RESUMO
The cellular response to hypoxia involves several signalling pathways that mediate adaptation and survival. REDD1 (regulated in development and DNA damage responses 1), a hypoxia-inducible factor-1 target gene, has a crucial role in inhibiting mammalian target of rapamycin complex 1 (mTORC1) signalling during hypoxic stress. However, little is known about the signalling pathways and post-translational modifications that regulate REDD1 function. Here, we show that REDD1 is subject to ubiquitin-mediated degradation mediated by the CUL4A-DDB1-ROC1-beta-TRCP E3 ligase complex and through the activity of glycogen synthase kinase 3beta. Furthermore, REDD1 degradation is crucially required for the restoration of mTOR signalling as cells recover from hypoxic stress. Our findings define a mechanism underlying REDD1 degradation and its importance for regulating mTOR signalling.
Assuntos
Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Quinases/metabolismo , Fatores de Transcrição/fisiologia , Proteínas de Transporte/metabolismo , Hipóxia Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Proteínas de Ligação a DNA/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Immunoblotting , Fosforilação , Estabilidade Proteica , Inibidores da Síntese de Proteínas/farmacologia , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismoRESUMO
p27, an important cell cycle regulator, blocks the G(1)/S transition in cells by binding and inhibiting Cdk2/cyclin A and Cdk2/cyclin E complexes (Cdk2/E). Ubiquitination and subsequent degradation play a critical role in regulating the levels of p27 during cell cycle progression. Here we provide evidence suggesting that both Cdk2/E and phosphorylation of Thr(187) on p27 are essential for the recognition of p27 by the SCF(Skp2/Cks1) complex, the ubiquitin-protein isopeptide ligase (E3). Cdk2/E provides a high affinity binding site, whereas the phosphorylated Thr(187) provides a low affinity binding site for the Skp2/Cks1 complex. Furthermore, binding of phosphorylated p27/Cdk2/E to the E3 complex showed positive cooperativity. Consistently, p27 is also ubiquitinated in a similarly cooperative manner. In the absence of p27, Cdk2/E and Cks1 increase Skp2 phosphorylation. This phosphorylation enhances Skp2 auto-ubiquitination, whereas p27 inhibits both phosphorylation and auto-ubiquitination of Skp2.
Assuntos
Proteínas de Transporte/química , Quinases Ciclina-Dependentes/química , Complexos Multiproteicos/química , Processamento de Proteína Pós-Traducional , Proteínas Quinases Associadas a Fase S/química , Ubiquitina-Proteína Ligases/química , Animais , Quinases relacionadas a CDC2 e CDC28 , Proteínas de Transporte/metabolismo , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Ciclina A/química , Ciclina A/metabolismo , Ciclina E/química , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/química , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Fase G1/fisiologia , Humanos , Complexos Multiproteicos/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fase S/fisiologia , Proteínas Quinases Associadas a Fase S/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Several approaches have been used in an effort to identify proteins that interact with beta-amyloid precursor protein (APP). However, few studies have addressed the identification of proteins associated with APP in brain tissue from patients with Alzheimer's disease. We report the results of a pilot proteomic study performed on complexes immunoprecipitated with APP in brain samples of patients with Alzheimer's disease and normal control subjects. The 21 proteins identified could be grouped into five functional classes: molecular chaperones, cytoskeletal and structural proteins, proteins involved in trafficking, adaptors, and enzymes. Among the proteins identified, six had been reported previously as direct, indirect, or genetically inferred APP interactors. The other 15 proteins immunoprecipitated with APP were novel potential partners. We confirmed the APP interaction by Western blotting and coimmunolocalization in brain tissues, for 5 of the 21 interactors. In agreement with previous studies, our results are compatible with an involvement of APP in axonal transport and vesicular trafficking, and with a potential association of APP with cellular protein folding/protein degradation systems.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteômica/métodos , Precursor de Proteína beta-Amiloide/química , Western Blotting/métodos , Encéfalo/patologia , Cristalinas/metabolismo , Dinaminas/metabolismo , Eletroforese em Gel Bidimensional/métodos , Humanos , Imuno-Histoquímica/métodos , Imunoprecipitação/métodos , Projetos PilotoRESUMO
Relaxation values reflecting residue-specific line broadening revealed amino acids in the DNA-binding domain of PU.1 on a surface potentially involved in protein-protein interactions. Mutation of these amino acids did not cause protein unfolding but destabilized PU.1-DNA binding. Addition of IFN response factor 4 to form the ternary complex recovered binding stability. Fluorescence quenching experiments proved that this surface of PU.1 interacts with IFN response factor 4 during binding. Our results provide evidence that residues that display increased conformational exchange can be used to predict areas of protein-protein interactions.
