Deprivation of protein or amino acid induces C/EBPβ synthesis and binding to amino acid response elements, but its action is not an absolute requirement for enhanced transcription

Michelle M. Thiaville, Elizabeth E. Dudenhausen, Can Zhong, Yuan Xiang Pan, Michael S. Kilberg

Research output: Contribution to journalArticlepeer-review

Abstract

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/EBPβ (CCAAT/enhancer-binding protein β) expression is up-regulated following activation of the AAR. DNA-binding studies, both in vitro and in vivo, have revealed increased C/EBPβ 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/EBPβ 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/EBPβ was located in the nucleus. Consistent with these observations, amino acid limitation caused an increase in C/EBPβ DNA-binding activity in nuclear extracts and chromatin immunoprecipitation revealed an increase in C/EBPβ 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/EBPβ protein was phosphorylated on Thr235 and the phospho-C/EBPβ did bind to an AARE. Induction of AARE-enhanced transcription was slightly greater in C/EBPβ-deficient MEFs (mouse embryonic fibroblasts) or C/EBPβ siRNA (small interfering RNA)-treated HepG2 cells compared with the corresponding control cells. Transient expression of LAP*, LAP or LIP in C/EBPβ-deficient fibroblasts caused suppression of increased transcription from an AARE-driven reporter gene. Collectively, the results demonstrate that C/EBPβ 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.

Original languageEnglish (US)
Pages (from-to)473-484
Number of pages12
JournalBiochemical Journal
Volume410
Issue number3
DOIs
StatePublished - Mar 15 2008
Externally publishedYes

Keywords

  • 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)

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint

Dive into the research topics of 'Deprivation of protein or amino acid induces C/EBPβ synthesis and binding to amino acid response elements, but its action is not an absolute requirement for enhanced transcription'. Together they form a unique fingerprint.

Cite this