RESUMO
Small molecule inhibition of the BET family of proteins, which bind acetylated lysines within histones, has been shown to have a marked therapeutic benefit in pre-clinical models of mixed lineage leukemia (MLL) fusion protein-driven leukemias. Here, we report that I-BET151, a highly specific BET family bromodomain inhibitor, leads to growth inhibition in a human erythroleukemic (HEL) cell line as well as in erythroid precursors isolated from polycythemia vera patients. One of the genes most highly downregulated by I-BET151 was LMO2, an important oncogenic regulator of hematopoietic stem cell development and erythropoiesis. We previously reported that LMO2 transcription is dependent upon Janus kinase 2 (JAK2) kinase activity in HEL cells. Here, we show that the transcriptional changes induced by a JAK2 inhibitor (TG101209) and I-BET151 in HEL cells are significantly over-lapping, suggesting a common pathway of action. We generated JAK2 inhibitor resistant HEL cells and showed that these retain sensitivity to I-BET151. These data highlight I-BET151 as a potential alternative treatment against myeloproliferative neoplasms driven by constitutively active JAK2 kinase.
Assuntos
Neoplasias Hematológicas/patologia , Janus Quinase 2/metabolismo , Transtornos Mieloproliferativos/patologia , Proteínas Oncogênicas/antagonistas & inibidores , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Neoplasias Hematológicas/enzimologia , Neoplasias Hematológicas/metabolismo , Humanos , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The role of the transcriptional coactivator p300 in cell cycle control has not been analysed in detail due to the lack of appropriate experimental systems. We have now examined cell cycle progression of p300-deficient cancer cell lines, where p300 was disrupted either by gene targeting (p300(-) cells) or knocked down using RNAi. Despite significant proliferation defects under normal growth conditions, p300-deficient cells progressed rapidly through G1 with premature S-phase entry. Accelerated G1/S transition was associated with early retinoblastoma (RB) hyperphosphorylation and activation of E2F targets. The p300-acetylase activity was dispensable since expression of a HAT-deficient p300 mutant reversed these changes. Co-immunoprecipitation showed p300/RB interaction occurs in vivo during G1, and this interaction has two peaks: in early G1 with unphosphorylated RB and in late G1 with phosphorylated RB. In vitro kinase assays showed that p300 directly inhibits cdk6-mediated RB phosphorylation, suggesting p300 acts in early G1 to prevent RB hyperphosphorylation and delay premature S-phase entry. Paradoxically, continued cycling of p300(-) cells despite prolonged serum depletion was observed, and this occurred in association with persistent RB hyperphosphorylation. Altogether, these results suggest that p300 has an important role in G1/S control, possibly by modulating RB phosphorylation.
Assuntos
Fase G1 , Neoplasias/patologia , Fase S , Fatores de Transcrição de p300-CBP/fisiologia , Sequência de Bases , Divisão Celular , Primers do DNA , Humanos , Fosforilação , Proteína do Retinoblastoma/metabolismo , Células Tumorais CultivadasRESUMO
In eukaryotic cells the histone methylase SUV39H1 and the methyl-lysine binding protein HP1 functionally interact to repress transcription at heterochromatic sites. Lysine 9 of histone H3 is methylated by SUV39H1 (ref. 2), creating a binding site for the chromo domain of HP1 (refs 3, 4). Here we show that SUV39H1 and HP1 are both involved in the repressive functions of the retinoblastoma (Rb) protein. Rb associates with SUV39H1 and HP1 in vivo by means of its pocket domain. SUV39H1 cooperates with Rb to repress the cyclin E promoter, and in fibroblasts that are disrupted for SUV39, the activity of the cyclin E and cyclin A2 genes are specifically elevated. Chromatin immunoprecipitations show that Rb is necessary to direct methylation of histone H3, and is necessary for binding of HP1 to the cyclin E promoter. These results indicate that the SUV39H1-HP1 complex is not only involved in heterochromatic silencing but also has a role in repression of euchromatic genes by Rb and perhaps other co-repressor proteins.
Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Metiltransferases/fisiologia , Regiões Promotoras Genéticas , Proteínas Repressoras/fisiologia , Proteína do Retinoblastoma/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Homólogo 5 da Proteína Cromobox , Ciclina E/genética , Escherichia coli , Feminino , Células HeLa , Histona Metiltransferases , Humanos , Lisina/metabolismo , Metilação , Metiltransferases/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Proteínas Metiltransferases , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Heterochromatin protein 1 (HP1) is localized at heterochromatin sites where it mediates gene silencing. The chromo domain of HP1 is necessary for both targeting and transcriptional repression. In the fission yeast Schizosaccharomyces pombe, the correct localization of Swi6 (the HP1 equivalent) depends on Clr4, a homologue of the mammalian SUV39H1 histone methylase. Both Clr4 and SUV39H1 methylate specifically lysine 9 of histone H3 (ref. 6). Here we show that HP1 can bind with high affinity to histone H3 methylated at lysine 9 but not at lysine 4. The chromo domain of HP1 is identified as its methyl-lysine-binding domain. A point mutation in the chromo domain, which destroys the gene silencing activity of HP1 in Drosophila, abolishes methyl-lysine-binding activity. Genetic and biochemical analysis in S. pombe shows that the methylase activity of Clr4 is necessary for the correct localization of Swi6 at centromeric heterochromatin and for gene silencing. These results provide a stepwise model for the formation of a transcriptionally silent heterochromatin: SUV39H1 places a 'methyl marker' on histone H3, which is then recognized by HP1 through its chromo domain. This model may also explain the stable inheritance of the heterochromatic state.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Metiltransferases , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Sequência de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromatina/metabolismo , Homólogo 5 da Proteína Cromobox , Proteínas Fúngicas/metabolismo , Histona-Lisina N-Metiltransferase , Humanos , Metilação , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Schizosaccharomyces/metabolismo , Fatores de Transcrição/metabolismoRESUMO
In the nucleus, DNA is tightly packaged into higher-order structures, generating an environment that is highly repressive towards DNA processes such as gene transcription. Acetylation of lysine residues within proteins has recently emerged as a major mechanism used by the cell to overcome this repression. Acetylation of non-histone proteins, including transcription factors, as well as histones, appears to be involved in this process. Like phosphorylation, acetylation is a dynamic process that can regulate protein-DNA and protein-protein interactions. Moreover, a conserved domain, the bromodomain, has been implicated in the binding of acetylated peptides, suggesting a role for acetylation in intracellular signalling.
Assuntos
Regulação da Expressão Gênica , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Acetilação , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Histona Acetiltransferases , Histonas/química , Humanos , Dados de Sequência Molecular , Nucleossomos/genética , Nucleossomos/ultraestruturaRESUMO
The major immediate-early proteins of human cytomegalovirus (HCMV) play a pivotal role in controlling viral and cellular gene expression during productive infection. As well as negatively autoregulating its own promoter, the HCMV 86-kDa major immediate early protein (IE86) activates viral early gene expression and is known to be a promiscuous transcriptional regulator of cellular genes. IE86 appears to act as a multimodal transcription factor. It is able to bind directly to target promoters to activate transcription but is also able to bridge between upstream binding factors such as CREB/ATF and the basal transcription complex as well as interacting directly with general transcription factors such as TATA-binding protein and TFIIB. We now show that IE86 is also able to interact directly with histone acetyltransferases during infection. At least one of these factors is the histone acetyltransferase CBP-associated factor (P/CAF). Furthermore, we show that this interaction results in synergistic transactivation by IE86 of IE86-responsive promoters. Recruitment of such chromatin-remodeling factors to target promoters by IE86 may help explain the ability of this viral protein to act as a promiscuous transactivator of cellular genes.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Glicoproteínas de Membrana , Proteínas de Saccharomyces cerevisiae , Transativadores , Ativação Transcricional , Proteínas do Envelope Viral , Proteínas Virais , Acetiltransferases/genética , Proteínas de Ciclo Celular/genética , Citomegalovirus/genética , Regulação da Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Histona Acetiltransferases , Humanos , Proteínas Imediatamente Precoces/genética , Testes de Precipitina , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição , Transfecção , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido , Fatores de Transcrição de p300-CBPRESUMO
Histone acetylases were originally identified because of their ability to acetylate histone substrates [1] [2] [3]. Acetylases can also target other proteins such as transcription factors [4] [5] [6] [7]. We asked whether the acetylase CREB-binding protein (CBP) could acetylate proteins not directly involved in transcription. A large panel of proteins, involved in a variety of cellular processes, were tested as substrates for recombinant CBP. This screen identified two proteins involved in nuclear import, Rch1 (human importin-alpha) and importin-alpha7, as targets for CBP. The acetylation site within Rch1 was mapped to a single residue, Lys22. By comparing the context of Lys22 with the sequences of other known substrates of CBP and the closely related acetylase p300, we identified G/SK (in the single-letter amino acid code) as a consensus acetylation motif. Mutagenesis of the glycine, as well as the lysine, severely impaired Rch1 acetylation, supporting the view that GK is part of a recognition motif for acetylation by CBP/p300. Using an antibody raised against an acetylated Rch1 peptide, we show that Rch1 was acetylated at Lys22 in vivo and that CBP or p300 could mediate this reaction. Lys22 lies within the binding site for a second nuclear import factor, importin-beta. Acetylation of Lys22 promoted interaction with importin-beta in vitro. Collectively, these results demonstrate that acetylation is not unique to proteins involved in transcription. Acetylation may regulate a variety of biological processes, including nuclear import.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , alfa Carioferinas , Acetilação , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteína de Ligação a CREB , Proteínas de Transporte/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Sequência Consenso , Glicina/química , Glicina/genética , Histona Acetiltransferases , Humanos , Carioferinas , Lisina/química , Lisina/genética , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Alinhamento de Sequência , Transativadores/química , Transativadores/genética , Fatores de Transcrição , Transformação Genética , Fatores de Transcrição de p300-CBPRESUMO
Acetyltransferases are essential enzymes for a wide variety of cellular processes and mutations in acetyltransferase genes have been associated with the development of certain cancers. For this reason, we conducted a computerized sequence homology search for novel acetyltransferases. Here, we show that the putative tumour suppressor protein Fus-2 has homology to the catalytic domain of acetyltransferases. We demonstrate that Fus-2 can acetylate the N-terminus of proteins using a ping-pong mechanism and that it has a specificity for substrates. Consistent with other N-acetyltransferases, Fus-2 localizes to the cytoplasm, as shown by GFP-tag experiments. Since the Fus-2 gene maps to the chromosomal region 3p21.3, which contains at least one tumour suppressor gene, the N-acetyltransferase functions of Fus-2 may be relevant to its potential role in cancer.
Assuntos
Arilamina N-Acetiltransferase/fisiologia , Genes Supressores de Tumor , Sequência de Aminoácidos , Arilamina N-Acetiltransferase/genética , Humanos , Dados de Sequência MolecularRESUMO
NF-ATc, an inducibly expressed transcription factor, controls gene expression in T lymphocytes and cardiomyocytes. We show here that the transcriptional co-activators CBP/p300 bind to and control the activity of the inducible N-terminal transactivation domain of NF-ATc, TAD-A. Similar to the N terminal transactivation domain of c-Jun, TAD-A is inducibly phosphorylated, but this phosphorylation is dispensable for the interaction with CBP/p300. Constitutive active versions of c-Raf and Rac synergistically enhance the CBP/p300-mediated increase of TAD-A activity, indicating the important role CBP/p300 plays in the integration of T cell activation signals. Since a mutation of CBP abolishing HAT activity is almost as active as wild-type CBP in T cells, functions of CBP/p300 other than histone acetylation appear to control the NF-AT-dependent transcription in T cells.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Proteínas Nucleares/fisiologia , Linfócitos T/imunologia , Transativadores/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/farmacologia , Proteínas de Ligação a DNA/fisiologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Transdução de Sinais , Fator de Transcrição AP-1/fisiologia , Proteínas rac de Ligação ao GTPRESUMO
E7 is the main transforming protein of human papilloma virus type 16 (HPV16) which is implicated in the formation of cervical cancer. The transforming activity of E7 has been attributed to its interaction with the retinoblastoma (Rb) tumour suppressor. However, Rb binding is not sufficient for transformation by E7. Mutations within a zinc finger domain, which is dispensable for Rb binding, also abolish E7 transformation functions. Here we show that HPV16 E7 associates with histone deacetylase in vitro and in vivo, via its zinc finger domain. Using a genetic screen, we identify Mi2beta, a component of the recently identified NURD histone deacetylase complex, as a protein that binds directly to the E7 zinc finger. A zinc finger point mutant which is unable to bind Mi2beta and histone deacetylase but is still able to bind Rb fails to overcome cell cycle arrest in osteosarcoma cells. Our results suggest that the binding to a histone deacetylase complex is an important parameter for the growthpromoting activity of the human papilloma virus E7 protein. This provides the first indication that viral oncoproteins control cell proliferation by targeting deacetylation pathways.
Assuntos
Adenosina Trifosfatases , Autoantígenos/metabolismo , DNA Helicases , Histona Desacetilases/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Sequência de Aminoácidos , Transformação Celular Neoplásica , Transformação Celular Viral , Histona Desacetilase 1 , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Dados de Sequência Molecular , Proteínas E7 de Papillomavirus , Ligação Proteica , Proteína do Retinoblastoma/metabolismo , Dedos de ZincoRESUMO
The P/CAF protein has intrinsic histone acetyltransferase (HAT) activity and is capable of binding the transcriptional co-activator CBP. Here we show that P/CAF can regulate transcription and that this function is independent of its binding to CBP. The HAT domain of P/CAF has transcriptional activation potential in yeast. In mammalian cells P/CAF can stimulate transcription of the RSV promoter, using the activity of its HAT domain. We show that the adenovirus protein E1A targets P/CAF and sequesters its transcriptional activity. Binding of E1A to P/CAF is direct, independent of CBP and requires residues within E1A conserved region 1. We find that the P/CAF binding residues in E1A are within a motif shown to be essential for efficient disruption of myogenesis by E1A. The fact that E1A can directly bind and regulate the activity of P/CAF, independently of its regulation of CBP, highlights an important role for P/CAF in the process of cell differentiation.
Assuntos
Acetiltransferases/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Proteínas de Saccharomyces cerevisiae , Acetiltransferases/biossíntese , Acetiltransferases/fisiologia , Proteínas E1A de Adenovirus/fisiologia , Proteína de Ligação a CREB , Linhagem Celular , Ativação Enzimática , Repressão Enzimática , Histona Acetiltransferases , Humanos , Proteínas Nucleares/fisiologia , Ligação Proteica , Proteínas Repressoras/fisiologia , Transativadores/fisiologia , Transcrição Gênica , Células Tumorais CultivadasRESUMO
The CBP co-activator protein possesses an intrinsic acetyltransferase (AT) activity capable of acetylating nucleosomal histones, as well as other proteins such as the transcription factors TFIIE and TFIIF. In addition, CBP associates with two other TSs, P/CAF and SRC1. We set out to establish whether the intrinsic AT activity of CBP contributes to transcriptional activation. We show that a region of CBP, encompassing the previously defined histone AT (HAT) domain, can stimulate transcription when tethered to a promoter. The stimulatory effect of this activation domain shows some promoter preference and is dependent on AT activity. Analysis of 14 point mutations reveals a direct correlation between CBP's ability to acetylate histones in vitro and to activate transcription in vivo. We also find that the HAT domains of CBP and P/CAF share sequence similarity. Four conserved motifs are identified, three of which are analogous to motifs A, B and D, found in other N-acetyltransferases. The fourth motif, termed E, is unique to CBP and P/CAF. Mutagenesis shows that all four motifs in CBP contribute to its HAT activity in vitro and its ability to activate transcription in vivo. These results demonstrate that the AT activity of CBP is directly involved in stimulating gene transcription. The identification of specific HAT domain motifs, conserved between CBP and P/CAF, should facilitate the identification of other members of this AT family.
Assuntos
Acetiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores , Fatores de Transcrição TFII , Fatores de Transcrição/metabolismo , Transcrição Gênica , Acetiltransferases/genética , Adenoviridae/genética , Sequência de Aminoácidos , Sítios de Ligação , Proteína de Ligação a CREB , Linhagem Celular , Histona Acetiltransferases , Humanos , Dados de Sequência Molecular , Mutagênese , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TATA Box , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
Recent discoveries of plant retinoblastoma (Rb) protein homologues and D-type cyclins suggest that control of the onset of cell division in plants may have stronger parallels with mammalian G1/S controls than with yeasts. In mammals, the Rb protein interacts specifically with D-type cyclins and regulates cell proliferation by binding and inhibiting E2F transcription factors. However, the developmental role of Rb in plants and its potential interaction with cell cycle regulators is unknown. We show that the maize Rb homologue ZmRb-1 is temporally and spatially regulated during maize leaf development. ZmRb-1 is highly expressed in differentiating cells, but almost undetectable in proliferating cells. In vitro, both ZmRb-1 and human Rb bind all classes of plant D-type cyclins with the involvement of a conserved N-terminal Leu-x-Cys-x-Glu (LxCxE) Rb-interaction motif. This binding is strongly reduced by mutation of the conserved Cys-470 of ZmRb-1. ZmRb-1 binds human and Drosophila E2F, and inhibits transcriptional activation of human E2F. We also show that ZmRb-1 is a good in vitro substrate for all human G1/S protein kinases. The functional conservation of proteins that control the G1/S transition in mammals and plants points to the existence of plant E2F homologues. We conclude that evolution of Rb and cyclin D proteins occurred after separation of the fungi from the higher eukaryotic lineage, but preceded the divergence of plant and animal kingdoms.
Assuntos
Proteínas de Ciclo Celular , Ciclinas/metabolismo , Regulação da Expressão Gênica de Plantas , Interfase/fisiologia , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Sítios de Ligação , Células Cultivadas , Ciclina D , Ciclinas/genética , Proteínas de Drosophila , Fase G1/fisiologia , Humanos , Modelos Biológicos , Família Multigênica , Fosfoproteínas/metabolismo , Fosforilação , Proteínas de Plantas/genética , Testes de Precipitina , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Fase S/fisiologia , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Supressão Genética , Transcrição Gênica , Zea mays/crescimento & desenvolvimentoRESUMO
The retinoblastoma protein (Rb) silences specific genes that are active in the S phase of the cell cycle and which are regulated by E2F transcription factors. Rb binds to the activation domain of E2F and then actively represses the promoter by a mechanism that is poorly understood. Here we show that Rb associates with a histone deacetylase, HDAC1, through the Rb 'pocket' domain. Association with the deacetylase is reduced by naturally occurring mutations in the pocket and by binding of the human papilloma virus oncoprotein E7. We find that Rb can recruit histone deacetylase to E2F and that Rb cooperates with HDAC1 to repress the E2F-regulated promoter of the gene encoding the cell-cycle protein cyclin E. Inhibition of histone deacetylase activity by trichostatin A (TSA) inhibits Rb-mediated repression of a chromosomally integrated E2F-regulated promoter. Our results indicate that histone deacetylases are important for regulating the cell cycle and that active transcriptional repression by Rb may involve the modification of chromatin structure.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Proteína do Retinoblastoma/fisiologia , Transcrição Gênica , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Ciclina E/genética , Fatores de Transcrição E2F , Células HeLa , Histona Desacetilase 1 , Humanos , Dados de Sequência Molecular , Ligação Proteica , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismoAssuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Fatores de Transcrição E2F , Fator de Transcrição E2F6 , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteína 1 de Ligação ao Retinoblastoma , Homologia de Sequência de Aminoácidos , Fator de Transcrição DP1 , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
We have investigated the relationship between c-Jun N-terminal kinase (JNK) activity, apoptosis, and the potential of survival factors to rescue primary rat sympathetic neurones deprived of trophic support. Incubation of sympathetic neurones in the absence of nerve growth factor (NGF) caused a time-dependent increase in JNK activity, which became apparent by 3 h and attained maximal levels that were three- to fourfold higher than activity measured in neurones maintained for the same periods with NGF. Continuous culture in the presence of either NGF or the cyclic AMP analogue 4-(8-chlorophenylthio) cyclic AMP (CPTcAMP) not only prevented JNK activation from occurring, but also suppressed JNK activity that had been elevated by prior culture of the neurones in the absence of trophic support. When either NGF or CPTcAMP was added to cultures that had been initially deprived of neurotrophic support for up to 10 h, this resulted in complete suppression of total JNK activity, arrest of apoptosis, and rescue of >90% of the neurones that did not display apoptotic morphology by this time. However, when either agent was added after more protracted periods of initial neurotrophin deprivation (> or = 14 h), although this also resulted in near-complete suppression of total JNK activity and short-term arrest of apoptosis, not all of the neurones that appeared to be nonapoptotic at the time of agent addition were rescued. The lack of death commitment after 10 h of maintained JNK activity was not due to a late induction of c-Jun expression, because the majority of newly isolated sympathetic neurones had already been expressing high levels of c-Jun in their nuclei for several hours, yet were capable of being rescued by NGF. Elevation of JNK activity as a result of neurotrophic-factor deprivation was also associated with enhanced phosphorylation of c-Jun, assessed by immunoblot analysis and immunocytochemistry, and addition of NGF to cultures previously deprived of neurotrophic support resulted in a reversion of the state of phospho-c-Jun to that observed in cultures that had been maintained in the continuous presence of trophic support. We conclude that activation of JNK and c-Jun phosphorylation are not necessarily rate-limiting for apoptosis induction. In some neurones undergoing prolonged NGF deprivation, suppression of JNK activity and c-Jun dephosphorylation by NGF may be insufficient to effect their rescue. Thus, if c-Jun mediates death by increasing the expression of "death" genes, these must become effective very close to the death commitment point.
Assuntos
Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Proteínas Proto-Oncogênicas c-jun/metabolismo , Gânglio Cervical Superior/metabolismo , Animais , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Immunoblotting , Proteínas Quinases JNK Ativadas por Mitógeno , Cinética , Fatores de Crescimento Neural/administração & dosagem , Fatores de Crescimento Neural/farmacologia , Fosforilação , Ratos , Gânglio Cervical Superior/citologia , Tionucleotídeos/farmacologiaRESUMO
A yeast two-hybrid screen has identified HBP1 as a transcription factor capable of interacting with the pocket protein family. We show that HBP1 can interact with one of these, RB, both in vitro and in mammalian cells. Two distinct RB binding sites are present within HBP1--a high affinity binding site, mediated by an LXCXE motif and a separate low affinity binding site present within an activation domain. GAL4-fusion experiments indicate that HBP1 contains a masked activation domain. Deletion of two independent N- and C-terminal inhibitor domains unmasks an activation domain which is 100-fold more active than the full length protein. The released activation capacity is repressed by RB, p130 and p107. In addition, E1A can repress the activity of HBP1 via conserved region 1 sequences in a manner independent of the CBP co-activator. We show by stable expression in NIH3T3 cells that HBP1 has the capacity to induce morphological transformation of cells in culture.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Fosfoproteínas/metabolismo , Proteínas , Proteínas Repressoras/metabolismo , Células 3T3 , Animais , Humanos , Camundongos , Ligação Proteica , Proteína p130 Retinoblastoma-Like , Células Tumorais CultivadasRESUMO
The transcription initiation factor TFIID is a multimeric protein complex composed of TATA box-binding protein (TBP) and many TBP-associated factors (TAF(II)s). TAF(II)s are important cofactors that mediate activated transcription by providing interaction sites for distinct activators. Here, we present evidence that human TAF(II)250 and its homologs in Drosophila and yeast have histone acetyltransferase (HAT) activity in vitro. HAT activity maps to the central, most conserved portion of dTAF(II)230 and yTAF(II)130. The HAT activity of dTAF(II)230 resembles that of yeast and human GCN5 in that it is specific for histones H3 and H4 in vitro. Our findings suggest that targeted histone acetylation at specific promoters by TAF(II)250 may be involved in mechanisms by which TFIID gains access to transcriptionally repressed chromatin.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Ligação a DNA , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Galinhas , Drosophila melanogaster , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica , Células HeLa , Histona Acetiltransferases , Humanos , Proteínas de Insetos/metabolismo , Substâncias Macromoleculares , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Peptídeos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Recombinantes , Saccharomyces cerevisiae , Deleção de Sequência , Relação Estrutura-Atividade , Especificidade por Substrato , Fator de Transcrição TFIID , Transcrição GênicaRESUMO
The CBP protein acts as a transcriptional adaptor for many different transcription factors by directly contacting DNA-bound activators. One mechanism by which CBP is thought to stimulate transcription is by recruiting the histone acetyltransferase (HAT) P/CAF to the promoter. Here we show that CBP has intrinsic HAT activity. The HAT domain of CBP is adjacent to the binding site for the transcriptional activator E1A. Although E1A displaces P/CAF from CBP, it does not disrupt the CBP-associated HAT activity. Thus E1A carries HAT activity when complexed with CBP. Targeting CBP-associated HAT activity to specific promoters may therefore be a mechanism by which E1A acts as a transcriptional activator.
Assuntos
Acetiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores , Fatores de Transcrição/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Animais , Ligação Competitiva , Células COS , Proteína de Ligação a CREB , Linhagem Celular , Escherichia coli , Histona Acetiltransferases , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The CBP protein mediates PKA induced transcription by binding to the PKA phosphorylated activation domain of CREB. Here we show that CBP also stimulates the activity of both c-Jun and v-Jun in vivo. The CREB binding domain of CBP is sufficient to contact to c-Jun in vitro. When this domain of CBP is linked to the activation domain of VP16 and expressed in vivo it stimulates c-Jun dependent transcription. Deletion analysis of c-Jun indicate that the CBP binding site is within the N-terminal activation domain. Loss of binding to CBP in vitro correlates with severely reduced transactivation capacity in vivo. Mutation of Ser63/73 in c-Jun, or the corresponding position in v-Jun (Ser36/46) leads to reduced binding to CBP in vitro and abolishes augmentation of transcription in vivo. These data are consistent with a mechanism by which CBP acts as a co-activator protein for Jun dependent transcription by interacting with the Jun N-terminal activation domain.