RESUMO
The prostate apoptosis response-4 (Par-4) protein has been shown to function as an effector of cell death in response to various apoptotic stimuli, and down-regulation of this protein has been suggested to be a key event during tumorigenesis. Several studies suggest an essential function for the COOH-terminal leucine repeats/death domain of Par-4 in mediating apoptosis. We investigated the biological role of this domain in vivo by generating knock-out mice expressing a Par-4 mutant protein lacking the COOH terminus domain. We found that the Par-4 mutant mice are viable and fertile with no overt phenotype, thus excluding an essential role for the COOH terminus domain of Par-4 in embryogenesis and developmental apoptosis. To determine the requirement of Par-4 for apoptosis, we treated primary fibroblasts with various stimuli that trigger mitochondria and membrane receptor cell death pathways. Fibroblasts isolated from Par-4 mutant mice are as sensitive as the wild-type cells to these apoptosis-inducing agents. Similar effects were observed following RNA interference (RNAi)-mediated knockdown of Par-4 in these cells. In contrast, RNAi-mediated depletion of Par-4 in HeLa cells resulted in a significant inhibition of apoptosis induced by various proapoptotic agents. Taken together, our findings provide strong genetic evidence that the proapoptotic function of Par-4 is dependent on the cellular context and raise the possibility that alterations of Par-4 function may occur during carcinogenesis.
Assuntos
Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/fisiologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Calcimicina/farmacologia , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Células HeLa , Humanos , Ionomicina/farmacologia , Camundongos , Camundongos Knockout , Estrutura Terciária de Proteína , Interferência de RNA , Estaurosporina/farmacologiaRESUMO
Yin Yang 1 (YY1) is a transcription factor that plays an essential role in development. However, the full spectrum of YY1's functions and mechanism of action remains unclear. We find that YY1 ablation results in p53 accumulation due to a reduction of p53 ubiquitination in vivo. Conversely, YY1 overexpression stimulates p53 ubiquitination and degradation. Significantly, recombinant YY1 is sufficient to induce Hdm2-mediated p53 polyubiquitination in vitro, suggesting that this function of YY1 is independent of its transcriptional activity. We identify direct physical interactions of YY1 with Hdm2 and p53 and show that the basis for YY1-regulating p53 ubiquitination is its ability to facilitate Hdm2-p53 interaction. Importantly, the tumor suppressor p14ARF compromises the Hdm2-YY1 interaction, which is important for YY1 regulation of p53. Taken together, these findings identify YY1 as a potential cofactor for Hdm2 in the regulation of p53 homeostasis and suggest a possible role for YY1 in tumorigenesis.
Assuntos
Linfócitos B/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-bcl-2 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Apoptose , Linfócitos B/citologia , Western Blotting , Divisão Celular , Linhagem Celular Tumoral , Galinhas , Deleção de Genes , Glutationa Transferase/metabolismo , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/deficiência , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , RNA Interferente Pequeno/genética , Proteínas Recombinantes/metabolismo , Retroviridae/genética , Fatores de Transcrição/química , Fatores de Transcrição/deficiência , Transgenes , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Ubiquitina/metabolismo , Proteína X Associada a bcl-2RESUMO
Lymphoid enhancer factor/T-cell factor (LEF/TCF) are transcription factors that mediate the Wnt signaling pathway, and have crucial roles during embryonic development in various organisms. Here we report that acetylation enhances nuclear retention of POP-1, the Caenorhabditis elegans LEF/TCF homolog, through increasing nuclear import and blocking nuclear export. We identify three lysines that are acetylated in vivo, and demonstrate their essential requirement for proper nuclear localization and biological activity of POP-1 during C. elegans embryogenesis. The conservation of these lysines among other LEF/TCF family members suggests that acetylation may be an important, evolutionarily conserved mechanism regulating subcellular distribution of LEF/TCF factors.