Abstract
Cells respond to physiological stress by phosphorylating the alpha subunit of the translation initiation factor eIF2. This adaptive response inhibits protein synthesis and up-regulates genes essential for cell survival. Cat-1, the transporter for the essential amino acids, arginine and lysine, is one of the up-regulated genes. We previously showed that stress increases cat-1 expression by coordinated stabilization of the mRNA and increased mRNA translation. This induction is triggered by amino acid depletion and the unfolded protein response (UPR), which is caused by unfolded proteins in the endoplasmic reticulum. We show here that cat-1 gene transcription is also increased by cellular stress. Our studies demonstrate that the cat-1 gene promoter/regulatory region is TATA-less and is located in a region that includes 94 bases of the first exon. Transcription from this promoter is stimulated 8-fold by cellular stress. An amino acid response element within the first exon is shown to be required for the response to amino acid depletion but not to the UPR. The stimulation of transcription by amino acid depletion requires activation of GCN2 kinase, which phosphorylates eIF2alpha. This phosphorylation also induces translation of the cat-1 mRNA, demonstrating that stress-induced transcriptional and translational control of cat-1 are downstream targets of a signaling pathway initiating with eIF2alpha phosphorylation. Our studies show that the increase in cat-1 gene expression by cellular stress involves at least three types of coordinate regulation: regulation of transcription, regulation of mRNA stability, and regulation of mRNA translation.
Highlights
Mammalian cells have response mechanisms that allow cells to survive amino acid limitation [1, 2]
We have previously shown that expression of the cat-1 gene is increased by amino acid starvation [6]. cat-1 gene expression is induced by the unfolded protein response (UPR),1 which is triggered by the accumulation of unfolded proteins in the endoplasmic reticulum (ER) [7]
To prove that the increase in cat-1 mRNA during starvation is due directly to amino acid starvation, we examined the effects of removing single essential amino acids from the medium (Fig. 1A)
Summary
Mammalian cells have response mechanisms that allow cells to survive amino acid limitation [1, 2]. Our results demonstrate that regulation of cat-1 gene expression at the level of transcription, translation, and mRNA stability is part of the adaptive response of cells to stress. To study the cat-1 gene promoter, we prepared a chimeric vector (PA1.4/NheI) that contains 1.4 kb of genomic DNA upstream of the transcription start site and 94 bp of the first exon linked to a LUC reporter (Fig. 3A).
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