The discovery of the function of the receptor for the ecotropic retrovirus as a membrane transporter for the essential amino acids lysine and arginine was a landmark finding in the field of molecular nutrition. This finding indicated that cationic amino acid transporters (CATs) act pathologically as viral receptors. The importance of this transporter was further supported by knockout mice that were not viable after birth. CAT-1 was the first amino acid transporter to be cloned; several other CATs were later characterized biochemically and molecularly. These transporters mediate the bidirectional transport of cationic amino acids, thus supporting important metabolic functions, such as synthesis of proteins, nitric oxide (NO) synthesis, polyamine biosynthesis, and interorgan amino acid flow. This review briefly describes the advances in the regulation of cationic amino acid transport, focusing on the molecular mechanisms that regulate the CAT-1 transporter. Of particular interest to this review is the regulation of CAT-1 by nutritional stresses, such as amino acid availability. The studies that are reviewed conclude that the CAT-1 gene is essential for cell survival during stress because it allows cells to resume growth as soon as amino acids become available.

The cationic amino acid transporter, Cat-1, facilitates the uptake of the essential amino acids arginine and lysine. Amino acid starvation causes accumulation and increased translation of cat-1 mRNA, resulting in a 58-fold increase in protein levels and increased arginine uptake. A bicistronic mRNA expression system was used to demonstrate the presence of an internal ribosomal entry sequence (IRES) within the 5-untranslated region of the cat-1 mRNA. This study shows that IRES-mediated translation of the cat-1 mRNA is regulated by amino acid availability. This IRES causes an increase in translation under conditions of amino acid starvation. In contrast, cap-dependent protein synthesis is inhibited during amino acid starvation, which is well correlated with decreased phosphorylation of the cap-binding protein, eIF4E. These findings reveal a new aspect of mammalian gene expression and regulation that provides a cellular stress response; when the nutrient supply is limited, the activation of IRES-mediated translation of mammalian mRNAs results in the synthesis of proteins essential for cell survival.Amino acids are essential nutrients for cell growth and maintenance. Mammalian cells have developed an adaptive response to changes in amino acid availability (1). When the amino acid supply is limited, protein synthesis decreases, and there are increases in catabolism of cellular proteins, amino acid biosynthesis, and amino acid transport across the plasma membrane. Together these responses provide cells with amino acids needed for survival. A significant part of this adaptive response is the increased expression of the cat-1 gene, which encodes the transporter for the essential cationic amino acids, lysine and arginine (2). We have shown that the level of the Cat-1 protein and the transport of cationic amino acids increase in amino acid-depleted cells (3). Because amino acid starvation inhibits cap-dependent initiation of protein synthesis (4), we hypothesized that the cat-1 mRNA is translated in amino aciddepleted cells through a cap-independent mechanism. This translation would involve an internal ribosomal entry sequence (IRES 1 ) within the 5Ј-untranslated region (5Ј-UTR). IRES sequences have been implicated in the translation of viral mRNAs in infected cells, where cap-dependent translation of cellular mRNAs is inhibited (5). These sequences are also important in the translation of several mammalian mRNAs encoding regulatory proteins involved in growth and differentiation (6 -11). Furthermore, a role of IRES sequences in cell cycle-dependent translation of mammalian mRNAs has been demonstrated recently (6,(12)(13)(14). This study provides support for our hypothesis by demonstrating that the 5Ј-UTR of the cat-1 mRNA contains an IRES sequence. Moreover, translation from this IRES is stimulated in amino acid-starved cells, when cap-dependent translation is decreased. These findings suggest a mechanism for synthesis of proteins required for amino acid accumulation in starved cells when total protein synthesis is inhib...

Initiation of translation from most cellular mRNAs occurs via scanning; the 40 S ribosomal subunit binds to the m 7 G-cap and then moves along the mRNA until an initiation codon is encountered. Some cellular mRNAs contain internal ribosome entry sequences (IRESs) within their 5-untranslated regions, which allow initiation independently of the 5-cap. This study investigated the ability of cellular stress to regulate the activity of IRESs in cellular mRNAs. Three stresses were studied that cause the phosphorylation of the translation initiation factor, eIF2␣, by activating specific kinases: (i) amino acid starvation, which activates GCN2; (ii) endoplasmic reticulum (ER) stress, which activates PKR-like ER kinase, PERK kinase; and (iii) doublestranded RNA, which activates double-stranded RNAdependent protein kinase (PKR) by mimicking viral infection. Amino acid starvation and ER stress caused transient phosphorylation of eIF2␣ during the first hour of treatment, whereas double-stranded RNA caused a sustained phosphorylation of eIF2␣ after 2 h. The effects of these treatments on IRES-mediated initiation were investigated using bicistronic mRNA expression vectors. No effect was seen for the IRESs from the mRNAs for the chaperone BiP and the protein kinase Pim-1. In contrast, translation mediated by the IRESs from the cationic amino acid transporter, cat-1, and of the cricket paralysis virus intergenic region, were stimulated 3-to 10-fold by all three treatments. eIF2␣ phosphorylation was required for the response because inactivation of phosphorylation prevented the stimulation. It is concluded that cellular stress can stimulate translation from some cellular IRESs via a mechanism that requires the phosphorylation of eIF2␣. Moreover, there are distinct regulatory patterns for different cellular mRNAs that contain IRESs within their 5-untranslated regions.

The cationic amino acid transporter, Cat-1, is a high affinity transporter of the essential amino acids, arginine and lysine. Expression of the cat-1 gene increases during nutritional stress as part of the adaptive response to starvation. Amino acid limitation induces coordinate increases in stability and translation of the cat-1 mRNA, at a time when global protein synthesis decreases. It is shown here that increased cat-1 mRNA stability requires an 11 nucleotide AU-rich element within the distal 217 bases of the 3-untranslated region. When this 217-nucleotide nutrient sensor AU-rich element (NS-ARE) is present in a chimeric mRNA it confers mRNA stabilization during amino acid starvation. HuR is a member of the ELAV family of RNA-binding proteins that has been implicated in regulating the stability of ARE-containing mRNAs. We show here that the cytoplasmic concentration of HuR increases during amino acid starvation, at a time when total cellular HuR levels decrease. In addition, RNA gel shift experiments in vitro demonstrated that HuR binds to the NS-ARE and binding was dependent on the 11 residue AU-rich element. Moreover, HuR binding to the NS-ARE in extracts from amino acid-starved cells increased in parallel with the accumulation of cytoplasmic HuR. It is proposed that an adaptive response of cells to nutritional stress results in increased mRNA stability mediated by HuR binding to the NS-ARE.

Adaptation to amino acid deficiency is critical for cell survival. In yeast, this adaptation involves phosphorylation of the translation eukaryotic initiation factor (eIF) 2␣ by the kinase GCN2. This leads to the increased translation of the transcription factor GCN4, which in turn increases transcription of amino acid biosynthetic genes, at a time when expression of most genes decreases. Here it is shown that translation of the arginine/lysine transporter cat-1 mRNA increases during amino acid starvation of mammalian cells. This increase requires both GCN2 phosphorylation of eIF2␣ and the translation of a 48-amino acid upstream open reading frame (uORF) present within the 5-leader of the transporter mRNA. When this 5-leader was placed in a bicistronic mRNA expression vector, it functioned as an internal ribosomal entry sequence and its regulated activity was dependent on uORF translation. Amino acid starvation also induced translation of monocistronic mRNAs containing the cat-1 5-leader, in a manner dependent on eIF2␣ phosphorylation and translation of the 48-amino acid uORF. This is the first example of mammalian regulation of internal ribosomal entry sequence-mediated translation by eIF2␣ phosphorylation during amino acid starvation, suggesting that the mechanism of induced Cat-1 protein synthesis is part of the adaptive response of cells to amino acid limitation.