A feedback transcriptional mechanism controls the level of the arginine/lysine transporter cat-1 during amino acid starvation

Abstract

The adaptive response to amino acid limitation in mammalian cells inhibits global protein synthesis and promotes the expression of proteins that protect cells from stress. The arginine/lysine transporter, cat-1, is induced during amino acid starvation by transcriptional and post-transcriptional mechanisms. It is shown in the present study that the transient induction of cat-1 transcription is regulated by the stress response pathway that involves phosphorylation of the translation initiation factor, eIF2 (eukaryotic initiation factor-2). This phosphorylation induces expression of the bZIP (basic leucine zipper protein) transcription factors C/EBP (CCAAT/enhancer-binding protein)-beta and ATF (activating transcription factor) 4, which in turn induces ATF3. Transfection experiments in control and mutant cells, and chromatin immunoprecipitations showed that ATF4 activates, whereas ATF3 represses cat-1 transcription, via an AARE (amino acid response element), TGATGAAAC, in the first exon of the cat-1 gene, which functions both in the endogenous and in a heterologous promoter. ATF4 and C/EBPbeta activated transcription when expressed in transfected cells and they bound as heterodimers to the AARE in vitro. The induction of transcription by ATF4 was inhibited by ATF3, which also bound to the AARE as a heterodimer with C/EBPbeta. These results suggest that the transient increase in cat-1 transcription is due to transcriptional activation caused by ATF4 followed by transcriptional repression by ATF3 via a feedback mechanism.

Figure 1. Induction of the cat-1 mRNA level in MEF cells by amino acid starvation is inhibited by mutations in eIF2α and ATF4 but not ATF3

Wild-type and mutant MEF cells were starved of amino acids for the indicated times and RNA levels were determined by Northern blot analysis as described in the Materials and methods section for the following RNAs: cat-1, AS, GAPDH and 18S ribosomal RNA as a loading control. (A) Analysis of wild-type MEF and cells homozygous for the eIF2α S51A mutation (A/A). (B) Analysis of wild-type MEF and cells with homozygous disruptions in ATF3 or ATF4.

Figure 2. Time course of bZIP transcription factor accumulation during amino acid starvation in rat C6 glioma cells

(A) C6 glioma cells were starved of amino acids for the indicated times and protein levels in nuclear extracts were determined by Western blotting as described in the Materials and methods section. The arrow indicates the location of the CHOP protein. (B) Protein levels in (A) were quantified and normalized to the level in cells prior to starvation. (C) Protein levels in whole cell extracts were analysed by Western blotting.

Figure 3. bZIP transcription factors associate with the cat-1 promoter during amino acid starvation

(A and B) C6 glioma cells were starved of amino acids for the indicated times and ChIP was performed with antibodies to RNA polymerase II (Pol II), Sp1, ATF4, ATF3 and C/EBPβ as described in the Materials and methods section. In (A), PCR was used to amplify bases −37 to +156, relative to the cat-1 transcription start site. Samples that used input DNA as a template are also shown. In (B), samples precipitated with ATF4 antibody were amplified with the indicated primers. Representative gels are shown. (C) Time course of endogenous cat-1 mRNA accumulation during amino acid starvation. Cells were starved of amino acids for the indicated times and the 3.4 and 7.9 kb forms of the cat-1 mRNA were determined by Northern blot analysis. These two forms arise by the use of alternate polyadenylation sites. 18S ribosomal RNA was measured by ethidium bromide staining as a loading control. (D) ATF3 is required for a decrease in cat-1 transcription late in starvation. Wild-type and ATF3^−/− MEF cells were starved for amino acids and the level of cat-1 mRNA was analysed by reverse transcription and real-time PCR as described in the Materials and methods section. The means±S.E.M. from three experiments is shown. **Significantly different from wild type (P<.001). (E) Wild-type and ATF3^−/− MEF cells were starved of amino acids for the indicated times and protein levels in nuclear extracts were determined by Western blotting as described in the Materials and methods section.

Figure 4. bZiP transcription factors regulate cat-1 gene transcription through the AARE

(A) Constructs used in the present study. The PA1.4/UTR constructs contain 1.4 kb of cat-1 genomic DNA upstream of the transcription start site, the entire cat-1 mRNA leader and the LUC open reading frame. The transcription start site (arrow) and the open reading frame in the mRNA leader (upstream open reading frame) are shown. Point mutations are shown by shaded boxes. (B and C) C6 cells were transiently transfected with wild-type PA1.4/UTR (B) or the indicated PA1.4/UTR constructs (C) and plasmids encoding transcription factors. LUC activity was measured in extracts from fed cells. The graphs show the means±S.E.M. of LUC activity/μg protein from three independent samples normalized to the values in cells transfected with wild-type PA1.4/UTR without transcription factors. Significant differences from samples without transfected transcription factor are indicated: *P<0.01, **P<0.001.

Figure 5. Transcription of a heterologous promoter containing three copies of the cat-1 AARE is induced by amino acid starvation

(A) The 3x-AARE construct contains three repeats of the AARE (underlined) in the enhancer site of the pGL3-promoter vector. Point mutations mut1–mut6 were also introduced into the AARE repeats. (B) C6 glioma cells were transiently transfected with the indicated 3x-AARE constructs and expression plasmids. The LUC activity in fed cells was measured and normalized to the level in cells transfected with the pGL3-promoter vector without cat-1 promoter sequences. The graph shows relative LUC activity normalized to samples without transfected transcription factor cDNA. Data from three independent samples are shown. *Significantly different from cells without transcription factor (P<0.01).

Figure 6. Recombinant ATF4 and C/EBPβ proteins bind to the cat-1 AARE in vitro

EMSA assays were performed by incubating a ³²P-labelled double-stranded oligonucleotide containing the AARE with the indicated recombinant proteins and antibodies. Samples were analysed by gel electrophoresis as described in the Materials and methods section. (A) Sequence of the oligonucleotide probe. (B and C) Samples contained the indicated recombinant proteins (100 ng each) and antibodies as described in the Materials and methods section. In (B), the image for lanes 1–4 is 3 times darker than the other lanes to show the positions of the complex with ATF4 (*) and the supershifted complex (**).

Figure 7. Homodimers and heterodimers of ATF4 and ATF3 with C/EBPβ bind wild-type but not mutant AARE's

EMSA assays were performed by incubating recombinant proteins with the ³²P-labelled oligonucleotide containing the wild-type AARE and a 10-fold excess of the indicated wild-type and mutant unlabelled oligonucleotides. Experimental analysis is as described in the legend to Figure 6 and in the Materials and methods section.