RESUMO
The PBX1 homeodomain transcription factor is converted by t(1;19) chromosomal translocations in acute leukemia into the chimeric E2A-PBX1 oncoprotein. Fusion with E2A confers potent transcriptional activation and constitutive nuclear localization, bypassing the need for dimerization with protein partners that normally stabilize and regulate import of PBX1 into the nucleus, but the mechanisms underlying its oncogenic activation are incompletely defined. We demonstrate here that E2A-PBX1 self-associates through the PBX1 PBC-B domain of the chimeric protein to form higher-order oligomers in t(1;19) human leukemia cells, and that this property is required for oncogenic activity. Structural and functional studies indicate that self-association facilitates the binding of E2A-PBX1 to DNA. Mutants unable to self-associate are transformation defective, however their oncogenic activity is rescued by the synthetic oligomerization domain of FKBP, which confers conditional transformation properties on E2A-PBX1. In contrast to self-association, PBX1 protein domains that mediate interactions with HOX DNA-binding partners are dispensable. These studies suggest that oligomeric self-association may compensate for the inability of monomeric E2A-PBX1 to stably bind DNA and circumvents protein interactions that otherwise modulate PBX1 stability, nuclear localization, DNA binding, and transcriptional activity. The unique dependence on self-association for E2A-PBX1 oncogenic activity suggests potential approaches for mechanism-based targeted therapies.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinogênese/genética , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/genética , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Cromossomos Humanos Par 1/química , Cromossomos Humanos Par 19/química , DNA de Neoplasias/metabolismo , Células HEK293 , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas de Fusão Oncogênica/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo , Transcrição Gênica , Translocação GenéticaRESUMO
Human monogenic obesity syndromes, including Bardet-Biedl syndrome (BBS), implicate neuronal primary cilia in regulation of energy homeostasis. Cilia in hypothalamic neurons have been hypothesized to sense and regulate systemic energy status, but the molecular mechanism of this signaling remains unknown. Here, we report a comprehensive localization screen of 42 G-protein-coupled receptors (GPCR) revealing seven ciliary GPCRs, including the neuropeptide Y (NPY) receptors NPY2R and NPY5R. We show that mice modeling BBS disease or obese tubby mice fail to localize NPY2R to cilia in the hypothalamus and that BBS mutant mice fail to activate c-fos or decrease food intake in response to the NPY2R ligand PYY3-36. We find that cells with ciliary NPY2R show augmented PYY3-36-dependent cAMP signaling. Our data demonstrate that ciliary targeting of NPY receptors is important for controlling energy balance in mammals, revealing a physiologically defined ligand-receptor pathway signaling within neuronal cilia.
Assuntos
Síndrome de Bardet-Biedl/metabolismo , Neurônios/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Transdução de Sinais , Animais , Depressores do Apetite/farmacologia , Síndrome de Bardet-Biedl/genética , Linhagem Celular , Células Cultivadas , Cílios/metabolismo , AMP Cíclico/metabolismo , Ingestão de Alimentos/efeitos dos fármacos , Humanos , Hipotálamo/citologia , Hipotálamo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/farmacologia , Peptídeo YY/farmacologia , Transporte Proteico , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The primary cilium is required for Sonic hedgehog (Shh) signaling in vertebrates. In contrast to mutants affecting ciliary assembly, mutations in the intraflagellar transport complex A (IFT-A) paradoxically cause increased Shh signaling. We previously showed that the IFT-A complex, in addition to its canonical role in retrograde IFT, binds to the tubby-like protein, Tulp3, and recruits it to cilia. Here, we describe a conserved vertebrate G-protein-coupled receptor, Gpr161, which localizes to primary cilia in a Tulp3/IFT-A-dependent manner. Complete loss of Gpr161 in mouse causes midgestation lethality and increased Shh signaling in the neural tube, phenocopying Tulp3/IFT-A mutants. Constitutive Gpr161 activity increases cAMP levels and represses Shh signaling by determining the processing of Gli3 to its repressor form. Conversely, Shh signaling directs Gpr161 to be internalized from cilia, preventing its activity. Thus, Gpr161 defines a morphogenetic pathway coupling protein kinase A activation to Shh signaling during neural tube development.
Assuntos
Cílios/metabolismo , Embrião de Mamíferos/metabolismo , Tubo Neural/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Dados de Sequência Molecular , Filogenia , Proteínas/metabolismo , Receptores Acoplados a Proteínas G/química , Alinhamento de SequênciaRESUMO
The anaphase-promoting complex/cyclosome (APC/C) inhibitor Emi1 controls progression to S phase and mitosis by stabilizing key APC/C ubiquitination substrates, including cyclin A. Examining Emi1 binding proteins, we identified the Evi5 oncogene as a regulator of Emi1 accumulation. Evi5 antagonizes SCF(betaTrCP)-dependent Emi1 ubiquitination and destruction by binding to a site adjacent to Emi1's DSGxxS degron and blocking both degron phosphorylation by Polo-like kinases and subsequent betaTrCP binding. Thus, Evi5 functions as a stabilizing factor maintaining Emi1 levels in S/G2 phase. Evi5 protein accumulates in early G1 following Plk1 destruction and is degraded in a Plk1- and ubiquitin-dependent manner in early mitosis. Ablation of Evi5 induces precocious degradation of Emi1 by the Plk/SCF(betaTrCP) pathway, causing premature APC/C activation; cyclin destruction; cell-cycle arrest; centrosome overduplication; and, finally, mitotic catastrophe. We propose that the balance of Evi5 and Polo-like kinase activities determines the timely accumulation of Emi1 and cyclin, ensuring mitotic fidelity.
Assuntos
Anáfase/fisiologia , Proteínas de Ciclo Celular/metabolismo , Ciclina A/metabolismo , Proteínas Nucleares/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/farmacologia , Linhagem Celular , Proteínas F-Box , Proteínas Ativadoras de GTPase , Células HeLa , Humanos , Interfase , Modelos Biológicos , Proteínas Nucleares/genética , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Xenopus , Quinase 1 Polo-LikeRESUMO
Unfertilized vertebrate eggs are arrested in metaphase of meiosis II with high cyclin B/Cdc2 activity to prevent parthenogenesis. Until fertilization, exit from metaphase is blocked by an activity called cytostatic factor (CSF), which stabilizes cyclin B by inhibiting the anaphase-promoting complex (APC) ubiquitin ligase. The APC inhibitor early mitotic inhibitor 1 (Emi1) was recently found to be required for maintenance of CSF arrest. We show here that exogenous Emi1 is unstable in CSF-arrested Xenopus eggs and is destroyed by the SCF(betaTrCP) ubiquitin ligase, suggesting that endogenous Emi1, an apparent 44-kDa protein, requires a stabilizing factor. However, anti-Emi1 antibodies crossreact with native Emi2/Erp1/FBXO43, a homolog of Emi1 and conserved APC inhibitor. Emi2 is stable in CSF-arrested eggs, is sufficient to prevent CSF release, and is rapidly degraded in a Polo-like kinase 1-dependent manner in response to calcium-mediated egg activation. These results identify Emi2 as a candidate CSF maintenance protein.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-mos/metabolismo , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Anáfase/fisiologia , Ciclossomo-Complexo Promotor de Anáfase , Animais , Sinalização do Cálcio , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/imunologia , Reações Cruzadas , Ciclina B/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/imunologia , Feminino , Técnicas In Vitro , Meiose/fisiologia , Dados de Sequência Molecular , Oócitos/crescimento & desenvolvimento , Xenopus/genética , Xenopus/crescimento & desenvolvimento , Proteínas de Xenopus/genética , Proteínas de Xenopus/imunologiaRESUMO
Progression through mitosis requires activation of cyclin B/Cdk1 and its downstream targets, including Polo-like kinase and the anaphase-promoting complex (APC), the ubiquitin ligase directing degradation of cyclins A and B. Recent evidence shows that APC activation requires destruction of the APC inhibitor Emi1. In prophase, phosphorylation of Emi1 generates a D-pS-G-X-X-pS degron to recruit the SCF(betaTrCP) ubiquitin ligase, causing Emi1 destruction and allowing progression beyond prometaphase, but the kinases directing this phosphorylation remain undefined. We show here that the polo-like kinase Plk1 is strictly required for Emi1 destruction and that overexpression of Plk1 is sufficient to trigger Emi1 destruction. Plk1 stimulates Emi1 phosphorylation, betaTrCP binding, and ubiquitination in vitro and cyclin B/Cdk1 enhances these effects. Plk1 binds to Emi1 in mitosis and the two proteins colocalize on the mitotic spindle poles, suggesting that Plk1 may spatially control Emi1 destruction. These data support the hypothesis that Plk1 activates the APC by directing the SCF-dependent destruction of Emi1 in prophase.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Complexos Ubiquitina-Proteína Ligase/metabolismo , Anáfase , Ciclossomo-Complexo Promotor de Anáfase , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Polaridade Celular , Ativação Enzimática , Proteínas F-Box , Humanos , Mitose , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/genética , Fuso Acromático/metabolismo , Fatores de Tempo , Ubiquitina/metabolismo , Proteínas de Xenopus , Xenopus laevis , Quinase 1 Polo-LikeRESUMO
Progression through mitosis occurs because cyclin B/Cdc2 activation induces the anaphase promoting complex (APC) to cause cyclin B destruction and mitotic exit. To ensure that cyclin B/Cdc2 does not prematurely activate the APC in early mitosis, there must be a mechanism delaying APC activation. Emi1 is a protein capable of inhibiting the APC in S and G2. We show here that Emi1 is phosphorylated by Cdc2, and on a DSGxxS consensus site, is subsequently recognized by the SCF(betaTrCP/Slimb) ubiquitin ligase and destroyed, thus providing a delay for APC activation. Failure of betaTrCP-dependent Emi1 destruction stabilizes APC substrates and results in mitotic catastrophe including centrosome overduplication, potentially explaining mitotic deficiencies in Drosophila Slimb/betaTrCP mutants. We hypothesize that Emi1 destruction relieves a late prophase checkpoint for APC activation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Mitose , Peptídeo Sintases/metabolismo , Proteínas de Xenopus , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Sequência Consenso , Ciclina A/metabolismo , Ciclina B/metabolismo , Proteínas de Drosophila , Ativação Enzimática , Proteínas F-Box , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Humanos , Mitose/efeitos dos fármacos , Modelos Biológicos , Mutação , Nocodazol/farmacologia , Oócitos/citologia , Oócitos/fisiologia , Fosforilação , Proteínas Ligases SKP Culina F-Box , Suínos , Fatores de Tempo , Xenopus , Proteínas Contendo Repetições de beta-TransducinaRESUMO
The AP2 transcription factor family is a set of developmentally regulated, retinoic acid inducible genes composed of four related factors, AP2alpha, AP2beta, AP2gamma, and AP2delta. AP2 factors orchestrate a variety of cell processes including apoptosis, cell growth, and tissue differentiation during embryogenesis. In studies of primary malignancies, AP2alpha has been shown to function as a tumor suppressor in breast cancer, colon cancer, and malignant melanoma. In cell culture models, overexpression of AP2alpha inhibits cell division and stable colony formation, whereas, a dominant-negative AP2alpha mutant increases invasiveness and tumorigenicity. Here we show that AP2alpha targets the p53 tumor suppressor protein. Studies with chromatin immunoprecipitation demonstrate that AP2alpha is brought to p53 binding sites in p53-regulated promoters. The interaction between AP2alpha and p53 augments p53-mediated transcriptional activation, which results in up-regulation of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). AP2alpha is able to induce G(1) and G(2) cell cycle arrest only in the presence of wild-type p53. Thus, we conclude that the tumor suppressor activity of AP2alpha is mediated through a direct interaction with p53. These results also provide a mechanism to explain patterns of gene expression in cancers where AP2alpha is known to function as a tumor suppressor.