ATF4-dependent transcription mediates signaling of amino acid limitation

Abstract

Mammals respond to dietary nutrient fluctuations; for example, deficiency of dietary protein or an imbalance of essential amino acids activates an amino acid response (AAR) signal transduction pathway, consisting of detection of uncharged tRNA by the GCN2 kinase, eIF2alpha phosphorylation and ATF4 expression. In concert with heterodimerization partners, ATF4 activates specific genes via a CCAAT-enhancer binding protein-activating transcription factor response element (CARE). This review outlines the ATF4-dependent transcriptional mechanisms associated with the AAR, focusing on progress during the past 5 years. Recent evidence suggests that maternal nutrient deprivation not only has immediate metabolic effects on the fetus, but also triggers gene expression changes in adulthood, possibly through epigenetic mechanisms. Therefore, understanding the transcriptional programs initiated by amino acid limitation is crucial and timely.

Figure 1

Several cellular stress signals are transduced through four eukaryotic initiation factor 2α (eIF2α) kinases. (i) The eIF2α kinases general non-derepressible 2 (GCN2), double-stranded RNA-activated protein kinase (PKR), double-stranded RNA activated protein kinase-like ER kinase (PERK), and heme-regulated inhibitor kinase (HRI) are activated by a wide spectrum of cell stress signals and all phosphorylate eIF2α on serine 51. (ii) Phospho-eIF2α binds eIF2B in a non-functional complex and leads to (iii) suppression of global translation, but a paradoxical increase in translation of specific mRNA species, such as that for activating transcription factor 4 (ATF4). De-phosphorylation of eIF2α is mediated by protein phosphatase 1 (PP1) which is targeted to eIF2α by growth arrest and DNA damage-inducible 34 (GADD34).

Figure 2

Synthesis of activating transcription factor 4 (ATF4) is increased in response to cell stress. (a) The mRNA for ATF4 contains two short upstream open reading frames (uORF). (b) In normal protein/amino acid conditions, ribosomal scanning leads to translation of uORF1 and re-initiation at uORF2, which is out-of-frame and overlaps with the ATF4 coding sequence. Thus, little or no ATF4 is synthesized. (c) During low protein/amino acid conditions, general control non-derepressible 2 (GCN2) phosphorylates eIF2α, which in turn depletes the available GDP-GTP exchange factor, eukaryotic initiation factor 2B (eIF2B), by non-functional binding. Consequently, there is a reduction in active eIF2-GTP and a slower rate of re-initiation after uORF1 translation. As a result, ribosome assembly occurs after the AUG in uORF2 and coincident with translation of the ORF encoding ATF4. In this way, increased ATF4 protein production occurs only during periods of cellular stress.

Figure 3

The genomic C/EBP-ATF (CCAAT-enhancer binding protein-activating transcription factor) composite sites that function as ATF4-responsive C/EBP-ATF response elements (CARE) are composed of half sites for C/EBP and ATF family members. Shown are some examples of genes for which CARE activity has been demonstrated and the gene location for the C/EBP-ATF sites. The genes are as follows: ASNS, asparagine synthetase; Cat-1, cationic amino acid transporter 1; xCT, sodium-independent aspartate/glutamate/cystine transporter; CHOP, C/EBP homology protein; SNAT2, System A neutral amino acid transporter 2; TRB3, Tribbles 3; HERP, homocysteine-induced ER protein; ATF3, activating transcription factor 3; VEGF, vascular endothelial growth factor; 4E-BP1, eukaryotic initiation factor 4E binding protein 1.

Figure 4

(a) Model for activating transcription factor 4 (ATF4) self-limiting regulation of the asparagine synthetase (ASNS) gene. (b) During the initial 2-6 h of amino acid limitation, enhanced translational control of ATF4 protein production results in increased binding of ATF4 to the AARE. A low level of CCAAT-enhancer binding protein β (C/EBPβ) is constitutively bound. In parallel with ATF4 binding, histone acetylation occurs and the general transcription machinery is recruited to the promoter. Beyond 6 h, ATF4 action has caused increased expression of C/EBPβ and ATF3 proteins, and their binding to the C/EBP-ATF response element (CARE) increases coincident with a decline in the transcription activity of the ASNS gene. The C/EBP homology protein (CHOP) gene represents a variation on this general theme given that it also requires phospho-ATF2 and p300/CBP associated factor (PCAF). Ac, acetylated (histone); CHOP, C/EBP homology protein Inr, initiator sequence; Pol II, RNA polymerase II; TAF, TBP associated factors; TBP, TATA binding protein; TATA, TBP binding sequence; TFIIA/B/D/E/F/H, RNA Pol II associated general transcription factors.