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
Trang 1Functional 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, a PERK
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 vitroeIF2a 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) is a well 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
Severo Ochoa, 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 inhibifac-tor 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)
Trang 2mammalian 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-32P]ATP and [a-32P]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 usingBLAST[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 start as it matches the Drosophila 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¢- and 3¢-UTRs in their cDNA are 57 and 337 nucleotides shorter, respectively
Northern blot analysis Poly(A)+RNA was prepared as described previously [22] Fifteen lg of Poly(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-32P]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: 94C 3 min, 1 cycle, then 94 C 45 s, 50 C 45 s and 68C 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 RNAin situ hybridization Localization of RNA in whole mount embryos with antisense digoxigenin-labeled RNA probes was performed
as described [24]
Trang 3Based 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 ofDrosophila eIF2a The D melanogaster eIF2a ORF [26] was subcloned into
a pRSETB vector The pRSETB-eIF2a-S50A mutant was generated using the QuickChangeTM 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).
Trang 4pYXDPERK-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: 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]) and J82 (MATa gcn2D ura3–52 leu2–3
leu2–112 trp1-D63 sui2D [SUI2-S51A LEU2]) b y the LiAc
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 30C 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 melanogasterScheneider 2 (S2) cells were maintained in
Complete DESTM Expression Medium (Invitrogen)
con-taining 10% (v/v) fetal bovine serum When specified, S2 cells
were treated with either 0.5, 1 or 2 lMthapsigargin (Sigma)
for 80 min or 150, 250 or 500 lMdithiothreitol 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/lacZ was
provided by Invitrogen Cells were transfected with 19 lg of
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 lM 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 lg per dish)
were transfected by the calcium phosphate method, as
described [7]
Immunoprecipitation, eIF2a kinase assay and immunoblotting
All cells were washed once with NaCl/Pi (137 mM NaCl, 2.6 mMKCl, 4 mM Na2HPO4, 1.8 mMKH2PO4, pH 7.4) and lysed in lysis buffer [20 mMTris/HCl, pH 7.8, 200 mM NaCl, 10% (v/v) glycerol, 1% (v/v) Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Complete, 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/Pi and two more times with kinase buffer (20 mM Tris/HCl pH 7.6, 50 mM NaCl, 10 mM MgCl2 and 1 mMdithiothreitol) The immunoprecipitates were preincubated for 15 min at 32C in the presence of kinase buffer with 0.1 mM ATP and 0.25 mgÆmL)1BSA All samples were subsequently incubated for 15 min at
32C 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 [c32P]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/Pi and harvested in homogenization buffer (5 mM Hepes/KOH, pH 7.4, 2 mM MgCl2, 1 mM phenylmethanesulfonyl fluoride and the protease inhibitor cocktail) After homogenization, using 20 strokes of a Dounce homogenizer, one volume of a buffer containing
40 mM Hepes/KOH, pH 7.4, 2 mM EDTA and 0.5M sucrose, was added All operations were performed at
4C 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 mM Hepes/KOH, pH 7.4, 1 mM EDTA, 0.25M sucrose) and the supernatant was centrifuged at
180 000 g for 90 min to obtain a low density microsomal
Trang 5(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 theD 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 aPROSITE
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) or most of the
regula-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 Experimental procedures, revealed a single fragment of 1.1 kbin 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.
Trang 6accumulation 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 phosphorylatesDrosophila eIF2a at S50 in vitro
and mediates translational control inS 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 Experimental procedures 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].
Trang 7Therefore, 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
Experimental procedures 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 Experimental procedures 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.
Trang 8These 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.
Trang 9proportion 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 cerevisiaecells, 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 Experimental
proce-dures 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.
Trang 10results 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 S2 cells 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.