Amino acid limitation induces expression of ATF5 mRNA at the post-transcriptional level

Abstract

ATF5 is a transcription factor in the cAMP response element (CRE)-binding protein/activating transcription factor (CREB/ATF) family. We studied the effect of amino acid limitation on ATF5 mRNA levels in a mammalian cell line. Northern-blot analysis demonstrated that limitation of a single amino acid, glutamine, methionine, or leucine, resulted in increased ATF5 mRNA levels in HeLaS3 cells. This resulted, at least in part, from increased half-life of the ATF5 mRNA transcript. Cycloheximide inhibited the increase in ATF5 mRNA expression induced by glutamine limitation, indicating that it was dependent on de novo protein synthesis. Moreover, rapamycin had no effect on basal ATF5 mRNA expression or on increased expression induced by glutamine limitation. These results indicate that amino acid limitation regulates ATF5 mRNA expression during post-transcription in a rapamycin-independent manner. The potential role for ATF5 in protecting cells from amino acid-limitation is of considerable interest.

Introduction

All cells regulate gene expression in response to external environmental stresses such as temperature, oxygen tension, and heavy metal and nutrient availability. In mammals, plasma amino acids are highly sensitive to the nutritional state (Jousse et al., 2004). Limiting amino acid availability is known to upregulate the expression of many genes, including c-Jun, c-myc, ornithine decarboxylase, (Pohjanpelto and Hölttä, 1990), asparagine synthetase (AS) (Gong et al., 1991), cationic amino acid transporter-1 (Aulak et al., 1999), CCAAT/enhancer-binding protein (C/EBP), homologous protein (CHOP, also called GADD153) (Bruhat et al., 1997), and FoxO4 (Imae et al., 2003). These genes are involved in amino acid biosynthesis as well as other cellular processes.

Barbosa-Tessmann et al. have identified two cis-elements in the AS gene promoter, which is a nutrient-sensing response unit (NSRU) containing nutrient-sensing response elements (NSRE)-1 and NSRE-2 (a region from − 75 to − 34) that are responsible for amino acid-dependent transcriptional regulation (Barbosa-Tessmann et al., 2000). Transcription factors, ATF4 and C/EBPβ, were shown to regulate transcription of the AS gene in response to amino acid limitation. The CHOP gene encodes a transcription factor related to the C/EBP family. Amino acid-dependent CHOP gene expression is regulated at both the transcriptional and post-transcriptional level (Bruhat et al., 1997). A region from − 313 to − 295 in the CHOP promoter is an amino acid-response element (AARE), and induction of CHOP gene expression in response to amino acid limitation requires both ATF4 and ATF2 (Averous et al., 2004). While it is known that amino acid limitation regulates ATF4 expression and ATF2 phosphorylation, few examples of transcription factors that are regulated by amino acid availability have been reported.

ATF5 (formerly designated ATFx) is a transcription factor in the CREB/ATF family that was first identified as a protein that binds to the lipopolysaccharide response element (GPE-1) on the granulocyte-colony-stimulating factor (G-CSF) gene along with C/EBPγ (Nishizawa and Nagata, 1992). It contains a DNA-binding and dimerization domain (bZIP domain) and regulates processes that are involved in cellular differentiation (Angelastro et al., 2003), the cell cycle (Pati et al., 1999), and apoptosis (Devireddy et al., 2001, Persengiev et al., 2002). ATF5 represses cAMP-induced transcription in cultured cells (Pati et al., 1999) and is shown to inhibit apoptosis (Persengiev et al., 2002). Angelastro et al. (2003) demonstrated that ATF5 represses CRE-mediated expression of neural genes and represses neural differentiation. Cdc34 is the G2 check-point gene, and ATF5 is a target of Cdc34-dependent ubiquitin-mediated proteolysis (Pati et al., 1999) whose expression is affected by the cell cycle.

The amino acid sequence of ATF5 is closely related to the sequence of ATF4, another member of the CREB/ATF transcription factor family. ATF4 expression is affected by amino acid availability and regulates target genes in an amino acid-dependent manner (Averous et al., 2004). The purpose of this study was to evaluate the effect of amino acid availability on ATF5 expression. We selected glutamine, methionine, or leucine to conduct the amino acid limitation study, because: (1) these amino acids are reported to induce CHOP mRNA expression (Jousse et al., 1999), and (2) leucine and methionine are essential amino acids while glutamine is a non-essential amino acid. Thus, we could examine the effect of two types of amino acids on ATF5 mRNA expression. We observed that amino acid limitation resulted in a marked increase in ATF5 mRNA expression in HeLaS3 cells. This increase was the result, at least in part, of the increased stabilization of ATF5 mRNA. Moreover, rapamycin had no effect on the basal level of ATF5 mRNA expression or on increased expression induced by amino acid limitation. These results indicate that ATF5 may be involved in rapamycin (mTOR)-independent nutrient control of gene expression, and that ATF5 induction may be a protective response of cells that are deprived of amino acids.