RESUMEN
Overexpression of the transcriptional coregulators C-terminal binding proteins 1 and 2 (CtBP1 and 2) occurs in many human solid tumors and is associated with poor prognosis. CtBP modulates oncogenic gene expression programs and is an emerging drug target, but its oncogenic role is unclear. Consistent with this oncogenic potential, exogenous CtBP2 transformed primary mouse and human cells to anchorage independence similarly to mutant H-Ras. To investigate CtBP's contribution to in vivo tumorigenesis, Apcmin/+ mice, which succumb to massive intestinal polyposis, were bred to Ctbp2+/- mice. CtBP interacts with adenomatous polyposis coli (APC) protein, and is stabilized in both APC-mutated human colon cancers and Apcmin/+ intestinal polyps. Ctbp2 heterozygosity increased the median survival of Apcmin/+ mice from 21 to 48 weeks, and reduced polyp formation by 90%, with Ctbp2+/- polyps exhibiting reduced levels of ß-catenin and its oncogenic transcriptional target, cyclin D1. CtBP's potential as a therapeutic target was studied by treating Apcmin/+ mice with the CtBP small-molecule inhibitors 4-methylthio-2-oxobutyric acid and 2-hydroxy-imino phenylpyruvic acid, both of which reduced polyposis by more than half compared with vehicle treatment. Phenocopying Ctbp2 deletion, both Ctbp inhibitors caused substantial decreases in the protein level of Ctbp2, as well its oncogenic partner ß-catenin, and the effects of the inhibitors on CtBP and ß-catenin levels could be modeled in an APC-mutated human colon cancer cell line. CtBP2 is thus a druggable transforming oncoprotein critical for the evolution of neoplasia driven by Apc mutation.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Poliposis Adenomatosa del Colon/terapia , Oxidorreductasas de Alcohol/metabolismo , Carcinogénesis , Proteínas del Tejido Nervioso/metabolismo , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/metabolismo , Poliposis Adenomatosa del Colon/patología , Oxidorreductasas de Alcohol/antagonistas & inhibidores , Oxidorreductasas de Alcohol/genética , Animales , Línea Celular Tumoral , Proteínas Co-Represoras , Neoplasias del Colon/genética , Ciclina D/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Fibroblastos , Humanos , Metionina/análogos & derivados , Metionina/uso terapéutico , Ratones , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , beta Catenina/metabolismoRESUMEN
The alternative reading frame (ARF) tumor suppressor exerts both p53-dependent and p53-independent functions. The corepressor C-terminal binding protein (CtBP) interacts with ARF, resulting in proteasome-mediated degradation of CtBP. ARF can induce apoptosis in p53-null colon cancer cells, in a manner dependent on ARF interaction with CtBP. Bik was uniquely identified in an apoptotic gene array as coordinately upregulated in colon cancer cells after either CtBP2 knockdown or ARF overexpression. Validating the array findings, ARF induced Bik mRNA and protein expression, and this activity required an intact CtBP binding domain. Apoptosis induced by CtBP deficiency was substantially impaired when Bik expression was simultaneously silenced. An analysis of the Bik promoter revealed binding sites for the CtBP-interacting basic Kruppel-like factor (BKLF). A Bik promoter luciferase reporter was repressed by BKLF and CtBP2, and ARF reversed CtBP-associated repression. Chromatin immunoprecipitation analyses showed that CtBP was recruited to the Bik promoter largely by BKLF. Expression profiling of BH3-only gene expression in ARF-expressing or CtBP-deficient cells revealed that Bik was uniquely regulated by ARF/CtBP in colon cancer cells, whereas additional BH3-only proteins (Bim, Bmf) showed CtBP-dependent repression in osteosarcoma cells. ARF antagonism of CtBP repression of Bik and other BH3-only genes may have a critical role in ARF-induced p53-independent apoptosis and tumor suppression.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas de la Membrana/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Oxidorreductasas de Alcohol/genética , Animales , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Humanos , Proteínas de la Membrana/genética , Ratones , Proteínas Mitocondriales , Regiones Promotoras Genéticas , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal/fisiología , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
The tumor suppressor p53 preserves genome integrity by inducing transcription of genes controlling growth arrest or apoptosis. Transcriptional activation involves nucleosomal perturbation by chromatin remodeling enzymes. Mammalian SWI/SNF remodeling complexes incorporate either the Brahma-related gene 1 (BRG1) or Brahma (Brm) as the ATPase subunit. The observation that tumor cell lines harboring wild-type p53 specifically maintain expression of BRG1 and that BRG1 complexes with p53 prompted us to examine the role of BRG1 in regulation of p53. Remarkably, RNAi depletion of BRG1, but not Brm, led to the activation of endogenous wild-type p53 and cell senescence. We found a proline-rich region unique to BRG1 was required for binding to the histone acetyl transferase protein, CBP, as well as to p53. Ectopic expression of a proline-rich region deletion mutant BRG1 that is defective for CBP binding inhibited p53 destabilization. Importantly, RNAi knockdown of BRG1 and CBP reduced p53 poly-ubiquitination in vivo. In support of p53 inactivation by the combined activities of BRG1 and CBP, we show that DNA damage signals promoted disassociation of BRG1 from CBP, thereby allowing p53 accumulation. Our data demonstrate a novel function of the evolutionarily conserved chromatin remodeling subunit BRG1, which cooperates with CBP to constrain p53 activity and permit cancer cell proliferation.
Asunto(s)
Proliferación Celular , ADN Helicasas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Sitios de Unión , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Daño del ADN , ADN Helicasas/genética , Células HeLa , Humanos , Immunoblotting , Inmunoprecipitación , Células Jurkat , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Nucleares/genética , Poliubiquitina/metabolismo , Unión Proteica , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Transfección , Proteína p53 Supresora de Tumor/genética , Ubiquitinación , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Rad23 proteins function in both DNA repair and protein stability regulation. As ubiquitinated forms of p53 are stabilized after DNA damage in concert with p53 functional activation, and human Rad23 proteins (hHR23A and B) regulate p53 stability in unstressed cells, the role of hHR23B in post-genotoxin regulation of p53 was investigated. Depletion of hHR23B by specific short interfering RNA before genotoxic exposure attenuated p53, p21 and bax induction, abrogated the accumulation of ubiquitinated p53 and suppressed apoptosis. Expression of ubiquitin derivatives with all lysines mutated except K48 or K63 demonstrated that K48-linked p53-ubiquitin conjugates were specifically induced after DNA damage. hHR23B, along with native and ubiquitinated p53, accumulated in chromatin after genotoxic exposure, and the accumulation of ubiquitinated p53 in chromatin was prevented by hHR23B depletion. Chromatin immunoprecipitation analysis demonstrated that hHR23B and p53 both localized to the p21 promoter shortly after DNA damage. hHR23B thus plays a critical role in the activation and function of p53 after specific genotoxic exposures.
Asunto(s)
Apoptosis , Daño del ADN , Enzimas Reparadoras del ADN/fisiología , Proteínas de Unión al ADN/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/genética , Cromatina/química , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , ADN/efectos de los fármacos , ADN/efectos de la radiación , Enzimas Reparadoras del ADN/análisis , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Humanos , Lisina/química , Lisina/genética , Mutación , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Interferencia de ARN , Proteína p53 Supresora de Tumor/análisis , Proteína p53 Supresora de Tumor/genética , Ubiquitina/metabolismoRESUMEN
PTEN is a tumor suppressor protein that functions, in large part, by dephosphorylating the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate and by doing so antagonizing the action of phosphoinositide 3-kinase. PTEN structural domains include an N-terminal phosphatase domain, a lipid-binding C2 domain, and a 50-amino acid C-terminal tail that contains a PDZ binding sequence. We showed previously that phosphorylation of the PTEN tail negatively regulates PTEN activity. We now show that phosphorylated PTEN exists in a monomeric "closed" conformation and has low affinity for PDZ domain-containing proteins. Conversely, when unphosphorylated, PTEN is in an "open" conformation, is recruited into a high molecular weight complex (PTEN-associated complex), and strongly interacts with PDZ-containing proteins such as MAGI-2. As a consequence, when compared with wild-type PTEN, the phosphorylation-deficient mutant form of PTEN strongly cooperates with MAGI-2 to block Akt activation. These results indicate that phosphorylation of the PTEN tail causes a conformational change that results in the masking of the PDZ binding domain. Consequently, the ability of PTEN to bind to PDZ domain-containing proteins is reduced dramatically. These data suggest that phosphorylation of the PTEN tail suppresses the activity of PTEN by controlling the recruitment of PTEN into the PTEN-associated complex.
Asunto(s)
Receptores de Activinas Tipo II , Proteínas Portadoras/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Línea Celular , Guanilato-Quinasas , Humanos , Isoenzimas , Modelos Biológicos , Complejos Multienzimáticos/metabolismo , Nucleósido-Fosfato Quinasa/metabolismo , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/química , Fosforilación , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Proteínas Supresoras de Tumor/químicaRESUMEN
Substantial evidence points to a critical role for the p300/CREB binding protein (CBP) coactivators in p53 responses to DNA damage. p300/CBP and the associated protein P/CAF bind to and acetylate p53 during the DNA damage response, and are needed for full p53 transactivation as well as downstream p53 effects of growth arrest and/or apoptosis. Beyond this simplistic model, p300/CBP appear to be complex integrators of signals that regulate p53, and biochemically, the multipartite p53/p300/CBP interaction is equally complex. Through physical interaction with p53, p300/CBP can both positively and negatively regulate p53 transactivation, as well as p53 protein turnover depending on cellular context and environmental stimuli, such as DNA damage.
Asunto(s)
Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Animales , Proteína de Unión a CREB , Ciclo Celular , Daño del ADN , Regulación de la Expresión Génica , Humanos , Modelos Biológicos , Unión Proteica , Transducción de Señal , Activación Transcripcional , Proteína p53 Supresora de Tumor/genéticaRESUMEN
p19ARF suppresses the growth of cells lacking p53 through an unknown mechanism. p19ARF was found to complex with transcription factors E2F1, -2, and -3. Levels of endogenous or ectopically expressed E2F1, -2, and -3, but not E2F6, were reduced after synthesis of p19ARF, through a mechanism involving increased turnover. p19ARF-induced degradation of E2F1 depended on a functional proteasome, and E2F1 was relocalized to nucleoli when coexpressed with p19ARF. Consistent with reduced levels of E2F1 and E2F3, the proliferation of cells defective for p53 function was suppressed by p19ARF, and the effect was partially reversed by ectopic overexpression of E2F1. These results suggest a broader role for p19ARF as a tumor suppressor, in which targeting of certain E2F species may cooperate with stimulation of the p53 pathway to counteract oncogenic growth signals.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Proteínas/metabolismo , Factores de Transcripción/metabolismo , Northern Blotting , Western Blotting , División Celular , Línea Celular , Nucléolo Celular/metabolismo , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Factor de Transcripción E2F3 , Factor de Transcripción E2F6 , Humanos , Hidrólisis , Unión Proteica , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Proteína p14ARF Supresora de Tumor , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Control of proliferation and differentiation by the retinoblastoma tumor suppressor protein (pRB) and related family members depends upon their interactions with key cellular substrates. Efforts to identify such cellular targets led to the isolation of a novel protein, EID-1 (for E1A-like inhibitor of differentiation 1). Here, we show that EID-1 is a potent inhibitor of differentiation and link this activity to its ability to inhibit p300 (and the highly related molecule, CREB-binding protein, or CBP) histone acetylation activity. EID-1 is rapidly degraded by the proteasome as cells exit the cell cycle. Ubiquitination of EID-1 requires an intact C-terminal region that is also necessary for stable binding to p300 and pRB, two proteins that bind to the ubiquitin ligase MDM2. A pRB variant that can bind to EID1, but not MDM2, stabilizes EID-1 in cells. Thus, EID-1 may act at a nodal point that couples cell cycle exit to the transcriptional activation of genes required for differentiation.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Ciclo Celular/fisiología , Proteínas de Saccharomyces cerevisiae , Acetiltransferasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Proteína de Unión a CREB , Proteínas de Ciclo Celular , Diferenciación Celular , Clonación Molecular , Regulación hacia Abajo , Histona Acetiltransferasas , Datos de Secuencia Molecular , Proteínas Nucleares/antagonistas & inhibidores , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Proteínas Represoras , Proteína de Retinoblastoma/metabolismo , Transactivadores/antagonistas & inhibidores , Activación Transcripcional , Técnicas del Sistema de Dos Híbridos , Ubiquitinas/metabolismoRESUMEN
The Epstein-Barr virus (EBV) nuclear protein 2 (EBNA2) and herpes simplex virion protein 16 (VP16) acidic domains that mediate transcriptional activation now are found to have affinity for p300, CBP, and PCAF histone acetyltransferases (HATs). Transcriptionally inactive point mutations in these domains lack affinity for p300, CBP, or PCAF. P300 and CBP copurify with the principal HAT activities that bind to EBNA2 or VP16 acidic domains through velocity sedimentation and anion-exchange chromatography. EBNA2 binds to both the N- and C-terminal domains of p300 and coimmune-precipitates from transfected 293T cells with p300. In EBV-infected Akata Burkitt's tumor cells that do not express the EBV encoded oncoproteins EBNA2 or LMP1, p300 expression enhances the ability of EBNA2 to up-regulate LMP1 expression. Through its intrinsic HAT activity, PCAF can further potentiate the p300 effect. In 293 T cells, P300 and CBP (but not PCAF) can also coactivate transcription mediated by the EBNA2 or VP16 acidic domains and HAT-negative mutants of p300 have partial activity. Thus, the EBNA2 and VP16 acidic domains can utilize the intrinsic HAT or scaffolding properties of p300 to activate transcription.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr , Herpesvirus Humano 4/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Transactivadores/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas Virales/metabolismo , Linfoma de Burkitt , Proteína de Unión a CREB , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Viral de la Expresión Génica/genética , Genes Reporteros , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Histona Acetiltransferasas , Humanos , Mutación , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Células Tumorales Cultivadas , Factores de Transcripción p300-CBPRESUMEN
Control of p53 turnover is critical to p53 function. E1A binding to p300/CBP translates into enhanced p53 stability, implying that these coactivator proteins normally operate in p53 turnover control. In this regard, the p300 C/H1 region serves as a specific in vivo binding site for both p53 and MDM2, a naturally occurring p53 destabilizer. Moreover, most of the endogenous MDM2 is bound to p300, and genetic analysis implies that specific interactions of p53 and MDM2 with p300 C/H1 are important steps in the MDM2-directed turnover of p53. A specific role for p300 in endogenous p53 degradation is underscored by the p53-stabilizing effect of overproducing the p300 C/H1 domain. Taken together, the data indicate that specific interactions between p300/CBP C/H1, p53, and MDM2 are intimately involved in the MDM2-mediated control of p53 abundance.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular , Humanos , Datos de Secuencia Molecular , Mutagénesis/fisiología , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Oncogénicas Virales/genética , Unión Proteica/fisiología , Proteínas Proto-Oncogénicas c-mdm2 , Homología de Secuencia de Aminoácido , Transactivadores/genética , Ubiquitinas/metabolismoRESUMEN
The adenovirus E1A and SV40 large-T-antigen oncoproteins bind to members of the p300/CBP transcriptional coactivator family. Binding of p300/CBP is implicated in the transforming mechanisms of E1A and T-antigen oncoproteins. A common region of the T antigen is critical for binding both p300/CBP and the tumour suppressor p53, suggesting a link between the functions of p53 and p300. Here we report that p300/CBP binds to p53 in the absence of viral oncoproteins, and that p300 and p53 colocalize within the nucleus and coexist in a stable DNA-binding complex. Consistent with its ability to bind to p300, E1A disrupted functions mediated by p53. It reduced p53-mediated activation of the p21 and bax promoters, and suppressed p53-induced cell-cycle arrest and apoptosis. We conclude that members of the p300/CBP family are transcriptional adaptors for p53, modulating its checkpoint function in the G1 phase of the cell cycle and its induction of apoptosis. Disruption of p300/p53-dependent growth control may be part of the mechanism by which E1A induces cell transformation. These results help to explain how p53 mediates growth and checkpoint control, and how members of the p300/CBP family affect progression from G1 to the S phase of the cell cycle.
Asunto(s)
Proteínas Nucleares/metabolismo , Transactivadores , Factores de Transcripción/metabolismo , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismo , Células 3T3 , Proteínas E1A de Adenovirus/metabolismo , Animales , Apoptosis , Proteína de Unión a CREB , Ciclo Celular , Línea Celular , Núcleo Celular/metabolismo , ADN/metabolismo , Proteína p300 Asociada a E1A , Fase G1 , Ratones , Regiones Promotoras Genéticas , TransfecciónRESUMEN
Although genetic and biochemical data indicate that the cell protein RBPJkappa is a mediator of EBNA-2 and EBNA-3C effects on transcriptional regulatory elements, the extent of association of these Epstein-Barr virus nuclear proteins with RBPJkappa in transformed B lymphocytes has not been determined. We now report that most of the EBNA-2 and at least 20% of the EBNA-3C coimmunoprecipitated with RBPJkappa from extracts of transformed B lymphocytes that contained most of the cellular EBNA-2 and EBNA3C. Both proteins are associated preferentially with the smaller of the two RBPJkappa isoforms. EBNA-2-RBPJkappa complexes do not contain EBNA-3C, and EBNA-3C-RBPJkappa complexes do not contain EBNA-2. Although EBNA-2 and EBNA-3C are extensively associated with RBPJkappa, a fraction of RBPJkappa appears to be free of EBNAs after repeated immunoprecipitations with anti-EBNA, Epstein-Barr virus-immune, human antibody. Promoters with RBPJkappa sites in their regulatory elements are likely to be differentially regulated by these RBPJkappa-EBNA-2 and RBPJkappa-EBNA-3 complexes.
Asunto(s)
Antígenos Virales/metabolismo , Linfocitos B/inmunología , Proteínas de Unión al ADN/metabolismo , Herpesvirus Humano 4/fisiología , Activación de Linfocitos , Proteínas Nucleares , Antígenos Nucleares del Virus de Epstein-Barr , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas , Pruebas de PrecipitinaAsunto(s)
Papillomavirus Bovino 1/química , Proteínas de Unión al ADN/química , Antígenos Nucleares del Virus de Epstein-Barr/química , Herpesvirus Humano 4/química , Origen de Réplica , Proteínas Virales/química , Secuencia de Aminoácidos , Animales , Bovinos , Humanos , Datos de Secuencia Molecular , Conformación Proteica , Alineación de SecuenciaRESUMEN
Expression of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) oncogene is regulated by the EBV nuclear protein 2 (EBNA-2) transactivator. EBNA-2 is known to interact with the cellular DNA-binding protein J kappa and is recruited to promoters containing the GTGGGAA J kappa recognition sequence. The minimal EBNA-2-responsive LMP-1 promoter includes one J kappa-binding site, and we now show that mutation of that site, such that J kappa cannot bind, reduces EBNA-2 responsiveness by 60%. To identify other factors which interact with the LMP-1 EBNA-2 response element (E2RE), a -236/-145 minimal E2RE was used as a probe in an electrophoretic mobility shift assay. The previously characterized factors J kappa, PU.1, and AML1 bind to the LMP-1 E2RE, along with six other unidentified factors (LBF2 to LBF7). Binding sites were mapped for each factor. LBF4 is B- and T-cell specific and recognizes the PU.1 GGAA core sequence as shown by methylation interference. LBF4 has a molecular mass of 105 kDa and is probably unrelated to PU.1. LBF2 was found only in epithelial cell lines, whereas LBF3, LBF5, LBF6, and LBF7 were not cell type specific. Mutations of the AML1- or LBF4-binding sites had no effect on EBNA-2 transactivation, whereas mutation of the PU.1-binding site completely eliminated EBNA-2 responses. A gst-EBNA-2 fusion protein specifically depleted PU.1 from nuclear extracts and bound in vitro translated PU.1, providing biochemical evidence for a direct EBNA-2-PU.1 interaction. Thus, EBNA-2 transactivation of the LMP-1 promoter is dependent on interaction with at least two distinct sequence-specific DNA-binding proteins, J kappa and PU.1. LBF3, LBF5, LBF6, or LBF7 may also be involved, since their binding sites also contribute to EBNA-2 responsiveness.
Asunto(s)
Antígenos Virales/genética , Proteínas de Unión al ADN/genética , Proteínas Nucleares , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas , Factores de Transcripción , Activación Transcripcional , Proteínas de la Matriz Viral/genética , Secuencia de Bases , Línea Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Células Epiteliales , Epitelio/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas , Linfocitos/metabolismo , Datos de Secuencia Molecular , Mutación , Proteínas de Neoplasias/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Oncogénicas de Retroviridae , TransactivadoresRESUMEN
Epstein-Barr virus nuclear antigen 2 (EBNA-2) plays an essential role in primary B-lymphocyte growth transformation. EBNA-2 is an acidic transcriptional transactivator that is brought to virus and cell EBNA-2 response elements by interaction with a factor that recognizes the double-stranded sequence MNYYGTGGGAA, where M is A or C, N is any nucleotide, and Y is a pyrimidine. A 63-kDa protein that recognizes this DNA sequence has now been purified by S-Sepharose and oligonucleotide affinity chromatography. p63 peptide sequence is identical to the predicted amino acid sequence for the human J kappa immunoglobulin recombination signal binding protein. Purified or recombinant in vitro-translated J kappa binds to the MNYYGTGGGAA EBNA-2 response element sequence and interacts with EBNA-2. Surprisingly, J kappa does not bind to the J kappa 1 heptamer recombination signal sequence (CACTGTG), and its prior identification as a heptamer binding protein was most likely due to the addition of a BamHI restriction site to the native heptamer creating a near EBNA-2 response element consensus (CACTGTGGGAT).
Asunto(s)
Antígenos Virales/metabolismo , Proteínas de Unión al ADN/metabolismo , Herpesvirus Humano 4/genética , Proteínas Nucleares/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Secuencia de Aminoácidos , Linfocitos B/metabolismo , Secuencia de Bases , Proteínas de Unión al ADN/genética , Epitelio/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes/metabolismo , Recombinación Genética , Linfocitos T/metabolismoRESUMEN
The papillomavirus E2 protein is a DNA binding protein that regulates viral transcription and replication. E2 binds DNA as a dimer. Recent crystallographic data for E2 complexed to DNA revealed that novel peptide structures in E2 mediated dimerization and DNA binding. To identify important features of these motifs we have used limited proteolysis and urea denaturation as biochemical probes for structure, applying these techniques to E2 alone, E2 bound to DNA, cross-linked products, and mutants that were targeted at Trp360, a contact point along the dimer interface. DNA binding stabilized E2 structure, shifting the point at which it denatures from 5 to 7.6 M urea. In contrast, Trp360 mutant proteins, while dimeric, were more sensitive to denaturation by urea when bound to DNA. The most striking results came from uv cross-linking studies in which Trp360 was targeted as the site of cross-linking. Ultraviolet cross-linking dramatically increased the resistance of E2 to proteolysis regardless of the protease tested and with no deleterious effect on the affinity of E2 for DNA. Cross-linking through Cys356 with bismaleimidohexane did not promote stabilization. The ability to stabilize or destabilize E2 by Trp360-targeted modifications demonstrates the importance of the Trp360-Trp360 interaction, which may represent a general feature of the beta-barrel motif.
Asunto(s)
Papillomavirus Bovino 1/metabolismo , Proteínas de Unión al ADN/metabolismo , Triptófano , Proteínas Virales/metabolismo , Secuencia de Bases , Sitios de Unión , Quimotripsina , Cartilla de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/efectos de la radiación , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos/metabolismo , Fragmentos de Péptidos/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efectos de la radiación , Tripsina/metabolismo , Rayos Ultravioleta , Proteínas Virales/genética , Proteínas Virales/efectos de la radiaciónRESUMEN
Human papillomavirus type 18 (HPV-18) E7 proteins bind zinc through Cys-X-X-Cys repeats located at the C terminus of the protein. In order to examine the role of these cysteine motifs in E7 function, we expressed the HPV-18 E7 protein in bacteria and found that purified E7 forms a dimer through interactions with zinc. Mutants with single mutations within the Cys-X-X-Cys motifs bound a reduced level of zinc in a zinc blot assay, while a double mutant lost all zinc-binding activity. When expressed in vivo, none of the mutants cooperated with an activated ras oncogene to transform primary rat embryo fibroblasts, but all mutants retained nearly wild-type Rb-binding activity. The results indicate that the cysteine motifs play an important role in transformation by HPV-18 E7 but do not contribute to Rb binding.
Asunto(s)
Transformación Celular Viral , Cisteína/metabolismo , Proteínas de Unión al ADN , Proteínas Oncogénicas Virales/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Rubidio/metabolismo , Zinc/metabolismo , Proteínas E1A de Adenovirus/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , Secuencia Conservada , Análisis Mutacional de ADN , Ácido Edético/farmacología , Escherichia coli/genética , Semivida , Modelos Químicos , Datos de Secuencia Molecular , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/aislamiento & purificación , Proteínas Oncogénicas Virales/farmacocinética , Conformación Proteica/efectos de los fármacos , Ratas , Proteínas Recombinantes/metabolismo , Proteína de Retinoblastoma/metabolismoRESUMEN
The dominant transcriptional regulator of the papillomaviruses, E2, binds to its specific DNA target through a previously unobserved dimeric antiparallel beta-barrel. The DNA is severely but smoothly bent over the barrel by the interaction of successive major grooves with a pair of symmetrically disposed alpha-helices. The specific interface is an 'interwoven' network of interactions where the identifying base pairs of the target contact more than one amino-acid side chain and the discriminating amino acids interact with more than one base pair.
Asunto(s)
Papillomavirus Bovino 1/genética , Proteínas de Unión al ADN/química , ADN/metabolismo , Proteínas Virales/química , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cristalización , ADN/química , Proteínas de Unión al ADN/metabolismo , Enlace de Hidrógeno , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Conformación de Ácido Nucleico , Pliegue de Proteína , Proteínas Virales/metabolismoRESUMEN
The bovine papillomavirus E2 protein regulates viral transcription by binding as a dimer to the DNA sequence ACCGN4CGGT. The dimerization and DNA-binding properties are localized within its carboxy-terminal 85 amino acids (325-410). Utilizing random mutagenesis coupled with phenotypic selection in yeast, functionally important amino acids in the DNA-binding domain were identified. Four trans-activation defective point mutants within a short segment (amino acids 337-344) were DNA binding defective but dimeric. The mutation of a conserved tryptophan to serine also eliminated DNA binding, but loss of dimerization was implicated because addition of dimeric monoclonal antibody complemented this defect. A simple assay for E2 dimerization was developed using UV irradiation to produce an interchain cross-link within a dimer. No heterodimeric complexes were formed when pools of E2 of varying lengths were mixed, and only proteins with tryptophan at position 360 could be UV cross-linked. Peptide mapping of irradiated E2 protein localized the cross-link to an 18-amino-acid region bracketing this tryptophan. Substitutions for this tryptophan demonstrated the requirement for a hydrophobic residue at this position, but surprisingly, even alanine was functional. Replacement of this tryptophan with three polar amino acids or glycine eliminated DNA-binding activity, but addition of dimeric monoclonal antibody restored this function. The amino acids that were identified as being involved in DNA contact and dimerization imply that these functions are mediated by novel binding motifs.
Asunto(s)
Papillomavirus Bovino 1/metabolismo , Proteínas de Unión al ADN/metabolismo , Transactivadores/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Papillomavirus Bovino 1/genética , Clonación Molecular , ADN/metabolismo , Análisis Mutacional de ADN , Proteínas de Unión al ADN/química , Elementos de Facilitación Genéticos/genética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mapeo Peptídico , Transactivadores/química , Proteínas Virales/químicaRESUMEN
The DNA-binding properties of purified full-length E2 protein from bovine papillomavirus type 1 have been investigated by utilizing a quantitative gel shift analysis. By using a recombinant baculovirus which express the E2 open reading frame from the polyhedrin promoter, the full-length E2 protein was synthesized in insect cells and purified to homogeneity by using an E2 binding site (ACCGN4CGGT)-specific oligonucleotide column. The Kd of E2 binding to a 41-bp oligonucleotide containing a single binding site was found to be 2 x 10(-11) M. When two binding sites were included on an oligonucleotide, cooperative binding to these sites by the E2 protein was observed. A cooperativity parameter of 8.5 was determined for E2 binding to two sites. An 86-amino-acid peptide encompassing the C terminus of the protein retains the ability to bind E2 binding sites with a Kd of 4 x 10(-10) M but exhibits slight cooperativity of binding to two adjacent sites. A major determinant for cooperative binding of the full-length E2 protein is thus encoded by the N-terminal amino acids outside the minimal DNA binding domain.