From the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122
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ABSTRACT |
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A family of protein kinases regulate translation
initiation in response to cellular stresses by phosphorylation of
eukaryotic initiation factor-2 (eIF-2). One family member from yeast,
GCN2, contains a region homologous to histidyl-tRNA synthetases
juxtaposed to the kinase catalytic domain. It is thought that uncharged
tRNA accumulating during amino acid starvation binds to the
synthetase-related sequences and stimulates phosphorylation of the subunit of eIF-2. In this report, we define another domain in GCN2 that
functions to target the kinase to ribosomes. A truncated version of
GCN2 containing only amino acid residues 1467 to 1590 can independently associate with the translational machinery. Interestingly, this region
of GCN2 shares sequence similarities with the core of the double-stranded RNA-binding domain (DRBD). Substitutions of the lysine
residues conserved among DRBD sequences block association of GCN2 with
ribosomes and impaired the ability of the kinase to stimulate
translational control in response to amino acid limitation. Additionally, as found for other DRBD sequences, recombinant protein containing GCN2 residues 1467-1590 can bind double-stranded RNA in vitro, suggesting that interaction with rRNA mediates
ribosome targeting. These results indicate that appropriate ribosome
localization of the kinase is an obligate step in the mechanism leading
to translational control by GCN2.
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INTRODUCTION |
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Targeting of proteins to different compartments in the cell is an important mechanism regulating protein function. Proteins can associate with organelles, membranes, or components in the soluble fraction of the cell, providing proteins access to substrates or regulatory ligands. GCN2 is a member of a family of protein kinases that regulate translation by phosphorylation of eukaryotic translation initiation factor-2 (eIF-2)1 (1-4). Localization of GCN2 protein kinase to ribosomes appears to be a critical step leading to phosphorylation of eIF-2 in response to cellular stress.
Phosphorylation of eIF-2 is a well characterized mechanism regulating
eukaryotic protein synthesis. The eIF-2 is a three-subunit protein that
couples with Met-tRNAiMet and
participates in ribosomal selection of the start codon (5). During this
initiation process, GTP bound to eIF-2 is hydrolyzed to GDP.
Phosphorylation of the subunit of eIF-2 at serine 51 impedes
recycling of eIF-2-GDP to the active form, eIF-2-GTP. Currently, three
protein kinases that phosphorylate this regulated site of eIF-2 have
been characterized and their cDNAs cloned (1-3). Two of the
proteins regulate protein synthesis in mammalian cells. The
RNA-dependent protein kinase, PKR, participates in the
antiviral defense mechanism mediated by interferon (6) and is also
thought to function as a suppressor of cell proliferation and
tumorigenesis (7-9), and the heme-regulated inhibitor kinase, HRI, is
expressed predominately in reticulocytes and bone marrow and couples
the synthesis of globin, the principal translation product in these tissues, to hemin availability (10). The third eIF-2 kinase, GCN2,
functions in the general amino acid control pathway of yeast Saccharomyces cerevisiae. In response to starvation for any
one of several different amino acids, GCN2 phosphorylation of eIF-2 stimulates the translation of GCN4 (2, 4, 11, 12). The GCN4
protein is a transcriptional activator of more than 30 genes involved
in amino acid biosynthesis.
This report centers on the regulation of the GCN2 protein kinase. The kinase catalytic domain of GCN2 shares sequence and structural similarities with the PKR and HRI that are distinguishable from other eukaryotic protein kinases (2, 13, 14). Adjacent to the kinase catalytic domain, GCN2 contains a region homologous to histidyl-tRNA synthetase (HisRS) that binds uncharged tRNA (12, 15). It is proposed that different uncharged tRNAs, which accumulate during amino acid starvation conditions, can interact with the synthetase-related domain of GCN2, resulting in activation of the kinase and phosphorylation of eIF-2 (2, 4, 12, 15, 16).
Another domain that is important for regulation of GCN2 involves
targeting of the kinase to ribosomes. Ramirez et al. (17) showed by several criteria that GCN2 was associated with ribosomes. GCN2 co-migrated with free 40 S and 60 S ribosomal subunits, 80 S
particles, and polysomes separated by sucrose gradient centrifugation. When ribosomes were dissociated into 40 S and 60 S subunits by omitting
MgCl2 from the extract preparation, GCN2 remained
associated with 60 S ribosomal subunits (17). GCN2 was also complexed
with ribosomal subunits after electrophoresis in a composite
agarose-acrylamide gel under nondenaturing conditions. The related
eIF-2 kinase, PKR, was also found to interact with the ribosomal
machinery based on biochemical fractionation (3, 18-20) and
immunofluorescent staining (21). Two regions in the amino
terminus of PKR, designated dsRNA-binding domains (DRBDs),
contain several basic amino acids in a predicted -helical structure
that are related to a family of RNA-binding proteins (22-25). In
addition to regulating kinase activity by dsRNA, the DRBD sequences
facilitate PKR association with ribosomes (20). PKR targeting to
ribosomes is proposed to enhance in vivo phosphorylation of
eIF-2 by providing the kinase access to this substrate.
In this report, we describe the finding that GCN2 residues 1467-1590 can bind independently to ribosomes. This GCN2 domain contains a lysine-rich sequence with features similar to the core of the DRBDs. Alteration of these conserved lysine residues blocked both GCN2 interaction with ribosomes and stimulation of GCN4 expression in response to starvation for amino acids. As previously observed for the DRBDs, the lysine residues are essential for binding to dsRNA in vitro, suggesting that interaction with rRNA mediates ribosome targeting. This cellular localization may provide GCN2 access to its eIF-2 substrate or to regulatory ligands. Furthermore, these findings indicate that related RNA-binding sequences facilitates the in vivo activities of both GCN2 and PKR in response to cellular stresses.
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MATERIALS AND METHODS |
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Yeast Strains and Plasmid Constructions--
Yeast strains H1149
(MAT gcn2::LEU2 ino1 ura3-52 leu2-3, -112 HIS4-lacZ) (15) and H1816 (MATa ura3-52
leu2-3,-112
gcn2
sui2 GCN4-lacZ p1097
[SUI2, LEU2]) (11) were transformed with different alleles of GCN2 in the following low copy number
URA3-based plasmids: GCN2 in p722 (26);
gcn2-m2 in p299 (12); GCN2 in pC102-2 (27); and
p560 (15) is a derivative of pC102-2 containing a unique
SacI restriction site introduced into GCN2 after
the codon for residue 1467. There were no detectable differences in the
GCN2 phenotype between strains transformed with plasmids
p722, pC102-2, or p560. Plasmid p630 (26) contains GCN2
inserted into the high copy number URA3 plasmid YEp24.
Plasmids p332 (12) and p644 (26) are derivatives of p630 containing the
gcn2-m2 and gcn2-K559R alleles, respectively. To
construct the gcn2-605 mutation, three lysine residues in
the carboxyl terminus of GCN2, at positions 1483, 1484, and 1487, were
altered by polymerase chain reaction to CTG, ATA, and ATA encoding
leucine, isoleucine, and isoleucine, respectively. The
gcn2-605 mutation was introduced into plasmid p560 to
generate the low copy number plasmid pSZ-6, and into high copy number
plasmid p630 to generate pSZ-15.
Assay for GCN4-lacZ and His4-lacZ Enzyme
Activity--
-Galactosidase activity was measured as described
previously (30). For repressing conditions, saturated cultures were
diluted 1:50 in the synthetic dextrose (SD) medium (31) supplemented with essential amino acids and cells were harvested after growth for
5 h at 30 °C. For starvation conditions, cells were grown for
2 h under repressing conditions, 10 mM 3-aminotriazole
was added to the medium and the culture was incubated for an additional 5 h at 30 °C. Values reported here are the averages from three independent assays.
-Galactosidase activities were expressed as
nanomoles of
o-nitrophenyl-
-D-galactopyranoside hydrolyzed per min/mg of protein.
GCN2 Immunoblot and Immunoprecipitation Kinase Assay-- Immunoblots were carried out as described previously (26). Full-length GCN2 and gcn2-502-1054 were detected using rabbit polyclonal antiserum prepared against a TrpE-GCN2 fusion protein (15) and 125I-protein A. The polyhistidine tagged gcn2-1467-1590 was measured by immunoblot analysis using rabbit polyclonal antibody prepared against the carboxyl terminus of GCN2 and horseradish peroxidase-labeled secondary antibody. The gcn2-1467-1590 fusion protein, with a molecular weight of 16,000, was also detected using rabbit polyclonal antisera prepared against the polyhistidine tag encoded in pET-15b and was absent in strain WY294 containing the pYCDE2 vector alone. Relative amounts of GCN2 proteins were compared by measuring band intensities from autoradiographs of different length exposures using a Bio-Rad Model GS-670 Imaging Densitometer. GCN2 immunoprecipitation kinase assays were performed as described previously with the immunoprecipitation reactions carried out in the presence of 0.1% SDS, 1.0% Triton X-100, and 0.5% sodium deoxycholate (17, 26).
Ribosome Association-- Yeast strain H1149 was transformed with pC102-2, low copy number plasmid encoding GCN2; p630, high copy GCN2; p299, low copy gcn2-m2; p332, high copy gcn2-m2; pSZ-6, low copy gcn2-605; pSZ-15, high copy gcn2-605; or p644, high copy gcn2-K559R. No differences were detected in GCN2 ribosome association when the kinase was expressed from low copy or high copy number plasmids (17). The gcn2-1467-1590 protein was expressed using strain WY294 containing pSZ-31 and gcn2-502-1054 was expressed using H1816 containing p408 (28). Cells were grown under repressing conditions and 50 µg/ml cycloheximide was added to the culture 5 min before harvesting. Cells were collected by centrifugation and washed once with Breaking Solution (20 mM Tris-HCl, pH 7.9, 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 50 µg/ml cycloheximide, and 200 µg/ml heparin). Cells were resuspended in Breaking Solution in the presence of protease inhibitors (1 µM pepstatin, 1 µM leupeptin, 0.15 µM aprotinin, and 100 µM phenylmethylsulfonyl fluoride), lysed using glass beads, and clarified. Supernatant samples containing 20 A260 units were loaded onto a 5-47% sucrose gradient in Breaking Solution without heparin as described previously (17) and ultracentrifugation was performed using a Beckman rotor SW41 at 39,000 rpm for 3 h. Gradients were fractionated using an ISCO UA-6 absorbance monitor set at 254 nm and 0.5-ml aliquots were collected. Sucrose gradient analysis of the gcn2-1467-1590 fusion was also performed without cycloheximide and Mg2+ using a modified Breaking Solution (20 mM Tris-HCl, pH 7.9, 50 mM NaCl, 1 mM dithiothreitol, and 1 mM EDTA) in the presence or absence of 0.5 M KCl as described (17).
Expression of Recombinant Carboxyl-terminal GCN2
Protein--
E. coli strain BL21 (DE3)
(F ompT rB
mB
containing lysogen DE3) transformed with either
expression plasmid pSZ-3 or pSZ-5 was grown at 30 °C in LB medium
supplemented with 100 µg/ml ampicillin until mid-logarithmic phase
and 0.5 mM
isopropyl-1-thio-
-D-galactopyranoside was added to the
culture and incubated for an additional 3 h. The cell pellet was
collected by centrifugation and washed once with a solution of 20 mM Tris-HCl, pH 7.9, and 500 mM NaCl. Cells were then resuspended in solution A (20 mM Tris-HCl, pH
7.9, 500 mM NaCl, 10% glycerol, and 1 mM
phenylmethylsulfonyl fluoride) with 5 mM imidazole and
lysed using a French press. Lysates were clarified by centrifugation at
39,000 × g and supernatant was loaded onto a column
containing nickel chelation resin (Qiagen, Hilden, Germany) that binds
to the polyhistidine tag of the fusion proteins. After washing the
column with solution A containing 180 mM imidazole,
gcn2-1467-1590 fusion proteins were eluted with 400 mM
imidazole in solution A. The molecular weight of the gcn2-1467-1590 fusion protein was 16,000, in agreement with that predicted from the
DNA sequence. This protein was absent from an identically prepared
extract from BL21(DE3) transformed with vector pET-15b. Additionally,
both GCN2 and gcn2-605 recombinant proteins were recognized by
polyclonal antiserum prepared against the carboxyl terminus of
GCN2.
dsRNA Binding Assay-- To measure binding of the gcn2-1467-1590 recombinant to dsRNA, we followed a procedure similar to that described by O'Malley et al. (32). Briefly, 5 µg of GCN2 or gcn2-605 recombinant protein were mixed with poly(I)·poly(C) bound to Sepharose 4B (Pharmacia Biotech) or Sepharose 4B alone in a 100-µl solution of binding buffer (20 mM HEPES, pH 7.5, 200 mM KCl, 10 mM MgCl2, and 0.5% Nonidet P-40). After incubating the binding mixtures for 30 min at room temperature, the beads were collected by brief centrifugation at 10,000 × g. Beads were washed three times in binding buffer and recombinant protein bound to the beads were eluted by added SDS sample buffer, followed by boiling for 5 min. Equal aliquots of each sample were analyzed by gel electrophoresis in a 15% SDS-polyacrylamide gel and the recombinant protein was visualized by staining the gel with Coomassie Blue.
Circular Dichroism Spectrapolarimetry-- CD spectra were measured at 25 °C using a Jasco J-720 spectropolarimeter. Samples were prepared in 25 mM potassium phosphate buffer (pH 7.5). Three scans were averaged and the spectra were recorded with a 0.1-cm path length cuvette from 190 to 280 nm at a speed of 50 nm/min and with an increment of 1 nm. The mean residue ellipticities were calculated per amide bond. Secondary structure contents were estimated using the reference spectra of Yang et al. (33) and the SSE-338 program (Japan Spectroscopic Co., Tokyo, Japan).
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RESULTS |
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Carboxyl Terminus of GCN2 Functions as a Ribosome-binding
Domain--
GCN2 interaction with ribosomes is proposed to facilitate
stimulation of GCN4 translation in response to amino acid
starvation. Ribosomal association appears to involve the
carboxyl-terminal portion of GCN2 since deletion of this region reduced
interaction of the kinase with ribosomes (17). To directly address
whether the carboxyl terminus of GCN2 functions independently as a
ribosome-binding domain, we expressed a polyhistidine fusion protein
containing only GCN2 residues 1467-1590 in yeast strain WY294. Cell
lysates were prepared as described under "Materials and Methods"
and analyzed by sucrose gradient sedimentation. The distribution of the
gcn2-1467-1590 in each gradient fraction was measured by immunoblot
using polyclonal antiserum prepared against the carboxyl terminus of
GCN2 (Fig. 1). The truncated GCN2 protein
co-sedimented with ribosomes, with over 80% of gcn2-1467-1590 found
in gradient fractions containing 60 S and 80 S particles and polysomes.
Similar results were found when the truncated GCN2 protein was
expressed in strain H1894 (gcn2) indicating that the
ribosomal association was independent of endogenous full-length GCN2.
By comparison, the GCN2 kinase catalytic sequences from 502 to 1054 expressed in yeast migrated free of ribosomes when analyzed in a
similar sucrose gradient (Fig. 1B).
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DRBD-related Sequences in the Carboxyl Terminus of GCN2 Are
Required for Stimulation of the General Amino Acid Control
Pathway--
Given that the DRBD sequences of PKR mediate association
of this mammalian eIF-2 kinase with ribosomes (20), we examined whether
there are sequence similarities between GCN2 residues 1467-1590 and
the RNA-binding regions of PKR. Interestingly, residues 1479 to 1498 in
GCN2 share sequence features similar to the core of the DRBD sequences
found in PKR and other members of this RNA-binding family (Fig.
2). Although the structure of the
RNA-binding regions in PKR have not yet been determined, the resolved
structures of DRBD sequences from Staufen protein in Drosophila
melanogaster and RNase III from E. coli show that this
core region is -helical (23, 34). The conserved lysine residues are
located at the amino-terminal portion of the helix and are proposed to
directly contact dsRNA (25, 35). Analysis of the GCN2 sequence also leads to a prediction of positively charged residues clustered in an
-helical secondary structure (Fig. 2).
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Association between GCN2 and Ribosomes Requires the DRBD-related
Sequences--
Alterations in the conserved lysine residues in the
DRBD-related region of GCN2 blocked the ability of the kinase to
stimulate GCN4 translation in response to histidine
starvation. To determine whether this lysine-rich sequence is required
for GCN2 association with ribosomes, we prepared cellular extracts from
yeast strain H1149 encoding gcn2-605 or other mutant alleles
of GCN2 and fractionated the lysates by centrifugation using
sucrose gradients. In an earlier study, Ramirez et al. (17)
measured GCN2 protein in each gradient fraction by immunoprecipitating
GCN2 and assaying for autophosphorylation activity by incubation of the
immune complex in the presence of [-32P]ATP. This
kinase assay which uses polyclonal antisera prepared against the GCN2
kinase domain was more sensitive than immunoblot assays and when
compared in parallel experiments was found to be an accurate measure of
steady-state protein levels. Consistent with this earlier study, we
found wild-type GCN2 kinase associated with free 40 S, 60 S, and 80 S
particles and polysomes. We observed a similar pattern of GCN2
distribution in the sucrose gradient when we fractionated a cellular
extract prepared from yeast cells expressing the kinase from a high
copy number plasmid and measured GCN2 protein by immunoblot analysis
(data not shown).
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The Carboxyl Terminus of GCN2 Can Bind dsRNA and Contains an
-Helical Structure--
Given the well characterized affinity of
the DRBD regions of PKR for dsRNA, we wanted to address directly
whether the carboxyl-terminal domain of GCN2 shared this binding
property. We overexpressed in E. coli a recombinant protein
containing the GCN2 sequence from residues 1467 to 1590 fused to an
amino-terminal sequence containing six contiguous histidine residues.
Nickel chelation resin was used to purify the recombinant fusion
protein, and in parallel, we overexpressed and purified a similar
fusion protein containing the gcn2-605 residue
substitutions. Both proteins were purified to apparent homogeneity as
judged by Coomassie staining of an SDS-polyacrylamide gel after
electrophoretic analysis (Fig. 5).
Purified GCN2 or gcn2-605 recombinant protein were mixed in a buffer
solution containing poly(I)·poly(C) bound to Sepharose or to
Sepharose alone. After incubating the samples, beads were collected by
a brief centrifugation and washed. Equal aliquots of proteins bound to
beads were analyzed by SDS-PAGE and visualized by staining with
Coomassie Blue (Fig. 5). Recombinant protein containing the
carboxyl-terminal domain from wild-type GCN2 was found to bind dsRNA,
whereas no association was found with Sepharose alone. The recombinant
gcn2-605 protein had no affinity for dsRNA. We conclude that the
ribosomal targeting domain of GCN2 can bind dsRNA.
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DISCUSSION |
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GCN2 is a multidomain protein kinase that regulates GCN4 translation initiation in response to amino acid starvation. In this report, we observed that the carboxyl terminus of GCN2 from residues 1467 to 1590 directly facilitate targeting of the kinase to the translation machinery. Interestingly, comparison of this region of GCN2 with DRBD sequences that facilitate ribosome interaction of a related eIF-2 kinase, PKR, revealed a lysine-rich sequence in GCN2 with features similar to the core of this RNA-binding domain. Substitutions of lysine residues conserved among DRBDs block association of GCN2 with ribosomes and impaired the ability of the kinase to stimulate the general control pathway in response to amino acid limitation. These results suggest that appropriate localization of GCN2 to the translational machinery is an obligate step in the mechanism leading to kinase phosphorylation of eIF-2.
Role of Ribosome Binding in Stimulating GCN2 Phosphorylation of
eIF-2 in Response to Amino Acid Starvation Conditions--
Three
mutant alleles of GCN2 were examined for their effects on association
of the kinase to ribosomes. While the gcn2-605 protein, containing
substitutions in the DRBD-related sequence, was not associated with
ribosomes, the kinase-defective gcn2-K559R protein showed a pattern of
ribosome interaction similar to wild-type GCN2 (Fig. 4). GCN2
interaction with uncharged tRNA also does not appear to be a
prerequisite step leading to ribosome association, since the gcn2-m2
mutant protein, containing substitutions in the HisRS-related domain
that greatly reduce binding of uncharged tRNA in vitro (12),
co-fractionates with ribosomes in the sucrose gradient (Fig. 4). These
results suggest that the DRBD-related sequence of GCN2 interacts with
ribosomes independent of autophosphorylation of GCN2 or stimulation of
kinase activity by uncharged tRNA that accumulates during amino acid
starvation conditions.
General Role of DRBD Sequences in Targeting Proteins to Ribosomes-- Over 20 different proteins have been identified that contain DRBD sequences (24). Many of these proteins have been characterized only by genomic sequencing projects and the role of DRBDs in facilitating their physiological functions are currently unclear. We have shown that DRBD-related sequences mediate association of the eIF-2 kinases, GCN2 and PKR, with ribosomes. Recently, another DRBD-containing protein, X1rbpa, that is the Xenopus homolog of TAR-RNA-binding protein, was also found to associate with ribosomes (43). Another likely example is the protein encoded by the YML3 gene from S. cerevisiae (44, 45) that contains a single DRBD and is associated with the large ribosomal subunit in mitochondria. The specific ribosomal locations that can accommodate by different DRBD sequences appear to be variable, with ribosomal dissociation experiments indicating that GCN2 and PKR are localized to 60 S and 40 S ribosomal subunits, respectively. These different ribosomal binding sites suggest that DRBD sequences bind to unique double-stranded regions in rRNA. Amino acid residue differences between the DRBDs would be expected to mediate this specificity for different RNA sequences and structures. Additionally, the fact that many proteins contain multiple DRBD sequences suggests that multiple RNA-binding elements may contribute to the affinity for unique ribosomal sites.
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ACKNOWLEDGEMENTS |
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We thank Peter Roach and Anna DePaoli-Roach for their comments on this manuscript, Eric Long for his assistance with the circular dichroism studies, and Minerva Garcia-Barrio and Alan Hinnebusch for GCN2 antiserum.
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FOOTNOTES |
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* This work was supported in part by United States Public Health Service Grant GM49164 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 317-274-0549;
Fax: 317-274-4686; E-mail: ron_wek{at}iucc.iupui.edu.
1 The abbreviations used are: eIF-2, eukaryotic initiation factor-2; ds, double-stranded; DRBD, double-stranded RNA-binding domain; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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