RESUMO
Microtubules have pivotal roles in fundamental cellular processes and are targets of antitubulin chemotherapeutics. Microtubule-targeted agents such as Taxol and vincristine are prescribed widely for various malignancies, including ovarian and breast adenocarcinomas, non-small-cell lung cancer, leukaemias and lymphomas. These agents arrest cells in mitosis and subsequently induce cell death through poorly defined mechanisms. The strategies that resistant tumour cells use to evade death induced by antitubulin agents are also unclear. Here we show that the pro-survival protein MCL1 (ref. 3) is a crucial regulator of apoptosis triggered by antitubulin chemotherapeutics. During mitotic arrest, MCL1 protein levels decline markedly, through a post-translational mechanism, potentiating cell death. Phosphorylation of MCL1 directs its interaction with the tumour-suppressor protein FBW7, which is the substrate-binding component of a ubiquitin ligase complex. The polyubiquitylation of MCL1 then targets it for proteasomal degradation. The degradation of MCL1 was blocked in patient-derived tumour cells that lacked FBW7 or had loss-of-function mutations in FBW7, conferring resistance to antitubulin agents and promoting chemotherapeutic-induced polyploidy. Additionally, primary tumour samples were enriched for FBW7 inactivation and elevated MCL1 levels, underscoring the prominent roles of these proteins in oncogenesis. Our findings suggest that profiling the FBW7 and MCL1 status of tumours, in terms of protein levels, messenger RNA levels and genetic status, could be useful to predict the response of patients to antitubulin chemotherapeutics.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Fibroblastos , Humanos , Camundongos , Mitose/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Paclitaxel/farmacologia , Farmacogenética , Fosforilação/efeitos dos fármacos , Poliploidia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Vincristina/farmacologiaRESUMO
NF-kappaB transcription factors mediate the effects of pro-inflammatory cytokines such as tumour necrosis factor-alpha and interleukin-1beta. Failure to downregulate NF-kappaB transcriptional activity results in chronic inflammation and cell death, as observed in A20-deficient mice. A20 is a potent inhibitor of NF-kappaB signalling, but its mechanism of action is unknown. Here we show that A20 downregulates NF-kappaB signalling through the cooperative activity of its two ubiquitin-editing domains. The amino-terminal domain of A20, which is a de-ubiquitinating (DUB) enzyme of the OTU (ovarian tumour) family, removes lysine-63 (K63)-linked ubiquitin chains from receptor interacting protein (RIP), an essential mediator of the proximal TNF receptor 1 (TNFR1) signalling complex. The carboxy-terminal domain of A20, composed of seven C2/C2 zinc fingers, then functions as a ubiquitin ligase by polyubiquitinating RIP with K48-linked ubiquitin chains, thereby targeting RIP for proteasomal degradation. Here we define a novel ubiquitin ligase domain and identify two sequential mechanisms by which A20 downregulates NF-kappaB signalling. We also provide an example of a protein containing separate ubiquitin ligase and DUB domains, both of which participate in mediating a distinct regulatory effect.
Assuntos
NF-kappa B/metabolismo , Proteínas/química , Proteínas/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA , Regulação para Baixo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Nucleares , Estrutura Terciária de Proteína , Proteínas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores , Fator 2 Associado a Receptor de TNF , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
The dual specificity (Tyr/Thr) phosphatase Cdc25A activates cyclin-dependent kinases (Cdks) to promote cell-cycle progression and has significant oncogenic potential. Cdc25A protein levels are regulated tightly in normal tissues, but many human cancers overexpress Cdc25A. The underlying mechanism for overexpression has been enigmatic. Here we show that Cdc25A is stabilized by the ubiquitin hydrolase Dub3. Upon binding Cdc25A, Dub3 removes the polyubiquitin modifications that mark Cdc25A for proteasomal degradation. Dub3 knockdown in cells increased Cdc25A ubiquitylation and degradation, resulting in reduced Cdk/Cyclin activity and arrest at G1/S and G2/M phases of the cell cycle. In contrast, acute Dub3 overexpression produced a signature response to oncogene induction: cells accumulated in S and G2 because of replication stress, and activated a DNA damage response. Dub3 also transformed NIH-3T3 cells and cooperated with activated H-Ras to promote growth in soft agar. Importantly, we show that Dub3 overexpression is responsible for an abnormally high level of Cdc25A in a subset of human breast cancers. Moreover, Dub3 knockdown significantly retarded the growth of breast tumour xenografts in nude mice. As a major regulator of Cdc25A, Dub3 is an example of a transforming ubiquitin hydrolase that subverts a key component of the cell cycle machinery.
Assuntos
Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica/metabolismo , Endopeptidases/metabolismo , Oncogenes , Processamento de Proteína Pós-Traducional , Fosfatases cdc25/metabolismo , Animais , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Dano ao DNA , Endopeptidases/genética , Estabilidade Enzimática , Feminino , Regulação Neoplásica da Expressão Gênica , Genes myc , Genes ras , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA , Fatores de Tempo , Transfecção , Transplante Heterólogo , Carga Tumoral , Ubiquitinação , Fosfatases cdc25/genéticaRESUMO
Production of type I interferon (IFN-I) is a critical host defense triggered by pattern-recognition receptors (PRRs) of the innate immune system. Deubiquitinating enzyme A (DUBA), an ovarian tumor domain-containing deubiquitinating enzyme, was discovered in a small interfering RNA-based screen as a regulator of IFN-I production. Reduction of DUBA augmented the PRR-induced IFN-I response, whereas ectopic expression of DUBA had the converse effect. DUBA bound tumor necrosis factor receptor-associated factor 3 (TRAF3), an adaptor protein essential for the IFN-I response. TRAF3 is an E3 ubiquitin ligase that preferentially assembled lysine-63-linked polyubiquitin chains. DUBA selectively cleaved the lysine-63-linked polyubiquitin chains on TRAF3, resulting in its dissociation from the downstream signaling complex containing TANK-binding kinase 1. A discrete ubiquitin interaction motif within DUBA was required for efficient deubiquitination of TRAF3 and optimal suppression of IFN-I. Our data identify DUBA as a negative regulator of innate immune responses.
Assuntos
Endopeptidases/metabolismo , Interferon Tipo I/biossíntese , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Interferon Tipo I/genética , Interferon-alfa/genética , Dados de Sequência Molecular , NF-kappa B/metabolismo , Estrutura Terciária de Proteína , RNA Interferente Pequeno , Transdução de Sinais , Fator 3 Associado a Receptor de TNF/metabolismo , Receptor 3 Toll-Like/metabolismo , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Arabidopsis thaliana De-etiolated-1 (AtDET1) is a highly conserved protein, with orthologs in vertebrate and invertebrate organisms. AtDET1 negatively regulates photomorphogenesis, but its biochemical mechanism and function in other species are unknown. We report that human DET1 (hDET1) promotes ubiquitination and degradation of the proto-oncogenic transcription factor c-Jun by assembling a multisubunit ubiquitin ligase containing DNA Damage Binding Protein-1 (DDB1), cullin 4A (CUL4A), Regulator of Cullins-1 (ROC1), and constitutively photomorphogenic-1. Ablation of any subunit by RNA interference stabilized c-Jun and increased c-Jun-activated transcription. These findings characterize a c-Jun ubiquitin ligase and define a specific function for hDET1 in mammalian cells.