Glutamine-dependent Antiapoptotic Interaction of Human Glutaminyl-tRNA Synthetase with Apoptosis Signal-regulating Kinase 1

Abstract

Glutamine has been known to be an apoptosis suppressor, since it blocks apoptosis induced by heat shock, irradiation, and c-Myc overexpression. Here, we demonstrated that HeLa cells were susceptible to Fas-mediated apoptosis under the condition of glutamine deprivation. Fas ligation activated apoptosis signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK; also known as stress-activated protein kinase (SAPK)) in Gln-deprived cells but not in normal cells, suggesting that Gln might be involved in the activity control of ASK1 and JNK/SAPK. As one of the possible mechanisms for the suppressive effect of Gln on ASK1, we investigated the molecular interaction between human glutaminyl-tRNA synthetase (QRS) and ASK1 and found the Gln-dependent association of the two molecules. While their association was enhanced by the elevation of Gln concentration, they were dissociated by Fas ligation within 5 min. The association involved the catalytic domains of the two enzymes. The ASK1 activity was inhibited by the interaction with QRS as determined by in vitro kinase and transcription assays. Finally, we have shown that QRS inhibited the cell death induced by ASK1, and this antiapoptotic function of QRS was weakened by the deprivation of Gln. Thus, the antiapoptotic interaction of QRS with ASK1 is controlled positively by the cellular concentration of Gln and negatively by Fas ligation. The results of this work provide one possible explanation for the working mechanism of the antiapoptotic activity of Gln and suggest a novel function of mammalian ARSs. Glutamine has been known to be an apoptosis suppressor, since it blocks apoptosis induced by heat shock, irradiation, and c-Myc overexpression. Here, we demonstrated that HeLa cells were susceptible to Fas-mediated apoptosis under the condition of glutamine deprivation. Fas ligation activated apoptosis signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK; also known as stress-activated protein kinase (SAPK)) in Gln-deprived cells but not in normal cells, suggesting that Gln might be involved in the activity control of ASK1 and JNK/SAPK. As one of the possible mechanisms for the suppressive effect of Gln on ASK1, we investigated the molecular interaction between human glutaminyl-tRNA synthetase (QRS) and ASK1 and found the Gln-dependent association of the two molecules. While their association was enhanced by the elevation of Gln concentration, they were dissociated by Fas ligation within 5 min. The association involved the catalytic domains of the two enzymes. The ASK1 activity was inhibited by the interaction with QRS as determined by in vitro kinase and transcription assays. Finally, we have shown that QRS inhibited the cell death induced by ASK1, and this antiapoptotic function of QRS was weakened by the deprivation of Gln. Thus, the antiapoptotic interaction of QRS with ASK1 is controlled positively by the cellular concentration of Gln and negatively by Fas ligation. The results of this work provide one possible explanation for the working mechanism of the antiapoptotic activity of Gln and suggest a novel function of mammalian ARSs. tumor necrosis factor aminoacyl-tRNA synthetase glutaminyl-tRNA synthetase apoptosis signal-regulating kinase 1 c-Jun N-terminal kinase stress-activated protein kinase mitogen-activating protein kinase hemagglutinin myelin basic protein Apoptosis is a genetically regulated process that is essential for correct morphogenesis during embryogenesis and the maintenance of tissue homeostasis (1Vaux D.L. Korsmeyer S.J. Cell. 1999; 96: 245-254Abstract Full Text Full Text PDF PubMed Scopus (1356) Google Scholar, 2Rich T. Watson C.J. Wyllie A. Nat. Cell Biol. 1999; 1: E69-E71Crossref PubMed Scopus (199) Google Scholar). Since dysregulation of apoptosis has been implicated in autoimmune disease and atherosclerosis as well as neurodegenerative disorders and cancer (3Chervonsky A.V. Curr. Opin. Immunol. 1999; 11: 684-688Crossref PubMed Scopus (53) Google Scholar, 4Bennett M.R. Boyle J.J. Atherosclerosis. 1998; 138: 3-9Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 5Lee J.M. Zipfel G.J. Choi D.W. Nature. 1999; 399: A7-A14Crossref PubMed Scopus (995) Google Scholar), tumor suppressor and proapoptotic genes should tightly regulate apoptosis (6King K.L. Cidlowski J.A. Annu. Rev. Physiol. 1998; 60: 601-617Crossref PubMed Scopus (424) Google Scholar). Apoptosis is induced by cytokines such as Fas ligand and tumor necrosis factor (TNF),1 growth factor withdrawal, ischemia, and amino acid deprivation (5Lee J.M. Zipfel G.J. Choi D.W. Nature. 1999; 399: A7-A14Crossref PubMed Scopus (995) Google Scholar, 7Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5112) Google Scholar, 8Papaconstantinou H.T. Hwang K.O. Rajaraman S. Hellmich M.R. Townsend Jr., C.M. Ko T.C. Surgery. 1998; 124: 152-160Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar).Glutamine is a nonessential amino acid, but it is heavily utilized as a major metabolic fuel as well as a precursor for nucleotide synthesis in fibroblasts, lymphocytes, and macrophages (9Neu J. Shenoy V. Chakrabarti R. FASEB. J. 1996; 10: 829-837Crossref PubMed Scopus (120) Google Scholar). Since Gln deprivation induces apoptosis in intestinal epithelial cells, whereas methionine deprivation does not (8Papaconstantinou H.T. Hwang K.O. Rajaraman S. Hellmich M.R. Townsend Jr., C.M. Ko T.C. Surgery. 1998; 124: 152-160Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), Gln might play an important role in protecting cells from apoptosis induced by different stimuli. For example, Gln supplementation reduces apoptosis induced by heat shock, irradiation, and c-Myc overexpression (10Chow A. Zhang R. J. Nutr. 1998; 128: 1296-1301Crossref PubMed Scopus (75) Google Scholar, 11Santoso J.T. Lucci III, J.A. Coleman R.L. Hatch S. Wong P. Miller D. Mathis J.M. Gynecol. Oncol. 1998; 71: 359-363Abstract Full Text PDF PubMed Scopus (6) Google Scholar, 12Xu Y. Nguyen Q. Lo D.C. Czaja M.J. J. Cell. Physiol. 1997; 170: 192-199Crossref PubMed Scopus (66) Google Scholar). Furthermore, Gln stimulates intestinal cell proliferation and activates mitogen-activated protein kinases such as p42/p44 MAPK and JNK/SAPK (13Rhoads J.M. Argenzio R.A. Chen W. Rippe R.A. Westwick J.K. Cox A.D. Berschneider H.M. Brenner D.A. Am. J. Physiol. 1997; 272: 943-953PubMed Google Scholar, 14Rhoads J.M. Argenzio R.A. Chen W. Graves L.M. Licato L.L. Blikslager A.T. Smith J. Gatzy J. Brenner D.A. Gastroenterology. 2000; 118: 90-100Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), suggesting that Gln could regulate signal transduction pathways for cellular proliferation and apoptosis. However, the molecular mechanism of Gln in suppressing apoptosis and stimulating cellular proliferation remains to be explained.Aminoacyl-tRNA synthetases (ARSs) catalyze aminoacylation of their cognate tRNAs and thus play an essential role in protein synthesis. ARSs have been found in cytoskeleton- or endoplasmic reticulum-associated structures or in cytoplasm, but they are also found in the nucleus and even in the nucleolus (15Dang C.V. Yang D.C. Pollard T.D. J. Cell Biol. 1983; 96: 1138-1147Crossref PubMed Scopus (78) Google Scholar, 16Schimmel P. Wang C.C. Trends. Biochem. Sci. 1999; 24: 127-128Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 17Ko Y.-G. Kang Y.-S. Kim E.-K. Park S.G. Kim S. J. Cell Biol. 2000; 149: 567-574Crossref PubMed Scopus (102) Google Scholar), suggesting that ARSs have various noncanonical functions in addition to tRNA aminoacylation from eukaryotes. In particular, methionyl-tRNA synthetase is involved in rRNA biogenesis at its localization site in the nucleolus (17Ko Y.-G. Kang Y.-S. Kim E.-K. Park S.G. Kim S. J. Cell Biol. 2000; 149: 567-574Crossref PubMed Scopus (102) Google Scholar).Upon cellular exposure to apoptosis condition, mammalian tyrosyl-tRNA synthetase is secreted and split into two fragments with distinct cytokine activities by leukocyte elastase and extracellular protease (18Wakasugi K. Schimmel P. Science. 1999; 284: 147-151Crossref PubMed Scopus (412) Google Scholar, 19Wakasugi K. Schimmel P. J. Biol. Chem. 1999; 274: 23155-23159Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The split cytokines contain an endothelial monocyte-activating polypeptide II-like domain and an interleukin-8-like domain. Thus, tyrosyl-tRNA synthetase has a potent apoptosis-inducing activity after proteolytic cleavage, since their split polypepetides stimulate the production of TNF and tissue factor from target cells and have leukocyte chemotaxis activity to scavenge apoptotic corpses. The precursor of endothelial monocyte-activating polypeptide II is associated with the N-terminal noncatalytic extension of arginyl-tRNA synthetase and enhances aminoacylation activity (20Park S.G. Jung K.H. Lee J.S. Jo Y.J. Motegi H. Kim S. Shiba K. J. Biol. Chem. 1999; 274: 16673-16676Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Like human tyrosyl-tRNA synthetase, the cytokine domain of this precursor is released upon apoptosis and exerts its proapoptotic function (21Knies U.E. Behrensdorf H.A. Mitchell C.A. Deutsch U. Risau W. Drexler H.C.A. Cluass M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12322-12327Crossref PubMed Scopus (139) Google Scholar, 22Schwarz M.A. Kandel J. Brett J. Li J. Hayward J. Schwarz R.E. Chappey O. Wautier J.-L. Chabot J. Gerfo P.L. Stern D. J. Exp. Med. 1999; 190: 341-353Crossref PubMed Scopus (115) Google Scholar).Here, we studied a novel regulatory role of human glutaminyl-tRNA synthetase (QRS) in inhibiting apoptosis signal-regulating kinase 1 (ASK1). We first searched for the effect of Gln in suppressing Fas-mediated apoptosis and reducing the activation of ASK1 as well as JNK/SAPK. We also observed that QRS inhibits ASK1 kinase activity and apoptosis by binding to ASK1. The molecular interaction of QRS and ASK1 was dependent on glutamine concentration, suggesting that glutamine could repress Fas-mediated apoptosis and ASK1 activation via QRS.DISCUSSIONAminoacyl-tRNA synthetases are a family of enzymes essential for protein synthesis. However, it has been discovered that these enzymes are actively involved in a broad repertoire of other critical cellular activities as well as protein synthesis (36Martinis S.A. Plateau P. Cavarelli J. Florentz C. EMBO J. 1999; 18: 4591-4596Crossref PubMed Scopus (109) Google Scholar). It has been previously demonstrated that ARSs have idiosyncratic distribution in cytoplasm, nucleus, and nucleolus (17Ko Y.-G. Kang Y.-S. Kim E.-K. Park S.G. Kim S. J. Cell Biol. 2000; 149: 567-574Crossref PubMed Scopus (102) Google Scholar, 37Popenko V.I. Ivanova J.L. Cherny N.E. Filonenko V.V. Beresten S.F. Wolfson A.D. Kisselev L.L. Eur. J. Cell Biol. 1994; 65: 60-69PubMed Google Scholar). For instance, methionyl-tRNA synthetase is localized in the nucleolus of rapidly proliferating mammalian cells and is responsible for ribosomal RNA biosynthesis (17Ko Y.-G. Kang Y.-S. Kim E.-K. Park S.G. Kim S. J. Cell Biol. 2000; 149: 567-574Crossref PubMed Scopus (102) Google Scholar). Seshaiah and Andrew (38Seshaiah P. Andrew D.J. Mol. Biol. Cell. 1999; 10: 1595-1608Crossref PubMed Scopus (32) Google Scholar) also showed that each ARS is uniquely expressed in different tissues and developmental stages ofDrosophila. Thus, the differential expression and cellular localization of each ARS imply that each ARS has its noncanonical function in addition to the catalytic activity for tRNA aminoacylation. Indeed, different ARSs play roles in tRNA maturation (proofreading and nuclear export), cytokine-like activity, mitochondrial RNA splicing, and transcriptional and translational regulation (36Martinis S.A. Plateau P. Cavarelli J. Florentz C. EMBO J. 1999; 18: 4591-4596Crossref PubMed Scopus (109) Google Scholar).Here, we found that human QRS is not only the enzyme for cell proliferation but also the protein that plays a regulatory role in cell death through an antagonistic interaction with ASK1, a protein kinase that plays a critical role in apoptosis. QRS has been classified as a component of the multi-tRNA synthetase complex (39Kisselev L.L. Wolfson A.D. Prog. Nucleic Acids Res. Mol. Biol. 1994; 48: 83-142Crossref PubMed Scopus (90) Google Scholar, 40Yang D.C.H. Curr. Top. Cell. Regul. 1996; 34: 101-136Crossref PubMed Scopus (50) Google Scholar). However, QRS not associated with the multi-tRNA synthetase complex has been found (41Pahuski E. Klekamp M. Condon T. Hampel A.E. J. Cell. Physiol. 1983; 114: 82-87Crossref PubMed Scopus (14) Google Scholar) and shown to be catalytically active (26Kim T. Park S.G. Kim J.E. Seol W. Ko Y.-G. Kim S. J. Biol. Chem. 2000; 275: 21768-21772Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Thus, QRS bound to ASK1 may be in dynamic equilibrium with that in the multi-tRNA synthetase complex.Extensive investigations have revealed important effects of exogenous Gln in nutritional rehabilitation, stimulating intestinal villus regrowth after damage from chemotherapy and improving intestinal barrier function. In addition, Gln blocks apoptosis induced by heat shock, irradiation, and c-Myc-overexpression. In Figs. 1 and 2, we show that Gln prevents the activation of stress kinases such as ASK1 and JNK/ASK1 and then apoptosis initiated by Fas ligation, suggesting that Gln is an apoptosis suppressor. The cellular susceptibility to Fas-mediated signaling by Gln deprivation could not be explained by the protein synthesis inhibition, because ASK1 and JNK/SAPK were activated within 10 min after Fas ligation in Gln-free cells but not in normal cells. Therefore, Gln could suppress apoptosis by inhibiting ASK1 and then JNK/SAPK.Although the of exogenous QRS was expressed in cells in and Gln-free the molecular interaction between QRS and ASK1 was and then the ASK1 activity was inhibited in the of Gln the effect of Gln on the interaction of the two molecules. It has been demonstrated that amino be signaling molecules. For instance, as a in because exogenous stimulates of mitochondrial function in cells P. Nature. 1999; PubMed Scopus Google Scholar). Here, we suggest that Gln could be a signaling for ASK1 activation and suppressing apoptosis via binding to of apoptosis are by the activation of kinase and of kinase by extracellular prevents apoptosis by and that are involved in the apoptosis A. Nature. 1999; PubMed Scopus Google Scholar, E. S. Science. 1998; PubMed Scopus Google Scholar, H. S. H. Y. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. M.J. P. M.J. J. Cell. 1999; 96: Full Text Full Text PDF PubMed Scopus Google Scholar, G.J. J.L. Nature. 1999; PubMed Scopus Google Scholar, J.A. L.M. Nature. 1999; PubMed Scopus Google Scholar, J.A. Nature. 1999; PubMed Scopus Google Scholar). Since Fas-mediated apoptosis is by MAPK through overexpression of and basic growth factor H. S. J. Cell Biol. 2000; PubMed Scopus (53) Google Scholar) and is by inhibiting MAPK with J.E. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar), MAPK could be signal inhibiting apoptosis. In addition to kinase and we that Gln and QRS are for apoptosis. investigations are to the mechanism and control for the role of QRS in the results of this work provide the first that one of the mammalian ARSs has a antagonistic interaction with a kinase that plays a role in apoptosis. Apoptosis is a genetically regulated process that is essential for correct morphogenesis during embryogenesis and the maintenance of tissue homeostasis (1Vaux D.L. Korsmeyer S.J. Cell. 1999; 96: 245-254Abstract Full Text Full Text PDF PubMed Scopus (1356) Google Scholar, 2Rich T. Watson C.J. Wyllie A. Nat. Cell Biol. 1999; 1: E69-E71Crossref PubMed Scopus (199) Google Scholar). Since dysregulation of apoptosis has been implicated in autoimmune disease and atherosclerosis as well as neurodegenerative disorders and cancer (3Chervonsky A.V. Curr. Opin. Immunol. 1999; 11: 684-688Crossref PubMed Scopus (53) Google Scholar, 4Bennett M.R. Boyle J.J. Atherosclerosis. 1998; 138: 3-9Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 5Lee J.M. Zipfel G.J. Choi D.W. Nature. 1999; 399: A7-A14Crossref PubMed Scopus (995) Google Scholar), tumor suppressor and proapoptotic genes should tightly regulate apoptosis (6King K.L. Cidlowski J.A. Annu. Rev. Physiol. 1998; 60: 601-617Crossref PubMed Scopus (424) Google Scholar). Apoptosis is induced by cytokines such as Fas ligand and tumor necrosis factor (TNF),1 growth factor withdrawal, ischemia, and amino acid deprivation (5Lee J.M. Zipfel G.J. Choi D.W. Nature. 1999; 399: A7-A14Crossref PubMed Scopus (995) Google Scholar, 7Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5112) Google Scholar, 8Papaconstantinou H.T. Hwang K.O. Rajaraman S. Hellmich M.R. Townsend Jr., C.M. Ko T.C. Surgery. 1998; 124: 152-160Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Glutamine is a nonessential amino acid, but it is heavily utilized as a major metabolic fuel as well as a precursor for nucleotide synthesis in fibroblasts, lymphocytes, and macrophages (9Neu J. Shenoy V. Chakrabarti R. FASEB. J. 1996; 10: 829-837Crossref PubMed Scopus (120) Google Scholar). Since Gln deprivation induces apoptosis in intestinal epithelial cells, whereas methionine deprivation does not (8Papaconstantinou H.T. Hwang K.O. Rajaraman S. Hellmich M.R. Townsend Jr., C.M. Ko T.C. Surgery. 1998; 124: 152-160Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), Gln might play an important role in protecting cells from apoptosis induced by different stimuli. For example, Gln supplementation reduces apoptosis induced by heat shock, irradiation, and c-Myc overexpression (10Chow A. Zhang R. J. Nutr. 1998; 128: 1296-1301Crossref PubMed Scopus (75) Google Scholar, 11Santoso J.T. Lucci III, J.A. Coleman R.L. Hatch S. Wong P. Miller D. Mathis J.M. Gynecol. Oncol. 1998; 71: 359-363Abstract Full Text PDF PubMed Scopus (6) Google Scholar, 12Xu Y. Nguyen Q. Lo D.C. Czaja M.J. J. Cell. Physiol. 1997; 170: 192-199Crossref PubMed Scopus (66) Google Scholar). Furthermore, Gln stimulates intestinal cell proliferation and activates mitogen-activated protein kinases such as p42/p44 MAPK and JNK/SAPK (13Rhoads J.M. Argenzio R.A. Chen W. Rippe R.A. Westwick J.K. Cox A.D. Berschneider H.M. Brenner D.A. Am. J. Physiol. 1997; 272: 943-953PubMed Google Scholar, 14Rhoads J.M. Argenzio R.A. Chen W. Graves L.M. Licato L.L. Blikslager A.T. Smith J. Gatzy J. Brenner D.A. Gastroenterology. 2000; 118: 90-100Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), suggesting that Gln could regulate signal transduction pathways for cellular proliferation and apoptosis. However, the molecular mechanism of Gln in suppressing apoptosis and stimulating cellular proliferation remains to be synthetases (ARSs) catalyze aminoacylation of their cognate tRNAs and thus play an essential role in protein synthesis. ARSs have been found in cytoskeleton- or endoplasmic reticulum-associated structures or in cytoplasm, but they are also found in the nucleus and even in the nucleolus (15Dang C.V. Yang D.C. Pollard T.D. J. Cell Biol. 1983; 96: 1138-1147Crossref PubMed Scopus (78) Google Scholar, 16Schimmel P. Wang C.C. Trends. Biochem. Sci. 1999; 24: 127-128Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 17Ko Y.-G. Kang Y.-S. Kim E.-K. Park S.G. Kim S. J. Cell Biol. 2000; 149: 567-574Crossref PubMed Scopus (102) Google Scholar), suggesting that ARSs have various noncanonical functions in addition to tRNA aminoacylation from eukaryotes. In particular, methionyl-tRNA synthetase is involved in rRNA biogenesis at its localization site in the nucleolus (17Ko Y.-G. Kang Y.-S. Kim E.-K. Park S.G. Kim S. J. Cell Biol. 2000; 149: 567-574Crossref PubMed Scopus (102) Google Scholar). cellular exposure to apoptosis condition, mammalian tyrosyl-tRNA synthetase is secreted and split into two fragments with distinct cytokine activities by leukocyte elastase and extracellular protease (18Wakasugi K. Schimmel P. Science. 1999; 284: 147-151Crossref PubMed Scopus (412) Google Scholar, 19Wakasugi K. Schimmel P. J. Biol. Chem. 1999; 274: 23155-23159Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The split cytokines contain an endothelial monocyte-activating polypeptide II-like domain and an interleukin-8-like domain. Thus, tyrosyl-tRNA synthetase has a potent apoptosis-inducing activity after proteolytic cleavage, since their split polypepetides stimulate the production of TNF and tissue factor from target cells and have leukocyte chemotaxis activity to scavenge apoptotic corpses. The precursor of endothelial monocyte-activating polypeptide II is associated with the N-terminal noncatalytic extension of arginyl-tRNA synthetase and enhances aminoacylation activity (20Park S.G. Jung K.H. Lee J.S. Jo Y.J. Motegi H. Kim S. Shiba K. J. Biol. Chem. 1999; 274: 16673-16676Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Like human tyrosyl-tRNA synthetase, the cytokine domain of this precursor is released upon apoptosis and exerts its proapoptotic function (21Knies U.E. Behrensdorf H.A. Mitchell C.A. Deutsch U. Risau W. Drexler H.C.A. Cluass M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12322-12327Crossref PubMed Scopus (139) Google Scholar, 22Schwarz M.A. Kandel J. Brett J. Li J. Hayward J. Schwarz R.E. Chappey O. Wautier J.-L. Chabot J. Gerfo P.L. Stern D. J. Exp. Med. 1999; 190: 341-353Crossref PubMed Scopus (115) Google Scholar). Here, we studied a novel regulatory role of human glutaminyl-tRNA synthetase (QRS) in inhibiting apoptosis signal-regulating kinase 1 (ASK1). We first searched for the effect of Gln in suppressing Fas-mediated apoptosis and reducing the activation of ASK1 as well as JNK/SAPK. We also observed that QRS inhibits ASK1 kinase activity and apoptosis by binding to ASK1. The molecular interaction of QRS and ASK1 was dependent on glutamine concentration, suggesting that glutamine could repress Fas-mediated apoptosis and ASK1 activation via synthetases are a family of enzymes essential for protein synthesis. However, it has been discovered that these enzymes are actively involved in a broad repertoire of other critical cellular activities as well as protein synthesis (36Martinis S.A. Plateau P. Cavarelli J. Florentz C. EMBO J. 1999; 18: 4591-4596Crossref PubMed Scopus (109) Google Scholar). It has been previously demonstrated that ARSs have idiosyncratic distribution in cytoplasm, nucleus, and nucleolus (17Ko Y.-G. Kang Y.-S. Kim E.-K. Park S.G. Kim S. J. Cell Biol. 2000; 149: 567-574Crossref PubMed Scopus (102) Google Scholar, 37Popenko V.I. Ivanova J.L. Cherny N.E. Filonenko V.V. Beresten S.F. Wolfson A.D. Kisselev L.L. Eur. J. Cell Biol. 1994; 65: 60-69PubMed Google Scholar). For instance, methionyl-tRNA synthetase is localized in the nucleolus of rapidly proliferating mammalian cells and is responsible for ribosomal RNA biosynthesis (17Ko Y.-G. Kang Y.-S. Kim E.-K. Park S.G. Kim S. J. Cell Biol. 2000; 149: 567-574Crossref PubMed Scopus (102) Google Scholar). Seshaiah and Andrew (38Seshaiah P. Andrew D.J. Mol. Biol. Cell. 1999; 10: 1595-1608Crossref PubMed Scopus (32) Google Scholar) also showed that each ARS is uniquely expressed in different tissues and developmental stages ofDrosophila. Thus, the differential expression and cellular localization of each ARS imply that each ARS has its noncanonical function in addition to the catalytic activity for tRNA aminoacylation. Indeed, different ARSs play roles in tRNA maturation (proofreading and nuclear export), cytokine-like activity, mitochondrial RNA splicing, and transcriptional and translational regulation (36Martinis S.A. Plateau P. Cavarelli J. Florentz C. EMBO J. 1999; 18: 4591-4596Crossref PubMed Scopus (109) Google Scholar).Here, we found that human QRS is not only the enzyme for cell proliferation but also the protein that plays a regulatory role in cell death through an antagonistic interaction with ASK1, a protein kinase that plays a critical role in apoptosis. QRS has been classified as a component of the multi-tRNA synthetase complex (39Kisselev L.L. Wolfson A.D. Prog. Nucleic Acids Res. Mol. Biol. 1994; 48: 83-142Crossref PubMed Scopus (90) Google Scholar, 40Yang D.C.H. Curr. Top. Cell. Regul. 1996; 34: 101-136Crossref PubMed Scopus (50) Google Scholar). However, QRS not associated with the multi-tRNA synthetase complex has been found (41Pahuski E. Klekamp M. Condon T. Hampel A.E. J. Cell. Physiol. 1983; 114: 82-87Crossref PubMed Scopus (14) Google Scholar) and shown to be catalytically active (26Kim T. Park S.G. Kim J.E. Seol W. Ko Y.-G. Kim S. J. Biol. Chem. 2000; 275: 21768-21772Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Thus, QRS bound to ASK1 may be in dynamic equilibrium with that in the multi-tRNA synthetase complex.Extensive investigations have revealed important effects of exogenous Gln in nutritional rehabilitation, stimulating intestinal villus regrowth after damage from chemotherapy and improving intestinal barrier function. In addition, Gln blocks apoptosis induced by heat shock, irradiation, and c-Myc-overexpression. In Figs. 1 and 2, we show that Gln prevents the activation of stress kinases such as ASK1 and JNK/ASK1 and then apoptosis initiated by Fas ligation, suggesting that Gln is an apoptosis suppressor. The cellular susceptibility to Fas-mediated signaling by Gln deprivation could not be explained by the protein synthesis inhibition, because ASK1 and JNK/SAPK were activated within 10 min after Fas ligation in Gln-free cells but not in normal cells. Therefore, Gln could suppress apoptosis by inhibiting ASK1 and then JNK/SAPK.Although the of exogenous QRS was expressed in cells in and Gln-free the molecular interaction between QRS and ASK1 was and then the ASK1 activity was inhibited in the of Gln the effect of Gln on the interaction of the two molecules. It has been demonstrated that amino be signaling molecules. For instance, as a in because exogenous stimulates of mitochondrial function in cells P. Nature. 1999; PubMed Scopus Google Scholar). Here, we suggest that Gln could be a signaling for ASK1 activation and suppressing apoptosis via binding to of apoptosis are by the activation of kinase and of kinase by extracellular prevents apoptosis by and that are involved in the apoptosis A. Nature. 1999; PubMed Scopus Google Scholar, E. S. Science. 1998; PubMed Scopus Google Scholar, H. S. H. Y. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. M.J. P. M.J. J. Cell. 1999; 96: Full Text Full Text PDF PubMed Scopus Google Scholar, G.J. J.L. Nature. 1999; PubMed Scopus Google Scholar, J.A. L.M. Nature. 1999; PubMed Scopus Google Scholar, J.A. Nature. 1999; PubMed Scopus Google Scholar). Since Fas-mediated apoptosis is by MAPK through overexpression of and basic growth factor H. S. J. Cell Biol. 2000; PubMed Scopus (53) Google Scholar) and is by inhibiting MAPK with J.E. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar), MAPK could be signal inhibiting apoptosis. In addition to kinase and we that Gln and QRS are for apoptosis. investigations are to the mechanism and control for the role of QRS in the results of this work provide the first that one of the mammalian ARSs has a antagonistic interaction with a kinase that plays a role in apoptosis. synthetases are a family of enzymes essential for protein synthesis. However, it has been discovered that these enzymes are actively involved in a broad repertoire of other critical cellular activities as well as protein synthesis (36Martinis S.A. Plateau P. Cavarelli J. Florentz C. EMBO J. 1999; 18: 4591-4596Crossref PubMed Scopus (109) Google Scholar). It has been previously demonstrated that ARSs have idiosyncratic distribution in cytoplasm, nucleus, and nucleolus (17Ko Y.-G. Kang Y.-S. Kim E.-K. Park S.G. Kim S. J. Cell Biol. 2000; 149: 567-574Crossref PubMed Scopus (102) Google Scholar, 37Popenko V.I. Ivanova J.L. Cherny N.E. Filonenko V.V. Beresten S.F. Wolfson A.D. Kisselev L.L. Eur. J. Cell Biol. 1994; 65: 60-69PubMed Google Scholar). For instance, methionyl-tRNA synthetase is localized in the nucleolus of rapidly proliferating mammalian cells and is responsible for ribosomal RNA biosynthesis (17Ko Y.-G. Kang Y.-S. Kim E.-K. Park S.G. Kim S. J. Cell Biol. 2000; 149: 567-574Crossref PubMed Scopus (102) Google Scholar). Seshaiah and Andrew (38Seshaiah P. Andrew D.J. Mol. Biol. Cell. 1999; 10: 1595-1608Crossref PubMed Scopus (32) Google Scholar) also showed that each ARS is uniquely expressed in different tissues and developmental stages ofDrosophila. Thus, the differential expression and cellular localization of each ARS imply that each ARS has its noncanonical function in addition to the catalytic activity for tRNA aminoacylation. Indeed, different ARSs play roles in tRNA maturation (proofreading and nuclear export), cytokine-like activity, mitochondrial RNA splicing, and transcriptional and translational regulation (36Martinis S.A. Plateau P. Cavarelli J. Florentz C. EMBO J. 1999; 18: 4591-4596Crossref PubMed Scopus (109) Google Scholar). Here, we found that human QRS is not only the enzyme for cell proliferation but also the protein that plays a regulatory role in cell death through an antagonistic interaction with ASK1, a protein kinase that plays a critical role in apoptosis. QRS has been classified as a component of the multi-tRNA synthetase complex (39Kisselev L.L. Wolfson A.D. Prog. Nucleic Acids Res. Mol. Biol. 1994; 48: 83-142Crossref PubMed Scopus (90) Google Scholar, 40Yang D.C.H. Curr. Top. Cell. Regul. 1996; 34: 101-136Crossref PubMed Scopus (50) Google Scholar). However, QRS not associated with the multi-tRNA synthetase complex has been found (41Pahuski E. Klekamp M. Condon T. Hampel A.E. J. Cell. Physiol. 1983; 114: 82-87Crossref PubMed Scopus (14) Google Scholar) and shown to be catalytically active (26Kim T. Park S.G. Kim J.E. Seol W. Ko Y.-G. Kim S. J. Biol. Chem. 2000; 275: 21768-21772Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Thus, QRS bound to ASK1 may be in dynamic equilibrium with that in the multi-tRNA synthetase investigations have revealed important effects of exogenous Gln in nutritional rehabilitation, stimulating intestinal villus regrowth after damage from chemotherapy and improving intestinal barrier function. In addition, Gln blocks apoptosis induced by heat shock, irradiation, and c-Myc-overexpression. In Figs. 1 and 2, we show that Gln prevents the activation of stress kinases such as ASK1 and JNK/ASK1 and then apoptosis initiated by Fas ligation, suggesting that Gln is an apoptosis suppressor. The cellular susceptibility to Fas-mediated signaling by Gln deprivation could not be explained by the protein synthesis inhibition, because ASK1 and JNK/SAPK were activated within 10 min after Fas ligation in Gln-free cells but not in normal cells. Therefore, Gln could suppress apoptosis by inhibiting ASK1 and then JNK/SAPK. the of exogenous QRS was expressed in cells in and Gln-free the molecular interaction between QRS and ASK1 was and then the ASK1 activity was inhibited in the of Gln the effect of Gln on the interaction of the two molecules. It has been demonstrated that amino be signaling molecules. For instance, as a in because exogenous stimulates of mitochondrial function in cells P. Nature. 1999; PubMed Scopus Google Scholar). Here, we suggest that Gln could be a signaling for ASK1 activation and suppressing apoptosis via binding to of apoptosis are by the activation of kinase and of kinase by extracellular prevents apoptosis by and that are involved in the apoptosis A. Nature. 1999; PubMed Scopus Google Scholar, E. S. Science. 1998; PubMed Scopus Google Scholar, H. S. H. Y. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. M.J. P. M.J. J. 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