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*
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL 32610 and
Departamento de Bioquimica, Universidade Estadual de Maringa, Maringa, Brazil
3To whom correspondence should be addressed. E-mail: mkilberg{at}ufl.edu.
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ABSTRACT |
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 TOP ABSTRACT Amino Acids As Signal...Activation of the Human...The chop Amino Acid...Regulation of Asparagine...The Proximal Promoter of...The ERSR Pathway and...The Nutrient Sensing Response...Concluding Remarks.LITERATURE CITED |
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KEY WORDS: • transcription • nutrient control • gene expression • stress • nitrogen metabolism
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Amino Acids As Signal Molecules. |
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TOPABSTRACTAmino Acids As Signal...Activation of the Human...The chop Amino Acid...Regulation of Asparagine...The Proximal Promoter of...The ERSR Pathway and...The Nutrient Sensing Response...Concluding Remarks.LITERATURE CITED |
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Activation of the Human chop Gene by Nutrient Starvation. |
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TOPABSTRACTAmino Acids As Signal...Activation of the Human...The chop Amino Acid...Regulation of Asparagine...The Proximal Promoter of...The ERSR Pathway and...The Nutrient Sensing Response...Concluding Remarks.LITERATURE CITED |
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) and the AAR pathway, which is activated by amino acid limitation (2
). Originally, it was believed that CHOP formed heterodimers with other C/EBP family members to inhibit their action (3
), but CHOP-C/EBP heterodimers can also activate genes (4
).
Glucose starvation of mammalian cells results in abnormal accumulation of glycoproteins in the endoplasmic reticulum (ER) that causes the ER stress response (ERSR), known as the unfolded protein response (UPR) in yeast (5
). The ERSR pathway increases transcription of many genes, several of which are chaperones, such as GRP78, necessary for protein processing within the ER. Amino acid deprivation does not cause accumulation of unfolded proteins and therefore, does not induce ERSR-activated genes such as GRP78 (6
). Target genes for the mammalian ERSR pathway contain a highly conserved cis-element (ER stress element, ERSE) for which the consensus sequence is 5'-CCAAT-N9-CCACG-3' (7
,8
). For example, the human chop promoter contains a functional ERSE sequence at nucleotides (nt) -93 to -75 (1
). Transcription from the chop gene is also enhanced after amino acid limitation, but Jousse et al. (2
) used deletion analysis of the chop promoter to show that the amino acid response element (AARE), located at nt -302 to -310, was physically separated from the ERSE.
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The chop Amino Acid Response Element. |
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TOPABSTRACTAmino Acids As Signal...Activation of the Human...The chop Amino Acid...Regulation of Asparagine...The Proximal Promoter of...The ERSR Pathway and...The Nutrient Sensing Response...Concluding Remarks.LITERATURE CITED |
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) identified the chop amino acid response element (AARE) core sequence as 5'-TGATGCAAT-3', (nt -302 to -310) within the human chop promoter. Mouse embryonic fibroblasts (MEF), deficient for either ATF2 or C/EBPß, were then used to demonstrate activation by the ERSR pathway in both knockout cell lines. However, activation of chop transcription following amino acid limitation occurred in the C/EBPß deficient line, but not in those lacking ATF2. In addition, transfection of the ATF2-deficient cells with an ATF2 expression plasmid restored amino acid control and a dominant negative form of ATF2 suppressed the induction in wild-type MEF (9
). |
Regulation of Asparagine Synthetase by Amino Acids. |
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TOPABSTRACTAmino Acids As Signal...Activation of the Human...The chop Amino Acid...Regulation of Asparagine...The Proximal Promoter of...The ERSR Pathway and...The Nutrient Sensing Response...Concluding Remarks.LITERATURE CITED |
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). Arfin et al. (11
) demonstrated that incubation of Chinese hamster ovary cells in medium lacking asparagine decreased the aminoacylation of tRNAAsn and increased AS activity. Furthermore, cells containing defective tRNA synthetases exhibit increased AS activity (12
). Gong et al. (13
) and Hutson and Kilberg (14
) showed that the level of AS mRNA increased in cells deprived of individual amino acids, consistent with earlier activity analysis by Andrulis et al. (12
). The AS mRNA content of HepG2 cells is elevated approximately equally by depletion of all 20 amino acids or by a single one such as histidine (Fig. 1
). These results are consistent with the proposal that, similar to its yeast counterpart, the mammalian GCN2 kinase senses amino acid deprivation by binding a broad spectrum of uncharged tRNAs (15
,16
). The role of tRNA charging is also illustrated by the observation that the amino alcohol histidinol, which prevents the formation of histidinyl-tRNAHis (17
), increased AS mRNA to a level equal to that observed when cells were amino acid deprived (14
). That histidinol induces AS gene transcription without decreasing the cellular histidine concentration demonstrates that the AAR pathway is not monitoring the level of free amino acids directly.
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The Proximal Promoter of the Human AS Gene. |
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TOPABSTRACTAmino Acids As Signal...Activation of the Human...The chop Amino Acid...Regulation of Asparagine...The Proximal Promoter of...The ERSR Pathway and...The Nutrient Sensing Response...Concluding Remarks.LITERATURE CITED |
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) were the first to demonstrate that the amino acid-dependent regulation of AS expression was transcriptional and the first group to identify a mammalian AARE. Deletion analysis of the human AS proximal promoter showed that the sequence spanning from nt -164 to + 44 within the AS gene retained full inducibility by amino acid deprivation (18
). Further scanning of this region by mutagenesis revealed that an AARE was present at nt -70 to -64 (5'-CATGATG-3'). |
The ERSR Pathway and Control of AS Gene Expression. |
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TOPABSTRACTAmino Acids As Signal...Activation of the Human...The chop Amino Acid...Regulation of Asparagine...The Proximal Promoter of...The ERSR Pathway and...The Nutrient Sensing Response...Concluding Remarks.LITERATURE CITED |
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) and that this transcriptional induction is mediated via the ERSR pathway (19
). Barbosa-Tessmann et al. (19
) showed that expression of a reporter gene was significantly enhanced by several ERSR activators when it was driven by the human AS promoter. Deletion analysis indicated that the cis-elements responsible for the ERSR control of the AS gene were located within nt -111 to -34 of the AS promoter, but the known mammalian ERSE consensus sequence (5'-CCAAT-N9-CCACG-3'), present in all other ERSR-inducible genes previously identified, was not present. Barbosa-Tessmann et al. (20
) went on to demonstrate that the activation of AS gene transcription by starvation of either amino acids (AAR pathway) or glucose (ERSR pathway) is mediated through a common and unique set of genomic elements within the AS proximal promoter. |
The Nutrient Sensing Response Unit of the AS Gene. |
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TOPABSTRACTAmino Acids As Signal...Activation of the Human...The chop Amino Acid...Regulation of Asparagine...The Proximal Promoter of...The ERSR Pathway and...The Nutrient Sensing Response...Concluding Remarks.LITERATURE CITED |
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,in vivo footprinting documented that the human AS promoter contains six separate protein binding sites (20
). Of these, five have been implicated in nutrient control of the human AS gene: three GC boxes (GC-I, GC-II, and GC-III) and two nutrient sensing response elements (NSRE-1, -2). All three GC boxes are required to maintain basal transcription and to obtain maximal activation of the AS gene by amino acid limitation (21
). However, when functionally analyzed, there is a difference in their degree of importance with regard to transcription (GC-III > GC-II > GC-I). In vitro, two of the GC sequences formed protein-DNA complexes (GC-II and GC-III) with either Sp1 or Sp3, but the absolute amount of these complexes and the total pool of either Sp1 or Sp3 protein did not increase after amino acid limitation. In vivo expression of either Sp1 or Sp3 in Drosophila SL2 cells, which lack Sp proteins, increased AS promoter activity, but functional differences between the factors were observed (21
). Sp1 expression increased basal transcription from the AS promoter, but did not cause a further increase when SL2 cells were amino acid deprived. In contrast, Sp3 expression enhanced both the basal and the starvation-induced AS-driven transcription.
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). These two sites, originally labeled sites V and VI (20
), have been renamed (Fig. 3
) NSRE-1 and NSRE-2. The NSRE-1 sequence (5'-TGATGAAAC-3'), located from nt -68 to -60 within the AS proximal promoter, overlaps the sequence first identified by Guerrini et al. (18
) to have AARE activity. Given that the NSRE-1 sequence is necessary for induction of the AS gene after activation of not only the AAR pathway, but also the ERSR pathway (20
), it is clear that this element functions more broadly than simply as an AARE. Consistent with the in vivo footprinting data, electrophoresis mobility shift assays (EMSA) with nuclear extracts from amino acid-starved HepG2 cells revealed increased amounts of protein-NSRE-1 complexes (20
). Furthermore, NSRE-2 (5'-GTTACA-3', nt -48 to -43), a sequence 11 nt downstream of NSRE-1 (Fig. 3)
, is also required for induction of the AS gene after either amino acid or glucose starvation (20
). The term nutrient sensing response unit (NSRU) has been coined to describe the collective action of these elements.
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), whereas the chop promoter lacks an NSRE-2 sequence but contains an ERSE. The chop AARE and the AS NSRE-1 also appear to bind different transcription factors, because the chop ATF2 is functional (9
), but in the case of AS, the NSRE-1 sequence does not bind ATF2, but does require C/EBPß (22
). These differences may indicate that there is heterogeneity in the upstream signaling steps in a single AAR pathway or, possibly, that there are multiple AAR pathways. |
Concluding Remarks. |
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TOPABSTRACTAmino Acids As Signal...Activation of the Human...The chop Amino Acid...Regulation of Asparagine...The Proximal Promoter of...The ERSR Pathway and...The Nutrient Sensing Response...Concluding Remarks.LITERATURE CITED |
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,24
). Similar mechanisms may exist in animals, but an in vivo model of glucose starvation will be required to fully test this hypothesis.
Glucose starvation (or ER stress) may also initiate asparagine biosynthesis because this amino acid has an important, but not well understood, role in cell growth control. It is noteworthy that a defective AS gene blocks cells at the G1 step of the cell cycle (25
) and asparagine deprivation induces apoptosis (26
,27
). One wonders if asparagine limitation causes these effects simply through decreased availability for protein synthesis, or is it possible that asparagine serves another role, perhaps as a signal molecule? If so, both carbohydrate and amino acid starvation may trigger asparagine biosynthesis, because asparagine is an indicator of insufficient substrate for continued cell division.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Manuscript received 4 March 2002. Revision accepted 22 March 2002.
4 Abbreviations used: AAR(E), amino acid response (element); AS, asparagine synthetase; C/EBP, CCAAT/enhancer-binding protein; CHOP, C/EBP homology protein; EMSA, electrophoresis mobility shift assay; ER, endoplasmic reticulum; ERSR(E), endoplasmic reticulum stress response (element); GH, growth hormone; MEF, mouse embryonic fibroblasts; NSR(E), nutrient sensing response (element); nt, nucleotide; UPR, unfolded protein response.
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LITERATURE CITED |
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TOPABSTRACTAmino Acids As Signal...Activation of the Human...The chop Amino Acid...Regulation of Asparagine...The Proximal Promoter of...The ERSR Pathway and...The Nutrient Sensing Response...Concluding Remarks.LITERATURE CITED |
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1. Yoshida, H., Okada, T., Haze, K., Yanagi, H., Yura, T., Negishi, M. & Mori, K. (2000) ATF6 activated by proteolysis binds in the presence of NF-Y (CBF) directly to the cis-acting element responsible for the mammalian unfolded protein response. Mol. Cell Biol. 20:6755-6767.
2. Jousse, C., Bruhat, A., Harding, H. P., Ferrara, M., Ron, D. & Fafournoux, P. (1999) Amino acid limitation regulates CHOP expression through a specific pathway independent of the unfolded protein response. FEBS Lett 448:211-216.[Medline]
3. Ron, D. & Habener, J. F. (1992) CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Gene. Devel. 6:439-453.
4. Wang, X.-Z., Kuroda, M., Sok, J., Batchvarova, N., Kimmel, R., Chung, P., Zinszner, H. & Ron, D. (1998) Identification of novel stress-induced genes downstream of. chop. EMBO J. 17:3619-3630.[Medline]
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7. Yoshida, H., Haze, K., Yanagi, H., Yura, T. & Mori, K. (1998) Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins. J. Biol. Chem. 273:33741-33749.
8. Roy, B. & Lee, A. S. (1999) The mammalian endoplasmic reticulum stress response element consists of an evolutionarily conserved tripartite structure and interacts with a novel stress-inducible complex. Nucleic Acids Res 27:1437-1443.
9. Bruhat, A., Jousse, C., Carraro, V., Reimold, A. M., Ferrara, M. & Fafournoux, P. (2000) Amino acids control mammalian gene transcription: activating transcription factor 2 is essential for the amino acid responsiveness of the CHOP promoter. Mol. Cell Biol. 20:7192-7204.
10. Richards, N. G. J. & Schuster, S. M. (1998) Mechanistic issues in asparagine synthetase catalysis. Adv. Enzymol. Relat. Areas Mol. Biol. 72:145-198.[Medline]
11. Arfin, S. M., Simpson, D. R., Chiang, C. S., Andrulis, I. L. & Hatfield, G. W. (1977) A role for asparaginyl-tRNA in the regulation of asparagine synthetase in a mammalian cell line. Proc. Natl. Acad. Sci. USA 74:2367-2369.
12. Andrulis, I. L., Hatfield, G. W. & Arfin, S. M. (1979) Asparaginyl-tRNA aminoacylation levels and asparagine synthetase expression in cultured Chinese hamster ovary cells. J. Biol. Chem. 254:10629-10633.
13. Gong, S. S., Guerrini, L. & Basilico, C. (1991) Regulation of asparagine synthetase gene expression by amino acid starvation. Mol. Cell. Biol. 11:6059-6066.
14. Hutson, R. G. & Kilberg, M. S. (1994) Cloning of rat asparagine synthetase and specificity of the amino acid-dependent control of its mRNA content. Biochem. J. 303:745-750.
15. Berlanga, J. J., Santoyo, J. & De Haro, C. (1999) Characterization of a mammalian homolog of the GCN2 eukaryotic initiation factor 2
kinase. Eur. J. Biochem. 265:754-762.[Medline]
16. Sood, R., Porter, A. C., Olsen, D. A., Cavener, D. R. & Wek, R. C. (2000) A mammalian homologue of GCN2 protein kinase important for translational control by phosphorylation of eukaryotic initiation factor-2. Genetics 154:787-801.
17. Hansen, B. S., Vaughan, M. H. & Wang, L.-J. (1972) Reversible inhibition by histidinol of protein synthesis in human cells at the activation of histidine. J. Biol. Chem. 247:3854-3857.
18. Guerrini, L., Gong, S. S., Mangasarian, K. & Basilico, C. (1993) Cis- and trans-acting elements involved in amino acid regulation of asparagine synthetase gene expression. Mol. Cell. Biol. 13:3202-3212.
19. Barbosa-Tessmann, I. P., Chen, C., Zhong, C., Schuster, S. M., Nick, H. S. & Kilberg, M. S. (1999) Activation of the unfolded protein response pathway induces human asparagine synthetase gene expression. J. Biol. Chem. 274:31139-31144.
20. Barbosa-Tessmann, I. P., Chen, C., Zhong, C., Siu, F., Schuster, S. M., Nick, H. S. & Kilberg, M. S. (2000) Activation of the human asparagine synthetase gene by the amino acid response and the endoplasmic reticulum stress response pathways occurs by common genomic elements. J. Biol. Chem. 275:26976-26985.
21. Leung-Pineda, V. & Kilberg, M. S. (2002) Role of Sp1 and Sp3 in the nutrient-regulated expression of the human asparagine synthetase gene. J. Biol. Chem. 277:16585-16591.
22. Siu, F. Y., Chen, C., Zhong, C. & Kilberg, M. S. (2001) CCAAT/enhancer-binding protein ß (C/EBPb) is a mediator of the nutrient sensing response pathway that activates the human asparagine synthetase gene. J. Biol. Chem. 276:48100-48107.
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27. Nandy, P., Periclou, A. P. & Avramis, V. I. (1998) The synergism of 6-mercaptopurine plus cytosine arabinoside followed by PEG-asparaginase in human leukemia cell lines (CCRF/CEM/0 and (CCRF/CEM/ara-C/7A) is due to increased cellular apoptosis. Anticancer Res 18:727-738.[Medline]
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) represent constitutively protected guanine residues; closed circles (•) represent constitutively enhanced guanine residues; open triangles (
)represent guanine residues showing increased protection in amino acid-deprived cells. The arrowheads (
) represent enhanced adenine residues. Bars with roman numerals represent putative individual protein binding sites. The relative positions of the nts are indicated on the side of each sequence.