期刊
BIOCHEMICAL JOURNAL
卷 410, 期 -, 页码 473-484出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20071252
关键词
activating transcription factor 4 (ATF4); asparagine synthetase; basic leucine zipper (bZIP); CCAAT/enhancer-binding protein (C/EBP); nutrient regulation; System A neutral amino acid transporter 2 (SNAT2)
资金
- NIDDK NIH HHS [R01 DK070647, R01 DK052064, R01 DK052064-13, DK-70647] Funding Source: Medline
A nutrient stress signalling pathway is triggered in response to protein or amino acid deprivation, namely the AAR (amino acid response), and previous studies have shown that C/EBP beta (CCAAT/enhancer-binding protein beta) expression is up-regulated following activation of the AAR. DNA-binding studies, both in vitro and in vivo, have revealed increased C/EBP beta association with AARE (AAR element) sequences in AAR target genes, but its role is still unresolved. The present results show that in HepG2 human hepatoma cells, the total amount of C/EBP beta protein, both the activating [LAP* and LAP (liver-enriched activating protein)] and inhibitory [LIP (liver-enriched inhibitory)] isoforms, was increased in histidine-deprived cells. Immunoblotting of subcellular fractions and immunostaining revealed that most of the C/EBP beta was located in the nucleus. Consistent with these observations, amino acid limitation caused an increase in C/EBP beta DNA-binding activity in nuclear extracts and chromatin immunoprecipitation revealed an increase in C/EBP beta binding to the AARE region in vivo, but at a time when transcription from the target gene was declining. A constant fraction of the basal and increased C/EBP beta protein was phosphorylated on Thr(235) and the phospho-C/EBP beta did bind to an AARE. Induction of AARE-enhanced transcription was slightly greater in C/EBP beta-deficient MEFs (mouse embryonic fibroblasts) or C/EBP beta siRNA (small interfering RNA)-treated HepG2 cells compared with the corresponding control cells. Transient expression of LAP*, LAP or LIP in C/EBP beta-deficient fibroblasts caused suppression of increased transcription from an AARE-driven reporter gene. Collectively, the results demonstrate that C/EBP beta is not required for transcriptional activation by the AAR pathway but, when present, acts in concert with ATF3 (activating transcription factor 3) to suppress transcription during the latter stages of the response.
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