Functional characterization of Drosophila melanogaster PERK eukaryotic initiation factor 2a (eIF2a) kinase Natalia Pomar, Juan J. Berlanga*, Sonsoles Campuzano, Greco Herna ´ ndez † ,Mo ´ nica Elı ´ as and Ce ´ sar de Haro Centro de Biologı ´ a Molecular ‘Severo Ochoa’, Consejo Superior de Investigaciones Cientı ´ ficas, Universidad Auto ´ noma de Madrid, Cantoblanco, Madrid, Spain Four distinct eukaryotic initiation factor 2a (eIF2a)kinases phosphorylate eIF2a at S51 and regulate protein synthesis in response to various environmental stresses. These are the hemin-regulated inhibitor (HRI), the interferon-inducible dsRNA-dependent kinase (PKR), the endoplasmic reticu- lum (ER)-resident kinase (PERK) and the GCN2 protein kinase. Whereas HRI and PKR appear to be restricted to mammalian cells, GCN2 and PERK seem to be widely distributed in eukaryotes. In this study, we have character- ized the second eIF2a kinase found in Drosophila,aPERK homologue (DPERK). Expression of DPERK is developmentally regulated. During embryogenesis, DPERK expression becomes con- centrated in the endodermal cells of the gut and in the germ line precursor cells. Recombinant wild-type DPERK, but not the inactive DPERK-K671R mutant, exhibited an autokinase activity, specifically phosphorylated Drosophila eIF2a at S50, and functionally replaced the endogenous Saccharomyces cerevisiae GCN2. The full length protein, when expressed in 293T cells, located in the ER-enriched fraction, and its subcellular localization changed with dele- tion of different N-terminal fragments. Kinase activity assays with these DPERK deletion mutants suggested that DPERK localization facilitates its in vivo function. Similar to mammalian PERK, DPERK forms oligomers in vivo and DPERK activity appears to be regulated by ER stress. Furthermore, the stable complexes between wild-type DPERK and DPERK-K671R mutant were mediated through the N terminus of the proteins and exhibited an in vitro eIF2a kinase activity. Keywords:eIF2a kinases; Drosophila melanogaster;transla- tional control; PERK homologue; ER stress. Protein synthesis is mainly regulated at the initiation of mRNA translation. Phosphorylation of the a-subunit of eukaryotic translation initiation factor 2 (eIF2a)isawell characterized mechanism of translational control (reviewed in [1,2]). A family of protein kinases phosphorylate eIF2a at S51 in response to a variety of cellular stresses, including nutrient starvation, iron deficiency, heat shock, viral infec- tion and stress signals from the endoplasmic reticulum (ER) [1,2]. All known eIF2a kinases consist of a conserved catalytic domain linked to different regulatory regions which facilitate the different stress signals controlling each protein kinase. Included in this family are four mammalian eIF2a kinases: the hemin-regulated inhibitor (HRI), the double-stranded RNA (dsRNA)-dependent kinase (PKR), the GCN2 protein kinase and the ER-resident kinase (PERK, also known as PEK) [1,3]. Additionally, novel eIF2a kinases from the fission yeast Schizosaccharomyces pombe [4], and from the malarial parasite Plasmodium falciparum (PfPK4) [5] have been reported. The well characterized mammalian eIF2a kinase, HRI, is expressed most abundantly in erythroid cells [6], although we found its mRNA and kinase activity in non-erythroid tissues and in NIH 3T3 cells [7]. HRI becomes activated in response to heme deficiency and the activity of HRI seems to be modulated by its association with heat shock proteins [1,6]. PKR is an interferon-induced dsRNA-activated eIF2a kinase. It is thought to be activated by dsRNA generated during viral replication or gene expression [8]. A third Correspondence to C. de Haro, Centro de Biologı ´ a Molecular ÔSevero OchoaÕ, CSIC-UAM, Facultad de Ciencias, Cantoblanco, 28049 Madrid, Spain. Fax: + 34 91 3974799, Tel.: + 34 91 3978432, E-mail: cdeharo@cbm.uam.es Abbreviations: ATF4, activating transcription factor 4; eIF2a,the a-subunit (38 kDa) of eukaryotic polypeptide chain initiation fac- tor 2; HRI, heme regulated inhibitor kinase; PKR, double-stranded RNA-dependent eIF2a kinase; ER, endoplasmic reticulum; PERK, PKR-like ER kinase; GCN2, yeast general amino acid control eIF2a kinase; mHRI, mouse liver HRI; SEK1/2, Schizosaccharomyces pombe eIF2a kinases; 3-AT, 3-aminotriazole; EST, expressed sequence tag; ORF, open reading frame; TEMED, N,N,N¢,N¢- tetramethylethylenediamine; HDM, high density microsomal fraction; LDM, low density microsomal fraction; SP, signal peptide; TM, transmembrane; pc, pole cells. *Present address: Department of Biochemistry, McGill University, H3G 1Y6 Montreal, Canada. Present address: Department of Molecular Biology, Gene Expression Laboratory, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Go ¨ ttingen, Germany. Note: The nucleotide sequence(s) data reported in this paper have been submitted to the EMBL Database and are available under the accession number(s) AJ 313085. (Received 6 June 2002, revised 22 October 2002, accepted 25 November 2002) Eur. J. Biochem. 270, 293–306 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03383.x mammalian eIF2a kinase, termed GCN2, was originally characterized in Saccharomyces cerevisiae as being required for the amino acid control of GCN4 mRNA translation. It is activated by uncharged tRNA under amino acid starvation [9]. The identification of GCN2 homologues from Drosophila melanogaster [10,11], Neurospora crassa [12], and mammals [13,14], places GCN2 as one of the best evolutionarily conserved members of the eIF2a kinase family [3]. Finally, the mammalian PERK (also known as PEK) was originally identified in rat pancreatic islet cells [15]. Mouse PERK is activated by ER stress and contains a lumenal domain that is similar to the sensor domain of the ER-stress kinase, Ire1 [16]. PERK homologues have also been identified in humans and Caenorhabditis elegans [17]. In addition, sequence analyses have led to the identification of a putative PERK homologue from D. melanogaster [17]. In this study, we functionally characterized the Drosophila PERK eIF2a kinase (DPERK). Northern blot, RT-PCR and in situ hybridization analyses indicate that DPERK expression is developmentally regulated. During embryonic development, DPERK mRNA preferentially accumulates in the gut endoderm and in the pole cells, after the germinal band retraction has taken place. Similar to other members of the eIF2a kinase family, DPERK phosphorylates Drosophila eIF2a on S50 (S51 in mammals and yeast), and mediates translational control in yeast. This study provides evidence of a striking conservation in structure, function and ER-stress regulation between mammalian and Drosophila PERK and poses the question of whether DPERK might be involved, in the same way as mammalian PERK, in the regulation of gene expression in response to certain stress signals. Experimental procedures Materials All reagents were from Sigma except ammonium persulfate, [c- 32 P]ATP and [a- 32 P]dCTP from Amersham Pharmacia Biotech, and acrylamide, N,N¢-methylenebisacrylamide, N,N,N¢,N¢-tetramethylethylenediamine (TEMED) and SDS from Bio-Rad. Specific DNA primers were obtained from Isogen Bioscience. Cloning and sequence analysis The kinase domain sequences of DGCN2 [10] were compared with the Drosophila General-Bank database with the aim of finding new eIF2a kinases. All the sequence analyses were performed using BLAST [18], FASTA [19], GAP (Wisconsin Package, Genetics Computer Group, University of Wisconsin, Madison) and CLUSTAL W [20] programs. The embryonic expressed sequence tag (EST), with accession number AA390738, matched as a possible fragment of a putative PERK-like eIF2a kinase. The EST AA390738 contained a full length cDNA of 4625 nucle- otides that hybridizes in Northern blot to a unique transcript of 4.7 kb. This cDNA displays a large ORF of 3489 bp (nucleotides 282–3770) and a putative polyadeny- lation signal (AAT AAA, nucleotides 4568–4573). The 5¢-UTR contained three ATG codons followed by in-frame termination codons, located upstream from the putative ATG initiation codon (nucleotides 282–284). This methionine codon is likely to represent the translational startasitmatchestheDrosophila consensus for transla- tional initiators [21]. The comparison of the full length DPERK cDNA with the D. melanogaster genome revealed that DPERK genomic DNA is encoded within genomic scaffold 142000013386046 (accession number AE003602) located in region 83A-83A of chromosome 3R. Recently, the sequence of DPERK cDNA was published by Sood et al. [17] and was found to be almost identical to the DPERK cDNA that we have characterized. Within the ORF, a single nucleotide change was found in our cDNA (the triplet CAT, nucleotides 2166–8, is GAT in their sequence) and the 5¢-and3¢-UTRs in their cDNA are 57 and 337 nucleotides shorter, respectively. Northern blot analysis Poly(A) + RNA was prepared as described previously [22]. Fifteen lgofPoly(A) + RNA from each developmental stage were separated on a formaldehyde (6%)-agarose (1%) gel, transferred to a nylon membrane, probed with the full length DPERK and actin cDNAs radiolabeled with [a- 32 P]dCTP and analysed by autoradiography. RT-PCR Poly(A) + RNA was isolated from D. melanogaster (Oregon R) staged 0–18 h old embryos, 1st, 2nd and 3rd larvae instars, pupae and adults, by using first, the RNeasy Mini kit and then the Oligotex mRNA Midi Kit (Qiagen). Preparations were digested with RNAse-free DNAse I (Qiagen) to eliminate genomic DNA contamination. cDNA populations were generated by reverse transcription using the Marathon cDNA Amplification Kit (Clontech) accord- ing to the manufacturer’s instructions. Developmental analysis of mRNA was carried out by PCR using the Expand-Long Template PCR System (Roche Molecular Biochemicals) and 6 ng of cDNA from every different developmental stage as a template under the following conditions: 94 °C 3 min, 1 cycle, then 94 °C45 s,50°C45 s and 68 °C 60 s, for 25 or 40 cycles. Primers 5¢-CG CGAGGAGTACGACTACGATGAGGAAGAG-3¢ and 5¢-CACTGATGCGGCTCACTGGAGCTGCTGAAG-3¢ were used for every amplification experiment to amplify nucleotides 2646–3778 of the DPERK cDNA. One tenth of the PCR reaction was loaded on a 1% agarose gel. Primers 5¢-ATGACCATCCGCCCAGCATACAGGCCCAAG-3¢ and 5¢-TGAGAACGCAGGCGACCGTTGGGGTTGG TG-3¢ were used to amplify nucleotides 1–392 of the ribosomal protein rp49 ORF under the same conditions to control the amount of RNA loaded in each lane, as described previously [23]. Whole-mount embryo RNA in situ hybridization Localization of RNA in whole mount embryos with antisense digoxigenin-labeled RNA probes was performed as described [24]. 294 N. Pomar et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Antibodies Based on the DPERK cDNA coding sequence, a syn- thetic peptide (CG-PKSSGSDDANDDNK) was produced corresponding to amino acids 873–884 (Fig. 1B), with two additional residues (CG) at the N-terminal end. The peptide was synthesized as described by Santoyo et al. [10] and coupled at the terminal cysteine residue to keyhole limpet hemocyanin (Calbiochem). Rabbits were immunized as described by Me ´ ndez and de Haro [25]. For simplicity, the serum containing anti-DPERK peptide Igs will be referred to as anti-DPERK Igs. The rabbit polyclonal antibodies against the ER marker, protein disulfide isomerase (a-PDI) were the kind gift of J. Gonza ´ lez Castan ˜ o(Universidad Auto ´ noma de Madrid, Madrid, Spain). The polyclonal antibody against mannosidase II (Man II) and the mono- clonal antibody (15C8) against a Golgi integral membrane protein were kindly provided by G. Egea (Universidad de Barcelona, Barcelona, Spain) and I. Sandoval (CBMSO, Madrid, Spain), respectively. Prokaryotic expression of Drosophila eIF2a The D. melanogaster eIF2a ORF [26] was subcloned into a pRSETB vector. The pRSETB-eIF2a-S50A mutant was generated using the QuickChange TM Site-Directed Mutagenesis Kit (Stratagene). Prokaryotic expression was performed as described previously [7]. Expression of DPERK wild type and mutants in yeast Nucleotides 274–3767 of DPERK-wt cDNA were ampli- fied by PCR, together with a V5 tag and a polyhistidine metal-binding peptide followed by a stop codon in frame at the C-terminal and introduced into the vector pYX212 (R & D Systems, Inc., Minneapolis, MN, USA). Fig. 1. The DPERK gene, amino acid sequence and mutant proteins. (A) Genomic structure of the DPERK gene. Exons and introns are shown to scale as boxes and solid lines, respectively. The coding sequence is indicated by black boxes. (B) amino acid sequence of the DPERK protein. Amino acid numbering is shown on the left. Kinase subdomains are identified by Roman numerals directly above the appropriate regions. The predicted signal peptide (SP) and transmembrane domain (TM) are indicated by bars above the sequence. The asterisk denotes the predicted asparagine-linked glycosylation site. (C) Schematic diagram of DPERK and DPERK mutant proteins. The 1162 amino acid-long wild type DPERK coding sequence is illus- trated by the larger box. The figures are drawn to scale. The C-terminal eIF2a kinase domain contains the 12 catalytic subdomains of Ser/ Thr protein kinases (black boxes), with the conserved lysine residue (K671) and the insert region of eIF2a kinases (white box). The reg- ulatory region (stippled boxes) includes an SP, TM and the predicted N-linked glycosylation site (N260). Three deletion mutants are shown: DSP (in which the first 43 amino acids containing the signal peptide were deleted); DTM (in which amino acids 543–569, containing the transmembrane domain were deleted); and DNt (in which the first 569 amino acids containing most of the regulatory domain were deleted). Ó FEBS 2003 Molecular characterization of DPERK (Eur. J. Biochem. 270) 295 pYXDPERK-K671R and pYXDPERK-N260A mutants were created as described above. Deletion mutants were generated by PCR using primers to produce an ATG initiation codon in frame, at the following nucleotides of the DPERK cDNA: DPERK-DSP, nucleotide 410, DPERK- DNt, nucleotide 2003. DPERK-DTM was generated by PCR with oligonucleotides that produced a NotI site at nucleotides 1883–2006. All these deletion mutants were also in frame at the C-terminal end with a V5 tag and a polyhistidine metal- binding peptide. Yeast GCN2 in pEMBLXyex4 [27] was kindly provided by C. V. de Aldana. Plasmids encoding either different DPERK forms or an empty pYX212 vector were introduced into yeast strains J80 (MATa gcn2D ura3–52 leu2–3 leu2–112 trp1-D63 sui2D [SUI2-LEU2])andJ82(MATa gcn2D ura3–52 leu2–3 leu2–112 trp1-D63 sui2D [SUI2-S51A LEU2])bytheLiAc method as described [28]. Transformants were selected by uracil prototrophy and spotted on to agar plates with synthetic medium containing 0.67% yeast nitrogen base, 2% glucose and 40 mgÆL )1 tryptophan (SD) or SD supplemented with 3-aminotriazole (3-AT) [29]. Agar plates were incubated for 3 days at 30 °C and photographed. Yeast extracts Protein extracts from harvested yeast cells were made by trichloroacetic acid precipitation after glass bead lysis as described [30]. Cell cultures and transfections D. melanogaster Scheneider 2 (S2) cells were maintained in Complete DES TM Expression Medium (Invitrogen) con- taining 10% (v/v) fetal bovine serum. When specified, S2 cells were treated with either 0.5, 1 or 2 l M thapsigargin (Sigma) for 80 min or 150, 250 or 500 l M dithiothreitol for 5 h. HEK 293T cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum. For expression of the fruit fly PERK (DPERK) in S2 cells, the coding sequence from residues 274–3767 was subcloned into a pMT/V5-His vector (Invitrogen) in frame with a C-terminal tag encoding the V5 or Myc epitopes and a polyhistidine metal-binding peptide. Mutants pMTDPERK-K671R and pMTDPERK-N260A were gen- erated by introducing a fragment of either pYXDPERK- K671R or pYXDPERK-N260A containing the appropriate mutation into pMTDPERK-wt. pMT/V5-His/lacZwas provided by Invitrogen. Cells were transfected with 19 lgof plasmid DNA per 35-mm dish using the calcium phosphate method, as described in the manufacturer’s instructions (Invitrogen). For cotransfections, the same conditions were used with 19 lg of plasmid DNA from each construction. Expression was induced with 500 l M copper sulphate for 24 h. For expression in the mammalian cells, DPERK-wt and the indicated mutants were subcloned in vectors pEYFP-N1 (Clontech) or pcDNA3.1 (Invitrogen) in frame with the YFP signal or V5 epitopes, respectively. 293T cells were plated on 60-mm dishes at 10% conflu- ence, 12–24 h before transfection. Plasmids (5 lgperdish) were transfected by the calcium phosphate method, as described [7]. Immunoprecipitation, eIF2a kinase assay and immunoblotting All cells were washed once with NaCl/P i (137 m M NaCl, 2.6 m M KCl, 4 m M Na 2 HPO 4 ,1.8m M KH 2 PO 4 ,pH7.4) and lysed in lysis buffer [20 m M Tris/HCl, pH 7.8, 200 m M NaCl, 10% (v/v) glycerol, 1% (v/v) Nonidet P-40, 1 m M phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (CompleteÒ, Boehringer Mannheim)]. Cell debris was removed by centrifugation and the protein concentra- tion was determined according to Bradford [31]. The supernatants were subjected to immunoprecipitation or to SDS/PAGE and blotted onto 0.25 lm nitrocellulose membranes. Immunoprecipitations were carried out either with anti-V5 (0.5 lg of Ig2A) (Invitrogen), anti-DPERK (7 lL of antiserum) or anti-DGCN2 (0.5 lg of affinity- purified polyclonal antibody) [10] and protein A-Sepharose with or without competing peptide (5 lg). The immuno- precipitates were washed twice with lysis buffer, once with 0.5 M LiCl in NaCl/P i and two more times with kinase buffer (20 m M Tris/HCl pH 7.6, 50 m M NaCl, 10 m M MgCl 2 and 1 m M dithiothreitol). The immunoprecipitates were preincubated for 15 min at 32 °C in the presence of kinase buffer with 0.1 m M ATP and 0.25 mgÆmL )1 BSA. All samples were subsequently incubated for 15 min at 32 °C in the presence of recombinant Drosophila eIF2a-wt, Drosophila eIF2a-S50A or purified rabbit reticulocyte eIF2 (0.5 lg) as a substrate and 5 lCi of [c 32 P]ATP (3000 CiÆmmol )1 ), to assay their ability to phosphorylate eIF2a, as previously reported [10,25]. Incubations were terminated by addition of SDS sample buffer. Samples were analysed by electrophoresis on 10% SDS/PAGE, followed by autoradiography. In order to quantify the phosphate incorporation into eIF2a, the areas corres- ponding to the phosphorylated eIF2a were scanned at 633 nm in a computing 300A densitometer (Molecular Dynamics, Inc.). The membranes were probed with different antibodies as indicated in each case: mouse anti-V5 (Invitrogen), mouse anti-Myc (Invitrogen) or mouse Living Colors Antibody (Clontech), followed by mouse secondary antibody conju- gated with horseradish peroxidase. The immunoreactive bands were detected by enhanced chemiluminiscence (ECL, Amersham Pharmacia Biotech). Subcellular fractionation analysis At 48–72 h post-transfection, 293T cells were washed twice with NaCl/P i and harvested in homogenization buffer (5 m M Hepes/KOH, pH 7.4, 2 m M MgCl 2 ,1m M phenylmethanesulfonyl fluoride and the protease inhibitor cocktail). After homogenization, using 20 strokes of a Dounce homogenizer, one volume of a buffer containing 40 m M Hepes/KOH, pH 7.4, 2 m M EDTA and 0.5 M sucrose, was added. All operations were performed at 4 °C. The supernatant, taken from a 20-min 19 000 g centrifugation, was subsequently centrifuged at 45 000 g for 30 min to obtain a high density microsomal (HDM) pellet. The HDM pellet was resuspended in HES buffer (20 m M Hepes/KOH, pH 7.4, 1 m M EDTA, 0.25 M sucrose) and the supernatant was centrifuged at 180 000 g for 90 min to obtain a low density microsomal 296 N. Pomar et al.(Eur. J. Biochem. 270) Ó FEBS 2003 (LDM) pellet [32], which was also resuspended in HES buffer, and a supernatant containing all the soluble cytoplasm components. Protein concentration in the different fractions was determined by Bradford analysis [31] and equivalent amounts of protein were subjected to SDS/PAGE and immunoblotted using Living Colors Antibodies. Results Molecular characterization of the D. melanogaster PERK gene The discovery, through database searching, of a putative PERK-like kinase encouraged us to characterize it. The EST cDNA clone, accession number AA390738, contained a full-length cDNA. Sequence analysis indicated that the 4625 nucleotide-long Drosophila PERK cDNA contains 281 bp of 5¢ untranslated sequence, 3489 bp of open reading frame, and 855 bp of 3¢ untranslated sequence (GenBank accession number AJ313085). The sequence of DPERK cDNA was compared with the D. melanogaster genome [33] to verify sequence and determine the genomic structure of DPERK gene. This analysis revealed the existence of three introns of 1402, 62 and 84 nucleotides, respectively, in the DPERK gene (Fig. 1A). The full-length DPERK cDNA encodes a protein of 1162 amino acids (Fig. 1B), with a predicted molecular mass of 131 kDa as reported previously [17]. The C-terminus eIF2a kinase domain of DPERK (642–1162) contained all 12 conserved catalytic subdomains of eukaryotic Ser/Thr protein kinases [34] with an invariant Lys (residue 671) in subdomain II. The N terminus regulatory domain of DPERK (residues 1–641) showed characteristic features of PERK-like kinases [15,16]: the predicted signal peptide (SP, residues 16–40) and transmembrane (TM, residues 544–563) domains, obtained through hydropathicity [35] and surface probability [36] analyses of the protein, and the putative N-linked glycosylation site (residue 260), given by a PROSITE scan of the sequence (www.expasy.ch/scanprosite/) (Fig. 1B). These data suggested that DPERK may be a glycoprotein that is not transported to the distal Golgi complex and resides in the ER. To understand the role of the N-terminus regulatory domain of DPERK, we constructed a series of mutants (Fig. 1C), including a point mutation of the invariant Asn residue (DPERK-N260A) and deletions that removed: the signal peptide domain (DPERK-DSP); the entire trans- membrane domain (DPERK-DTM)ormostoftheregula- tory domain (DPERK-DNt). The DPERK constructs were confirmed by sequencing and subcloned into appropriate expression vectors for functional analysis. Developmental expression of DPERK Northern blot analysis of poly(A) + RNAs isolated from different developmental stages revealed a unique DPERK transcript of approximately 4.7 kb. DPERK is expressed throughout development, having two major peaks of expression in early embryo and adult stages (Fig. 2A). Such a developmental pattern of expression of DPERK was confirmed by RT-PCR (Fig. 2B). To determine the pattern of the DPERK mRNA localization during embryogenesis we performed in situ hybridization experiments with whole embryos of D. melanogaster by using a digoxigenin-labeled antisense RNA probe. DPERK transcripts showed a preferential accumulation in certain tissues at different developmental stages (Fig. 3). An even distribution was found in the early syncytial blastoderm (Fig. 3A). At later blastoderm stages, the transcripts concentrated in the so-called cortex (Fig. 3B) and also in the cytoplasm of the cells, save in the pole cells (pc), the precursors of germ line. At the beginning of the germ band retraction (stage 11), the DPERK transcripts accumulated at high levels in the pole cells located at the posteriormost region of the invaginated midgut rudiment (Fig. 3C,D). During stage 14 (Fig. 3E,F), and extending into later stages (Fig. 3G), accumulation of DPERK transcripts was seen throughout the endodermal cells of the anterior (amg) and posterior (pmg) midgut. In a dorsal view, the concentration of the DPERK transcripts in the gut is more evident (Fig. 3F, inset). The preferential Fig. 2. Developmental expression of DPERK. (A) Northern blot of poly(A) + RNA prepared from different stages of development was hybridized with full length DPERK cDNA (top panel). The devel- opmental stages include embryos (E), first instar larvae (L1), second instar larvae (L2), pupae (P) and adult flies (A). The arrow indicates the unique DPERK transcript of 4.7 kb. The filter was rehybridized with a Drosophila actin probe to control the amount of RNA loaded in each lane (bottom panel). DPERK transcript levels were much higher in embryos and adults than in the other stages. (B) RT-PCR analysis was performed by using poly(A) + RNA purified from Drosophila embryos (E), first instar larvae (L1), second instar larvae (L2), third instar larvae (L3), pupae (P) and adult (A) stages. Amplification with the DPERK-specific primers between nucleotides 2646 and 3778 of DPERK cDNA, as described under ÔExperimental proceduresÕ, revealed a single fragment of 1.1 kb in each developmental stage (top panel). Complementary primers to the ribosomal protein rp49 were used, under same conditions, to control the amount of RNA loaded in each case (bottom panel). DPERK transcript levels show the same pattern, confirming the Northern blot analysis. Ó FEBS 2003 Molecular characterization of DPERK (Eur. J. Biochem. 270) 297 accumulation in the pole cells was maintained at late stage 16, once the pole cells migrated to the gonads (go) (Fig. 3H). It should be noted that the DPERK expression was not apparent in the central nervous system where the other Drosophila eIF2a kinase (DGCN2) was preferentially expressed [10]. DPERK phosphorylates Drosophila eIF2a at S50 in vitro and mediates translational control in S. cerevisiae To verify that DPERK is an eIF2a kinase, we expressed wild-type DPERK or the presumably inactive DPERK mutant (DPERK-K671R) in Drosophila S2 cells as V5-tagged fusion proteins. Recombinant proteins were immunoprecipitated by using anti-V5 Igs and the immune complexes were subjected to eIF2a kinase assays as described under ÔExperimental proceduresÕ. As a positive control, the reticulocyte heme-reversible HRI [37] was assayed under the same conditions (Fig. 4A, lanes 1 and 2). DPERK wild-type immune complexes underwent autophosphorylation (Fig. 4A, lanes 3 and 4) and were fully active in phosphorylating eIF2a (lane 3). These phosphorylated DPERK proteins were detected using immunoblot analysis (Fig. 4A, bottom, lanes 3 and 4). Moreover, recombinant DPERK specifically phosphoryl- ated Drosophila eIF2a at S50 (S51 in mammals and yeast) (Fig. 4A, lane 3) as phosphorylation was not observed in the assay mixture containing the mutant substrate eIF2a- S50A (lane 4). Furthermore, we found that the mutant DPERK-K671R did not phosphorylate itself or eIF2a (Fig. 4A, lanes 5 and 6), although it was expressed at a much higher level than wild type DPERK (Fig. 4A, bottom, lanes 5 and 6). These results are comparable with those previously observed in both mammalian PERK [16,17] and mouse HRI [7]. We therefore conclude that DPERK is, indeed, an eIF2a kinase of D. melanogaster and has both autokinase and eIF2a kinase activities in vitro. It is well known that S. cerevisiae is a useful model system for studying the in vivo role of eIF2a kinases in translational control [29]. To address whether DPERK can functionally replace yeast GCN2, plasmids encoding wild- type DPERK, the inactive DPERK mutant (DPERK- K671R) or the vector alone were introduced into two isogenic yeast strains lacking the GCN2 kinase. Yeast cells expressing these eIF2a kinases were compared to cells containing plasmid-encoded yeast GCN2, as a positive control. As expected, all transformants grew to similar levels in synthetic medium (Fig. 4B, lower). However only the cells expressing an active eIF2a kinase, either wild type DPERK or yeast GCN2, presented normal growth under amino acid starvation produced by the medium supple- mented with 3-AT [29] (Fig. 4B, upper). As for the other eIF2a kinases, the mutation of the invariant lysine in the kinase subdomain II impaired the ability of DPERK to rescue growth of yeast lacking GCN2. Moreover, the use of strain J82 (Dgcn2 SUI2-S51A), isogenic to J80 (gcn2D), with an alanine substitution for S51 in eIF2a, revealed that this phosphorylation site was required to provide growth resistance in the presence of 3-AT (Fig. 4B, upper). Fig. 3. Expression of DPERK during embry- onic development. Embryos were hybridized in situ with antisense DPERK RNA probes. When appropriate, anterior is left and dorsal is up. (A) Preblastoderm stage embryo showing generalized distribution of DPERK mRNA (probably due to a maternal contribution). (B) Embryo at the cellular blastoderm stage presenting generalized distribution of DPERK mRNA apart from the pole cells (pc). (C) Lateral view of a stage 11 embryo dis- playing expression of DPERK almost exclu- sively at the pole cells (pc). (D) An enlarged dorsal view of a similar stage 11 embryo highlights the accumulation of DPERK mRNA in the pole cells (pc). At progressively older embryonic stages (E, and F, stage 14; G, stage 16) expression of DPERK is localized in the endodermal cells of the anterior (amg) and posterior (pmg) midgut. This is most easily visualized in a dorsal view (F and see also the inset, mg, midgut). (H) Expression of DPERK persists in the pole cells at late stage 16, once the pole cells have migrated to the gonads (go). Hybridization with a sense RNA probe did not give any appreciable signal (not shown). Embryonic stages were classified according to Campos-Ortega and Hartenstein [52]. 298 N. Pomar et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Therefore, DPERK, similar to the other eIF2a kinase family members, requires the regulatory site (S51) in eIF2a for translational control. DPERK, but not DGCN2, is activated by ER stress To date, only two eIF2a kinases have been identified in D. melanogaster. They are the homologues of yeast GCN2 and mammalian PERK. It is well known that GCN2 is activated in cells deprived of nutrients [7,9] whereas mammalian PERK is activated in cells treated with agents that induce ER stress [16]. We then sought to determine whether DPERK is specifically activated by some agents that promote ER stress: thapsigargin and dithiothreitol [38]. We immunoprecipitated either DPERK (Fig. 5A,D) or DGCN2 (Fig. 5B,E) from extracts of Drosophila S2 cells treated with increasing concentrations of thapsigargin (Fig. 5A,B) or dithiothreitol (Figs 5D,E) by using the appropriate antibodies. The isolated immune complexes were subjected to eIF2a kinase assays as described under ÔExperimental proceduresÕ. Immunoprecipitations were specific because of the fact that they were prevented by addition of the respective competing peptide immunogen in the immunoprecipitation assay (Fig. 5A,B,D,E, lanes 2 and 7). We found that both DPERK and DGCN2 immune complexes from untreated S2 cells phosphorylated the a subunit of eIF2 (Fig. 5A,B,D,E, lane 3), how- ever, DPERK, but not DGCN2, activity was increased in cells undergoing ER stress as shown by the in vitro eIF2a kinase assay of DPERK and DGCN2 immune complexes (Fig. 5C,F). These results suggest that like its mammalian homologue, the trigger for Drosophila DPERK activation is likely to be misfolded proteins in the ER [16]. Localization and eIF2a kinase activity of DPERK – the role of the N-terminal domain It has been proposed that the N-terminal regulatory domain of mammalian PERK is located in the ER [16]. Because DPERK also contained a signal peptide at the N terminus and a hydrophobic domain in the middle of the molecule (Fig. 1B), we considered the possibility that these similarities could reflect a similar localization of DPERK. DPERK-wt and the indicated DPERK deletion mutant cDNAs specified above (Fig. 1C) were expressed in human 293T fibroblasts as YFP fusion proteins. These DPERK- transfected cells were subjected to several differential centrifugations [32] followed by a Western blot analysis of the enriched high density microsomal (HDM), low density microsomal (LDM) and cytosolic (CYT) fractions, as described under ÔExperimental proceduresÕ. Cells expressing the full length DPERK-wt accumulated the protein in HDM (Fig. 6A), a fraction where several marker enzyme activities characteristic of membranes of the ER are mostly recovered [39]. By contrast, DPERK mutants in which either the signal peptide (DPERK-DSP) or the entire transmembrane domain (DPERK-DTM) were deleted, preferentially accumulated in LDM (Fig. 6A). It is known that LDM fractions isolated from 3T3-L1 adipocytes using this procedure mostly contain membranes of the Golgi apparatus, endosomes and other intracellular membranes [32]. Finally, a C-terminal YFP-tagged mutant, in which most of the regulatory domain was deleted (DPERK-DNt) preferentially accumulated in the cytosolic enriched fraction (Fig. 6A). Fig. 4. DPERK is an eIF2a kinase. (A) Autokinase and eIF2a kinase activities of recombinant DPERK in vitro. Wild type DPERK spe- cifically phosphorylates the a-subunit of Drosophila eIF2 at residue S50 (S51 in mammals and yeast). S2 cells were transfected with plas- mids encoding DPERK-wt or DPERK-K671R containing the V5 epitope. Lysates were subjected to immunoprecipitation with an anti- V5 Ig, followed by an in vitro phosphorylation assay. These kinase reactions contained samples of purified HRI from rabbit reticulocyte lysates as a control (lanes 1 and 2), or either recombinant DPERK-wt (lanes 3 and 4) or else DPERK-K671R (lanes 5 and 6). The reactions also included either wild type Drosophila eIF2a (lanes 1, 3 and 5) or eIF2a-S50A (mut) (lanes 2, 4 and 6). Radiolabeled proteins were analysed by SDS/PAGE and transferred to an Inmobilon-P mem- brane followed by autoradiography (top panel). Positions of phos- phorylated DPERK, HRI, and eIF2a are indicated by arrows. The same membrane was probed with the anti-V5 Ig in a Western blot analysis (bottom panel). (B) DPERK functionally substitutes GCN2 in a yeast model system. Yeast J80 (Dgcn2) and J82 (Dgcn2, SUI2-S51A) strains were transformed with the indicated eIF2a kinases, or with the plasmid pYX212 (vector) as a negative control. S. cerevisiae GCN2 was used as a positive control. Patches of transformants were grown in the appropriate medium as indicated. Ó FEBS 2003 Molecular characterization of DPERK (Eur. J. Biochem. 270) 299 These studies were further extended to examine the relative organelle composition of the HDM, LDM and CYT fractions using antibodies specific to proteins in the ER and the Golgi apparatus. Thus, in agreement with recent studies [40], the ER marker protein disulfide isomerase (a-PDI) was mostly localized in the HDM fraction (Fig. 6B). In contrast, two distinct markers of the Golgi complex, a polyclonal antibody against mannosidase II (Man II) [41] and a monoclonal antibody (15C8), that recognizes an integral membrane protein located in the cis and medial Golgi cisternae [42], were found equally distributed within the HDM and LDM fractions (Fig. 6C). Previously, very similar results were obtained with fractions isolated from rat adipose cells [39]. A quantitative approach reveals that the HDM fraction is enriched in ER, relative to the other two fractions, but it also contains a significant Fig. 5. Endogenous DPERK, but not DGCN2, from S2 cells is activated by ER stress. Shown are the results of the in vitro phosphorylation assay of reticulocyte eIF2 by immune complexes obtained from extracts of S2 cells treated with increasing concentrations of thapsigargin (A and B) or dithiothreitol (D and E). Cell extracts were subjected to immunoprecipitation with either anti-DPERK (A and D) or anti-DGCN2 (B and E) Igs in the absence (lanes 3–6) or in the presence (lanes 2 and 7) of the respective competing peptide. Samples of purified HRI from rabbit reticulocytes were also assayed as a control to position phosphorylated eIF2a (lane 1). The amount of eIF2a phosphorylation was estimated by quantifying the corresponding band density of the autoradiogram in A and B (see panel C) and in D and E (see panel F). The intensity of the eIF2a band corresponding to untreated cells (lane 3) was defined as 100%. Similar results were obtained in at least two independent experiments. 300 N. Pomar et al.(Eur. J. Biochem. 270) Ó FEBS 2003 proportion of Golgi membranes. This apparent anomaly might be due to distinct membrane subspecies of the Golgi apparatus with sedimentation characteristics similar to those of the ER membranes. The results described above, together with preliminary immunofluorescence analyses of DPERK-wt (data not shown), strongly suggest that DPERK resides in the ER, similarly to its mammalian counterpart, and furthermore that the ER-targeting of DPERK is mediated by these two unique structural features. To our knowledge, this is the first report demonstrating a subcellular relocation of this eIF2a kinase in response to structural changes in the molecule. It has been proposed that protein targeting plays an important role in regulating enzymatic activity by providing access to local substrates or regulatory ligands. Consistent with the idea that the N-terminus of PERK is important for mediating activation of this eIF2a kinase, was the previ- ously reported finding that deletion of these sequences greatly reduces the catalytic activity of human PERK, but not that from the C. elegans homologue [17]. In an attempt to understand the role of the N-terminus domain, as well as that of the invariant aspargine residue (N260), a predicted N-linked glycosylation site, in the eIF2a kinase activity of DPERK, we expressed DPERK wild type and all the constructed mutants as V5-tagged derivatives, and immu- noprecipitated them by using anti-V5 antibodies. Because the expression of the DPERK deletion mutants in S2 cells was very inefficient, we used HEK 293T cells for expression of these mutants, under same conditions. These studies show that all of the immune complexes from either DPERK-wt or from distinct mutants containing an unmodified kinase catalytic domain underwent phosphory- lation and were fully active in phosphorylating eIF2a (Fig. 7A, top). As shown previously (Fig. 4A), replacing K671 from DPERK with arginine (K671R) abolished the ability of the protein to undergo autophosphorylation or to phosphorylate eIF2a (Fig. 7A, lane 3). We conclude that the N-terminus of DPERK is not required for in vitro catalytic activity. To characterize these DPERK mutants further we tested whether they would phosphorylate eIF2a at S51 in vivo and functionally replace yeast GCN2 when expressed in S. cerevisiae cells, as we previously observed in the wild-type DPERK (Fig. 4B). All constructs, with the exception of the plasmid encoding DPERK-DSP, were well expressed in the J82 strain, however, the expression of the active kinases (DPERK-wt and the DPERK-N260A mutant) in the isogenic strain J80 was significantly lower (Fig. 7C). An inhibition of its own synthesis promoted by the kinase activity could explain this effect. Even though deletion of the signal peptide or the transmembrane domain in DPERK seems not to affect the in vitro eIF2a kinase activity (Fig. 7A), DPERK mutants in those regions were not able to maintain eIF2a phosphorylation-dependent cell growth in yeast (Fig. 7B, upper). Interestingly, all of the mutant versions of DPERK, that were unable to support yeast growth under amino acid starvation conditions, were found to be preferentially accumulated in the LDM fraction (Fig. 6A), whereas either the wild type or the mutant DPERK-DNt that showed in vivo catalytic activity were found to be preferentially accumulated in the HDM or cytosolic fractions, respectively (Fig. 6A). Altogether, these Fig. 6. DPERK resides in the endoplasmic reticulum. (A) Distribution of DPERK-wt and the indicated DPERK mutants in HEK 293T fibroblasts from subcellular fractionation studies. DPERK-wt and its mutants were expressed in HEK 293T cells as YFP fusion proteins by transient transfection of the corresponding plasmids. Microsomal (HDM, LDM) and cytosolic (CYT) enriched fractions were obtained from the different lysates as described under ÔExperimental proce- duresÕ. Protein from cytoplasm or LDM fractions (50 lg), and 20– 30 lg of protein from the HDM fraction were separated on 7.5% SDS/PAGE and transferred to poly(vinylidene difluoride) membranes for immunoblotting with anti-YFP Ig. Results are representative of at least two independent experiments. (B) and (C). Lysates of HEK 293T fibroblasts were fractionated into cytosol, HDM and LDM as des- cribed in panel A. Fifty lg of protein from each fraction was resolved by SDS/PAGE and analysed by immunoblotting using a polyclonal antibody against protein desulfide isomerase (a-PDI), as a marker of ER (B) or a monoclonal antibody (15C8) and a polyclonal antibody against mannosidase II (Man II), as two distinct markers of the Golgi complex (C). The relative amount of each marker among the subcel- lular fractions was estimated by quantifying the corresponding band density of the immunoblots and indicated by the numbers below. The sum of these relative values was defined as 100. Similar results were obtained in three independent experiments. Ó FEBS 2003 Molecular characterization of DPERK (Eur. J. Biochem. 270) 301 results suggest that mislocation of DPERK mutants, rather than a lack of catalytic activity, prevents eIF2a phosphory- lation and cell growth in yeast. The N-terminal regulatory domain is required for the oligomerization of DPERK It has been proposed that oligomerization has an important function in activation of ER stress-signal transducers [43]. Thus, the oligomerization involving the N-terminal ER lumenal domain is necessary and sufficient to initiate Ire1 activation in yeast [44]. Although Ire1 and PERK share a weak sequence similarity in their lumenal domains, previous data suggest that they may use a similar mechanism to sense ER stress. In fact, treatment of cells with thapsigargin resulted in the rapid formation of a mammalian PERK- containing complex [45]. To identify possible in vivo complexes between wild type DPERK and the inactive DPERK-K671R mutant, we coexpressed either V5-tagged DPERK-WT or DPERK-K671R with wild-type or mutant forms of Myc- tagged derivatives in Drosophila S2 cells, as indicated in Fig. 7. Expression and activity of DPERK mutants. (A) Autokinase and eIF2a kinase activities of recombinant DPERK mutants in vitro.S2cells were transfected with plasmids encoding DPERK-wt, DPERK-K671R and DPERK-N260A, while 293T cells were transfected with plasmids encoding DPERK-DSP, DPERK-DTM, and DPERK-DNt. All the constructs contained the V5 epitope. In vitro kinase reactions contained the anti-V5 immune complexes prepared from lysates of different transfected cells and purified rabbit reticulocyte eIF2. Purified HRI from rabbit reticulocyte lysates was included as a control for positioning of phosphorylated eIF2a (lane 1). All of the samples were assayed as described in Fig. 4A. Thus, after autoradiography (upper panel), the same membrane was probed with monoclonal anti-V5 (lower panel). Positions of phosphorylated DPERK, HRI and eIF2a are indicated by arrows. Molecular mass markers are indicated on the left. (B) In vivo eIF2a kinase activity of recombinant DPERK mutants in a yeast model system. Yeast J80 and J82 strains were transformed with high-copy-number plasmids encoding for the indicated eIF2a kinases, or the plasmid pYX212 (vector) alone. All transformants were analysed as described for Fig. 4B. (C) To determine the expression of DPERK-wt and its mutants in the J80 and J82 transformants, equal amounts of protein from each cell extract were resolved by SDS/PAGE and analysed by immunoblot using monoclonal anti-V5 antibodies. Molecular mass markers are indicated on the left. 302 N. Pomar et al.(Eur. J. Biochem. 270) Ó FEBS 2003 [...]... (1997) Cloning and characterization of a cDNA encoding a protein synthesis initiation factor- 2a (eIF -2a) kinase from Drosophila melanogaster Homology to yeast GCN2 protein kinase J Biol Chem 272, 12544–12550 11 Olsen, D.S., Jordan, B., Chen, D., Wek, R.C & Cavener, D.R (1998) Isolation of the gene encoding the Drosophila melanogaster homolog of the Saccharomyces cerevisiae GCN2 eIF2a kinase Genetics 149,... from PERK, resulting in the activation of this kinase [44,45] We have shown here that recombinant DPERK, when expressed in 293T cells, also localized in the membranes of the ER It is interesting to note that the two highly hydrophobic regions of this eIF2a kinase are required for its appropriate subcellular localization of DPERK Thus, in the absence of each one of these two sequences, the majority of. .. Cavener, D.R & Wek, R.C (2000) A mammalian homologue of GCN2 protein kinase important for translational control by phosphorylation of eukaryotic factor- 2a Genetics 154, 787–801 15 Shi, Y., Vattem, K.M., Sood, R., An, J., Liang, J., Stramm, L & Wek, R.C (1998) Identification and characterization of pancreatic eukaryotic initiation factor- 2a subunit kinase, PEK, involved in translational control Mol Cell... phosphorylate the regulatory site, S50, in Drosophila eIF2a in vitro and in vivo and, furthermore, can functionally replace the endogenous 304 N Pomar et al (Eur J Biochem 270) yeast GCN2 kinase by a process requiring the S51 phosphorylation site in yeast eIF2a These results allow unambiguous classification of DPERK as an eIF2a kinase Drosophila DPERK is 49% identical to rat PERK within the catalytic domain and... Molecular characterization of DPERK (Eur J Biochem 270) 303 Fig 8 Oligomerization of DPERK in vivo and eIF2a kinase activity of these oligomers in vitro (A) Coimmunoprecipitation with anti-V5 Ig of DPERK-wt and the inactive mutant (K671R) from lysates containing the indicated proteins, tagged with either V5 or Myc epitopes, as shown in the figure Immune complexes were subjected to in vitro eIF2a kinase. .. N-terminal regulatory domain of DPERK is required for DPERK oligomerization, whereas the transmembrane domain, at least, is dispensable Discussion Four distinct protein kinases are known to phosphorylate eIF2a on S51 in mammals So far only two of them, the homologues of GCN2 and PERK (DGCN2 and DPERK), have been identified in D melanogaster [10,11,17 and this report] Here we show that DPERK can phosphorylate... (1998) cpc-3, the Neurospora crassa homologue of yeast GCN2, encodes a polypeptide with juxtaposed eIF2a kinase and histidyl-tRNA synthetase-related domains required for general amino acid control J Biol Chem 273, 20404–20416 13 Berlanga, J.J., Santoyo, J & de Haro, C (1999) Characterization of a mammalian homolog of the GCN2 eukaryotic initiation factor- 2a kinase Eur J Biochem 265, 754–762 14 Sood, R.,... of gene expression might also be conserved In this respect, the Drosophila homologue of the mammalian ATF4, the cryptocephal gene, controls molting and metamorphosis in Drosophila [51] Therefore, it will be of great interest to determine whether the Drosophila eIF2a kinases are involved in the translational control of mRNAs that encode key growth regulating proteins We speculate that the role of DPERK... 5, 1881–1891 ´ 25 Mendez, R & de Haro, C (1994) Casein kinase II is implicated in the regulation of heme-controlled translational inhibitor of reticulocyte lysates J Biol Chem 269, 6170–6176 26 Qu, S & Cavener, D.R (1994) Isolation and characterization of the Drosophila melanogaster eIF -2a gene encoding the alpha subunit of translation initiation factor eIF-2 Gene 140, 239– 242 27 Cesareni, G & Murray,... mediates PERK oligomerization in mammalian cells [50] It is possible that, like other eIF2a kinases, DPERK oligomerization mediated by its regulatory domain is both necessary and sufficient for activation of its eIF2a kinase activity in vivo DPERK is dynamically expressed during embryogenesis At later stages, DPERK expression concentrates in the gut and the gonads Interestingly, the highest levels of mammalian . Functional characterization of Drosophila melanogaster PERK eukaryotic initiation factor 2a (eIF2a) kinase Natalia Pomar, Juan J. Berlanga*, Sonsoles. fraction (Fig. 6A). Fig. 4. DPERK is an eIF2a kinase. (A) Autokinase and eIF2a kinase activities of recombinant DPERK in vitro. Wild type DPERK spe- cifically phosphorylates the a-subunit of Drosophila eIF2. Phosphorylation of the a-subunit of eukaryotic translation initiation factor 2 (eIF2a)isawell characterized mechanism of translational control (reviewed in [1,2]). A family of protein kinases phosphorylate