FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2034195215> ?p ?o ?g. }

• W2034195215 endingPage "2058" @default.
• W2034195215 startingPage "2050" @default.
• W2034195215 abstract "Adaptation to amino acid deficiency is critical for cell survival. In yeast, this adaptation involves phosphorylation of the translation eukaryotic initiation factor (eIF) 2α 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 eIF2α 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 eIF2α 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 eIF2α 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. Adaptation to amino acid deficiency is critical for cell survival. In yeast, this adaptation involves phosphorylation of the translation eukaryotic initiation factor (eIF) 2α 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 eIF2α 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 eIF2α 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 eIF2α 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. eukaryotic initiation factor luciferase open reading frame upstream open reading frame untranslated region Dulbecco's modified Eagle's medium fetal bovine serum amino acid-containing fed starved internal ribosome entry site IRES-specific trans-acting factor hairpin endoplasmic reticulum chloramphenicol acetyltransferase Amino acid starvation of yeast and mammalian cells induces phosphorylation of the translation initiation factor eIF2α,1 altering the pattern of gene expression to remedy the stress and conserve resources for the starving cells (1Pain V.M. Biochimie (Paris). 1994; 76: 718-728Crossref PubMed Scopus (50) Google Scholar). However, stress-response proteins are synthesized under conditions when global protein synthesis is inhibited (2Hinnebusch A.G. Trends Biochem. Sci. 1994; 19: 409-414Abstract Full Text PDF PubMed Scopus (157) Google Scholar). An adaptive response to amino acid starvation for any single amino acid has been extensively characterized in yeast; translation of the transcription factor GCN4 increases during total or single amino acid starvation, causing a transcriptional induction of the amino acid biosynthetic genes (2Hinnebusch A.G. Trends Biochem. Sci. 1994; 19: 409-414Abstract Full Text PDF PubMed Scopus (157) Google Scholar, 3Yang R. Wek S.A. Wek R.C. Mol. Cell. Biol. 2000; 20: 2706-2717Crossref PubMed Scopus (144) Google Scholar, 4Hinnebusch A.G. Mol. Microbiol. 1993; 10: 215-223Crossref PubMed Scopus (120) Google Scholar, 5Hinnebusch A.G. Semin. Cell Biol. 1994; 5: 417-426Crossref PubMed Scopus (123) Google Scholar). Yeast have developed a sophisticated mechanism to increase translation of the GCN4 mRNA when eIF2α is phosphorylated and levels of eIF2·GTP·Met-tRNAMet ternary complexes decrease (6Hinnebusch A.G. CRC Crit. Rev. Biochem. 1986; 21: 277-317Crossref PubMed Scopus (76) Google Scholar); initiation at the GCN4 ORF is inversely related to the concentration of ternary complexes in the cell (5Hinnebusch A.G. Semin. Cell Biol. 1994; 5: 417-426Crossref PubMed Scopus (123) Google Scholar). The regulatory system that mediates the response of yeast to amino acid starvation is known as a general amino acid control mechanism. Mammalian cells have also developed an adaptive response to changes in amino acid availability (7Laine R.O. Hutson R.G. Kilberg M.S. Prog. Nucleic Acids Res. Mol. Biol. 1996; 53: 219-248Crossref PubMed Scopus (29) Google Scholar). Given that essential amino acids have to be transported into cells via membrane-spanning amino acid transporter proteins, part of the adaptive response involves the increased expression of amino acid biosynthetic and transporter genes (8Malandro M.S. Kilberg M.S. Annu. Rev. Biochem. 1996; 65: 305-336Crossref PubMed Scopus (181) Google Scholar). A significant part of this adaptive response is the increased expression of the cationic amino acid transporter 1 gene (cat-1), the main transporter of the essential amino acids arginine and lysine (9Hyatt S.L. Aulak K.S. Malandro M. Kilberg M.S. Hatzoglou M. J. Biol. Chem. 1997; 272: 19951-19957Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). It has been shown previously that amino acid starvation induces the stability (10Aulak K.S. Mishra R. Zhou L. Hyatt S.L. de Jonge W. Lamers W. Snider M. Hatzoglou M. J. Biol. Chem. 1999; 274: 30424-30432Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) and translation of the cat-1 mRNA (11Fernandez J. Yaman I. Mishra R. Merrick W.C. Snider M.D. Lamers W.H. Hatzoglou M. J. Biol. Chem. 2001; 276: 12285-12291Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). This report demonstrates that the mechanism of increased translation of the cat-1 mRNA by amino acid limitation shares features with the general amino acid control mechanism as described in yeast. All the cat-15′-UTR-containing bicistronic mRNA expression vectors were generated by PCR amplification of the cat-1 5′-UTR cDNAs using the SalI/NcoI site of the pSVCAT/ICS/LUC plasmid (12Macejak D.G. Sarnow P. Nature. 1991; 353: 90-94Crossref PubMed Scopus (423) Google Scholar) by replacing the ICS DNA sequence. The plasmids containing the hairpin were constructed by replacing the ICS within the pSVhpCAT/ICS/LUC (12Macejak D.G. Sarnow P. Nature. 1991; 353: 90-94Crossref PubMed Scopus (423) Google Scholar) vector, at the SalI/NcoI sites. The cat-1 5′-UTRf mutants were generated by PCR-directed mutagenesis. The mutf-stop was generated by mutating the uORF stop codon (Fig. 1A, TGA to TTA within thecat-1 5′-UTRf) thus placing the uORF/ATG in frame with the LUC start codon. The mutf-start was generated by mutating the uORF/ATG from ATG to TTG within thecat-1 5′-UTRf and cat-15′-UTRt DNAs. The cat-1 5′-UTRf andcat-1 5′-UTRt have an NcoI site at the 3′ end, which contains the start codon ATG for the LUC cistron. The context of the A+1TG LUC/start codon within the mutf-stop and mutf-start vectors is AGCGCCCA+1TG (compare this sequence with Fig.1A). The monocistronic expression vectors, cat-15′-UTRf/LUC and cat-15′-UTRmutf/LUC, were generated by cloning theSalI/XbaI cat-15′-UTRf/LUC and cat-1 5′-UTRmutf/LUC fragments from the bicistronic vectors into theEcoRI/XbaI site of the pUHD10–3 vector. In this vector, transcription is directed by the minimal cytomegalovirus promoter (13Gossen M. Freundlieb S. Bender G. Muller G. Hillen W. Bujard H. Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2051) Google Scholar). The EcoRI cloning site is 76 nucleotides downstream of the transcription start site (13Gossen M. Freundlieb S. Bender G. Muller G. Hillen W. Bujard H. Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2051) Google Scholar). Expression vectors for the PERK, PERK-mut, GCN2, eIF2α S-A, and eIF2α S-D were kindly provided by D. Ron (New York University School of Medicine, New York, NY). All vectors contained the corresponding inserts within theXbaI/HindIII site of pCDNA3. In this vector, transcription is directed by the cytomegalovirus promoter and the selectable marker gene was replaced with the CD2 cell surface marker gene (14Brewer J.W. Diehl J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12625-12630Crossref PubMed Scopus (364) Google Scholar). The GCN2mut expression vector contained the GCN2mut cDNA (15Sood R. Porter A.C. Olsen D.A. Cavener D.R. Wek R.C. Genetics. 2000; 154: 787-801Crossref PubMed Google Scholar) within the same vector system as the rest. All cells were maintained in DMEM/F-12 medium supplemented with 10% FBS (CON). Plasmid DNA was transfected into C6 rat glioma cells using the calcium phosphate technique (9Hyatt S.L. Aulak K.S. Malandro M. Kilberg M.S. Hatzoglou M. J. Biol. Chem. 1997; 272: 19951-19957Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Cotransfections were performed with equimolar amounts of plasmid DNA. Amino acid-fed (F) cells were incubated in DMEM/F-12 supplemented with FBS dialyzed against phosphate-buffered saline (9Hyatt S.L. Aulak K.S. Malandro M. Kilberg M.S. Hatzoglou M. J. Biol. Chem. 1997; 272: 19951-19957Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Amino acid-starved cells (S) were incubated in Krebs-Ringer buffer supplemented with dialyzed FBS (9Hyatt S.L. Aulak K.S. Malandro M. Kilberg M.S. Hatzoglou M. J. Biol. Chem. 1997; 272: 19951-19957Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). No difference in the regulation of the cat-1 gene by amino acid starvation was observed when Krebs-Ringer buffer containing all amino acids was used in place of DMEM/F-12 medium (9Hyatt S.L. Aulak K.S. Malandro M. Kilberg M.S. Hatzoglou M. J. Biol. Chem. 1997; 272: 19951-19957Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Treatments were performed by culturing cells (5 × 105 cells/35-mm dish) for 48 h in growth medium followed by culture under fed or starved conditions for the appropriate times. Thapsigargin was added to 400 nm as described (16Harding H.P. Zhang Y. Bertolotti A. Zeng H. Ron D. Mol. Cell. 2000; 5: 897-904Abstract Full Text Full Text PDF PubMed Scopus (1570) Google Scholar). Stable C6-GCN2-mut cell lines were generated by transfecting the GCN2-mut-containing expression vector and aneo-expressing vector in a molar ratio of 10:1. Mass cultures were generated from cells selected in 0.1% G418. RNA was isolated from transfected cells with the bicistronic mRNA expression vectors and analyzed by Northern blotting using a LUC-specific hybridization probe. The correct size RNAs for the full-length bicistronic transcripts were observed for all vectors tested in this study. Cell extracts were prepared and analyzed for LUC (Tropix luciferase assay kit) and CAT activities as described previously (17Leahy P. Crawford D.R. Grossman G. Gronostajski R.M. Hanson R.W. J. Biol. Chem. 1999; 274: 8813-8822Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The activities were normalized to the protein content of the cell extracts, which was measured using the Bio-Rad DC assay. Expression of the eIF2αs was evaluated as described in detail previously (18Kaufman R.J. Davies M.V. Pathak V.K. Hershey J.W. Mol. Cell. Biol. 1989; 9: 946-958Crossref PubMed Scopus (333) Google Scholar). Briefly, cells at 36 h after transfection were incubated with [35S]methionine (100 μCi/ml) and cell extracts were prepared in RIPA buffer. Equal amounts of protein extracts were analyzed on an SDS-acrylamide gel and prepared for autoradiography. Details of the phosphorylation status of the eIF2αs and regulation of translation have been described (18Kaufman R.J. Davies M.V. Pathak V.K. Hershey J.W. Mol. Cell. Biol. 1989; 9: 946-958Crossref PubMed Scopus (333) Google Scholar,19Choi S.Y. Scherer B.J. Schnier J. Davies M.V. Kaufman R.J. Hershey J.W. J. Biol. Chem. 1992; 267: 286-293Abstract Full Text PDF PubMed Google Scholar). The previously described leader (5′-UTR) for thecat-1 mRNA was 224 bases (cat-15′-UTRt) and was incomplete (11Fernandez J. Yaman I. Mishra R. Merrick W.C. Snider M.D. Lamers W.H. Hatzoglou M. J. Biol. Chem. 2001; 276: 12285-12291Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). However, this truncatedcat-1 5′ UTRt functioned as an IRES and was regulated by amino acid starvation (11Fernandez J. Yaman I. Mishra R. Merrick W.C. Snider M.D. Lamers W.H. Hatzoglou M. J. Biol. Chem. 2001; 276: 12285-12291Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). To further study this regulation, a full-length 5′-UTR of the cat-1 mRNA was obtained by isolating a rat genomic DNA clone that contained the promoter region, the first exon, and the first intron of thecat-1 gene. 2J. Fernandez, R. Mishra, A. Aulak, D. Robinson, and M. Hatzoglou, manuscript in preparation. The transcription start site was determined in C6 cells (data not shown). This full-length UTR (Fig. 1A,cat-1 5′-UTRf) consists of exon 1 (154 nucleotides, in blue), exon 2 (98 nucleotides, in red), and the 5′ end of exon 3 (18 nucleotides, in red). An interesting feature of thecat-1 5′-UTR is the presence of a uORF. Bothcat-1 5′-UTRt and cat-15′-UTRf contained a 48-amino acid uORF, starting 46 bases 3′ to the mRNA cap site (Fig. 1, A andB). The ability of the cat-1 5′-UTRf sequence to mediate internal translation initiation was tested in a bicistronic expression vector (12Macejak D.G. Sarnow P. Nature. 1991; 353: 90-94Crossref PubMed Scopus (423) Google Scholar). In these vectors (Fig.2A), the CAT enzyme is translated from the first cistron by a cap-dependent scanning mechanism (12Macejak D.G. Sarnow P. Nature. 1991; 353: 90-94Crossref PubMed Scopus (423) Google Scholar). The second cistron, encoding the firefly LUC enzyme, is efficiently translated only if it is preceded by an IRES in the intercistronic spacer region (Fig. 2A). Translation of the second cistron should be independent of translation of the first cistron. A vector that contained a stable RNA hairpin (hp) upstream of the CAT cistron (Fig. 2A) was used to demonstrate that when translation of the CAT cistron is inhibited, translation of the LUC cistron is unaffected. Two bicistronic expression vectors were generated by introducing the cat-1 5′-UTRf into the intercistronic space of the empty expression vector: CAT/cat-1 5′-UTRf/LUC and hpCAT/cat-15′-UTRf/LUC (Fig. 2A). The LUC/CAT ratio was 20-fold higher for the hpCAT/cat-1 5′ UTRf/LUC when compared with the CAT/cat-1 5′-UTRf/LUC. C6 cells were transiently transfected with these vectors, and LUC and CAT activities were analyzed. This increase was because of decreased CAT and sustained LUC activities in the hairpin-containing vector (data not shown). These data demonstrated that the full-length cat-15′-UTRf leader has IRES activity. Transfection of these vectors into C6 cells demonstrated that translational control mediated by the full-length cat-1 5′-UTRf was indistinguishable (data not shown) to the previously tested truncatedcat-1 5′-UTRt (11Fernandez J. Yaman I. Mishra R. Merrick W.C. Snider M.D. Lamers W.H. Hatzoglou M. J. Biol. Chem. 2001; 276: 12285-12291Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Notice that bothcat-1 5′-UTRf and cat-15′-UTRt contain the uORF (Fig. 1). The following studies have been performed using both the truncated (11Fernandez J. Yaman I. Mishra R. Merrick W.C. Snider M.D. Lamers W.H. Hatzoglou M. J. Biol. Chem. 2001; 276: 12285-12291Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) and the full-length (Fig. 2A) UTRs within the bicistronic expression vectors (12Macejak D.G. Sarnow P. Nature. 1991; 353: 90-94Crossref PubMed Scopus (423) Google Scholar). It was shown recently that cat-1 5′-UTRt/IRES-mediated translation increased during amino acid starvation (11Fernandez J. Yaman I. Mishra R. Merrick W.C. Snider M.D. Lamers W.H. Hatzoglou M. J. Biol. Chem. 2001; 276: 12285-12291Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Interestingly, the kinetics of induction of IRES-mediated translation during amino acid starvation were different from the kinetics of induction of eIF2α phosphorylation. Phosphorylation of eIF2α transiently increased during the first hour of amino acid starvation (11Fernandez J. Yaman I. Mishra R. Merrick W.C. Snider M.D. Lamers W.H. Hatzoglou M. J. Biol. Chem. 2001; 276: 12285-12291Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), whereascat-1 IRES-mediated translation increased between 6 and 12 h and declined thereafter (11Fernandez J. Yaman I. Mishra R. Merrick W.C. Snider M.D. Lamers W.H. Hatzoglou M. J. Biol. Chem. 2001; 276: 12285-12291Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). These studies suggested that eIF2α phosphorylation does not directly regulate cat-1IRES activity. This is in contrast to the regulation of theGCN4 and other mammalian mRNAs (5Hinnebusch A.G. Semin. Cell Biol. 1994; 5: 417-426Crossref PubMed Scopus (123) Google Scholar, 20Harding H.P. Novoa I.I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2431) Google Scholar). In these cases, mRNA translation increases at the time when eIF2·GTP·Met-tRNAMet ternary complexes decrease (5Hinnebusch A.G. Semin. Cell Biol. 1994; 5: 417-426Crossref PubMed Scopus (123) Google Scholar). In yeast, uncharged tRNAs, which accumulate in cells depleted by any single amino acid, bind to and activate the yeast GCN2 cellular kinase, leading to phosphorylation of eIF2α and translational control (21Qiu H. Garcia-Barrio M.T. Hinnebusch A.G. Mol. Cell. Biol. 1998; 18: 2697-2711Crossref PubMed Scopus (51) Google Scholar). To gain an insight on the involvement of eIF2α phosphorylation on the increased cat-1 mRNA translation during amino acid starvation, the regulation of cat-1 IRES-mediated translation by individual amino acids was studied in C6 cells. Depletion of any single essential amino acid increased cat-1IRES activity with the same kinetics and to the same degree as described for total amino acid starvation (Fig. 2B and TableI). This is in agreement with translational control of the GCN4 mRNA by amino acid depletion (2Hinnebusch A.G. Trends Biochem. Sci. 1994; 19: 409-414Abstract Full Text PDF PubMed Scopus (157) Google Scholar) and supports the hypothesis that eIF2α phosphorylation is involved indirectly in cat-1 IRES translational control.Table IAbsolute values of LUC and CAT activities from experiments described in Fig. 2ExperimentsCATLUCLUC/CATunits/μg proteinunits/μg proteinExp. from Fig. 2B Control97 ± 498 ± 191.0 ± 0.2 -Arg73 ± 2993 ± 4613 ± 0.3 -Cys56 ± 6998 ± 8017 ± 2.4 -His57 ± 6974 ± 10017 ± 2.0 -Ile64 ± 21061 ± 3916 ± 1.0 -Leu59 ± 31016 ± 6317 ± 0.8 -Lys57 ± 3903 ± 9916 ± 0.1 -Met56 ± 2925 ± 9516 ± 1.1 -Phe59 ± 4923 ± 2715 ± 0.6 -Thr61 ± 31036 ± 10717 ± 1.7 -Trp58 ± 1943 ± 4216 ± 0.6 -Tyr64 ± 41120 ± 6518 ± 1.0 -Val55 ± 4871 ± 8516 ± 0.5Exp. from Fig. 2C ControlFed72 ± 3158 ± 282.2 ± 0.4Starved49 ± 61341 ± 25428 ± 6.8 GCN2Fed73 ± 9119 ± 361.7 ± 0.5Starved50 ± 31950 ± 21139 ± 2.5 GCN2-mutFed74 ± 1186 ± 161.2 ± 0.1Starved57 ± 2166 ± 332.9 ± 0.6 PERKFed69 ± 16190 ± 122.8 ± 0.4Starved42 ± 21478 ± 37134 ± 8.1 PERK-mutFed77 ± 6103 ± 161.3 ± 0.2Starved59 ± 3795 ± 13613 ± 2.2 PKR+/+Fed31 ± 3247 ± 558.0 ± 0.7Starved22 ± 21048 ± 7247 ± 1.8 PKR0/0Fed32 ± 2232 ± 277.2 ± 0.5Starved20 ± 1925 ± 7546 ± 4.5 Open table in a new tab We therefore tested whether eIF2α phosphorylation and mammalian GCN2 kinase activation are involved in cat-1 IRES activation during amino acid starvation. To test this hypothesis, C6 cells were transfected with the bicistronic expression vector containing thecat-1 5′-UTRt along with expression vectors expressing either the wild type mammalian GCN2 kinase (22Berlanga J.J. Santoyo J. De Haro C. Eur. J. Biochem. 1999; 265: 754-762Crossref PubMed Scopus (221) Google Scholar, 23Sood R. Porter A.C. Ma K. Quilliam L.A. Wek R.C. Biochem. J. 2000; 346: 281-293Crossref PubMed Scopus (134) Google Scholar) or a mutant GCN2, which lacked kinase activity (23Sood R. Porter A.C. Ma K. Quilliam L.A. Wek R.C. Biochem. J. 2000; 346: 281-293Crossref PubMed Scopus (134) Google Scholar). The mutant kinase forms inactive heterodimers with the wild type GCN2 kinase. Therefore, activation of the endogenous wild type GCN2 (23Sood R. Porter A.C. Ma K. Quilliam L.A. Wek R.C. Biochem. J. 2000; 346: 281-293Crossref PubMed Scopus (134) Google Scholar) is inhibited, leading to inhibition of eIF2α phosphorylation by amino acid starvation. The transfected wild type and mutant GCN2 kinases contained the FLAG epitope (15Sood R. Porter A.C. Olsen D.A. Cavener D.R. Wek R.C. Genetics. 2000; 154: 787-801Crossref PubMed Google Scholar), which allowed us to monitor expression of the mutant proteins by Western blot analysis (data not shown). Following 36 h of transfection, cells were incubated in either amino acid-containing (F) or amino acid-depleted (S) medium for 9 h as described previously (11Fernandez J. Yaman I. Mishra R. Merrick W.C. Snider M.D. Lamers W.H. Hatzoglou M. J. Biol. Chem. 2001; 276: 12285-12291Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). As shown, expression of the mutant kinase inhibitedcat-1 IRES activation by amino acid starvation (Fig.2C, compare the -fold change of the ratio LUC/CAT). Induction in LUC activity was 9-fold for control, 15-fold for the GCN2 wild type, and 2-fold for GCN2 mutant-expressing cells (Table I). The increases in LUC/CAT ratios were 13-, 21-, and 2.5-fold respectively (Fig. 2C and Table I). The CAT activity decreased by 30% (Table I). When the bicistronic cat-15′-UTRf-containing mRNA was tested, regulation of translation of the LUC cistron was indistinguishable from thecat-1 5′-UTRt (data not shown). It was concluded that cat-1 IRES translational activation by amino acid starvation depends on GCN2 activity. However, these experiments were transient transfections and did not allow the evaluation of the degree of eIF2α phosphorylation by amino acid starvation in cells expressing the mutant GCN2 kinase. This was demonstrated in a stable C6 cell line that expressed the mutant GCN2 kinase (Fig.3A). Amino acid starvation of this cell line caused a small increase in phospho-eIF2α levels (phospho-eIF2α levels increased 3-fold in control (data not shown) and 1.4-fold in GCN2-mut cells). However, cat-1IRES-mediated translation did not increase (Fig. 3B and Table II). The ability of eIF2α to be phosphorylated in the GCN2-mut cells was tested by treating them with the endoplasmic reticulum (ER) stress-causing agent thapsigargin. As expected (16Harding H.P. Zhang Y. Bertolotti A. Zeng H. Ron D. Mol. Cell. 2000; 5: 897-904Abstract Full Text Full Text PDF PubMed Scopus (1570) Google Scholar), thapsigargin treatment of cells induced eIF2α phosphorylation by 3-fold (Fig. 3A). The expression of the GCN2 mutant kinase decreased during the time course of amino acid starvation, as was expected, because of the decreased cap-dependent translation of the GCN2-mut mRNA (Fig.3A). 4E-BP-1 is dephosphorylated during the time course of amino acid starvation, thus decreasing cap-dependent translation by sequestering the cap-binding protein eIF4E (Fig.3A). However, expression of the mutant kinase is critical the first hour of amino acid starvation when the endogenous GCN2 kinase is activated (15Sood R. Porter A.C. Olsen D.A. Cavener D.R. Wek R.C. Genetics. 2000; 154: 787-801Crossref PubMed Google Scholar) and phosphorylates eIF2α. At 1 h of amino acid starvation, the mutant kinase was expressed at the same level as in amino acid-fed cells (Fig. 3A). These data further demonstrate that decreasing cap-dependent translation is not the cause for increased cat-1 IRES-activation. This is in agreement with our previous finding that treatment of cells with rapamycin did not alter cat-1 IRES-mediated translation (11Fernandez J. Yaman I. Mishra R. Merrick W.C. Snider M.D. Lamers W.H. Hatzoglou M. J. Biol. Chem. 2001; 276: 12285-12291Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). We therefore conclude that eIF2α phosphorylation by the GCN2 kinase is required for increased cat-1 IRES-mediated translation by amino acid starvation. This finding is in agreement with the regulation of translation of the yeast GCN4 mRNA by the yeast GCN2 kinase (5Hinnebusch A.G. Semin. Cell Biol. 1994; 5: 417-426Crossref PubMed Scopus (123) Google Scholar). However, in contrast to the yeast paradigm, eIF2α phosphorylation regulates cat-1 IRES-mediated translation indirectly.Table IIAbsolute values of LUC and CAT activities from experiments described in Fig. 3Fig. 3BexperimentCATLUCLUC/CATunits/μg proteinunits/μg proteincat-1 5′-UTRf CON412 ± 56772 ± 1241.9 ± 0.1 S3405 ± 14970 ± 232.3 ± 0.1 S6397 ± 241246 ± 463.1 ± 0.0 S9410 ± 271037 ± 732.5 ± 0.2 Open table in a new tab At least four kinases are known to phosphorylate eIF2α: GCN2, PERK, PKR, and HRI (24de Haro C. Mendez R. Santoyo J. FASEB J. 1996; 10: 1378-1387Crossref PubMed Scopus (239) Google Scholar). To determine the specific requirement for GCN2, the ER stress-induced ER-resident transmembrane kinase PERK (16)was tested. We transfected C6 cells with the bicistronic mRNA expression vector that contained the cat-1 5′-UTRt along with expression vectors expressing either the wild type PERK (14Brewer J.W. Diehl J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12625-12630Crossref PubMed Scopus (364) Google Scholar) or a mutant PERK, which lacked kinase activity (14Brewer J.W. Diehl J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12625-12630Crossref PubMed Scopus (364) Google Scholar). As with GCN2, overexpression of the mutant kinase forms dimers with the wild type PERK; however, this should not affect the activation of GCN2 by amino acid starvation. The expression of the mutant kinase was monitored by Western blot analysis of cell extracts prepared from transfected cells using an antibody against a Myc epitope (14Brewer J.W. Diehl J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12625-12630Crossref PubMed Scopus (364) Google Scholar) that was contained at the NH2 terminus of the kinase (data not shown). The levels of the mutant PERK kinase were sustained at the same level for the first 6 h of amino acid starvation and gradually declined thereafter (data not shown). Following 36 h of transfection, cells were incubated in either amino acid-fed or amino acid-starved medium for 9 h as described previously (11Fernandez J. Yaman I. Mishra R. Merrick W.C. Snider M.D. Lamers W.H. Hatzoglou M. J. Biol. Chem. 2001; 276: 12285-12291Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Expression of the PERK kinases did not affect induction of cat-1 IRES activity by amino acid starvation (Fig. 2C, compare the -fold change of the ratio LUC/CAT in PERK and PERK-mut cells). Amino acid starvation increased the LUC activity by 8-fold and decreased the CAT activity by 35 and 25%, respectively, in PERK wild type- and PERK mutant-expressing cells (Table I). The LUC/CAT ratio increased 12-fold for the PERK and 11-fold for the PERK-mut cells (Fig. 2C). It should be noted that the expression of the mutant PERK kinase decreased basal cat-1 IRES activity. This was because of a decrease in basal eIF2α phosphorylation levels in these cells (data not shown). The involvement of the PKR eIF2α kinase was tested next. PKR is activated by binding double-stranded RNA (25Clemens M.J. Bommer U.A. Int. J. Biochem. Cell Biol. 1999; 31: 1-23Crossref PubMed Scopus (206) Google Scholar, 26Proud C.G. Trends Biochem. Sci. 1995; 20: 241-246Abstract Full Text PDF PubMed Scopus (200) Google Scholar). To address the role of the PKR kinase, mouse embryo fibroblasts that either expressed the endogenous PKR kinase (PKR+/+) or had the kinase inactivated (PKR0/0) by homologous recombination were used (27Yang Y.L. Reis L.F. Pavlovic J. Aguzzi A. Schafer R. Kumar A. Williams B.R. Aguet M. Weissmann C. EMBO J. 1995; 14: 6095-6106Crossref PubMed Scopus (568) Google Scholar). cat-1 IRES-mediated translation was measured following 9 h of amino acid starvation. The basal activity for LUC and CAT were similar in PKR+/+ and PKR0/0 cells (Fig.2C and Table I). Amino acid starvation of cells induced the LUC activity by 4-fold and decreased the CAT by 40% in both cell lines (Fig. 2C and Table I). The LUC/CAT ratio increased 6-fold in the PKR+/+ and 5.5-fold in the PKR0/0 cells (Fig. 2C). The increase of cat-1 IRES activity was lower than C6 cells (4-fold as compared with 7-fold in C6 cells). The lower induction in PKR cells may be because of cell type differences. The latter is supported by the fact that the basal LUC activity in PKR cells was 4-fold higher than the equivalent in C6 cells (Table I). These data suggest that eIF2α phosphorylation andcat-1 IRES translational induction during amino acid starvation depend on GCN2 activation. To further demonstrate that phosphorylation of eIF2α is required for cat-1 IRES activation, we overexpressed an eIF2α that can either not be phosphorylated (Ser-51 changed to Ala) or mim" @default.
• W2034195215 created "2016-06-24" @default.
• W2034195215 creator A5002284937 @default.
• W2034195215 creator A5009709313 @default.
• W2034195215 creator A5035546319 @default.
• W2034195215 creator A5058869043 @default.
• W2034195215 creator A5078698929 @default.
• W2034195215 creator A5082229689 @default.
• W2034195215 creator A5082256441 @default.
• W2034195215 creator A5083775097 @default.
• W2034195215 date "2002-01-01" @default.
• W2034195215 modified "2023-09-29" @default.
• W2034195215 title "Regulation of Internal Ribosome Entry Site-mediated Translation by Eukaryotic Initiation Factor-2α Phosphorylation and Translation of a Small Upstream Open Reading Frame" @default.
• W2034195215 cites W1510227168 @default.
• W2034195215 cites W1569857352 @default.
• W2034195215 cites W1581963365 @default.
• W2034195215 cites W1822195738 @default.
• W2034195215 cites W1915631065 @default.
• W2034195215 cites W1971140237 @default.
• W2034195215 cites W1972835861 @default.
• W2034195215 cites W1976641851 @default.
• W2034195215 cites W1981577767 @default.
• W2034195215 cites W1991999080 @default.
• W2034195215 cites W1996084661 @default.
• W2034195215 cites W2003241548 @default.
• W2034195215 cites W2005275397 @default.
• W2034195215 cites W2007793133 @default.
• W2034195215 cites W2022846624 @default.
• W2034195215 cites W2027195993 @default.
• W2034195215 cites W2033084354 @default.
• W2034195215 cites W2045433502 @default.
• W2034195215 cites W2052416392 @default.
• W2034195215 cites W2060164191 @default.
• W2034195215 cites W2066825851 @default.
• W2034195215 cites W2076139045 @default.
• W2034195215 cites W2082546954 @default.
• W2034195215 cites W2088959549 @default.
• W2034195215 cites W2093158584 @default.
• W2034195215 cites W2096470485 @default.
• W2034195215 cites W2114079476 @default.
• W2034195215 cites W2121401282 @default.
• W2034195215 cites W2125833733 @default.
• W2034195215 cites W2127848114 @default.
• W2034195215 cites W2140549895 @default.
• W2034195215 cites W2145806440 @default.
• W2034195215 cites W2147336345 @default.
• W2034195215 cites W2149918090 @default.
• W2034195215 cites W2150518523 @default.
• W2034195215 cites W2170598671 @default.
• W2034195215 cites W2174879004 @default.
• W2034195215 cites W2214383802 @default.
• W2034195215 cites W2330638116 @default.
• W2034195215 cites W4242150291 @default.
• W2034195215 doi "https://doi.org/10.1074/jbc.m109199200" @default.
• W2034195215 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11684693" @default.
• W2034195215 hasPublicationYear "2002" @default.
• W2034195215 type Work @default.
• W2034195215 sameAs 2034195215 @default.
• W2034195215 citedByCount "98" @default.
• W2034195215 countsByYear W20341952152012 @default.
• W2034195215 countsByYear W20341952152013 @default.
• W2034195215 countsByYear W20341952152014 @default.
• W2034195215 countsByYear W20341952152015 @default.
• W2034195215 countsByYear W20341952152016 @default.
• W2034195215 countsByYear W20341952152017 @default.
• W2034195215 countsByYear W20341952152018 @default.
• W2034195215 countsByYear W20341952152019 @default.
• W2034195215 countsByYear W20341952152020 @default.
• W2034195215 crossrefType "journal-article" @default.
• W2034195215 hasAuthorship W2034195215A5002284937 @default.
• W2034195215 hasAuthorship W2034195215A5009709313 @default.
• W2034195215 hasAuthorship W2034195215A5035546319 @default.
• W2034195215 hasAuthorship W2034195215A5058869043 @default.
• W2034195215 hasAuthorship W2034195215A5078698929 @default.
• W2034195215 hasAuthorship W2034195215A5082229689 @default.
• W2034195215 hasAuthorship W2034195215A5082256441 @default.
• W2034195215 hasAuthorship W2034195215A5083775097 @default.
• W2034195215 hasBestOaLocation W20341952151 @default.
• W2034195215 hasConcept C104317684 @default.
• W2034195215 hasConcept C105580179 @default.
• W2034195215 hasConcept C11960822 @default.
• W2034195215 hasConcept C149364088 @default.
• W2034195215 hasConcept C166258313 @default.
• W2034195215 hasConcept C167625842 @default.
• W2034195215 hasConcept C185592680 @default.
• W2034195215 hasConcept C39685955 @default.
• W2034195215 hasConcept C4718897 @default.
• W2034195215 hasConcept C47289529 @default.
• W2034195215 hasConcept C55493867 @default.
• W2034195215 hasConcept C62493599 @default.
• W2034195215 hasConcept C67705224 @default.
• W2034195215 hasConcept C77430603 @default.
• W2034195215 hasConcept C86803240 @default.
• W2034195215 hasConcept C88478588 @default.
• W2034195215 hasConcept C95444343 @default.
• W2034195215 hasConcept C97154109 @default.
• W2034195215 hasConceptScore W2034195215C104317684 @default.
• W2034195215 hasConceptScore W2034195215C105580179 @default.

• next