From the
Division of Genome Biology, ¶Division of Experimental Therapeutics, and ||Center for the Development of Molecular Target Drugs, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, and the **Institute for Bioinformatics Research and Development (BIRD), Japan Science and Technology Corporation (JST), 5-3 Yonbancho, Chiyoda-ku, Tokyo 102-0081, Japan
Received for publication, March 27, 2003
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ABSTRACT |
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INTRODUCTION |
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The phosphorylation of eIF2 constitutes an evolutionarily conserved mechanism for eukaryotic translational control (3). In contrast with mammals bearing four eIF2
kinases, the budding yeast has the sole kinase GCN2, which is activated by accumulation of uncharged tRNAs due to amino acid starvation. GCN2 binds uncharged tRNAs via its histidyl-tRNA synthetase (His-RS)-related region. While GCN2 is activated by various stresses other than amino acid starvation, including purine deprivation, glucose limitation, high salinity, and exposure to an alkylating agent, the His-RS-related region has proven essential in all of the cases examined (2).
When supplied with enough nutrients, the yeast cells activate translation for cell growth and proliferation, in which the target of rapamycin (TOR) signaling pathway plays a central role (4, 5, 6). The TOR protein, a member of the phosphatidylinositol 3-kinase superfamily, is involved in regulation of various cellular processes, including transcription, stabilization of mRNAs, translation initiation and elongation, biogenesis of tRNAs and ribosome, turnover of nutrient transporters, protein kinase C signaling, and autophagy (4, 5). For the translation initiation, TOR proteins are known to facilitate the assembly of eIF4F, a heterotrimeric complex composed of eIF4A, -4E, and -4G (6). The eIF4F binds the 7-methylguanosine-containing cap structure via eIF4E to recruit mRNA to ribosome.
Since both general control and the TOR signaling play important roles in translational response to nutritional variation, it is likely that these pathways are coordinately regulated. We thus examined a possible cross-talk between the two pathways and obtained evidence that GCN2 is activated by rapamycin via a novel mechanism independent of an increment of uncharged tRNAs.
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EXPERIMENTAL PROCEDURES |
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PlasmidsThe plasmid p180 is a low copy number reporter construct to monitor translational derepression of GCN4 (7). It encodes for an mRNA bearing GCN4-lacZ fusion ORF preceded by the entire 5'-leader region of GCN4 mRNA, which contains four upstream ORFs responsible for the derepression (1). The plasmid pYC is a centromeretype variant of pYES2, an expression vector using GAL1 promoter (Invitrogen). The pYC01 expressing GCN2 under a control of GAL1 promoter was constructed by gap-repair cloning of the ORF for GCN2. The pYC02 and pYC03, expressing heme-regulated inhibitor (HRI) and double-stranded RNA-activated protein kinase (PKR), were constructed by transferring the SphI-KpnI fragment of p1246 and p1420 to pYC, respectively (8). The pYC04 bearing gcn2-m2 allele (9) was constructed from pYC01 by site-directed mutagenesis. The WH2 cells bearing these eIF2 kinase plasmids were cultivated in SCR (synthetic complete medium containing raffinose instead of glucose) lacking uracil and shifted to SCR Ura His + 20 mM 3-aminotriazole (3AT) or SCR Ura + 200 ng/ml rapamycin.
Reporter Assay for Translational Derepression of GCN4 mRNAThe yeast cells bearing p180 were treated with either 200 ng/ml rapamycin or 20 mM 3AT in SC Ura or SC Ura His, respectively, and assayed for -galactosidase as described previously (10, 11).
Immunoblotting Analysis of eIF2Immunoblotting analysis of eIF2
was performed using an anti-eIF2
peptide antibody and the one specific to eIF2
phosphorylated at serine 51 (eIF2
[pS51]) (BIOSOURCE) as described previously (11). Isoelectric focusing gel electrophoresis was performed as described (12). Immunoblots were developed using a kit for enhanced chemiluminescence (ECL, Amarsham Biosciences).
Reverse Transcription-PCRIsolation of total RNAs, reverse transcription, and PCR were performed as described previously (13).
Analysis of tRNAsAminoacylated tRNAs were analyzed as described (14). Briefly, the yeast cells were disrupted by vigorous shaking with glass beads in AE buffer (50 mM sodium acetate (pH 5.3), 10 mM EDTA) at 4 °C. Total RNAs were extracted with AE-saturated phenol and precipitated with ethanol. Deacylated tRNAs were prepared by heating at 75 °C in 0.1 M Tris-HCl (pH 8.0) for 5 min. Total RNAs (10 µg) were separated at 4 °C by electrophoresis on a 10% polyacrylamide gel containing 8 M urea in 100 mM sodium acetate buffer (pH 5.0). Separated RNAs were blotted onto Hybond N+ membrane (Amersham Biosciences) and hybridized with radiolabeled probe for each tRNA species.
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RESULTS AND DISCUSSION |
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Rapamycin-induced Translation of GCN4 mRNA Requires GCN2-mediated Phosphorylation of eIF2Translational derepression of GCN4 mRNA does not always require the eIF2
kinase GCN2; several alternative mechanisms independent of GCN2 have been reported. For instance, a shift from amino acid-rich to minimal medium was shown to induce GCN4 translation independently of GCN2 (16). The reduction in amount or impairment in base modification of the initiator tRNA also leads to GCN2-independent derepression of GCN4 translation (1, 17). Similarly, overexpression of the RNA component of RNase MRP or tRNA pseudouridine 55 synthetase results in GCN4 derepression without activating GCN2 (18, 19). More recently, fusel alcohols such as butanol were shown to translationally induce GCN4 in a GCN2-independent manner (20).
It is thus important to examine whether rapamycin-induced GCN4 translation accompanies an increase in phosphorylated eIF2. We performed an immunoblot analysis using an antibody specific to eIF2
[pS51]. The results indicated that rapamycin, similar to 3AT, induces a significant increase in eIF2
[pS51], but, importantly, does not affect total eIF2
levels (Fig. 1C). Judging from these results and previous studies on 3AT-treated cells (1, 2), we assume that
50% of eIF2
can be phosphorylated by rapamycin-treatment. Interestingly, rapamycin, but not 3AT, failed to increase eIF2
[pS51] in the cells bearing TOR11 allele, which we had shown to be incapable of derepressing translation of GCN4 mRNA in response to rapamycin (Fig. 1C).
We next intended to know whether the observed increase in eIF2[pS51] is required for the rapamycin-induced translation of GCN4 mRNA. For this purpose, we examined the yeast cells deleted for GCN2, because the gcn2
cell contains no eIF2
kinase activity and thus lacks eIF2
[pS51] (Fig. 1C). As shown in Fig. 1C, rapamycin, similar to 3AT, totally failed to induce
-galatosidase activity in the gcn2
cells.
To further confirm the importance of the phosphorylation, we examined a strain deleted for GCN1, which encodes a GCN2-binding protein required for in vivo activation of the eIF2 kinase (10, 11, 21, 22). The gcn1
cells contain GCN2 in an amount comparable with that in the wild type cells, but fail to activate its kinase activity (8). As shown in Fig. 1C, the gcn1
cells could not induce the reporter activity upon rapamycin treatment. While a novel mode of GCN2 action independent of eIF2
phosphorylation was recently proposed (23), rapamycin-induced GCN4 translation requires the eIF2
kinase activity.
Neither Histidine Starvation nor Rapamycin Inhibits Dephosphorylation of eIF2[pS51]Theoretically, an increment of eIF2
[pS51] can be achieved not only by activation of GCN2 but also by impeded dephosphorylation of eIF2
[pS51]. Involvement of both mechanisms would be also plausible. We thus intended to examine a possible role for compromised dephosphorylation in the increment of eIF2
[pS51] induced by amino acid starvation and rapamycin. For this purpose, we generated strains lacking GCN2 but instead expressing a mammalian non-GCN2 eIF2
kinase, namely HRI or PKR (3). While overexpression of these eIF2
kinases was previously reported to induce a slow growth phenotype (8), our gcn2
strain expressing HRI from a single copy vector displays no apparent growth defect in a raffinose medium. On the other hand, the growth of the PKR-expressing strain was impaired. While the expression of these kinases did not affect total eIF2
levels (Ref. 8 and data not shown), both strains showed an induced level of eIF2
[pS51] comparable with that in the parental GCN2 cells subject to histidine starvation or rapamycin treatment (Fig. 2). These results suggest that the overexpressed eIF2
kinases are somehow disregulated and hence partly activated, as also shown in the previous study (8).
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If amino acid starvation leads to inhibition of eIF2[pS51] phosphatase(s), the steady-state level of eIF2
[pS51] is expected to increase even in the cells expressing the non-GCN2 kinases that are not activated by amino acid starvation (24, 25). We made the HRI-expressing cells starved for histidine and examined the amount of eIF2
[pS51]. In contrast with GCN2-expressing cells, histidine starvation did not increase eIF2
[pS51] in the HRI-expressing cells (Fig. 2). We also examined the PKR-expressing cells to find no change in the amount of eIF2
[pS51] (Fig. 2). It thus seems that amino acid starvation does not affect dephosphorylation of eIF2
[pS51].
We next examined the effect of rapamycin on dephosphorylation of eIF2[pS51] using the same strains. We failed to detect any noticeable change in the amount of eIF2
[pS51] in the rapamycin-treated HRI-expressing cells (Fig. 2). Similarly, the PKR-expressing cells failed to increase eIF2
[pS51] upon rapamycin treatment (Fig. 2). While no evidence has ever been provided for or against rapamycin-induced modulation of HRI or PKR activity, it is highly unlikely that rapamycin affects both the kinases and phosphatase so finely as to keep the steady-state level of eIF2
[pS51] unaffected. It is thus strongly suggested that rapamycin, similar to amino acid starvation, does not inhibit dephosphorylation of eIF2
[pS51].
Rapamycin-induced Activation of GCN2 Requires Binding to Uncharged tRNAsThe results described above indicate that rapamycin induces an increase in eIF2[pS51] via activation of the kinase GCN2. To be activated by amino acid starvation and other various stimuli, GCN2 has to bind uncharged tRNA via its His-RS-like domain (2, 9). We thus examined whether rapamycin-induced GCN2 activation requires an interaction with tRNA using gcn2-m2 mutant defective in tRNA binding (9). As shown in Fig. 3, the gcn2-m2 cells, maintaining a comparable eIF2
level to wild type cells (Ref. 9 and data not shown), respond neither to histidine starvation nor to rapamycin. Thus, the activation of GCN2 by rapamycin, like that by amino acid starvation, requires an interaction of the kinase with uncharged tRNA.
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Rapamycin Activates GCN2 without Increasing Uncharged tRNAsConfirming the requirement for tRNA binding, we next examined whether rapamycin induces an increase in the amount of uncharged tRNAs. Total tRNAs were isolated from histidine-starved and rapamycin-treated cells under a cooled acidic condition so as to minimize deacylation and analyzed by acid-urea polyacrylamide gel electrophoresis. In this electrophoresis, aminoacylated tRNAs display more retarded mobility than uncharged ones, which were obtained through deacylation by a heat treatment under an alkaline condition (Fig. 4). As expected, 3AT-treatment facilitated deacylation of tRNA for histidine to prove the principle of the assay (Fig. 4).
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Replenishment of the 3AT-treated cells with histidine induced a rapid aminoacylation of the tRNA, thereby ceasing GCN2 activation to decline eIF2[pS51]. Intriguingly, the aminoacylation induced by refeding of the starved amino acid was observed even in the cells pretreated with rapamycin. It thus seems that rapamycin does not affect de novo aminoacylation of tRNA.
We next examined tRNAs isolated from the cells treated with rapamycin in a rich medium. Rapamycin-treatment increased eIF2 phosphorylation but failed to induce no apparent change in electrophoretic pattern of bulk tRNAs: most displayed more retarded mobility than alkaline-treated ones, indicative of aminoacylated states (data not shown). Such constitutive aminoacylation was more clearly demonstrated by visualization of specific tRNA on Northern blot (Fig. 4). Therefore, in contrast with amino acid starvation, rapamycin can activate GCN2 without increasing uncharged tRNAs.
Ser-577 Plays a Role in Rapamycin-induced Activation of GCN2In the context described above, it is quite intriguing to note that phosphorylation of GCN2 at Ser-577 was recently reported to reduce its binding affinity to uncharged tRNAs (26). We thus examined gcn2-S577A mutant cells to know whether this residue plays a role in rapamycin-induced activation of GCN2. As expected, the mutant cells showed a significantly higher basal GCN2 activity than its parental cells (Fig. 5). Notably, rapamycin failed to further derepress GCN4 translation in the mutant cells (Fig. 5). The cells bearing gcn2-S577E allele, which had been shown to only partly mimic Ser-577-phosphorylated GCN2 (26), also displayed a similar defective response to rapamycin (Fig. 5). It is thus likely that dephosphorylation of this residue is involved in rapamycin-induced GCN2 activation.
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ConclusionsHere we demonstrated that rapamycin, similar to amino acid starvation, activates the eIF2 kinase GCN2 in an uncharged tRNA-dependent manner (Fig. 3), with constitutive dephosphorylation of eIF2
[pS51] (Fig. 2), to increase the amount of eIF2
[pS51], thereby leading to translational derepression of GCN4 mRNA (Fig. 1). However, in contrast with amino acid starvation, rapamycin can do so without increasing the amount of uncharged tRNAs (Fig. 4), but presumably by acting via dephosphorylation of Ser-577 to increase its tRNA-binding affinity (Fig. 5). Further pursuit of the mechanism for TOR-mediated inhibition of GCN2 would help us better understand not only the cross-talk between the two important pathways for nutritional stress response but the mode of action for rapamycin and its derivatives, which are currently used as immunosuppressants and potential anti-cancer drugs.
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FOOTNOTES |
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Recipient of postdoctoral fellowship from JSPS.
To whom correspondence should be addressed: Division of Genome Biology, Cancer Research Inst., Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan. Tel.: 81-76-265-2726; Fax: 81-76-234-4508; E-mail: titolab{at}kenroku.kanazawa-u.ac.jp.
1 The abbreviations used are: ORF, open reading frame; eIF, eukaryotic initiation factor; His-RS, histidyl-tRNA synthetase; TOR, target of rapamycin; HRI, heme-regulated inhibitor; PKR, double-stranded RNA-activated protein kinase; SCR, synthetic complete medium containing 2% raffinose; 3AT, 3-aminotriazole; SC, synthetic complete medium; eIF2[pS51], eIF2
phosphorylated at Ser-51.
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ACKNOWLEDGMENTS |
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REFERENCES |
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