPR) that may require PSs for their activation. Here, we have analyzed whether ATF6 cleavage requires a PS-dependent gamma-secretase activity and whether inhibition of gamma-secretase activity would affect the UPR. Endoproteolysis of ATF6 was observed in the presence of the highly potent gamma-secretase inhibitor L-685,458. ATF6 processing also occurred in the presence of functionally inactive dominant negative mutants of PS1 (PS1 D385N) and PS2 (PS2 D366A) that do not support endoproteolysis of beta APP and Notch. Our results therefore demonstrate that ATF6 is not a substrate for PS mediated gamma-secretase-like endoproteolysis. This finding indicates that gamma-secretase inhibitors, which are currently developed as therapeutic agents to lower the A beta burden in brains of AD patients, do not interfere with the UPR response.

Asunto(s)

Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Unión al ADN/metabolismo , Endopeptidasas/metabolismo , Proteínas de la Membrana/metabolismo , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 6 , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/genética , Ácido Aspártico Endopeptidasas/metabolismo , Carbamatos/farmacología , Línea Celular , Proteínas de Unión al ADN/genética , Dipéptidos/farmacología , Humanos , Riñón/citología , Proteínas de la Membrana/genética , Mutagénesis/fisiología , Placa Amiloide/metabolismo , Presenilina-1 , Presenilina-2 , Inhibidores de Proteasas/farmacología , Receptores Notch , Factores de Transcripción/genética , Transfección

RESUMEN

ATF6 is a member of the basic-leucine zipper family of transcription factors. It contains a transmembrane domain and is located in membranes of the endoplasmic reticulum. ATF6 has been implicated in the endoplasmic reticulum (ER) stress response pathway since it can activate expression of GRP78 and other genes induced by the ER stress response. ER stress appears to activate ATF6 by cleavage from the ER membrane and translocation to the nucleus. However, direct DNA binding by ATF6 had not been demonstrated. In this report, we have identified a consensus DNA binding sequence for ATF6. This site is related to but distinct from ATF1/CREB binding sites. The site was placed in a reporter gene and was specifically activated by ATF6 overexpression and was strongly induced by the ER stress response. A dominant negative form of ATF6 blocked ER stress induction of both ATF6 site and GRP78 reporter genes. We further found that GAL4-ATF6 could be activated by ER stress. These results demonstrate that ATF6 is a direct target of the ER stress response. A proximal sensor of the ER stress response, human IRE1 (hIRE1), was sufficient to activate the ATF6 reporter gene, while a dominant negative form of hIRE1 blocked ER stress activation, suggesting that hIRE1 is upstream of ATF6 in the ER stress signaling pathway.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Choque Térmico , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 6 , Secuencia de Bases , Sitios de Unión , Proteínas Portadoras/genética , ADN/química , Proteínas de Unión al ADN/biosíntesis , Chaperón BiP del Retículo Endoplásmico , Células HeLa , Humanos , Chaperonas Moleculares/genética , Estrés Oxidativo , Factores de Transcripción/biosíntesis , Tunicamicina/farmacología

RESUMEN

The c-fos enhancer can be activated by many signaling pathways through distinct elements of the enhancer. The enhancer contains at its core the serum response element (SRE) that binds serum response factor (SRF). On the 5' side of the SRE is a site for p62TCF which binds only when SRF is bound as well. p62TCF is encoded by three ets-related genes, Elk-1, SAP1 and SAP2. Each of these factors contain a transcriptional activation domain that is activated by phosphorylation by MAP kinases. On the 3' side of the SRE is the 'c-fos AP1 site' (FAP1) whose role has been less clear. We find here that the FAP1 site contributes strongly to phorbol ester (TPA) and Erk MAP kinase activation of the c-fos enhancer and that both the p62TCF and FAP1 sites are required for effective activation of the enhancer. We further find that the FAP1 site binds ATF1 and CREB from HeLa nuclear extracts and that the phosphorylation of these factors is induced by TPA. ATF1 and CREB can be phosphorylated by Rsk2 which is a protein kinase directly activated by Erk MAP kinases. These results suggest a signaling pathway in which Erk MAP kinase activates the c-fos enhancer by direct phosphorylation of p62TCF and by activation of Rsk related kinases that phosphorylate ATF1 and CREB.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Genes fos , Sistema de Señalización de MAP Quinasas/genética , Proteínas Proto-Oncogénicas c-fos/genética , Factor de Transcripción AP-1/fisiología , Factor de Transcripción Activador 1 , Sitios de Unión/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/inmunología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Elementos de Facilitación Genéticos/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Genes fos/efectos de los fármacos , Células HeLa , Complejo Antigénico de Nefritis de Heymann , Humanos , Sueros Inmunes/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Quinasas S6 Ribosómicas/fisiología , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción AP-1/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Ca(2+) induction of a subset of cellular and viral immediate-early activation genes in lymphocytes has been previously mapped to response elements recognized by the MEF2 family of transcription factors. Here, we demonstrate that Ca(2+) activation of MEF2 response elements in T lymphocytes is mediated in synergy by two Ca(2+)/calmodulin-dependent enzymes, the phosphatase calcineurin, and the kinase type IV/Gr (CaMKIV/Gr), which promote transcription by the MEF2 family members MEF2A and MEF2D. Calcineurin up-regulates the activity of both factors by an NFAT-dependent mechanism, while CaMKIV/Gr selectively and independently activates MEF2D. These results identify MEF2 proteins as effectors of a pathway of gene induction in T lymphocytes which integrates diverse Ca(2+) activation signals and may be broadly operative in several tissues.

Asunto(s)

Señalización del Calcio , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Nucleares , Linfocitos T/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Calcineurina/metabolismo , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Humanos , Proteínas de Dominio MADS , Factores de Transcripción MEF2 , Ratones , Datos de Secuencia Molecular , Factores Reguladores Miogénicos , Factores de Transcripción NFATC , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Unión Proteica , Receptores Citoplasmáticos y Nucleares , Receptores de Esteroides , Proteínas Recombinantes/metabolismo , Elementos de Respuesta , Activación Transcripcional

RESUMEN

ATF6 is a membrane-bound transcription factor that activates genes in the endoplasmic reticulum (ER) stress response. When unfolded proteins accumulate in the ER, ATF6 is cleaved to release its cytoplasmic domain, which enters the nucleus. Here, we show that ATF6 is processed by Site-1 protease (S1P) and Site-2 protease (S2P), the enzymes that process SREBPs in response to cholesterol deprivation. ATF6 processing was blocked completely in cells lacking S2P and partially in cells lacking S1P. ATF6 processing required the RxxL and asparagine/proline motifs, known requirements for S1P and S2P processing, respectively. Cells lacking S2P failed to induce GRP78, an ATF6 target, in response to ER stress. ATF6 processing did not require SCAP, which is essential for SREBP processing. We conclude that S1P and S2P are required for the ER stress response as well as for lipid synthesis.

Asunto(s)

Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas de Unión al ADN/metabolismo , Endopeptidasas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Choque Térmico , Membranas Intracelulares/metabolismo , Proproteína Convertasas , Serina Endopeptidasas/metabolismo , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 6 , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Endopeptidasas/genética , Chaperón BiP del Retículo Endoplásmico , Humanos , Immunoblotting , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Mutación , Unión Proteica , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Serina Endopeptidasas/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Tapsigargina/farmacología , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacos , Tunicamicina/farmacología

RESUMEN

T cell receptor (TCR)-induced apoptosis of thymocytes is mediated by calcium-dependent expression of the steroid receptors Nur77 and Nor1. Nur77 expression is controlled by the transcription factor myocyte enhancer factor 2 (MEF2), but how MEF2 is activated by calcium signaling is still obscure. Cabin1, a calcineurin inhibitor, was found to regulate MEF2. MEF2 was normally sequestered by Cabin1 in a transcriptionally inactive state. TCR engagement led to an increase in intracellular calcium concentration and the dissociation of MEF2 from Cabin1, as a result of competitive binding of activated calmodulin to Cabin1. The interplay between Cabin1, MEF2, and calmodulin defines a distinct signaling pathway from the TCR to the Nur77 promoter during T cell apoptosis.

Asunto(s)

Apoptosis , Señalización del Calcio , Proteínas de Unión al ADN/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Calcineurina/química , Calcineurina/genética , Calcineurina/metabolismo , Calcineurina/farmacología , Calcio/metabolismo , Calmodulina/metabolismo , Línea Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Expresión Génica , Genes Reporteros , Humanos , Células Jurkat , Factores de Transcripción MEF2 , Factores Reguladores Miogénicos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/farmacología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Citoplasmáticos y Nucleares , Receptores de Esteroides , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción Genética , Técnicas del Sistema de Dos Híbridos

RESUMEN

The cdc25A gene encodes a tyrosine phosphatase which activates cyclin-dependent kinase activity in the G1 phase of the cell cycle. cdc25A RNA levels are induced from 3 to 6 h after serum induction of serum-starved NIH 3T3 cells, suggesting that the cdc25A gene is a delayed-early gene. Analysis of cdc25A promoter constructs showed that the cdc25A promoter is sufficient for serum induction. Surprisingly for a gene expressed in early to mid-G1, serum induction of the promoter requires an E2F site at position -62 in the promoter. Deletion or point mutation of the E2F site resulted in activation of expression in serum-starved cells and no further induction by serum treatment. E2F factors were found to bind to the cdc25A E2F site along with the retinoblastoma protein (Rb) family members p130 and p107. A shift in mobility of the E2F-p107 complex in extracts of cells induced for 6 h correlated with induction of cdc25A expression. These results suggest that serum induction of cdc25A expression is mediated by inactivation of p107 or p130, both of which repress transcription when bound to the promoter through E2F.

Asunto(s)

Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Proteínas Tirosina Fosfatasas/genética , Proteínas , Factores de Transcripción/metabolismo , Fosfatasas cdc25 , Células 3T3 , Animales , Secuencia de Bases , Bovinos , Ciclina E/genética , ADN Complementario , Factores de Transcripción E2F , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero , Proteína de Retinoblastoma/metabolismo , Proteína 1 de Unión a Retinoblastoma , Proteína p107 Similar a la del Retinoblastoma , Proteína p130 Similar a la del Retinoblastoma , Albúmina Sérica Bovina , Factor de Transcripción DP1

RESUMEN

Myocyte enhancer factor 2 (MEF2) transcriptional regulatory proteins are key regulators of muscle-specific gene expression and also play a general role in the cellular response to growth factors, cytokines and environmental stressors. To identify signaling pathway components that might mediate these events, the potential role of MAP kinase and PKC signaling in the modulation of MEF2A phosphorylation and transcriptional activity were therefore studied. In transient transfection reporter assays, activated p38 MAP kinase potently increased MEF2A trans -activating potential, PKC[delta] and [epsiv] isotypes enhanced MEF2A transactivation to a lesser extent, while the ERK1/2 and JNK/SAPK pathways were without effect. A GAL4-based assay system showed that p38 MAP kinase and PKC[delta] target the MEF2A transactivation domain. We also observed an increase in p38 MAP kinase activity in congruence with the increase in MEF2A expression in differentiating primary muscle cells. COS cells overexpressing MEF2A alone or with one of the kinases were metabolically labeled with [32P]orthophosphate and MEF2A was immunoprecipitated using specific anti-MEF2A antibodies. MEF2A from cells co-transfected with activated p38 MAP kinase showed a decreased electrophoretic mobility due to phosphorylation. Subsequent phosphopeptide mapping and phosphoamino acid analysis indicated the appearance of several phoshopeptides due to p38 MAP kinase activation of MEF2A which were due to phosphorylation on serine and threonine residues. These studies position MEF2A as a nuclear target for the p38 MAP kinase signaling pathway.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Procesamiento Proteico-Postraduccional , Transducción de Señal , Factores de Transcripción/metabolismo , Animales , Células COS , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Proteínas de Dominio MADS , Factores de Transcripción MEF2 , Factores Reguladores Miogénicos , Fosforilación , Proteína Quinasa C/metabolismo , Transducción de Señal/genética , Factores de Transcripción/genética , Activación Transcripcional , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

Protein kinase C (PKC) is a multigene family of enzymes consisting of at least 11 isoforms. It has been implicated in the induction of c-fos and other immediate response genes by various mitogens. The serum response element (SRE) in the c-fos promoter is necessary and sufficient for induction of transcription of c-fos by serum, growth factors, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). It forms a complex with the ternary complex factor (TCF) and with a dimer of the serum response factor (SRF). TCF is the target of several signal transduction pathways and SRF is the target of the rhoA pathway. In this study we generated dominant-negative and constitutively active mutants of PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta to determine the roles of individual isoforms of PKC in activation of the SRE. Transient-transfection assays with NIH 3T3 cells, using an SRE-driven luciferase reporter plasmid, indicated that PKC-alpha and PKC-epsilon, but not PKC-delta or PKC-zeta, mediate SRE activation. TPA-induced activation of the SRE was partially inhibited by dominant negative c-Raf, ERK1, or ERK2, and constitutively active mutants of PKC-alpha and PKC-epsilon activated the transactivation domain of Elk-1. TPA-induced activation of the SRE was also partially inhibited by a dominant-negative MEKK1. Furthermore, TPA treatment of serum-starved NIH 3T3 cells led to phosphorylation of SEK1, and constitutively active mutants of PKC-alpha and PKC-epsilon activated the transactivation domain of c-Jun, a major substrate of JNK. Constitutively active mutants of PKC-alpha and PKC-epsilon could also induce a mutant c-fos promoter which lacks the TCF binding site, and they also induce transactivation activity of the SRF. Furthermore, rhoA-mediated SRE activation was blocked by dominant negative mutants of PKC-alpha or PKC-epsilon. Taken together, these findings indicate that PKC-alpha and PKC-epsilon can enhance the activities of at least three signaling pathways that converge on the SRE: c-Raf-MEK1-ERK-TCF, MEKK1-SEK1-JNK-TCF, and rhoA-SRF. Thus, specific isoforms of PKC may play a role in integrating networks of signal transduction pathways that control gene expression.

Asunto(s)

Genes fos , Isoenzimas/genética , Isoenzimas/metabolismo , MAP Quinasa Quinasa 4 , Quinasa 1 de Quinasa de Quinasa MAP , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión/genética , Células COS , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , MAP Quinasa Quinasa 1 , Ratones , Mutación , Plásmidos/genética , Regiones Promotoras Genéticas , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

Serum response factor (SRF) plays a central role during myogenesis, being required for the expression of striated alpha-actin genes. As shown here, the small GTPase RhoA-dependent activation of SRF results in the expression of muscle-specific genes, thereby promoting myogenic differentiation in myoblast cell lines. Co-expression of activated V14-RhoA and SRF results in an approximately 10-fold activation of the skeletal alpha-actin promoter in replicating myoblasts, while SRFpm1, a dominant negative SRF mutant, blocks RhoA dependent skeletal alpha-actin promoter activity. Serum withdrawal further potentiates RhoA- and SRF-mediated activation of alpha-actin promoter to about 30-fold in differentiated myotubes. In addition, the proximal SRE1 in the skeletal alpha-actin promoter is sufficient to mediate RhoA signaling via SRF. Furthermore, SRFpm1 and to a lesser extent dominant negative N19-RhoA inhibit myoblast fusion, postreplicative myogenic differentiation, and expression of direct SRF targets such as skeletal alpha-actin and indirect targets such as myogenin and alpha-myosin heavy chain. Moreover, RhoA also stimulates the autoregulatable murine SRF gene promoter in myoblasts, and the expression level of SRF is reduced in myoblasts overexpressing N19-RhoA. Our study supports the concept that RhoA signaling via SRF serves as an obligatory muscle differentiation regulatory pathway.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Proteínas de Unión al GTP/fisiología , Músculo Esquelético/citología , Proteínas Nucleares/fisiología , Transducción de Señal , Actinas/genética , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Proteínas de Unión al ADN/genética , Ratones , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-fos/genética , Factor de Respuesta Sérica , Activación Transcripcional , Proteína de Unión al GTP rhoA

RESUMEN

In various cell types certain stresses can stimulate p38 mitogen-activated protein kinase (p38 MAPK), leading to the transcriptional activation of genes that contribute to appropriate compensatory responses. In this report the mechanism of p38-activated transcription was studied in cardiac myocytes where this MAPK is a key regulator of the cell growth and the cardiac-specific gene induction that occurs in response to potentially stressful stimuli. In the cardiac atrial natriuretic factor (ANF) gene, a promoter-proximal serum response element (SRE), which binds serum response factor (SRF), was shown to be critical for ANF induction in primary cardiac myocytes transfected with the selective p38 MAPK activator, MKK6 (Glu). This ANF SRE does not possess sequences typically required for the binding of the Ets-related ternary complex factors (TCFs), such as Elk-1, indicating that p38-mediated induction through this element may take place independently of such TCFs. Although p38 did not phosphorylate SRF in vitro, it efficiently phosphorylated ATF6, a newly discovered SRF-binding protein that is believed to serve as a co-activator of SRF-inducible transcription at SREs. Expression of an ATF6 antisense RNA blocked p38-mediated ANF induction through the ANF SRE. Moreover, when fused to the Gal4 DNA-binding domain, an N-terminal 273-amino acid fragment of ATF6 was sufficient to support trans-activation of Gal4/luciferase expression in response to p38 but not the other stress kinase, N-terminal Jun kinase (JNK); p38-activating cardiac growth promoters also stimulated ATF6 trans-activation. These results indicate that through ATF6, p38 can augment SRE-mediated transcription independently of Ets-related TCFs, representing a novel mechanism of SRF-dependent transcription by MAP kinases.

Asunto(s)

Factor Natriurético Atrial/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Regulación de la Expresión Génica/genética , Proteínas Quinasas Activadas por Mitógenos , Miocardio/enzimología , Factor de Transcripción Activador 6 , Animales , Células Cultivadas , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endotelina-1/farmacología , Proteínas Nucleares/genética , Fenilefrina/farmacología , Fosforilación , Regiones Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN sin Sentido/farmacología , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Factor de Respuesta Sérica , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/fisiología , Transfección/genética , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

Many growth factors rapidly induce transcription of the c-fos proto-oncogene. We have investigated the pathways for induction of the c-fos promoter by serum and epidermal growth factor (EGF) in HeLa cells. Induction of the serum response element (SRE) of the c-fos promoter could be split into two parts, one involving the serum response factor-associated ternary complex factor (TCF) factors and the second mediated by core SRE sequences. Serum induction was mediated primarily by the core SRE, whereas EGF used both the TCF and core SRE pathways. Using activated and inhibitory signaling proteins, we found that phosphatidyl inositol 3-kinase (PI3K) and rho family members could mediate activation by serum. Activation by PI3K was mediated by core SRE sequences and was dependent upon rac and rho, suggesting a PI3K-to-rac-to-rho pathway for core SRE activation. The PI3K target Akt was also capable of activating the SRE but functioned through the TCF pathway, suggesting that Akt does not mediate the primary PI3K pathway to the SRE and that Akt is capable of activating TCF family members. Serum and EGF induction of the core SRE was partially inhibited by rho and PI3K inhibitors. The use of these inhibitors demonstrates the complexity of signaling pathways to the SRE and suggests that serum activates rho by PI3K-dependent and -independent pathways.

Asunto(s)

Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-fos/genética , Células 3T3 , Animales , Activación Enzimática , Factor de Crecimiento Epidérmico/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Ratones , Proto-Oncogenes Mas , Transducción de Señal , Proteínas de Unión al GTP rac , Proteínas de Unión al GTP rho

RESUMEN

The c-jun proto-oncogene encodes a transcription factor which is activated by mitogens both transcriptionally and by phosphorylation by Jun N-terminal kinase (JNK). We have investigated the cellular signalling pathways involved in epidermal growth factor (EGF) induction of the c-jun promoter. We find that two sequence elements, which bind ATF1 and MEF2D transcription factors, are required in HeLa cells, although they are not sufficient for maximal induction. Activated forms of Ras, RacI, Cdc42Hs, and MEKK increased expression of the c-jun promoter, while dominant negative forms of Ras, RacI, and MEK kinase (MEKK) inhibited EGF induction. These and previously published results suggest that EGF activates the c-jun promoter by a Ras-to-Rac-to-MEKK pathway. This pathway is similar to that used for posttranslational activation of c-jun by JNK.

Asunto(s)

Factor de Crecimiento Epidérmico/fisiología , Regulación de la Expresión Génica , Quinasa 1 de Quinasa de Quinasa MAP , Proteínas Quinasas Activadas por Mitógenos , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Factor de Transcripción Activador 1 , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al GTP/metabolismo , Regulación Enzimológica de la Expresión Génica , Células HeLa , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas de Dominio MADS , Factores de Transcripción MEF2 , Factores Reguladores Miogénicos , Fosforilación , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Proteína de Unión al GTP cdc42

RESUMEN

Serum response factor (SRF) is a transcription factor which binds to the serum response element (SRE) in the c-fos promoter. It is required for regulated expression of the c-fos gene as well as other immediate-early genes and some tissue-specific genes. To better understand the regulation of SRF, we used a yeast interaction assay to screen a human HeLa cell cDNA library for SRF-interacting proteins. ATF6, a basic-leucine zipper protein, was isolated by binding to SRF and in particular to its transcriptional activation domain. The binding of ATF6 to SRF was also detected in vitro. An ATF6-VP16 chimera activated expression of an SRE reporter gene in HeLa cells, suggesting that ATF6 can interact with endogenous SRF. More strikingly, an antisense ATF6 construct reduced serum induction of a c-fos reporter gene, suggesting that ATF6 is involved in activation of transcription by SRF. ATF6 was previously partially cloned as a member of the ATF family. The complete cDNA of ATF6 was isolated, and its expression pattern was described.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 6 , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Clonación Molecular , Huella de ADN , Proteínas de Unión al ADN/genética , Genes Reporteros , Genes fos , Células HeLa , Humanos , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Análisis de Secuencia de ADN , Factor de Respuesta Sérica , Factores de Transcripción/genética , Activación Transcripcional

RESUMEN

The c-Jun amino-terminal kinases (JNKs) are a subfamily of mitogen-activated protein kinases that phosphorylate c-Jun and ATF2, and it has been postulated that phosphorylated c-Jun enhances its own expression through AP-1 sites on the c-jun promoter. In this study, we asked whether signals activating JNK regulate the c-jun promoter. Using NIH 3T3 cells expressing G protein-coupled m1 acetylcholine receptors as an experimental model, we have recently shown that the cholinergic agonist carbachol, but not platelet-derived growth factor, potently elevates JNK activity. Consistent with these findings, carbachol, but not platelet-derived growth factor, increased the activity of a c-jun promoter-driven reporter gene (for chloramphenicol acetyltransferase). However, coexpression of JNK kinase kinase (MEKK) effectively increased JNK activity, but resulted in surprisingly limited induction of the c-jun promoter. This raised the possibility that pathway(s) distinct from JNK control the c-jun promoter, and prompted us to explore which of its regulatory elements participate in transcriptional control. We observed that deletion of the 3' AP-1 site diminished chloramphenicol acetyltransferase activity in response to carbachol, but only to a limited extent. In contrast, deletion of a MEF2 site dramatically reduced expression, and deletion of both the MEF2 and 3' AP-1 sites abolished induction. Furthermore, cotransfection with MEF2C and MEF2D cDNAs potently enhanced the activity of the c-jun promoter in response to carbachol, and stimulation of m1 receptors, but not direct JNK activation, induced expression of a MEF2-responsive plasmid. Taken together, these data strongly suggest that MEF2 mediates c-jun promoter expression by G protein-coupled receptors through a yet to be identified pathway, distinct from that of JNK.

Asunto(s)

Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas de Unión al GTP/fisiología , Genes jun , Proteínas Quinasas Activadas por Mitógenos , Regiones Promotoras Genéticas , Receptores Colinérgicos/fisiología , Factores de Transcripción/fisiología , Células 3T3 , Animales , Carbacol/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos , Factores de Transcripción MEF2 , Ratones , Factores Reguladores Miogénicos

Asunto(s)

ARN Polimerasa II/análisis , Transcripción Genética , Animales , Sistema Libre de Células/enzimología , Humanos

RESUMEN

Serum response factor (SRF) gene expression in avian embryonic muscle lineages plays a central role in activating alpha-actin gene activity. In early stage HH 6 avian embryos, SRF mRNA expression showed strong localization to the neural groove, primitive streak, lateral plate mesoderm, and Hensen's node, while distinct SRF expression was seen later in the neural folds and the somites by HH stage 8. SRF transcripts appeared in the precardiac splanchnic mesoderm in stage HH 9 embryos and was detected at higher levels in the myocardium, somites, and lateral mesoderm of HH 11 embryos. SRF antibody staining demonstrated significant SRF protein accumulation in the myocardium of the developing heart and the myotomal portion of somites. During primary myogenesis in culture, SRF transcripts and nuclear SRF protein content increased about 40-fold, as primary myoblasts withdrew from the cell cycle, reaching their highest levels prior to the upregulation of the skeletal alpha-actin gene. A dominant-negative SRF mutant, SRFpm1, which inhibited DNA binding, but not dimerization of monomeric SRF subunits, blocked transcriptional activation of a skeletal alpha-actin promoter-luciferase reporter gene during myogenesis. Transcriptional blockade was reversed by co-transfections of a wild-type SRF expression vector, but was not rescued by the expression of other myogenic factors, such as MyoD and Mef-2C. Thus, SRF displayed an embryonic expression pattern restricted primarily to striated muscle cell lineages, in which increased mass of nuclear SRF was obligatory for alpha-actin gene transcription.

Asunto(s)

Actinas/genética , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Músculos/embriología , Factores Reguladores Miogénicos , Proteínas Nucleares/genética , Factores de Transcripción/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Linaje de la Célula/genética , Embrión de Pollo , ADN Complementario/aislamiento & purificación , Proteínas de Unión al ADN/análisis , Inmunohistoquímica , Hibridación in Situ , Factores de Transcripción MEF2 , Datos de Secuencia Molecular , Músculos/química , Proteína MioD/genética , Proteínas Nucleares/análisis , Regiones Promotoras Genéticas/genética , ARN Mensajero/química , Factor de Respuesta Sérica , Factores de Transcripción/análisis

Asunto(s)

Proteínas de Unión al ADN/fisiología , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , ADN/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional , Factor de Respuesta Sérica , Transcripción Genética , Activación Transcripcional

RESUMEN

Serum induction of c-jun expression in HeLa cells requires a MEF2 site at -59 in the c-jun promoter. MEF2 sites, found in many muscle-specific enhancers, are bound by a family of transcription factors, MEF2A through -D, which are related to serum response factor in their DNA binding domains. We have found that MEF2D is the predominant protein in HeLa cells that binds to the c-jun MEF2 site. Serum induction of a MEF2 reporter gene was not observed in a line of NIH 3T3 cells which contain low MEF2 site binding activity. Transfection of MEF2D into NIH 3T3 cells reconstituted serum induction, demonstrating that MEF2D is required for the serum response. Deletion analysis of MEF2D showed that its DNA binding domain, when fused to a heterologous transcriptional activation domain, was sufficient for serum induction of a MEF2 reporter gene. This is the domain homologous to that in the serum response factor which is required for serum induction of the c-fos serum response element, suggesting that serum regulation of c-fos and c-jun may share a common mechanism.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión/genética , Proteínas Sanguíneas/farmacología , Proteínas de Unión al ADN/genética , Eliminación de Gen , Regulación de la Expresión Génica , Células HeLa , Humanos , Proteínas de Dominio MADS , Factores de Transcripción MEF2 , Ratones , Datos de Secuencia Molecular , Factores Reguladores Miogénicos , Plásmidos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-jun/genética , Factores de Transcripción/genética