RESUMO
B-Myb is one member of the vertebrate Myb family of transcription factors and is ubiquitously expressed. B-Myb activates transcription of a group of genes required for the G2/M cell cycle transition by forming the dREAM/Myb-MuvB-like complex, which was originally identified in Drosophila. Mutants of zebrafish B-myb and Drosophila myb exhibit defects in cell cycle progression and genome instability. Although the genome instability caused by a loss of B-Myb has been speculated to be due to abnormal cell cycle progression, the precise mechanism remains unknown. Here, we have purified a B-Myb complex containing clathrin and filamin (Myb-Clafi complex). This complex is required for normal localization of clathrin at the mitotic spindle, which was previously reported to stabilize kinetochore fibres. The Myb-Clafi complex is not tightly associated with the mitotic spindles, suggesting that this complex ferries clathrin to the mitotic spindles. Thus, identification of the Myb-Clafi complex reveals a previously unrecognized function of B-Myb that may contribute to its role in chromosome stability, possibly, tumour suppression.
Assuntos
Clatrina/metabolismo , Proteínas Contráteis/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Fuso Acromático/metabolismo , Animais , Clatrina/isolamento & purificação , Proteínas Contráteis/isolamento & purificação , Fibroblastos/metabolismo , Filaminas , Instabilidade Genômica , Células HeLa , Humanos , Camundongos , Proteínas dos Microfilamentos/isolamento & purificação , Mitose , Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-myb/isolamento & purificaçãoRESUMO
The c-myb proto-oncogene product (c-Myb) is degraded in response to Wnt-1 signaling. In this process, Fbxw7α, the F-box protein of the SCF complex, binds to c-Myb via its C-terminal WD40 domain, and induces the ubiquitination of c-Myb. Here, we report that Fbxw5, another F-box protein, enhances sumoylation of nuclear c-Myb. Fbxw5 enhanced c-Myb sumoylation via the DDB1-Cul4A-Rbx1 complex. Since the Fbxw5-DDB1-Cul4A-Rbx1 complex was shown to act as a ubiquitin ligase for tumor suppressor TSC2, our results suggest that this complex can function as a dual SUMO/ubiquitin ligase. Fbxw5, which is localized to both nucleus and cytosol, enhanced sumoylation of nuclear c-Myb and induced the localization of c-Myb to nuclear dot-like domains. Co-expression of Fbxw5 suppressed the trans-activation of c-myc promoter by wild-type c-Myb, but not by v-Myb, which lacks the sumoylation sites. These results suggest that multiple E3 ligases suppress c-Myb activity through sumoylation or ubiquitination, and that v-Myb is no longer subject to these negative regulations.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas F-Box/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular , Proteínas F-Box/genética , Células HEK293 , Humanos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myb/genéticaRESUMO
Adipocyte differentiation is an important component of obesity, but how hormonal cues mediate adipocyte differentiation remains elusive. BMP stimulates in vitro adipocyte differentiation, but the role of BMP in adipogenesis in vivo is unknown. Drosophila Schnurri (Shn) is required for the signaling of Decapentaplegic, a Drosophila BMP homolog, via interaction with the Mad/Medea transcription factors. Vertebrates have three Shn orthologs, Shn-1, -2, and -3. Here, we report that Shn-2(-/-) mice have reduced white adipose tissue and that Shn-2(-/-) mouse embryonic fibroblasts cannot efficiently differentiate into adipocytes in vitro. Shn-2 enters the nucleus upon BMP-2 stimulation and, in cooperation with Smad1/4 and C/EBPalpha, induces the expression of PPARgamma2, a key transcription factor for adipocyte differentiation. Shn-2 directly interacts with both Smad1/4 and C/EBPalpha on the PPARgamma2 promoter. These results indicate that Shn-2-mediated BMP signaling has a critical role in adipogenesis.
Assuntos
Adipogenia/fisiologia , Proteínas Morfogenéticas Ósseas/fisiologia , Proteínas de Ligação a DNA/fisiologia , Lipodistrofia/genética , Proteína Smad1/fisiologia , Proteína Smad4/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Adipogenia/genética , Tecido Adiposo/citologia , Tecido Adiposo/fisiologia , Animais , Proteína Morfogenética Óssea 2 , Proteína alfa Estimuladora de Ligação a CCAAT/fisiologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Técnicas In Vitro , Lipodistrofia/metabolismo , Masculino , Camundongos , PPAR gama/genética , PPAR gama/fisiologia , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/fisiologia , Transdução de Sinais/fisiologiaRESUMO
The c-myb proto-oncogene product (c-Myb) induces transcription of a group of target genes involved in the G1/S transition and in anti-apoptosis. The level of c-Myb is negatively regulated by the Wnt signal, but it remains obscure how c-Myb activity is positively regulated. We have found that ribosomal protein L4 (RPL4) binds to the DNA-binding domain of c-Myb. Co-immunoprecipitation experiments also indicated that RPL4 interacts with c-Myb. When c-Myb was overexpressed in CV-1 cells, significant amounts of RPL4 moved to the nucleoplasm from the nucleolus. RPL4 stimulated the c-Myb-dependent expression of a c-myc-luciferase reporter construct. Chromatin immunoprecipitation assays indicated that RPL4 binds to the 5'-regulatory region of the c-myc gene via c-Myb. Serum starvation and 2-deoxyglucose treatment of NIH3T3 cells induced the movement of RPL4 from the nucleoplasm to the nucleolus. Furthermore, c-myc mRNA levels were reduced by either serum starvation or 2-deoxyglucose treatment, and the degree of reduction in the c-myc mRNA level was correlated with the RPL4 level. These results suggest that growth factor and nutritional signals positively regulate c-Myb activity via its interaction with RPL4. Thus, RPL4 plays an important role in c-myc expression by interacting with c-Myb in response to growth factor and nutritional signals.
Assuntos
Proteínas Proto-Oncogênicas c-myb/metabolismo , Proteínas Ribossômicas/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Células NIH 3T3 , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myb/química , Proteínas Proto-Oncogênicas c-myb/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/genéticaRESUMO
Small ubiquitin-related modifiers (SUMOs) are proteins that are posttranslationally conjugated to diverse proteins. The c-myb proto-oncogene product (c-Myb) regulates proliferation and differentiation of hematopoietic cells. PIASy is the only known SUMO E3 ligase for c-Myb. Here, we report that TRAF7 binds to c-Myb and stimulates its sumoylation. TRAF7 bound to the DNA-binding domain of c-Myb via its WD40 repeats. TRAF7 has an E3 ubiquitin ligase activity for self-ubiquitination, but TRAF7 also stimulated the sumoylation of c-Myb at Lys-523 and Lys-499, which are the same sites as those used for PIASy-induced sumoylation. TRAF7 inhibited trans-activation induced by wild-type c-Myb, but not by the sumoylation site mutant of c-Myb. The expression of both c-myb and TRAF7 was down-regulated during differentiation of M1 cells. Endogenous TRAF7 localized to both the cytoplasm and nucleus of M1 cells. Consistent with this, significant amounts of sumoylated c-Myb were found in the cytoplasm of M1 cells, whereas nonsumoylated c-Myb was found predominantly in the nucleus. Overexpressed TRAF7 was localized in the cytoplasm of CV-1 cells, and sequestered c-Myb and SUMO1 in the cytosol, whereas PIASy was localized in the nucleus. Thus, TRAF7 negatively regulates c-Myb activity by sequestering c-Myb to the cytosol via sumoylation.
Assuntos
Proteínas Proto-Oncogênicas c-myb/metabolismo , Proteína SUMO-1/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/fisiologia , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Ligases/metabolismo , Camundongos , Ativação Transcricional/genéticaRESUMO
The c-myb proto-oncogene product (c-Myb) regulates proliferation and differentiation of hematopoietic cells. Recently we have shown that c-Myb is degraded in response to Wnt-1 stimulation via a pathway involving TAK1 (TGF-beta-activated kinase), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). NLK and HIPK2 bind directly to c-Myb and phosphorylate c-Myb at multiple sites, inducing its ubiquitination and proteasome-dependent degradation. The mammalian myb gene family contains two members in addition to c-myb, A-myb, and B-myb. Here, we report that the Wnt-NLK pathway also inhibits A-Myb activity, but by a different mechanism. As in the case of c-Myb, both NLK and HIPK2 bound directly to A-Myb and inhibited its activity. NLK phosphorylated A-Myb, but did not induce A-Myb degradation. Overexpression of NLK inhibited the association between A-Myb and the coactivator CBP, thus, blocking A-Myb-induced trans-activation. The kinase activity of NLK is required for the efficient inhibition of the association between A-Myb and CBP, although the kinase-negative form of NLK also partly inhibits the interaction between A-Myb and CBP. Furthermore, NLK induced the methylation of histone H3 at lysine-9 at A-Myb-bound promoter regions. Thus, the Wnt-NLK pathway inhibits the activity of each Myb family member by different mechanisms.
Assuntos
Proteína de Ligação a CREB/metabolismo , Histonas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Transativadores/metabolismo , Proteínas Wnt/fisiologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Chlorocebus aethiops , Humanos , Lisina , Metilação , Proteínas Quinases Ativadas por Mitógeno/genética , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Transativadores/genética , Ativação TranscricionalRESUMO
Tumor necrosis factor alpha (TNF-α) plays a role in apoptosis and proliferation in multiple types of cells, and defects in TNF-α-induced apoptosis are associated with various autoimmune diseases. Here, we show that TRIM27, a tripartite motif (TRIM) protein containing RING finger, B-box, and coiled-coil domains, positively regulates TNF-α-induced apoptosis. Trim27-deficient mice are resistant to TNF-α-d-galactosamine-induced hepatocyte apoptosis. Trim27-deficient mouse embryonic fibroblasts (MEFs) are also resistant to TNF-α-cycloheximide-induced apoptosis. TRIM27 forms a complex with and ubiquitinates the ubiquitin-specific protease USP7, which deubiquitinates receptor-interacting protein 1 (RIP1), resulting in the positive regulation of TNF-α-induced apoptosis. Our findings indicate that the ubiquitination-deubiquitination cascade mediated by the TRIM27-USP7 complex plays an important role in TNF-α-induced apoptosis.
Assuntos
Apoptose , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação , Animais , Cicloeximida/farmacologia , Fibroblastos/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Células HEK293 , Células Hep G2 , Hepatócitos/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Poliubiquitina/metabolismo , Multimerização Proteica , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico , Ubiquitina-Proteína Ligases , Peptidase 7 Específica de UbiquitinaRESUMO
Hedgehog (Hh) signalling plays an important role in various developmental processes by activating the Cubitus interruptus (Ci)/Glioblastoma (Gli) family of transcription factors. In the process of proper pattern formation, Ci activity is regulated by multiple mechanisms, including processing, trafficking, and degradation. However, it remains elusive how Ci distinctly recognizes the strong and moderate Hh signals. Roadkill (Rdx) induces Ci degradation in the anterior region of the Drosophila wing disc. Here, we report that Rdx inhibited Ci activity by two different mechanisms. In the region abutting the anterior/posterior boundary, which receives strong Hh signal, Rdx inhibited the nuclear import of Ci by releasing importin α3 from Ci. In this region, Rdx negatively regulated the expression of transcription factor Knot/Collier. In farther anterior regions receiving moderate levels of Hh signal, Rdx induced Ci degradation, as reported previously. Thus, two different mechanisms by which Rdx negatively regulates Ci may play an important role in the fine-tuning of Hh responses.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Cruzamentos Genéticos , Drosophila melanogaster , Genótipo , Modelos Genéticos , Mutação , Plasmídeos/metabolismo , Isoformas de Proteínas , Interferência de RNA , Técnicas do Sistema de Duplo-HíbridoRESUMO
The c-myb proto-oncogene product (c-Myb) is degraded in response to Wnt-1 signaling via a pathway involving TAK1 (transforming growth factor-beta-activated kinase 1), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). NLK directly binds to c-Myb, which results in the phosphorylation of c-Myb at multiple sites, and induces its ubiquitination and proteasome-dependent degradation. Here, we report that Fbxw7, the F-box protein of an SCF complex, targets c-Myb for degradation in a Wnt-1- and NLK-dependent manner. Fbxw7alpha directly binds to c-Myb via its C-terminal WD40 domain and induces the ubiquitination of c-Myb in the presence of NLK in vivo and in vitro. The c-Myb phosphorylation site mutant failed to interact with Fbxw7alpha, suggesting that the c-Myb/Fbxw7alpha interaction is enhanced by NLK phosphorylation of c-Myb. Treatment of M1 cells with Fbxw7 small interfering RNA (siRNA) rescued the Wnt-induced c-Myb degradation and also the Wnt-induced inhibition of cell proliferation. NLK bound to Cul1, a component of the SCF complex, while HIPK2 interacted with both Fbxw7alpha and Cul1, suggesting that both kinases enhance the c-Myb/SCF interaction. In contrast to c-Myb, the v-myb gene product (v-Myb) encoded by the avian myeloblastosis virus was resistant to NLK/Fbxw7alpha-induced degradation. Thus, Fbxw7 is an E3 ubiquitin ligase of c-Myb, and the increased c-Myb levels may contribute, at least partly, to transformation induced by mutation of Fbxw7.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proliferação de Células , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas F-Box/antagonistas & inibidores , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Proteínas Oncogênicas v-myb/genética , Proteínas Oncogênicas v-myb/metabolismo , Fosforilação/fisiologia , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica/fisiologia , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína/fisiologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myb/genética , RNA Interferente Pequeno/genética , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMO
Ski is a transcriptional co-repressor and is involved in the negative regulation of tumor growth factor-beta (TGF-beta) signaling. To understand more fully the role of Ski in TGF-beta signaling, we searched for novel Ski-interacting proteins. The identified C184M protein consists of 189 amino acids and contains the leucine-rich region. An association between Ski and C184M involving the leucine-rich region of C184M and the C-terminal coiled-coil motif of Ski was confirmed by glutathione S-transferase pull-down and immunoprecipitation assays. The C184M protein is located in the cytosol, and the C184M and Ski signals are co-localized in the cytoplasm when C184M was co-expressed with Ski in CV-1 cells. The cytoplasmic C184M-Ski complex inhibited the nuclear translocation of Smad2. Consistent with this, the activity of promoter containing the Smad-binding sites was repressed by C184M, and the TGF-beta-induced growth inhibition of mink lung Mv1Lu cells was attenuated by the ectopic expression of C184M. Thus, C184M inhibits TGF-beta signaling in concert with Ski. In hepatocytes, which express significant levels of C184M, the Ski signals were found only in the cytoplasm, supporting the notion that C184M forms a complex with Ski in the cytosol.
Assuntos
Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/fisiologia , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Coativadores de Receptor Nuclear , Ligação Proteica , Transporte Proteico , Proteínas Smad , Fatores de Transcrição , Técnicas do Sistema de Duplo-HíbridoRESUMO
Recently we have shown that the c-myb proto-oncogene product (c-Myb) is degraded in response to Wnt-1 signaling via the pathway involving TAK1 (transforming growth factor-beta-activated kinase), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). NLK and HIPK2 bind directly to c-Myb, which results in the phosphorylation of c-Myb at multiple sites, followed by its ubiquitination and proteasome-dependent degradation. The v-myb gene carried by avian myeloblastosis virus has a transforming capacity, but the c-myb proto-oncogene does not. Here, we report that two characteristics of v-Myb make it relatively resistant to Wnt-1-induced protein degradation. First, HIPK2 binds with a lower affinity to the DNA-binding domain of v-Myb than to that of c-Myb. The mutations of three hydrophobic amino acids on the surface of the DNA-binding domain in v-Myb decrease the affinity to HIPK2. Second, a loss of multiple NLK phosphorylation sites by truncation of the C-terminal region of c-Myb increases its stability. Among 15 putative NLK phosphorylation sites in mouse c-Myb, the phosphorylation sites in the C-terminal region are more critical than other sites for Wnt-1-induced protein degradation. The relative resistance of v-Myb to Wnt-1-induced degradation may explain, at least in part, the differential transforming capacity of v-Myb versus c-Myb.
Assuntos
Proteínas Oncogênicas v-myb/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Western Blotting , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Cloranfenicol O-Acetiltransferase/metabolismo , DNA/química , DNA/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Glutationa Transferase/metabolismo , Humanos , Leucina/química , Camundongos , Modelos Biológicos , Mutação , Fosforilação , Plasmídeos/metabolismo , Mutação Puntual , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proto-Oncogene Mas , Transdução de Sinais , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Proteínas Wnt , Proteína Wnt1RESUMO
Multiple co-repressors such as N-CoR/SMRT, mSin3, and the c-ski proto-oncogene product (c-Ski) mediate the transcriptional repression induced by Mad and the thyroid hormone receptor by recruiting the histone deacetylase complex. c-Ski also binds directly to Smad proteins, which are transcriptional activators in the transforming growth factor-beta (TGF-beta)/bone morphogenetic protein (BMP) signaling pathways, and inhibits TGF-beta/BMP-induced transcriptional activation. However, it remains unknown whether other co-repressor(s) are also involved with Ski in the negative regulation of the TGF-beta/BMP signaling pathways. Here, we report that the co-repressor homeodomain-interacting protein kinase 2 (HIPK2) directly binds to both c-Ski and Smad1. HIPK2 efficiently inhibited Smad1/4-induced transcription from the Smad site-containing promoter. A dominant negative form of HIPK2, in which the ATP binding motif in the kinase domain and the putative phosphorylation sites were mutated, enhanced Smad1/4-dependent transcription and the BMP-induced expression of alkaline phosphatase. Furthermore, the c-Ski-induced inhibition of the Smad1/4-dependent transcription was suppressed by a dominant negative form of HIPK2. The HIPK2 co-repressor activity may be regulated by an uncharacterized HIPK2 kinase. These results indicate that HIPK2, together with c-Ski, plays an important role in the negative regulation of BMP-induced transcriptional activation.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/fisiologia , Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Ativação Transcricional , Trifosfato de Adenosina/metabolismo , Fosfatase Alcalina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Divisão Celular , Linhagem Celular , Genes Dominantes , Glutationa Transferase/metabolismo , Histona Desacetilases/metabolismo , Humanos , Luciferases/metabolismo , Microscopia de Fluorescência , Modelos Genéticos , Dados de Sequência Molecular , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Proto-Oncogene Mas , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteínas Smad , Proteína Smad1 , Transativadores/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
PML-RARalpha, a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-alpha (RARalpha), causes acute promyelocytic leukemias (APL). Although the role of nuclear PML-RARalpha has been extensively studied, a significant amount of PML-RARalpha is in the cytoplasm. The role cytoplasmic PML-RARalpha plays in leukemogenesis is unknown. Here we report that PML-RARalpha induces the N-CoR accumulation in the endoplasmic reticulum (ER), leading to the induction of ER stress and the processing of activating transcription factor 6 (ATF6), the unfolded protein response. PML-RARalpha stimulates the ubiquitylation of N-CoR via Ubc6 that is involved in the protein quality control. This ER-associated degradation (ERAD) of N-CoR reduces the soluble N-CoR protein levels in the nucleus. The two N-CoR-interacting sites in PML-RARalpha are required for the ERAD of N-CoR, suggesting the aberrant binding of PML-RARalpha to N-CoR may induce the ERAD of N-CoR. Overexpression of N-CoR induces the differentiation of APL-derived NB4 cells, suggesting that the low levels of N-CoR in the nucleus may contribute at least partly to PML-RARalpha-mediated leukemogenesis.
Assuntos
Retículo Endoplasmático/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/fisiologia , Proteínas Repressoras/metabolismo , Divisão Celular , Linhagem Celular , Núcleo Celular/metabolismo , Transformação Celular Neoplásica , Retículo Endoplasmático/metabolismo , Citometria de Fluxo , Humanos , Hidrólise , Imuno-Histoquímica , Leucemia Promielocítica Aguda/patologia , Mutagênese , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Correpressor 1 de Receptor Nuclear , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismoRESUMO
The c-myb proto-oncogene product (c-Myb) regulates proliferation of hematopoietic cells by inducing the transcription of a group of target genes. Removal or mutations of the negative regulatory domain (NRD) in the C-terminal half of c-Myb leads to increased transactivating capacity and oncogenic activation. Here we report that TIF1beta directly binds to the NRD and negatively regulates the c-Myb-dependent trans-activation. In addition, three corepressors (Ski, N-CoR, and mSin3A) bind to the DNA-binding domain of c-Myb together with TIF1beta and recruit the histone deacetylase complex to c-Myb. Furthermore, the Drosophila TIF1beta homolog, Bonus, negatively regulates Drosophila Myb activity. The Ski corepressor competes with the coactivator CBP for binding to c-Myb, indicating that the selection of coactivators and corepressors is a key event for c-Myb-dependent transcription. Mutations or deletion of the NRD of c-Myb and the mutations found in the DNA-binding domain of v-Myb decrease the interaction with these corepressors and weaken the corepressor-induced negative regulation of Myb activity. These observations have conceptual implications for understanding how the nuclear oncogene is activated.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Genes myb/genética , Proteínas Nucleares , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Fatores de Transcrição , Animais , Drosophila , Camundongos , Mutação , Proteínas Repressoras/metabolismo , Ativação Transcricional , Proteína 28 com Motivo TripartidoRESUMO
p53 is known to repress transcription of a number of genes, but the mechanism of p53 recruitment to these target genes is unknown. The c-myb proto-oncogene product (c-Myb) positively regulates proliferation of immature hematopoietic cells, whereas p53 blocks cell cycle progression. Here, we demonstrate that p53 inhibits c-Myb-induced transcription and transformation by directly binding to c-Myb. The ability of c-Myb to maintain the undifferentiated state of M1 cells was also suppressed by p53. p53 did not affect the ability of c-Myb to bind to DNA but formed a ternary complex with the corepressor mSin3A and c-Myb. Thus, p53 antagonizes c-Myb by recruiting mSin3A to down-regulate specific Myb target genes.
Assuntos
Proteínas Proto-Oncogênicas c-myb/fisiologia , Proteínas Repressoras/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Linhagem Celular , Proliferação de Células , DNA/química , DNA/metabolismo , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica , Glutationa Transferase/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunoprecipitação , Interleucina-6/metabolismo , Luciferases/metabolismo , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Estrutura Terciária de Proteína , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myb/metabolismo , RNA/química , Complexo Correpressor Histona Desacetilase e Sin3 , Transcrição Gênica , Transfecção , Regulação para CimaRESUMO
Transcription factor Glioblastoma-3 (Gli3) is cleaved in the anterior region of the limb bud to generate its repressor form. In contrast, Sonic hedgehog (Shh) signaling from the posterior zone of polarizing activity blocks Gli3 processing and then induces the expression of Gli3 target genes, including Gli1. Here we report that the Ski corepressor binds to Gli3 and recruits the histone deacetylase complex. The Gli3-mediated repression was impaired by anti-Ski antibody and in Ski-deficient fibroblasts, and Shh-induced Gli1 gene transcription mediated by full-length Gli3 was inhibited by Ski. Furthermore, a Ski mutation enhanced the digit abnormalities caused by the Gli3 gene mutation. Thus, Ski plays an important role in pattern formation.
Assuntos
Aciltransferases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Proteínas do Tecido Nervoso , Fatores de Transcrição/metabolismo , Transcrição Gênica , Aciltransferases/genética , Aciltransferases/imunologia , Animais , Anticorpos/farmacologia , Sítios de Ligação , Células Cultivadas , Proteínas de Ligação a DNA/genética , Fibroblastos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas Hedgehog , Histona Desacetilases/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like , Deformidades Congênitas dos Membros/genética , Camundongos , Camundongos Mutantes , Mutação , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Regiões Promotoras Genéticas , Ratos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido , Proteína GLI1 em Dedos de Zinco , Proteína Gli3 com Dedos de ZincoRESUMO
The c-myb proto-oncogene product (c-Myb) regulates both the proliferation and apoptosis of hematopoietic cells by inducing the transcription of a group of target genes. However, the biologically relevant molecular mechanisms that regulate c-Myb activity remain unclear. Here we report that c-Myb protein is phosphorylated and degraded by Wnt-1 signal via the pathway involving TAK1 (TGF-beta-activated kinase), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). Wnt-1 signal causes the nuclear entry of TAK1, which then activates HIPK2 and the mitogen-activated protein (MAP) kinase-like kinase NLK. NLK binds directly to c-Myb together with HIPK2, which results in the phosphorylation of c-Myb at multiple sites, followed by its ubiquitination and proteasome-dependent degradation. Furthermore, overexpression of NLK in M1 cells abrogates the ability of c-Myb to maintain the undifferentiated state of these cells. The down-regulation of Myb by Wnt-1 signal may play an important role in a variety of developmental steps.