RESUMEN
Shiga toxins (Stxs) cause irreversible damage to eukaryotic ribosomes, yet cellular intoxication of intestinal epithelial cells (IECs) results in increased synthesis of selected proteins, notably cytokines. How mRNA translation is maintained in this circumstance is unclear. This study was designed to assess whether Stx-induced alterations in host signal transduction machinery permit translation despite protein synthesis inhibition. A key step of translation is recruitment of initiation machinery to the 5' mRNA cap. This event occurs in part via interaction of the 5' cap with the cap binding protein, eIF4E, whose activity is positively regulated by phosphorylation and negatively regulated by binding to the translational repressor 4E-BP1. Following Stx treatment of IECs, eIF4E phosphorylation was detected by Western blotting using phospho-specific antibodies. Treatment with the p38 inhibitor, SB202190, or either of the ERK1/2 inhibitors, PD98059 and U0126, partially blocked Stx1-induced eIF4E phosphorylation. The Mnk1 inhibitor, CGP57380, blocked both basal and Stx-induced eIF4E phosphorylation. Interestingly, pretreatment with CGP57380 did not alter basal protein synthesis, but diminished the ability of cells to maintain translation following Stx1 challenge. Stx1 also induced hyperphosphorylation of 4E-BP1 and phosphorylation of S6Kinase; both effects were blocked by rapamycin. These data are novel observations showing that Stxs regulate multiple signal transduction pathways controlling translation in host cells, and support a role for eIF4E phosphorylation in maintaining host cell translation despite ribosomal intoxication.
Asunto(s)
Mucosa Intestinal/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , Toxinas Shiga/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Compuestos de Anilina/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Humanos , Mucosa Intestinal/citología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Purinas/farmacología , ARN Mensajero/metabolismo , Proteínas Quinasas S6 Ribosómicas/genética , Proteínas Quinasas S6 Ribosómicas/metabolismo , Toxinas Shiga/farmacología , Sirolimus/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
TFII-I is a transcription factor that shuttles between the cytoplasm and nucleus and is regulated by serine and tyrosine phosphorylation. Tyrosine phosphorylation of TFII-I can be regulated in a signal-dependent manner in various cell types. In B lymphocytes, Bruton's tyrosine kinase has been identified as a TFII-I tyrosine kinase. Here we report that JAK2 can phosphorylate and regulate TFII-I in nonlymphoid cells. The activity of TFII-I on the c-fos promoter in response to serum can be abolished by dominant negative JAK2 or the specific JAK2 kinase inhibitor AG490. Consistent with this, we have also found that JAK2 is activated by serum stimulation of fibroblasts. Tyrosine 248 of TFII-I is phosphorylated in vivo upon serum stimulation or JAK2 overexpression, and mutation of tyrosine 248 to phenylalanine inhibits the ability of JAK2 to phosphorylate TFII-I in vitro. Tyrosine 248 of TFII-I is required for its interaction with and phosphorylation by ERK and its in vivo activity on the c-fos promoter. These results indicate that the interaction between TFII-I and ERK, which is essential for its activity, can be regulated by JAK2 through phosphorylation of TFII-I at tyrosine 248. Thus, like the STAT factors, TFII-I is a direct substrate of JAK2 and a signal-dependent transcription factor that integrates signals from both tyrosine kinase and mitogen-activated protein kinase pathways to regulate transcription.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Transducción de Señal , Factores de Transcripción TFII , Células 3T3 , Animales , Humanos , Janus Quinasa 2 , Ratones , FosforilaciónRESUMEN
The signal transducers and activators of transcription (STAT) transcription factors become phosphorylated on tyrosine and translocate to the nucleus after stimulation of cells with growth factors or cytokines. We show that the Rac1 guanosine triphosphatase can bind to and regulate STAT3 activity. Dominant negative Rac1 inhibited STAT3 activation by growth factors, whereas activated Rac1 stimulated STAT3 phosphorylation on both tyrosine and serine residues. Moreover, activated Rac1 formed a complex with STAT3 in mammalian cells. Yeast two-hybrid analysis indicated that STAT3 binds directly to active but not inactive Rac1 and that the interaction occurs via the effector domain. Rac1 may serve as an alternate mechanism for targeting STAT3 to tyrosine kinase signaling complexes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas , Transactivadores/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Sustitución de Aminoácidos , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Proteínas de Unión al ADN/genética , Activación Enzimática , Factor de Crecimiento Epidérmico/farmacología , Regulación de la Expresión Génica , Genes Reporteros , Vectores Genéticos , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Janus Quinasa 2 , Mutación , Proteínas de Neoplasias , Fosforilación , Fosfoserina/metabolismo , Fosfotirosina/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Ratas , Factor de Transcripción STAT3 , Transducción de Señal , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Transactivadores/genética , Transfección , Técnicas del Sistema de Dos Híbridos , Proteína de Unión al GTP rac1/genéticaRESUMEN
We have previously shown that TFII-I enhances transcriptional activation of the c-fos promoter through interactions with upstream elements in a signal-dependent manner. Here we demonstrate that activated Ras and RhoA synergize with TFII-I for c-fos promoter activation, whereas dominant-negative Ras and RhoA inhibit these effects of TFII-I. The Mek1 inhibitor, PD98059 abrogates the enhancement of the c-fos promoter by TFII-I, indicating that TFII-I function is dependent on an active mitogen-activated protein (MAP) kinase pathway. Analysis of the TFII-I protein sequence revealed that TFII-I contains a consensus MAP kinase interaction domain (D box). Consistent with this, we have found that TFII-I forms an in vivo complex with extracellular signal-related kinase (ERK). Point mutations within the consensus MAP kinase binding motif of TFII-I inhibit its ability to bind ERK and its ability to enhance the c-fos promoter. Therefore, the D box of TFII-I is required for its activity on the c-fos promoter. Moreover, the interaction between TFII-I and ERK can be regulated. Serum stimulation enhances complex formation between TFII-I and ERK, and dominant-negative Ras abrogates this interaction. In addition, TFII-I can be phosphorylated in vitro by ERK and mutation of consensus MAP kinase substrate sites at serines 627 and 633 impairs the phosphorylation of TFII-I by ERK and its activity on the c-fos promoter. These results suggest that ERK regulates the activity of TFII-I by direct phosphorylation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Genes fos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factores de Transcripción/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Humanos , Ratones , Proteínas Quinasas Activadas por Mitógenos/genética , Datos de Secuencia Molecular , Fosforilación , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Factores de Transcripción/genética , Proteínas ras/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Mammalian somatic cells are usually diploid. Occasional rare human tumors have been shown to have a hypodiploid karyotype. We have isolated a near-haploid subclone (P1-55) from a heterogeneous human leukemia cell line, KBM-7. These near-haploid cells have approximately half the human diploid DNA content and have a haploid karyotype except for a disomy of chromosome 8 (25, XY, +8, Ph(+)). This cell line maintains a majority of cells with a near-haploid karyotype for at least 12 weeks in culture. By serial subcloning, we have isolated near-haploid subclones that maintain ploidy for at least 8 months in culture. Near-haploid cells can also be efficiently isolated from mixed ploidy cultures by size selection. The availability of this human near-haploid cell line should facilitate the genetic analysis of cultured human cells.
Asunto(s)
Haploidia , Leucemia/genética , Leucemia/patología , Células Tumorales Cultivadas , Humanos , CariotipificaciónRESUMEN
Genetic screens in Drosophila have identified p50(cdc37) to be an essential component of the sevenless receptor/mitogen-activated kinase protein (MAPK) signaling pathway, but neither the function nor the target of p50(cdc37) in this pathway has been defined. In this study, we examined the role of p50(cdc37) and its Hsp90 chaperone partner in Raf/Mek/MAPK signaling biochemically. We found that coexpression of wild-type p50(cdc37) with Raf-1 resulted in robust and dose-dependent activation of Raf-1 in Sf9 cells. In addition, p50(cdc37) greatly potentiated v-Src-mediated Raf-1 activation. Moreover, we found that p50(cdc37) is the primary determinant of Hsp90 recruitment to Raf-1. Overexpression of a p50(cdc37) mutant which is unable to recruit Hsp90 into the Raf-1 complex inhibited Raf-1 and MAPK activation by growth factors. Similarly, pretreatment with geldanamycin (GA), an Hsp90-specific inhibitor, prevented both the association of Raf-1 with the p50(cdc37)-Hsp90 heterodimer and Raf-1 kinase activation by serum. Activation of Raf-1 via baculovirus coexpression with oncogenic Src or Ras in Sf9 cells was also strongly inhibited by dominant negative p50(cdc37) or by GA. Thus, formation of a ternary Raf-1-p50(cdc37)-Hsp90 complex is crucial for Raf-1 activity and MAPK pathway signaling. These results provide the first biochemical evidence for the requirement of the p50(cdc37)-Hsp90 complex in protein kinase regulation and for Raf-1 function in particular.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares , Proteínas Proto-Oncogénicas c-raf/metabolismo , Animales , Benzoquinonas , Células COS , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular , Chaperoninas , Pollos , Dimerización , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Proteínas HSP90 de Choque Térmico/genética , Humanos , Lactamas Macrocíclicas , Proteínas Proto-Oncogénicas c-raf/genética , Quinonas/farmacología , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , SpodopteraRESUMEN
Reactive oxygen species (ROS) play an important role in the pathogenesis of many human diseases, including the acute respiratory distress syndrome, Parkinson's disease, pulmonary fibrosis, and Alzheimer's disease. In mammalian cells, several genes known to be induced during the immediate early response to growth factors, including the protooncogenes c-fos and c-myc, have also been shown to be induced by ROS. We show that members of the STAT family of transcription factors, including STAT1 and STAT3, are activated in fibroblasts and A-431 carcinoma cells in response to H2O2. This activation occurs within 5 min, can be inhibited by antioxidants, and does not require protein synthesis. STAT activation in these cell lines is oxidant specific and does not occur in response to superoxide- or nitric oxide-generating stimuli. Buthionine sulfoximine, which depletes intracellular glutathione, also activates the STAT pathway. Moreover, H2O2 stimulates the activity of the known STAT kinases JAK2 and TYK2. Activation of STATs by platelet-derived growth factor (PDGF) is significantly inhibited by N-acetyl-L-cysteine and diphenylene iodonium, indicating that ROS production contributes to STAT activation in response to PDGF. These findings indicate that the JAK-STAT pathway responds to intracellular ROS and that PDGF uses ROS as a second messenger to regulate STAT activation.
Asunto(s)
Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas , Especies Reactivas de Oxígeno/fisiología , Transducción de Señal/fisiología , Transactivadores/fisiología , Células 3T3 , Animales , Antioxidantes/farmacología , Proteínas de Unión al ADN/fisiología , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Fibroblastos/metabolismo , Peróxido de Hidrógeno/farmacología , Janus Quinasa 2 , Ratones , Oxidantes/farmacología , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Transducción de Señal/efectos de los fármacos , TYK2 Quinasa , Células Tumorales CultivadasRESUMEN
Several protein kinases (e.g. pp60(src), v-Raf) exist in heterocomplexes with hsp90 and a 50-kDa protein that is the mammalian homolog of the yeast cell cycle control protein Cdc37. In contrast, unliganded steroid receptors exist in heterocomplexes with hsp90 and a tetratricopeptide repeat (TPR) domain protein, such as an immunophilin. Although p50(cdc37) and TPR domain proteins bind directly to hsp90, p50(cdc37) is not present in native steroid receptor.hsp90 heterocomplexes. To obtain some insight as to how v-Raf selects predominantly hsp90.p50(cdc37) heterocomplexes, rather than hsp90.TPR protein heterocomplexes, we have examined the binding of p50(cdc37) to hsp90 and to Raf. We show that p50(cdc37) exists in separate hsp90 heterocomplexes from the TPR domain proteins and that intact TPR proteins compete for p50(cdc37) binding to hsp90 but a protein fragment containing a TPR domain does not. This suggests that the binding site for p50(cdc37) lies topologically adjacent to the TPR acceptor site on the surface of hsp90. Also, we show that p50(cdc37) binds directly to v-Raf, with the catalytic domain of Raf being sufficient. We propose that the combination of exclusive binding of p50(cdc37) versus a TPR domain protein to hsp90 plus direct binding of p50(cdc37) to Raf allows the protein kinase to select for the dominant hsp90.p50(cdc37) composition that is observed with a variety of protein kinase heterocomplexes immunoadsorbed from cytosols.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Drosophila , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares , Animales , Sitios de Unión/fisiología , Proteínas Portadoras , Chaperoninas , Humanos , Quinasas Janus , Proteínas de Complejo Poro Nuclear , Proteínas Oncogénicas v-raf , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Conejos , Ratas , Proteínas Oncogénicas de Retroviridae/metabolismo , Factores de TranscripciónRESUMEN
The transcription factor TFII-I was initially isolated as a factor that can bind to initiator elements in core promoters. Recent evidence suggests that TFII-I may also have a role in signal transduction. We have found that overexpression of TFII-I can enhance the response of the wild-type c-fos promoter to a variety of stimuli. This effect depends on the c-fos c-sis-platelet-derived growth factor-inducible factor binding element (SIE) and serum response element (SRE). There is no effect of cotransfected TFII-I on the TATA box containing the c-fos basal promoter. Three TFII-I binding sites can be found in c-fos promoter. Two of these overlap the c-fos SIE and SRE, and another is located just upstream of the TATA box. Mutations that distinguish between serum response factor (SRF), STAT, and TFII-I binding to the c-fos SIE and SRE suggest that the binding of TFII-I to these elements is important for c-fos induction in conjunction with the SRF and STAT transcription factors. Moreover, TFII-I can form in vivo protein-protein complexes with the c-fos upstream activators SRF, STAT1, and STAT3. These results suggest that TFII-I may mediate the functional interdependence of the c-fos SIE and SRE elements. In addition, the ras pathway is required for TFII-I to exert its effects on the c-fos promoter, and growth factor stimulation enhances tyrosine phosphorylation of TFII-I. These results indicate that TFII-I is involved in signal transduction as well as transcriptional activation of the c-fos promoter.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-fos/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Células 3T3 , Animales , Sitios de Unión , ADN/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Ratones , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , TATA Box , Transactivadores/metabolismoRESUMEN
Signaling mechanisms leading to regulation of gene transcription by growth hormone (GH) and other molecules that signal via the cytokine receptor family have been elusive. Based upon recent findings that GH and interferons activate JAK family tyrosine kinases, we have identified a novel signaling pathway leading from the GH receptor to the nucleus. We report that in 3T3-F442A fibroblasts, GH stimulates tyrosyl phosphorylation of a protein recognized by antibody to p91, a component of DNA-binding complexes that are activated by tyrosyl phosphorylation in response to interferons alpha and gamma. In addition, a GH-inducible DNA binding factor (GHIF) is identified that binds to the c-sis-inducible element of the c-fos promoter. GHIF contains a protein antigenically related to p91 and is tyrosyl-phosphorylated. These findings indicate that in signaling between their receptors and the nucleus, GH and interferons utilize related or identical components, including JAK family tyrosine kinases and proteins in the p91 family. When combined with recent findings that many members of the cytokine receptor family activate JAK kinases, including some cytokines that activate p91-related proteins, these findings suggest that signaling pathways involving JAK kinases and p91 family members may be broadly distributed.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Hormona del Crecimiento/farmacología , Interferón gamma/farmacología , Factores de Transcripción/biosíntesis , Células 3T3 , Animales , Proteínas de Unión al ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Ratones , Peso Molecular , Fosforilación , Fosfotirosina , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes , Transducción de Señal , Factores de Transcripción/aislamiento & purificación , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
The c-fos protooncogene is transcriptionally activated by a wide variety of agents including serum, growth factors, and phorbol esters. This induction is rapid and transient and is mediated through a number of identified promoter elements. Growth hormone (GH) is also known to induce transcription of c-fos in a variety of cell types including NIH 3T3 fibroblasts and 3T3-F442A preadipocytes. To identify DNA sequences in the c-fos gene regulated by GH, this study sought to determine whether induction of c-fos by GH involves previously identified c-fos promoter elements. A plasmid containing a growth factor-sensitive fragment of the upstream region of the c-fos promoter from -361 to -264 bp was tested for GH sensitivity. The fragment was cloned upstream of a human c-fos reporter [designated FOS by Human Gene Mapping 11 (1991)], which included basal promoter elements. In transiently transfected mouse NIH 3T3 fibroblasts, the promoter fragment conferred GH sensitivity on the human c-fos reporter. To identify a specific GH-sensitive DNA sequence in this promoter, a serum response element (SRE)-reporter plasmid was tested and found to be stimulated by GH. GH was effective in inducing expression through the SRE over a range of physiological GH concentrations. Since GH was recently found to synergize with serum factors in inducing c-fos transcription, the effect of GH and serum on SRE function was examined for insight into the mechanism for such synergism. The combined effect of GH and serum to induce reporter expression through the SRE was greater than the added effects of GH and serum, indicating that the synergism between GH and serum in inducing c-fos involves the SRE sequence. These studies identify the SRE as one specific DNA sequence in the c-fos promoter functionally regulated by GH. It is notable that GH is effective at physiological concentrations. Furthermore, synergism in c-fos induction between GH and serum factors is evident through the SRE.
Asunto(s)
Hormona del Crecimiento/farmacología , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Transcripción Genética/efectos de los fármacos , Células 3T3 , Animales , Secuencia de Bases , Fenómenos Fisiológicos Sanguíneos , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-fos/genéticaRESUMEN
In cells in culture, specific stimuli induce selective patterns of immediate-early gene induction. In the present study, we tested for such selectivity of stimulated gene expression by monitoring the expression of fos/jun gene mRNAs in the striatum in rats treated in vivo with the indirect dopamine agonist cocaine. We found by Northern blot and in situ hybridization analysis that cocaine induces the coordinate expression of c-fos and jun B mRNAs in neurons of the rat's striatum. By contrast, another immediate-early gene of the leucine-zipper family, c-jun, was not induced in striatal neurons by cocaine at any time tested from 1 to 24 hr after treatment. With the same probe, we could detect the induction of c-jun mRNA (as well as that of c-fos and jun B mRNAs) in the hippocampus following administration of pentylenetetrazol. The induction of expression of c-fos and jun B was rapid and transient, with peak expression occurring at approximately 1 hr after cocaine administration, and the induction of the two genes was in similar striatal sites. These results establish that differential patterns of expression of fos/jun genes occur in striatal neurons following exposure to cocaine, a potent psychomotor stimulant. We suggest that these tissue-specific patterns of gene expression may contribute to the response specificity of striatal neurons to stimulation by monoamines including dopamine.
Asunto(s)
Cocaína/farmacología , Cuerpo Estriado/metabolismo , Expresión Génica/efectos de los fármacos , Genes fos/genética , Genes jun/genética , Animales , Northern Blotting , Cuerpo Estriado/efectos de los fármacos , Hibridación in Situ , Cinética , Masculino , Neuronas/metabolismo , Sondas de Oligonucleótidos , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-DawleyRESUMEN
From this brief overview of the regulation of the c-fos promoter, it can be seen that the regulation of early-response genes is a complex affair. Therefore, it is not easy to predict from the upstream sequence of a given early-response gene exactly which elements are responsible for responding to what signals in a given cell type. However, from studies of other early-response genes, it is clear that several of the elements found upstream of c-fos appear frequently and are important in the regulation of other early-response genes. For instance, the zif/268 gene has four separate CArG boxes that are similar to fos. These CArG boxes can function in the serum and TPA response, but interestingly, they are not imbedded in a region of dyad symmetry as in c-fos (Christy and Nathans 1989). Upstream of the c-jun oncogene is an AP-1 site that can modulate the expression and induction of this gene and is responsive to TPA (Angel et al. 1988). Moreover, other studies suggest that cAMP-mediated signals can repress induction of c-jun through this element (de Groot et al. 1991; Mechta et al. 1989). This would explain why in certain circumstances, such as depolarization of PC12 cells or in the striatum in response to cocaine, there is an uncoupling of the induction of c-jun and jun-B. Depolarization induces c-fos and jun-B but not c-jun; however, growth factors such as NGF can induce all three genes in the same cell (Bartel et al. 1989). Upstream of the jun-B gene there does not appear to be an SRE, but there is a new element that can be responsive to both cAMP and phorbol esters (de Groot et al. 1991). Genes such as c-myc, JE, and KC have no consensus SREs upstream, and the regulatory elements responsible for the induction of these genes have not been clearly identified (Rollins et al. 1988). However, there is some evidence from the c-myc gene that the E2F binding sites are important for its regulation by serum (Mudryj et al. 1990; Sacca and Cochran 1990). In addition, there are two SIF sites upstream of the c-myc proto-oncogene (B.H. Cochran and T.E. Hayes, unpublished results). Upstream of the nur-77 gene there are no SREs, but there are four potential calcium/CRE-like elements (Watson and Milbrandt 1989).(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Proto-Oncogenes/genética , Animales , Humanos , Sistema Nervioso/metabolismo , Proto-Oncogenes Mas , Proto-Oncogenes/efectos de los fármacosRESUMEN
In the yeast Saccharomyces cerevisiae, expression of alpha-specific genes is governed by the MAT alpha 1 and MCM1 gene products. MAT alpha 1 and MCM1 bind cooperatively to PQ elements upstream of alpha-specific genes. The PQ element not only directs alpha-specific expression but can also direct gene induction in response to treatment with a-mating pheromone. We have used gene fusions to investigate whether induction conferred by the PQ box is mediated through either MAT alpha 1 or MCM1, or a combination of both. When MCM1 is fused to the DNA-binding domain of the bacterial repressor LexA, this fusion protein is capable of trans-activating a lacZ reporter gene driven by a LexA operator. However, the transcriptional activity of the MCM1-LexA fusion is not further enhanced by treatment of cells with a-factor. A MAT alpha 1-LexA fusion protein is also capable of trans-activation through a LexA operator. Moreover, the activity of the MAT alpha 1-LexA fusion protein can be further induced by treatment with a-factor. When progressive deletions are made from the amino terminus of MAT alpha 1 in the fusion protein, the basal level of trans-activation progressively decreases, but the inducibility of the fusion protein increases. MAT alpha 1-LexA fusion proteins, which have greater than or equal to 57 amino acids deleted from the amino terminus of MAT alpha 1 are not capable of trans-activation. In addition, the activity of the MAT alpha 1-LexA fusion protein is dependent on the functions of the STE7, STE11, and STE12 genes that encode components of the pheromone response pathway.
Asunto(s)
Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Proteínas de Homeodominio , Péptidos/genética , Proteínas Represoras/genética , Saccharomyces cerevisiae/genética , Serina Endopeptidasas , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Deleción Cromosómica , Proteínas Fúngicas/metabolismo , Factor de Apareamiento , Modelos Genéticos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Feromonas/genética , Plásmidos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Moldes Genéticos , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
The c-fos proto-oncogene is rapidly and transiently induced by a variety of extracellular stimuli. We have previously shown that conditioned media from v-sis transformed NRK cells rapidly induces a DNA binding protein which binds to a conserved sequence upstream of the human c-fos gene. We now show that purified recombinant c-sis/PDGF can induce this binding activity which we have termed SIF, for sis-inducible factor. Oligonucleotides which bind to the SIF protein will confer sis/PDGF inducibility onto a truncated, unresponsive c-fos promoter. However, sequences lying between -100 and -57 of the c-fos gene are required for this induction. The sis-responsive element functions independently of a region of dyad symmetry previously identified as the serum responsive element (SRE). The time course of c-fos expression driven by the sis-responsive element is similar to that mediated by the SRE. Unlike the SRE, which can respond to signals generated by sis/PDGF, serum or phorbol esters, the SIF binding element mediates c-fos induction only in response to sis/PDGF. The SRE and SIF elements function in an additive manner to stimulate the transcription of the c-fos gene in response to sis/PDGF.
Asunto(s)
Proteínas de Unión al ADN/genética , Oncogenes , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Receptores de Superficie Celular/genética , Animales , Secuencia de Bases , Células Cultivadas , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-fos , Receptores del Factor de Crecimiento Derivado de Plaquetas , Factor de Respuesta SéricaRESUMEN
Saccharomyces cerevisiae mating pheromones function by binding to cell surface receptors and activating signal transduction processes which regulate gene expression. In this report, we have analyzed the minimum sequence requirements for conferring both a and alpha mating pheromone inducibilities onto a heterologous promoter. Here we show that the repetitive pheromone response element (PRE) which binds to STE12 protein is sufficient to confer pheromone responsiveness only when present in multiple copies. Moreover, by itself, it is preferentially responsive to alpha factor in a cells. In contrast, a single copy of the PQ box of the STE3 upstream activation sequence (UAS) is sufficient to confer a-factor responsiveness in alpha cells. The PQ box binds both MCM1 and MAT alpha 1 in a cooperative manner, and neither the P nor Q site alone is sufficient to confer a-factor responsiveness. In a cells, however, even multiple copies of the PQ box fail to confer alpha-factor responsiveness. Therefore, the PRE and the PQ box are functionally distinct pheromone-responsive elements with opposite cell type specificities. Moreover, these results indicate that the MCM1 protein functions in a signal transduction pathway in a manner analogous to that of its mammalian homolog, the serum response factor, which regulates the expression of the c-fos proto-oncogene in mammals.
Asunto(s)
Genes Fúngicos , Péptidos/fisiología , Feromonas/fisiología , Saccharomyces cerevisiae/genética , Secuencia de Bases , Factor de Apareamiento , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Secuencias Repetitivas de Ácidos Nucleicos , Saccharomyces cerevisiae/fisiologíaRESUMEN
To investigate early events in GH action, we examined whether GH altered the expression of c-fos and c-jun mRNA in 3T3-F442A cells. The rapid induction of these early response protooncogenes is one of the initial events in stimulation of cell proliferation and differentiation by a variety of growth factors. In 3T3-F442A preadipocytes, which differentiate in response to GH, treatment with GH for 15-30 min transiently increased the expression of c-fos and c-jun mRNA at GH concentrations in the physiological range (10-500 ng/ml). Both genes were superinduced by GH in the presence of cycloheximide. Nuclear run-on experiments indicated that the effect of GH on c-fos and c-jun occurred at the level of gene transcription, indicating that in the 3T3-F442A preadipocytes, GH acts similarly to other growth factors which induce c-fos and c-jun. Chronic preincubation with the phorbol ester phorbol 12-myristate 13-acetate interfered with the induction of c-fos by GH, suggesting that diacylglycerol and protein kinase C are involved in this effect of GH. Insulin-like growth factor-I and insulin were less effective than equimolar concentrations of GH in inducing c-fos expression. Taken together, these findings are consistent with coordinated expression of c-fos and c-jun playing an early role in responses to GH in 3T3-F442A cells.
Asunto(s)
Proteínas de Unión al ADN/genética , Hormona del Crecimiento/farmacología , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes/efectos de los fármacos , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacos , Tejido Adiposo/efectos de los fármacos , Animales , Línea Celular , Cicloheximida/farmacología , Expresión Génica/efectos de los fármacos , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Cinética , Ratones , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-fos , Proteínas Proto-Oncogénicas c-jun , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The c-myc gene is rapidly induced in quiescent Balb/c-3T3 cells in response to the platelet-derived growth factor (PDGF). In order to study the mechanisms by which growth factors regulate induction of c-myc, we have attempted to identify growth factor-responsive elements in the murine c-myc locus. Various fragments of the c-myc gene linked to a bacterial CAT reporter gene were stably transfected into Balb/c-3T3 cells. A construct which includes the P1 promoter and 424 bp of upstream sequences shows a 3-5 fold induction of CAT RNA expression in response to sis/PDGF. S1 nuclease mapping experiments demonstrate that this mRNA initiates from the myc P1 promoter. Nuclear runoff transcription experiments performed with this myc/CAT construct show that this induction occurs at the transcriptional level. Deletion analysis led to the identification of an 81 bp segment in the first exon between the c-myc P1 and P2 promoters which is necessary to confer growth factor responsiveness in this construct.
Asunto(s)
Genes myc/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Animales , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Deleción Cromosómica , Ratones , Ratones Endogámicos BALB C , Plásmidos , Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas/genética , Mapeo Restrictivo , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
One of the elements that mediates growth factor and serum inducibility of the human c-fos gene is a region of dyad symmetry that lies between nucleotides -320 and -299 of the human gene. A mammalian protein specifically binds to this sequence element and has been termed the serum response factor (SRF). Gel-shift analysis and competition experiments demonstrate that there is a factor in the yeast Saccharomyces cerevisiae that binds specifically to the human c-fos SRE. The methylation interference pattern of the yeast factor is identical to that of the mammalian SRF. Regulatory elements of cell-type-specific genes in yeast have homologies to the c-fos SRE and complete for binding of both the mammalian and yeast factors to the SRE. Antisera to the gene product of the MCM1 locus react with the yeast SRE-binding factor. These data suggest that this yeast protein is closely related or identical to the factors [general regulator of mating type (GRM) and pheromone/receptor transcription factor (PRTF)] that are required for the regulation of cell-type-specific genes in yeast.