Asparagine synthetase: regulation by cell stress and involvement in tumor biology

Abstract

Asparagine synthetase (ASNS) catalyzes the conversion of aspartate and glutamine to asparagine and glutamate in an ATP-dependent reaction. The enzyme is ubiquitous in its organ distribution in mammals, but basal expression is relatively low in tissues other than the exocrine pancreas. Human ASNS activity is highly regulated in response to cell stress, primarily by increased transcription from a single gene located on chromosome 7. Among the genomic elements that control ASNS transcription is the C/EBP-ATF response element (CARE) within the promoter. Protein limitation or an imbalanced dietary amino acid composition activate the ASNS gene through the amino acid response (AAR), a process that is replicated in cell culture through limitation for any single essential amino acid. Endoplasmic reticulum stress also increases ASNS transcription through the PERK-eIF2-ATF4 arm of the unfolded protein response (UPR). Both the AAR and UPR lead to increased synthesis of ATF4, which binds to the CARE and induces ASNS transcription. Elevated expression of ASNS protein is associated with resistance to asparaginase therapy in childhood acute lymphoblastic leukemia and may be a predictive factor in drug sensitivity for certain solid tumors as well. Activation of the GCN2-eIF2-ATF4 signaling pathway, leading to increased ASNS expression appears to be a component of solid tumor adaptation to nutrient deprivation and/or hypoxia. Identifying the roles of ASNS in fetal development, tissue differentiation, and tumor growth may reveal that ASNS function extends beyond asparagine biosynthesis.

Figures

Fig. 1.

Expression of asparagine synthetase (ASNS) protein in rat tissues. The indicated tissues were harvested from rats fed a control chow, and immunoblotting of the resulting protein extracts was used to illustrate the basal expression of ASNS. These data are consistent with those of Milman et al. (63), who measured the tissue distribution of ASNS enzyme activity across many species, including humans.

Fig. 2.

Gene structure and proximal promoter sequence for human ASNS. A: exon-intron structure and size of the human ASNS gene. The translation start site is within exon 3 and the stop site within exon 13, so tan exon boxes represent 3′- and 5′-untranslated regions of the mRNA, and protein coding exons are shown in green. B: sequence of the proximal 173 bp for the human ASNS promoter. Designated are a number of transcription factor binding sites that have been identified by in vivo footprinting and single nucleotide mutagenesis to contribute to either basal or stress-induced transcription (15, 57, 101). Details of the role for each of these sequences are discussed in the text.

Fig. 3.

Multiple signal pathways that make up the amino acid response (AAR) in mammals. The AAR is a collection of signaling pathways that result in an integrated transcriptional program. Whereas uncharged tRNA activation of the GCN2 kinase has been documented to be the AA sensor for the pathways leading to increased NF-κB activity (51) and ATF4 synthesis (42), the sensor that leads to MEK (90) and GPCR12 (22) activation has not been identified (indicated by dashed lines). AA transporters have been proposed as possible sensor molecules (89).

Fig. 4.

eIF2 kinase sensors for the AAR vs. the UPR. Limitation for one or more AA causes an increased abundance of the corresponding uncharged tRNA that in turn binds to and activates the GCN2 kinase. On the other hand, other cellular stress events including glucose starvation or tunicamycin treatment that leads to glycoprotein accumulation, thapsigargin-induced inhibition of calcium homeostasis, viral overload, oxidative stress, and a wide range of disease states or drug treatments trigger endoplasmic reticulum (ER) stress (43). The resulting ER stress causes stimulation of a collection of signaling pathways collectively referred to as the unfolded protein response (UPR). One of these pathways involves activation of the ER-bound kinase PERK. Both GCN2 and PERK are members of a family of kinases that phosphorylate the eukaryotic translation initiation factor eIF2 (95). Phospho-(p-)eIF2 leads to suppression of global protein synthesis, but enhanced translation of select mRNA species, such as transcription factors ATF4 and ATF5, that contain short upstream opening reading frames that serve as regulatory sequences. Among the hundreds of ATF4 target genes is GADD34, which directs protein phosphatase-1 (PP1) to p-eIF2 and thus returns the translation factor to its dephosphorylated state and the promotion of global translation. As an ATF4-responsive enhancer element, the CARE sequence in ASNS, and many other CARE-containing genes, results in activation of transcription during either the AAR or the UPR.