RESUMO
We propose a biochemical mechanism by which Daxx modulates NF-kappaB transcriptional activity. Both chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA) have confirmed Daxx-mediated repression of transcriptional competence of NF-kappaB in HeLa cells. Overexpression of Daxx repressed the expression of NF-kappaB-regulated genes such as I kappa B alpha and IL8. Co-immunoprecipitation assay revealed the existence of intermolecular association between endogenous Daxx and p65 subunit of NF-kappaB stimulated by TNFalpha. Here, we suggest that Daxx-mediated repression of NF-kappaB transactivation correlates with the inhibition of p65 acetylation by Daxx. Based on the finding that the Daxx binding N-terminal side of p65 includes the major sites of acetylation mediated by p300/CBP, we further propose that the physical interaction between Daxx and p65 provides a functional framework for the inhibition of p65 acetylation by p300/CBP and subsequent repression of NF-kappaB transcriptional activity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Nucleares/metabolismo , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/genética , Ativação Transcricional/genética , Acetilação , Núcleo Celular/metabolismo , Proteínas Correpressoras , Proteína p300 Associada a E1A/metabolismo , Células HeLa , Humanos , Chaperonas Moleculares , Fosforilação , Ligação Proteica , Transporte Proteico , Frações Subcelulares/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
Daxx is a multifunctional protein that regulates a variety of cellular processes, including transcription, cell cycle, and apoptosis. SPOP is a BTB (Bric-a-brac/Tramtrack/Broad complex) protein that constitutes Cul3-based ubiquitin ligases. Here we show that SPOP serves as an adaptor of Daxx for the ubiquitination by Cul3-based ubiquitin ligase and subsequent degradation by the proteasome. Expression of SPOP with Cul3 markedly reduced Daxx level, and this degradation was blocked by SPOP-specific short hairpin RNAs. Inhibition of the proteasome by MG132 caused the prevention of Daxx degradation in parallel with the accumulation of ubiquitinated Daxx. Expression of SPOP with Cul3 reversed Daxx-mediated repression of ETS1- and p53-dependent transcription, and short hairpin RNA-mediated knock down of SPOP blocked the recovery of their transcriptional activation. Furthermore, Daxx degradation led to the cleavage of poly(ADP-ribose) polymerase and the increase in the number of terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end-labeling-positive apoptotic cells. These results suggest that SPOP/Cul3-ubiquitin ligase plays an essential role in the control of Daxx level and, thus, in the regulation of Daxx-mediated cellular processes, including transcriptional regulation and apoptosis.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas Culina/química , Proteínas Nucleares/fisiologia , Proteínas Repressoras/fisiologia , Ubiquitina-Proteína Ligases/química , Ubiquitina/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose , Células COS , Chlorocebus aethiops , Proteínas Correpressoras , Células HeLa , Humanos , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Terciária de Proteína , Proteína Supressora de Tumor p53/metabolismoRESUMO
Daxx-mediated transcriptional repression was modulated by a speckled POZ domain protein SPOP which was first identified as an autoantigen from the serum of a scleroderma patient. This is the first report on the biochemical and functional interactions between Daxx and SPOP. The COOH-terminal region of Daxx interacts with the NH2-terminal region of SPOP. SPOP reversed the transcriptional repression mediated by Daxx which binds with ETS1 transcription factor to repress ETS1-responsive gene expression. Mutagenesis study suggests that the ability of SPOP to self-associate as well as its ability to bind with Daxx was important for the modulation of Daxx-mediated transcriptional repression.
Assuntos
Rim/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Proteínas Nucleares/metabolismo , Transcrição Gênica/genética , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Regulação da Expressão Gênica/fisiologia , Humanos , Metaloproteinase 1 da Matriz/genética , Proteínas Nucleares/genética , Proteínas Repressoras , Relação Estrutura-AtividadeRESUMO
The 14-3-3 epsilon protein was identified as one of the caspase-3 substrates by the modified yeast two-hybrid system. The cellular 14-3-3 epsilon protein was also cleaved in response to the treatment of apoptosis inducers in cultured mammalian cells. Asp238 of the 14-3-3 epsilon protein was determined as the site of cleavage by caspase-3. The affinity of the cleaved 14-3-3 mutant protein (D238) to Bad, a death-promoting Bcl-2 family protein, was lower than that of wild type or the uncleavable mutant 14-3-3 epsilon protein (D238A). However, Bad associated with the cellular Bcl-x(L) more effectively in human 293T cells co-expressing Bad with the truncated form of the 14-3-3 epsilon protein (D238) than in control cells co-expressing Bad with wild type or the uncleavable mutant 14-3-3 epsilon protein (D238A). The present study suggests that the cleavage of 14-3-3 protein during apoptosis promotes cell death by releasing the associated Bad from the 14-3-3 protein and facilitates Bad translocation to the mitochondria and its interaction with Bcl-x(L).