RESUMO
Notch receptors and their ligands play important roles in both normal animal development and pathogenesis. We show here that the F-box/WD40 repeat protein SEL-10 negatively regulates Notch receptor activity by targeting the intracellular domain of Notch receptors for ubiquitin-mediated protein degradation. Blocking of endogenous SEL-10 activity was done by expression of a dominant-negative form containing only the WD40 repeats. In the case of Notch1, this block leads to an increase in Notch signaling stimulated by either an activated form of the Notch1 receptor or Jagged1-induced signaling through Notch1. Expression of dominant-negative SEL-10 leads to stabilization of the intracellular domain of Notch1. The Notch4 intracellular domain bound to SEL-10, but its activity was not increased as a result of dominant-negative SEL-10 expression. SEL-10 bound Notch4 via the WD40 repeats and bound preferentially to a phosphorylated form of Notch4 in cells. We mapped the region of Notch4 essential for SEL-10 binding to the C-terminal region downstream of the ankyrin repeats. When this C-terminal fragment of Notch4 was expressed in cells, it was highly labile but could be stabilized by the expression of dominant-negative SEL-10. Ubiquitination of Notch1 and Notch4 intracellular domains in vitro was dependent on SEL-10. Although SEL-10 interacts with the intracellular domains of both Notch1 and Notch4, these proteins respond differently to interference with SEL-10 function. Thus, SEL-10 functions to promote the ubiquitination of Notch proteins; however, the fates of these proteins may differ.
Assuntos
Proteínas de Caenorhabditis elegans , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Proteínas de Helminto/metabolismo , Proteínas de Helminto/fisiologia , Proteínas de Membrana/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Animais , Western Blotting , Linhagem Celular , Cisteína Endopeptidases , Relação Dose-Resposta a Droga , Deleção de Genes , Genes Dominantes , Vetores Genéticos , Humanos , Insetos , Ligantes , Luciferases/metabolismo , Modelos Genéticos , Complexos Multienzimáticos/antagonistas & inibidores , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/metabolismo , Receptores NotchRESUMO
BACKGROUND: The function of the fission yeast cullins Pcu1p and Pcu4p requires modification by the ubiquitin-related peptide Ned8p. A recent report by Lyapina et al. shows that the COP9/signalosome (CSN), a multifunctional eight subunit complex, regulates Ned8p modification of Pcu1p. Disruption of caa1/csn1, which encodes subunit 1 of the putative S. pombe CSN, results in accumulation of Pcu1p exclusively in the modified form. However, it remained unclear whether this reflects global control of all cullins by the entire CSN complex. RESULTS: We demonstrate that multiple CSN subunits control Ned8p modification of Pcu3p, another fission yeast cullin, which, in complex with the RING domain protein Pip1p, forms a ubiquitin ligase that functions in cellular stress response. Pcu3p is modified by Ned8p on Lys 729 and accumulates exclusively in the neddylated form in cells lacking the CSN subunits 1, 3, 4, and 5. These CSN subunits co-elute with Pcu3p in gel filtration fractions corresponding to approximately 550 kDa and specifically bind both native and Ned8p-modified Pcu3p in vivo. While CSN does not influence the subcellular localization of Pcu3p, Pcu3p-associated in vitro ubiquitin ligase activity is stimulated in the absence of CSN. CONCLUSIONS: Taken together, our data suggest that CSN is a global regulator of Ned8p modification of multiple cullins and potentially other proteins involved in cellular regulation.
Assuntos
Ligases/metabolismo , Proteínas/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/metabolismo , Ubiquitinas/metabolismo , Complexo do Signalossomo COP9 , Sequência Conservada , Cisteína/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Teste de Complementação Genética , Ligases/análise , Ligases/química , Lisina/metabolismo , Substâncias Macromoleculares , Complexos Multiproteicos , Mutação , Peptídeo Hidrolases , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de Sinais , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
The COP9 signalosome is an evolutionary conserved multiprotein complex of unknown function that acts as a negative regulator of photomorphogenic seedling development in Arabidopsis. Here, we show that plants with reduced COP9 signalosome levels had decreased auxin response similar to loss-of-function mutants of the E3 ubiquitin ligase SCFTIR1. Furthermore, we found that the COP9 signalosome and SCFTIR1 interacted in vivo and that the COP9 signalosome was required for efficient degradation of PSIAA6, a candidate substrate of SCFTIR1. Thus, the COP9 signalosome may play an important role in mediating E3 ubiquitin ligase-mediated responses.
Assuntos
Arabidopsis/efeitos dos fármacos , Ácidos Indolacéticos/farmacologia , Ligases/metabolismo , Proteínas de Plantas/metabolismo , Proteínas/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Brassica , Complexo do Signalossomo COP9 , Escuridão , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes Reporter/genética , Ligases/genética , Complexos Multiproteicos , Mutação/genética , Pisum sativum , Peptídeo Hidrolases , Fenótipo , Proteínas de Plantas/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Testes de Precipitina , Ligação Proteica , Biossíntese de Proteínas , Subunidades Proteicas , Proteínas/genética , RNA Antissenso/genética , RNA de Plantas/genética , RNA de Plantas/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína LigasesRESUMO
SCF ubiquitin ligases control various processes by marking regulatory proteins for ubiquitin-dependent proteolysis. To illuminate how SCF complexes are regulated, we sought proteins that interact with the human SCF component CUL1. The COP9 signalosome (CSN), a suppressor of plant photomorphogenesis, associated with multiple cullins and promoted cleavage of the ubiquitin-like protein NEDD8 from Schizosaccharomyces pombe CUL1 in vivo and in vitro. Multiple NEDD8-modified proteins uniquely accumulated in CSN-deficient S. pombe cells. We propose that the broad spectrum of activities previously attributed to CSN subunits--including repression of photomorphogenesis, activation of JUN, and activation of p27 nuclear export--underscores the importance of dynamic cycles of NEDD8 attachment and removal in biological regulation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Culina , Proteínas/metabolismo , Ubiquitinas/metabolismo , Células 3T3 , Animais , Western Blotting , Complexo do Signalossomo COP9 , Proteínas de Ciclo Celular/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Células HeLa , Humanos , Espectrometria de Massas , Camundongos , Complexos Multiproteicos , Mutação/genética , Proteína NEDD8 , Peptídeo Hidrolases , Peptídeo Sintases/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Subunidades Proteicas , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ligases SKP Culina F-Box , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Especificidade por Substrato , Suínos , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Ubiquitinas/genéticaRESUMO
Centrosomes organize the mitotic spindle to ensure accurate segregation of the chromosomes in mitosis. The mechanism that ensures accurate duplication and separation of the centrosomes underlies the fidelity of chromosome segregation, but remains unknown. In Saccharomyces cerevisiae, entry into S phase and separation of spindle pole bodies each require CDC4 and CDC34, which encode components of an SCF (Skp1-cullin-F-box) ubiquitin ligase, but a direct (SCF) connection to the spindle pole body is unknown. Using immunofluorescence microscopy, we show that in mammalian cells the Skp1 protein and the cullin Cul1 are localized to interphase and mitotic centrosomes and to the cytoplasm and nucleus. Deconvolution and immunoelectron microscopy suggest that Skp1 forms an extended pericentriolar structure that may function to organize the centrosome. Purified centrosomes also contain Skp1, and Cul1 modified by the ubiquitin-like molecule NEDD8, suggesting a role for NEDD8 in targeting. Using an in vitro assay for centriole separation in Xenopus extracts, antibodies to Skp1 or Cul1 block separation. Proteasome inhibitors block both centriole separation in vitro and centrosome duplication in Xenopus embryos. We identify candidate centrosomal F-box proteins, suggesting that distinct SCF complexes may direct proteolysis of factors mediating multiple steps in the centrosome cycle.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/genética , Centrossomo/enzimologia , Proteínas F-Box , Peptídeo Sintases/metabolismo , Complexos Ubiquitina-Proteína Ligase , Ubiquitina-Proteína Ligases , Células 3T3 , Ciclossomo-Complexo Promotor de Anáfase , Animais , Células CHO , Proteínas de Ciclo Celular/genética , Centríolos/fisiologia , Centríolos/ultraestrutura , Centrossomo/ultraestrutura , Cricetinae , Proteína 7 com Repetições F-Box-WD , Feminino , Ligases/genética , Ligases/metabolismo , Camundongos , Proteína NEDD8 , Óvulo , Fase S , Proteínas Quinases Associadas a Fase S , Proteínas Ligases SKP Culina F-Box , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae , Fuso Acromático/fisiologia , Fuso Acromático/ultraestrutura , Extratos de Tecidos/fisiologia , Enzimas de Conjugação de Ubiquitina , Ubiquitinas/metabolismo , Xenopus laevisRESUMO
SCFCdc4 (Skp1, Cdc53/cullin, F-box protein) defines a family of modular ubiquitin ligases (E3s) that regulate diverse processes including cell cycle, immune response, and development. Mass spectrometric analysis of proteins copurifying with Cdc53 identified the RING-H2 finger protein Hrt1 as a subunit of SCF. Hrt1 shows striking similarity to the Apc11 subunit of anaphase-promoting complex. Conditional inactivation of hrt1(ts) results in stabilization of the SCFCdc4 substrates Sic1 and Cln2 and cell cycle arrest at G1/S. Hrt1 assembles into recombinant SCF complexes and individually binds Cdc4, Cdc53 and Cdc34, but not Skp1. Hrt1 stimulates the E3 activity of recombinant SCF potently and enables the reconstitution of Cln2 ubiquitination by recombinant SCFGrr1. Surprisingly, SCF and the Cdc53/Hrt1 subcomplex activate autoubiquitination of Cdc34 E2 enzyme by a mechanism that does not appear to require a reactive thiol. The highly conserved human HRT1 complements the lethality of hrt1Delta, and human HRT2 binds CUL-1. We conclude that Cdc53/Hrt1 comprise a highly conserved module that serves as the functional core of a broad variety of heteromeric ubiquitin ligases.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Culina , Proteínas F-Box , Ligases/metabolismo , Proteínas de Saccharomyces cerevisiae , Complexos Ubiquitina-Proteína Ligase , Ubiquitina-Proteína Ligases , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Animais , Subunidade Apc11 do Ciclossomo-Complexo Promotor de Anáfase , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Ativação Enzimática , Proteína 7 com Repetições F-Box-WD , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases Associadas a Fase S , Proteínas Ligases SKP Culina F-Box , Enzimas de Conjugação de UbiquitinaRESUMO
The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by catalyzing ubiquitination of the S phase cyclin-dependent kinase inhibitor SIC1. SCF is composed of three proteins-ySKP1, CDC53 (Cullin), and the F-box protein CDC4-that are conserved from yeast to humans. As part of an effort to identify components and substrates of a putative human SCF complex, we isolated hSKP1 in a two-hybrid screen with hCUL1, the closest human homologue of CDC53. Here, we show that hCUL1 associates with hSKP1 in vivo and directly interacts with both hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-like particle. Moreover, hCUL1 complements the growth defect of yeast cdc53(ts) mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. Taken together, these data suggest that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. Further application of biochemical assays similar to those described here can now be used to identify regulators/components of hCUL1-based SCF complexes, to determine whether the hCUL2-hCUL5 proteins also are components of ubiquitin ligase complexes in human cells, and to screen for chemical compounds that modulate the activities of the hSKP1 and hCUL1 proteins.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular/metabolismo , Proteínas Culina , Proteínas F-Box , Ligases/metabolismo , Proteínas de Saccharomyces cerevisiae , Sequência Conservada , Quinase 2 Dependente de Ciclina , Proteínas Inibidoras de Quinase Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Inibidores Enzimáticos/metabolismo , Evolução Molecular , Proteína 7 com Repetições F-Box-WD , Proteínas Fúngicas/metabolismo , Células HeLa , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Associadas a Fase S , Ubiquitina-Proteína Ligases , Ubiquitinas/metabolismoRESUMO
Possible reasons for retarded intracellular diffusion of ADP were investigated. The isolated skinned cardiac fibers were used to study apparent kinetic parameters for externally added ADP in control of mitochondrial respiration. Participation of myosin-ATPase in binding of ADP within cells as it was supposed earlier (Saks, V.A., Belikova, Yu.O. and Kuznetsov, A.V. (1991) Biochim. Biophys. Acta 1074, 302-311) was completely excluded, since myosin-deprived skinned cardiac fibers ('ghosts') displayed the same kinetic parameters as intact ones (Kmapp for ADP about 300 microM). Significantly lower apparent Km values were obtained for fibers with osmotically disrupted outer mitochondrial membrane (25-35 microM), which was close to that observed for isolated heart mitochondria. The data obtained are in favor of limitation of ADP movement via anion-selective low-conductance porine channels in the outer membrane of mitochondria. It is proposed that the permeability of this membrane is controlled by some unknown intracellular factor(s). In the presence of saturating concentrations of creatine (25 mM) the apparent Km for ADP significantly decreases due to coupling of creatine kinase and oxidative phosphorylation reactions in mitochondria. This coupling is not observed in KCl medium in which mitochondrial creatine kinase is detached from the membrane. It is concluded that in the cells in-vivo ADP movement between cytoplasm and intramitochondrial space is controlled by low-conductivity anion channels in the outer membrane. Thus, the mitochondrial creatine kinase reaction coupled to the adenine nucleotide translocase is an important mechanism in control of oxidative phosphorylation in vivo due to its ability to manifold amplify these very weak ADP signals from cytoplasm.