 |
INTRODUCTION |
It is generally accepted that protein biosynthesis in eukaryotes
is controlled at the level of polypeptide chain initiation. Mammalian
protein synthesis is promptly adjusted in response to a variety of
different environmental stimuli including nutrient starvation, heat
shock, and viral infection (1). One of the best studied mechanisms of
translational regulation involves the phosphorylation of the
-subunit of eukaryotic initiation factor-2 (eIF-2
)1 (2-6). During
translation initiation, eIF-2 catalyzes the binding of the initiator
Met-tRNA to the 40 S ribosomal subunit by hydrolyzing GTP to GDP. eIF-2
is released from the ribosome as an inactive form bound to GDP in a
binary complex (7). To participate in the next round of translation
initiation, the GDP-bound eIF-2 has to be converted to the active GTP
bound form. This is carried out by the guanine nucleotide exchange
factor eIF-2B, which is present at a lower molar concentration in the
cell. Phosphorylation of eIF-2
results in inhibition of the eIF-2B
activity, reducing the rate of nucleotide exchange, thus the rate of
polypeptide chain initiation (7).
Eukaryotic protein synthesis is subject to regulation by various
conditions where eIF-2
phosphorylation is in parallel with an
inhibition of translation initiation (1, 6). Three protein kinases have
been studied in detail based on their ability to phosphorylate
eIF-2
. These include the double-stranded RNA-dependent kinase (PKR) (8-13), heme-regulated inhibitor kinase (HRI) (14, 15),
and yeast and Drosophila GCN2 (16-19). The kinases differ from other families of serine/threonine kinases by the presence of a
large insert between domains III and V (20). The eIF-2
kinases share
extensive homology within the catalytic domains and each of the kinases
has been demonstrated to phosphorylate eIF-2
specifically on the
Ser-51 residue (2, 5). In contrast, little homology is found in the
regulatory domains of the kinases, which mediate response to different
environmental stresses.
The presence of distinct regulatory domains within each eIF-2
kinase
allows for different physiological signals to regulate phosphorylation
of eIF-2
. PKR is regulated by two double-stranded RNA binding motifs
located in the N terminus of the protein (6). PKR is ubiquitously
expressed in various tissues at very low levels, and expression can be
induced by treatment with interferon or upon viral infection (21). The
kinase has been shown to participate in the cellular defense mechanism
against viral infection. PKR can be activated by double-stranded RNA
generated during the replicative cycle of certain viruses. Activation
of PKR results in phosphorylation of eIF-2
and down-regulation of
protein synthesis, preventing replication and spread of the virus to
neighboring cells. PKR has also been shown to play a role in regulating
cell growth and differentiation (22, 23). The kinase activity of HRI is
regulated by two heme regulatory motifs, which mediate the inhibition
by hemin (24). The kinase mediates inhibition of protein synthesis in
heme-deficient reticulocyte lysates. Although the exact molecular mechanism of such inhibition is not yet fully understood, this inhibition is thought to couple heme availability to synthesis of
globin, the major protein in reticulocytes. Interestingly, the HRI
kinase is also present in the human malarial parasite Plasmodium
falciparum. A large 559 amino acid insert between kinase domain IV
and V was identified in the parasite kinase, which distinguishes it
from the host kinase (25). In contrast to PKR, HRI is predominantly expressed in reticulocytes. Though a low level of mRNA is found in
nonerythroid tissues (13), the kinase was not detectable in these
tissues even under induction by anemia (26).
In contrast to PKR and HRI, which are believed to broadly regulate
protein synthesis within a given cell type, yeast GCN2 has been shown
to specifically regulate expression of GCN4 in response to amino acid
starvation (27). GCN4 is a transcription factor that coordinately
regulates more than 30 genes involved in amino acid biosynthesis in
yeast. Whereas activation of PKR and HRI results in inhibition of
translation initiation, phosphorylation of eIF-2
by GCN2 leads to
induction of GCN4 translation mediated by four short open
reading frames in the leader sequence of GCN4 mRNA.
Although little is known about the gene-specific nature of
translational control in higher organisms, recent cloning of a
Drosophila GCN2 homologue (18, 19) indicates that similar regulatory mechanisms may also exist in higher eukaryotes, such as
modulation of translation initiation by short upstream open reading
frames of certain eukaryotic genes (28). Identification of a
GCN2 homologue from Drosophila suggests that a
homologue may also exist in mammals, as amino acid starvation and
defective aminoacyl-tRNA synthetase has been reported to increase
eIF-2
phosphorylation in mammalian cells (29).
Because additional eIF-2
kinases have been implicated in regulating
protein synthesis in mammalian cells, not all the responses to stress
conditions are accounted for by the PKR and HRI kinases (6, 30-34).
Consequently, we have recently cloned and characterized a novel
eIF-2
kinase from rat pancreas, named PEK, as
pancreatic eIF-2
kinase (35).
In this study, we have isolated genomic and cDNA clones encoding
human PEK. We demonstrate that the PEK eIF-2
kinase activity is
separable from its autokinase activity. We show that the enzyme is
specifically expressed in pancreatic islets and co-localizes with
somatostatin, a pancreatic islet delta cell-specific hormone.
 |
MATERIALS AND METHODS |
Isolation of cDNA and Genomic Clones--
A 2.4-kb human PEK
cDNA fragment was generated by PCR amplification using cDNA
prepared from human testis (CLONTECH, Palo Alto,
CA) and oligonucleotide primers (5'-ACAACAAGAATATCCGCAAAA-3' and
5'-CCAAATGGATTGATTTCAGAA-3'). The 2.4-kb DNA fragment was labeled by
[
-32P]dCTP using a random primer-labeling kit (Life
Technologies, Inc.), and used as a probe to screen cDNA Uni-Zap XR
libraries (Stratagene, La Jolla, CA) prepared with mRNA from human
liver, pancreas, and testis. Plaque hybridization and purification was carried out according to a protocol recommended by Stratagene. After
purification by two subsequent rounds of screening, the cDNA
inserts from positive plaques were subcloned into plasmid pBluescript-SK by in vivo excision from the lambda phages as
described by Stratagene.
A 740-bp cDNA fragment was labeled by [
-32P]dCTP
using a random primer-labeling kit (Life Technologies, Inc.), and was
used as a probe to screen a human genomic library (Stratagene)
according to a protocol recommended by Stratagene on plaque
hybridization and purification. The 740-bp cDNA fragment was
generated by PCR amplification using oligonucleotide primers
(5'-GTGACTGTGGAGGACGCTGAGG-3' and 5'-AATGCCATAACTTTCCAGTCA-3') derived
from the 5'-end of the PEK cDNA, and using plasmid pEST0.9 as DNA
template. pEST0.9 is an expressed sequence tag (EST) clone
(GenBankTM accession number AA419589) that carries 900 bp
of the 5'-end coding region of human PEK cDNA. Isolation and
purification of genomic clones was carried out using a Wizard lambda
DNA purification system (Promega, Madison, WI) according to the
manufacturer's instruction.
Restriction site mapping and Southern blot analysis of the genomic
clones were carried out essentially as described by Sambrook et
al. (36). Briefly, lambda genomic DNAs were digested with a
combination of restriction enzymes including SstI,
SstII, and KpnI, and the genomic DNA fragments
were separated by agarose gel electrophoresis. After denaturation in
1.5 M NaCl, 0.5% N NaOH and neutralization
with 1 M Tris-HCl (pH 7.4), 1.5 M NaCl, the
genomic DNA fragments were transferred to a nylon membrane by using a
Turboblotter (Schleicher & Schuell) and probed with an oligonucleotide
(5'-GCCGCTGCTCCCACCTCAGCGACGCGAGTACCGGCGGCG-3') labeled by
[
-32P]ATP and T4 polynucleotide kinase (Life
Technologies, Inc.). DNA hybridization and washing of the membrane was
carried out using the same conditions used in cDNA library
screening. A 3.0-kb SstI genomic DNA fragment was subcloned
into the SstI site of pBluescript-SK, and the resulting
plasmid was named pBluescript-hPEK3.0.
In Vitro Mutagenesis of PEK--
Oligonucleotide-directed
in vitro mutagenesis was carried out using a QuickChange kit
(Stratagene) and complementary primers (5'-TGCAATTACGCCATCGCGCGCATCCGTCTCCCAAAC-3' and
5'-GTTTGGGAGACGGATGCGCGCGATGGCGTAATTGCA-3') to create a
substitution of the amino acid lysine to alanine at position 614 of the
rat PEK carried on plasmid pPCRscript-rPEK. The mutant PEK was fully
sequenced to verify the amino acid substitution and to ensure that no
additional alterations were made. The mutant PEK was excised with
EcoRI and KpnI from pPCRscript-rPEK and subcloned into the corresponding sites located on the baculoviral expression vector pFastBac-HTa (Life Technologies, Inc.). The resulting plasmid was named pHTa-K614A.
Assembly of Full-length cDNA and Baculoviral Expression of
Human PEK--
A 4.3-kb cDNA containing the full-length coding
region of human PEK and the 5'- and 3'-untranslated regions was
constructed by subcloning a 200-bp SstI/NotI
fragment isolated from pBluescript-hPEK3.0 and a 1.6-kb
NotI/EcoRV fragment from pEST4.1 (an EST clone,
accession number AA669109) into the SstI and
EcoRI sites of pBS-hPITK3.7. The 200-bp, 1.6-kb, and the
plasmid pBS-hPITK3.7 carries the 5'-end including the
5'-untranslated region, the middle coding region, and the 3'-end
including the 3'-untranslated region of the human PEK, respectively.
The resulting plasmid was named pBluescript-hPEK. The 4.3-kb insert was
released from the plasmid by restriction digestion with SstI
and KpnI and inserted into the corresponding sites in
the baculoviral expression vector pFastBac (Life Technologies, Inc.).
The resulting plasmid was named pFastBac-hPEK.
Generation of recombinant baculoviral clones that express wild type and
K614A mutant PEK was carried out using a Bac-to-Bac baculovirus
expression system (Life Technologies, Inc.). DH10Bac competent
Escherichia coli (Life Technologies, Inc.) were transformed with pFastBac-hPEK for the wild type PEK or with pHTa-K614A for the
mutant PEK. Culture of the Sf-9 insect cells, propagation of
recombinant baculovirus, and expression of PEK and mutant K614A proteins in Sf-9 cells were carried out according to the protocol provided by the manufacturer.
Immunoprecipitation Kinase Assay--
The activity of
recombinant rat PEK from Sf-9 cell lysate was assessed in
immune-complexed kinase assays using recombinant eIF-2
as a
substrate. Frozen pellets of Sf-9 cells expressing human PEK and
Lys-614 mutant PEK or an unrelated bacterial protein were resuspended
in cell lysis buffer (10 mM HEPES, pH 7.4, 1 mM
EGTA, 1 mM MgCl2, 1 mM
2-aminoethylisothiouronium bromide, 1× CompleteTM
(Boehringer Mannheim)), followed by centrifugation at 10,000 × g for 10 min to eliminate insoluble material. The
supernatants were precleared with 20 µl of rabbit preimmune serum,
followed by immunoprecipitation with 20 µl of affinity-purified
polyclonal rabbit anti-PEK peptide antibody PITK-289 at 4 °C for 90 min on a rocker. After incubation with 100 µl of protein A-Sepharose beads at 4 °C for 1 h with rocking, the immune complexes were washed twice with wash buffer (10 mM HEPES, pH 7.4, 10 mM benzamidine, 150 mM NaCl, 0.1 mg/ml bovine
serum albumin, 5 mM EDTA) and twice with kinase buffer (20 mM Tris-HCl, pH 7.9, 50 mM NaCl, 10 mM MgCl2, 0.1 mM ATP, 1 mM dithiothreitol). The eIF-2
kinase assay was carried
out by addition of 1, 2, and 4 µg of purified human eIF-2
and 20 µCi of [
-32P]ATP to the bead slurry and incubation
at 37 °C for 30 min. Reactions were terminated by boiling with equal
volume of 2× SDS-polyacrylamide gel electrophoresis sample buffer for
3 min and analyzed by SDS-polyacrylamide gel electrophoresis. The gels
were dried and subjected to autoradiography at
70 °C.
Immunoblot Assay of Recombinant PEK--
Immunoprecipitates from
the eIF-2
kinase activity assay were separated by SDS-polyacrylamide
gel electrophoresis and transferred to polyvinylidene difluoride
membranes (Bio-Rad). After blocking with 3% skim milk for 60 min in
TBST buffer (25 mM Tris, pH 7.5, 137 mM NaCl,
2.6 mM KCl, 0.1% Tween 20), the polyvinylidene difluoride membranes were incubated with 2 µl/ml purified rabbit anti-PEK antibody PITK-289 for 60 min in the TBST buffer plus 1.5% skim milk.
Following a brief wash in TBST buffer, the membranes were incubated for
60 min with 0.3-0.5 µg/ml horseradish peroxidase-goat anti-rabbit
IgG (Zymed Laboratories Inc., San Francisco, CA) in TBST buffer plus 1.5% skim milk. Detection of PEK proteins on the
membrane was carried out using an ECL detection system from Amersham
Pharmacia Biotech.
Northern Blot Analysis--
Human multiple tissue Northern blots
(2 µg of mRNA/lane) were purchased from CLONTECH
Laboratories (Palo Alto, CA). A 2.4-kb human PEK cDNA fragment from
PCR amplification was labeled by [
-32P]dCTP using a
random prime-labeling kit from Life Technologies, Inc. and was used in
the Northern blot analyses. Hybridization was carried out in
hybridization buffer (2× SSC, 0.5% SDS, 0.1% bovine serum albumin,
0.1% polyvinylpyrolidone, 0.1% Ficoll, 100 µg/ml heparin, and 1 mM EDTA) at 60 °C overnight, followed by three washes at
60 °C in 2x SSC buffer with 0.1% SDS. Expression levels of PEK
relative to
-actin was quantified by using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Immunofluorescence Microscopy--
Two polyclonal PEK
antibodies, PITK-217 (diluted 1:400) and PITK-289 (diluted 1:200), were
used in immunofluorescence microscopy. The antibodies were developed by
immunization of rabbits with synthetic peptides derived from two
regions of the predicted rat PEK protein sequence. The PITK-217
antibody was directed to a peptide sequence
(QMQLCRKENLKDWMNRRCSMEDREHRVCLH) in the kinase subdomain V, and the
PITK-289 antibody was directed to a peptide sequence
(ENAVFENLEFPGKTVLRQRS) derived from the C terminus of rat PEK. The
following antibodies were also used for immunocytochemistry; somatostatin (mouse monoclonal, diluted 1:100, Biogenesis, Sandown, NH), glucagon (rabbit polyclonal, diluted 1:400, Novocastra Lab Ltd.,
UK), insulin (mouse monoclonal, diluted 1:400, Biogenex, San Ramon,
CA), swine anti-rabbit fluorescein isothiocyanate-conjugated immunoglobulins (diluted 1:100, DAKO Corp., Carpinteria, CA), and
rabbit anti-mouse tetramethylrhodamine B isothiocyanate-conjugated immunoglobulins (diluted 1:100, DAKO Corp.). Isolated rat and human
tissues were immersed in 10% formalin for 2 h and embedded in
paraffin. Tissue sections were deparaffinized, rehydrated, and then
immersed in 0.1% hydrogen peroxide (H2O2) in
absolute methanol for 30 min to quench endogenous peroxidase activity. Immunohistochemical stains were performed using the Elite avidin-biotin immunoperoxidase complex kit (Vector Laboratories, Burlingame, CA). The
sections were rinsed briefly in phosphate-buffered saline, blocked with
nonspecific serum, and incubated for 60 min at room temperature with
rabbit PEK polyclonal antibodies PITK-217 or PITK-289 alone, or
together with each of the antibodies to somatostatin, glucagon, or
insulin, respectively, in the co-localization studies. After three
brief rinses with phosphate-buffered saline, the sections were
incubated with swine anti-rabbit fluorescein isothiocyanate-conjugated antibodies and rabbit anti-mouse tetramethylrhodamine B
isothiocyanate-conjugated antibodies at room temperature for 60 min.
The sections were rinsed three times in phosphate-buffered saline and
examined by fluorescent microscopy.
 |
RESULTS |
Isolation of the Gene Encoding Human PEK--
Multiple human
sequences were found to match the rat PEK cDNA sequence when the
rat sequence was used as a query to probe the EST data base. Using
primers derived from the human EST sequences and cDNA from human
testis, we amplified a 2.4-kb cDNA fragment by PCR. Sequence
analysis confirmed that the 2.4-kb cDNA fragment represented the
C-terminal part of the human PEK, and was used as a probe to screen a
cDNA library prepared with mRNA from human liver, pancreas, and
testis to obtain full-length human PEK cDNA. Multiple rounds of
screening of the libraries resulted in the isolation of a positive
clone, which carried a 3.7-kb cDNA insert that represented the
longest clone among 20 positives. Sequence comparison with rat PEK
confirmed that the 3.7-kb fragment carried the majority of the coding
region and 3'-untranslated sequences. A 400-bp sequence that overlaps
with the 5'-end of the 3.7-kb sequence was identified from two
independent EST clones (GenBankTM accession no. AA419589
and AA669109, IMAGE clone 746093 and 827392), which extend the cDNA
to 4.1 kb. Despite the cDNA insert of clone 746093 being
synthesized by priming at a site closer to the 5'-end of the PEK
mRNA, both clones terminated at the same site, indicating a strong
secondary structure at the 5'-end of the PEK mRNA. This is
confirmed by our effort to extend the 5'-end sequence by rapid
amplification of cDNA ends (RACE) using cDNA prepared from
different human tissues including pancreas, liver, testis, and ovary.
RACE yielded no additional sequence information on the 5'-end of the
PEK cDNA.
Genomic cloning was used to clone the rest of the human PEK cDNA. A
700-bp cDNA fragment derived from the 5'-end of the 4.1-kb cDNA
fragment was used as probe to screen a lambda FIX II (Stratagene) human
genomic library to isolate positive clones that carry the 5'-end of PEK
gene. Among more than 20 genomic positives, restriction mapping and
Northern blot analyses confirmed that only one clone, G5-2, carried
the extreme 5'-end of the cDNA sequence. A 3.0-kb SstI
genomic fragment was subcloned from G5-2 into the SstI site of pBluescript-SK. Sequence analyses confirmed that the 3.0-kb fragment
did contain the 5'-untranslated sequences and the 5'-end of the coding
region of human PEK that overlaps with the 4.1-kb cDNA sequence.
Comparison between the 3.0-kb human genomic sequence and the rat PEK
cDNA allowed us to extend the cDNA sequence to 4,325 nucleotides, which contains the full-length coding region of human
PEK.
PEK Gene Is Conserved from Caenorhabditis elegans to
Human--
Sequence analysis indicates that the 4,325 nucleotides
encoding the human PEK cDNA contains 72 bp of 5'-untranslated
sequence, 3,345 bp of open reading frame, and 908 bp of 3'-untranslated sequence. The sequences flanking the predicted start codon
(GACGCTGATGG) partially match the consensus
GCC(A/G)CCATGG described for translation initiation sites
(37). The single open reading frame predicts a 1,115-residue
polypeptide with a deduced molecular mass of 125 kDa (Fig.
1). A hydropathy plot of the protein
sequence indicates two highly hydrophobic regions (top panel
and boxed sequence, Fig. 1). One of the hydrophobic regions
is located at the N terminus, and the other one is located in the
middle of the peptide sequence. The N-terminal hydrophobic sequence
predicts a signal peptide with 99.5% probability to be a transmembrane
region or a leader sequence for secretion (Fig. 1, boxed
sequence).

View larger version (74K):
[in this window]
[in a new window]
|
Fig. 1.
A hydrophobicity plot (top
panel) and the predicted amino acid sequence of the human
PEK cDNA (bottom panel). The signal sequence
and the hydrophobic region are boxed, and the residues
composing the catalytic domain are underlined.
|
|
The predicted PEK protein shares 88.3% sequence identity and 93.4%
similarity with rat PEK. The two homologues mainly differ within the N
terminus regulatory domain where three clusters of 2-3 amino acid
inserts are present in the human PEK. The two hydrophobic regions,
signatures of a membrane protein, are also conserved between the human
and rat PEK. The kinase domain of human PEK appears to be more closely
related to PKR (43.6% identity) than the other two eIF-2
kinases,
HRI (37.2% identity) and yeast GCN2 (34.2% identity). In contrast,
the 550-residue N-terminal segment of the PEK is quite distinct,
presumably reflecting the different physiological signals that regulate
its activity. From a search of the protein data bases, the only
sequence that shows homology to the regulatory domain is with an
uncharacterized kinase recently identified from C. elegans.
The predicted C. elegans polypeptide is 1,085 (GenBankTM accession no. Z66563) residues in length and
shares 28.5% identity and 52.2% similarity with human PEK. In
contrast to restricted homologies within the kinase domain with HRI,
PKR, and GCN2, PEK shares homologies with the C. elegans
polypeptide in highly homologous clusters that span the entire
molecule. Two of the highly conserved regions are shown in Fig.
2. The nematode kinase may differ from the mammalian counterpart in subcellular localization, as the two
hydrophobic regions of human and rat PEK are not found in the C. elegans kinase.

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 2.
Analysis of amino acid sequence similarity
within the regulatory and kinase domains of human PEK with a threonine
kinase homologue from C. elegans. The human
sequence is indicated on the top line, and the C. elegans sequence is indicated on the bottom line.
Identical amino acids are indicated by a vertical bar, the
similarities are indicated by a colon, and the gaps are
indicated by a period.
|
|
Expression in Sf-9 Insect Cells and Measurement of eIF-2
Kinase
Activity--
The distinct homology shared between human PEK and other
eIF-2
kinases suggest that PEK is a novel eIF-2
kinase. To verify whether human PEK exhibits eIF-2
kinase activity, we assembled a
full-length cDNA encoding human PEK and expressed the cDNA in Sf-9 insect cells. Using a polyclonal peptide antibody directed to the
C terminus of PEK, we immunoprecipitated human PEK from Sf-9 cell
lysates expressing the recombinant protein. As a negative control for
the kinase assay, immunoprecipitation was also carried out using lysate
from Sf-9 cells expressing an unrelated bacterial protein. Addition of
1, 2, or 4 µg of purified human eIF-2
and [
-32P]ATP, respectively, to the immunoprecipitates of
human PEK resulted in a dose-dependent phosphorylation of
eIF-2
(Fig. 3, lanes
1-3, top panel, left arrow). In
contrast, no phosphorylation of eIF-2
was detected in
immunoprecipitates of the bacterial protein (Fig. 3, lane 4,
top panel). The recombinant human PEK protein appeared to be
autophosphorylated, as suggested by the presence of a phosphorylated peptide with the predicted molecular weight of human PEK, which is
absent in the negative control (Fig. 3, compare lanes
1-3 with lane 4, top panel, as
indicated by an arrow at the top left).

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 3.
Analysis of autophosphorylation and
eIF-2 kinase activity of the wild type and a
mutant PEK. Human eIF-2 kinase activity was measured from
immunoprecipitated PEK (top panel). Kinase activities were
assayed using purified eIF-2 in the immunoprecipitates from Sf-9
cells expressing recombinant human PEK (lanes 1-3), an
unrelated bacterial protein (lane 4), and a K614A mutant PEK
(lanes 5-7). The K614A mutant carries a lysine to alanine
substitution at position 614 as described under "Materials and
Methods." The assays contained 1 µg (lanes 1 and
5), 2 µg (lanes 2 and 6), and 4 µg
(lanes 3, 4, and 7) of purified human
eIF-2 protein. The left arrows indicate the positions of
autophosphorylated human PEK and phosphorylated human eIF-2 . The
right arrow indicates the position of the
autophosphorylated mutant PEK. Bottom panel, detection
of PEK proteins by Western blot analyses using anti-PEK antibody and
the same blot used in the top panel. The position of the
mutant PEK is indicated by a right arrow.
|
|
A Point Mutation Abolishes PEK Kinase Activity But Not
Autophosphorylation--
As indicated in Fig. 3, activation of PEK
enzymatic activity coincides with autophosphorylation of the PEK. To
address the issue of whether the autophosphorylation of PEK is a
prerequisite for its kinase activity, we have generated a mutant PEK in
which an alanine was substituted for a lysine at position 614, conserved among all the eIF-2
kinases. The mutant PEK, K614A, was
expressed in Sf-9 cells and was used in immunoprecipitation kinase
assays. In comparison with the wild type PEK, mutation of the conserved lysine residue has completely abolished its kinase activity, as no
eIF-2
phosphorylation was detected from immunoprecipitates from Sf-9
cells expressing K164A after addition of 1, 2, or 4 µg of purified
human eIF-2
and [
-32P]ATP to the kinase reactions
(Fig. 3, lanes 5-7, top panel). In
contrast, the mutant PEK remains autophosphorylated, evidenced by the
presence of a heavily phosphorylated protein band corresponding to the
predicted size of PEK (Fig. 3, right arrow, lanes
5-7, top panel). The mutation has also
caused a slight increase in mobility (Fig. 3, compare lanes
1-3 with lanes 5-7), presumably caused by changes in conformation of the mutant. The phosphorylated protein band was analyzed by Western blot analyses using PEK antibodies to verify the identity of the phosphorylated protein (Fig. 3, arrow, bottom panel). The result confirmed that
the phosphorylated protein was the PEK protein. Consistent with the
high level of autophosphorylation, the level of K614A mutant PEK
protein was expressed at a much higher level than that of the wild type
PEK, which was below the detection limit of the antibodies (Fig. 3, compare lanes 5-7 with lanes
1-3, bottom panel). The results suggest that
the PEK kinase activity can be separated from its autokinase activity.
Human PEK Is Predominantly Expressed in Pancreas--
The
expression of PEK mRNA in various human tissues was examined by
Northern blot analysis of poly(A)+ RNA using a cDNA
probe derived from the coding region of human PEK. This probe detected
a single ~5.2-kb mRNA transcript in all the tissues examined
(Fig. 4, arrow, upper
panel). No apparent alternative transcript sizes were detected in
any of the tissues. PEK mRNA was readily detected in pancreas and
placenta. PEK expression was also detectable at much lower levels in
other tissues including kidney, skeletal muscle, liver, lung, brain,
heart, peripheral blood leukocytes, colon, small intestine, ovary,
testis, prostate, thymus, and spleen. Normalization to the levels of
-actin (Fig. 4, arrow, lower panel) within
each tissue revealed that PEK was most abundantly expressed in pancreas
with an expression level that was more than 10-fold higher than
placenta and more than 20-fold higher than any of the other
tissues.

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 4.
Northern blot analysis of tissue distribution
of PEK mRNA. Multiple tissue Northern blots containing 2 µg
of mRNA from each human tissue were hybridized with a
[32P]dCTP-labeled cDNA probe of human PEK (top
panel). The membrane was then rehybridized with a -actin probe
(bottom panel). The names of the tissues are indicated at
the top. The positions of human PEK (~5.2 kb) and
-actin mRNAs are indicated with arrows.
|
|
Co-localization of Human PEK with Somatostatin in Pancreatic Islet
Delta Cells--
The pancreas consists of heterogeneous tissues, which
include exocrine acinar tissue and the endocrine islets. We have
developed two polyclonal peptide antibodies to PEK to investigate
tissue and cellular distribution. Although both antibodies yielded
similar results, antibody PITK-217 can be used at a much higher
dilution and was therefore used throughout the studies.
Immunohistochemical analysis was carried out to detect PEK protein
expression in human pancreas and various rat tissues including
pancreas, liver, spleen, small intestine, large intestine, stomach,
lymph node, uterus, heart, lung, kidney, brain, and skeletal muscle.
The peptide antibodies detected significant levels of PEK protein in
certain cell types within human and rat pancreatic islets (Fig.
5, panel A). In contrast, the
expression level in the exocrine pancreas and in other rat tissues was
not detectable by the antibodies (data not shown). The result is
consistent with the high level of mRNA levels detected from
pancreatic tissue by the Northern blot analysis.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 5.
Co-localization of human PEK with
somatostatin in the pancreatic islet. Immunofluorescence double
labeling was used for cellular co-localization of human PEK with
somatostatin in pancreatic islets using polyclonal PEK antibodies and a
monoclonal somatostatin antibody. The same section was viewed though a
fluorescein isothiocyanate filter for human PEK (panel A),
and the identical field was subsequently viewed through a rhodamine
filter to visualize human somatostatin (panel B).
Panel C shows the co-localization of both human PEK and
somatostatin. Panel D indicates staining of a pancreatic
islet with preimmune serum used as a negative control.
|
|
Pancreatic islets consist of four major cell types, including
insulin-secreting beta cells (82%), glucagon-secreting alpha cells
(13%), somatostatin-secreting delta cells (4%) and pancreatic polypeptide-secreting cells. To identify the cell type that expresses the PEK protein in pancreatic islets, immunofluorescence double labeling was used for cellular co-localization of PEK with
cell-specific hormones using antibodies against insulin, glucagon,
somatostatin, and pancreatic polypeptide. Each of the antibodies
detected cell-specific expression of the corresponding hormone (data
not shown). Only the cellular expression of somatostatin (Fig. 5,
panel B) co-localized with that of human PEK (Fig. 5,
panel C). None of the proteins were detected using the
pre-immune serum (Fig. 5, panel D). The results suggest that
PEK is predominantly expressed in pancreatic delta cells.
 |
DISCUSSION |
Previous reports indicate that not all eIF-2
kinase activity is
accounted for by HRI and PKR (6, 30-34). Thus it is not surprising to
learn that mice with targeted disruption of the PKR gene exhibit an
apparently normal phenotype with only mild impairment of resistance to
viral infection (38). In comparison to PKR and HRI, PEK is unusually
large, composed of 1,115 amino acids, with a unique large N terminus
upstream of the kinase domain. PEK carries a larger insert between
subdomain III and V, a feature which distinguishes eIF-2
kinases
from other families of serine/threonine kinases (20). The size of the
insert varies between different eIF-2
kinases, and has been shown to
be important in regulating eIF-2
kinase activity and substrate
specificity (24, 39).
eIF-2
phosphorylation is highly conserved from yeast to mammals. A
recent study shows that baculovirus adopts a strategy used by mammalian
viruses to overcome host defense responses to infection by expressing a
truncated version of eIF-2
kinase, which through a dominant-negative
mechanism can inhibit activity of eIF-2
kinases in Sf-9 insect cells
(40). In addition to yeast cells, insect cells also have been used to
study the translational control of mammalian eIF-2
kinases. Chen
et al. (57) showed that the expression of wild type HRI
caused severe inhibition of general protein synthesis in Sf9
insect cells. Such inhibition was relieved by co-expression of a mutant
eIF-2
, S51A, that carried an alanine replacement on the
phosphorylation site. It should be noted that the level of expression
of the wild type PEK in Sf-9 cells is very low when compared with the
level of mutant PEK. This is evident from the Western blot analysis of
the immunoprecipitated PEK proteins (Fig. 3). The low level of
expression is probably caused by the toxic effect of the wild type
kinase. We noted during the production of recombinant PEK in Sf-9 cells
that infection of the recombinant baculovirus expressing the wild type
PEK caused excessive cell death 48 h post-infection when compared
with cells infected with baculovirus expressing the mutant PEK (data
not shown). Hyperphosphorylation of eIF-2
by PKR was previously
shown to cause toxic effects in yeast cells (12, 41, 42). The toxic
effect of PEK may result from hyperphosphorylation of endogenous eIF-2
in the insect cells, because PEK have been shown to
functionally complement GCN2 in yeast (35). The toxic effect could also
result from translational arrest in Sf-9 cells caused by competition of
PEK for binding with PK2, as suggested by Dever et al.
(40).
One of the unique structural features of PEK is the presence of a
signal peptide at the extreme N terminus, which is conserved between
rat and human but is absent in the C. elegans homologue. Signal peptides are often associated with secretion or targeting of
proteins to different cellular compartments. Protein targeting plays an
important role in regulating kinase activity by providing access to
local substrates or regulatory ligands. Two double-stranded RNA-binding
domains located at the N terminus of PKR have been shown to facilitate
PKR association with the ribosome (43). Ribosomal association is
required for full activity of the kinase in vivo, even
though the double-stranded DNA-binding domain is not required for
in vitro phosphorylation of eIF-2
(43). Consistent with
the transcriptional activities observed with PKR, the kinase has also
been reported to be localized in the nucleus, though the domains
responsible for nuclear localization have not been characterized (44).
Similarly, GCN2 is also subjected to ribosomal targeting mediated by a
tyrosine-rich domain localized in the C terminus. Interestingly, the
tyrosine-rich sequence is conserved between GCN2 and PKR, but is
apparently absent in PEK. Mutations that change the lysine to other
amino acid sequences abolishes the ribosomal association and impairs
the kinase activity of PKR and yeast GCN2 in vivo (45). In
comparison, PEK does not carry any apparent regulatory motifs, such as
a double-stranded RNA-binding domain of PKR or hemin binding motif of
HRI. Its entire N-terminal 550 residue domain is quite unique and does
not share any significant homology with known peptide sequences in the
data bank except for an uncharacterized threonine kinase cloned
recently from C. elegans. Therefore, it is conceivable that
PEK is regulated by different mechanisms. It is possible that the
signal peptide and a hydrophobic domain localized in the middle of the
molecule may play important roles in targeting the kinase to
appropriate cellular compartment(s).
Previous studies (4, 24, 46-48) indicate that autophosphorylation is
essential for activation of the eIF-2
kinases. An invariant lysine
residue within the kinase domain II of eIF-2
kinases has been shown
to be important in regulating both the kinase activity and cell growth
and proliferation by PKR (6). However, the importance of the lysine
residue in regulating autokinase activity has not been clearly defined.
Substitution of the lysine residue with either an arginine or a proline
was shown to completely abolish PKR kinase activity. The mutations also
abolished PKR autophosphorylation both in vivo in NIH/3T3
cells (49) and in vitro in a cell-free system (50),
suggesting that autophosphorylation is required for eIF-2
kinase
activity. In contrast, the same mutation was reported to abolish only
the kinase activity without major effects on autophosphorylation (51).
We have generated a mutant PEK by replacing the invariant lysine at
position 614 with an alanine to study how PEK kinase activity is
regulated by the level of autophosphorylation. Consistent with the PKR
mutations, the lysine to alanine mutation completely abolished kinase
activity. In contrast, the mutant PEK remains autophosphorylated when
expressed in Sf-9 insect cells, suggesting that the kinase activity is
clearly separable from its autophosphorylation. Because a PEK homologue may also exist in Sf-9 insect cells, it is conceivable that the phosphorylation could potentially be carried out by an insect homologue
kinase in vivo. However, because the level of expression of
the recombinant mutant PEK is very high in Sf-9 cells, only a small
portion of the mutant PEK could be phosphorylated if an intermolecular
association is responsible. In contrast to PKR, our data suggest that
PEK kinase activity is not fully determined by the state of autophosphorylation.
It should be noted that even though the PEK mRNA is detected in
various tissues, immunohistochemical analysis only detected the PEK
protein in pancreatic islets. The pancreas is composed of both exocrine
ascinar tissue and endocrine islets that are scattered within the
exocrine tissue. Of importance, only 1-2% of pancreas mass in adult
human pancreas is endocrine islet (52). Therefore, the level of
expression of islet-specific genes can be masked by the vast majority
of exocrine pancreas when assayed in total pancreas. Islets of
Langerhans are composed of at least four cell types organized to form
micro-organs within the pancreas. About 70-80% of the islet cells are
insulin-producing
cells; 5% are somatostatin-producing delta
cells; 15-20% are glucagon-producing alpha cells or pancreatic
polypeptide-producing PP cells, and the remainder contains other
uncharacterized cell types. Thus the level of PEK mRNA in
pancreatic islets, especially in pancreatic delta cells, should be much
higher than that shown in total pancreas. This explains why protein
levels were not detectable in other tissues even though mRNA was
present. The islet-specific expression of PEK detected by
immunofluorescence is also consistent with our previous studies (35) on
PEK kinase activity in different tissues in which we showed that PEK
kinase activity was only detected in pancreas and pancreatic islets
among the various tissues examined.
The endocrine islet plays a role of paramount importance in maintaining
normoglycemia in mammals and humans. The islet functions by secreting
different hormones in a highly regulated fashion in response to changes
in environmental conditions. Among all the nutritional conditions, the
concentration of blood glucose is the single most important factor that
affects the synthesis and secretion of various islet hormones. The
coordinate action of several hormones including insulin and glucagon
helps to maintain the blood glucose in a narrow range under physiologic
conditions. Though not fully understood, somatostatin also plays an
important role in maintaining normal blood glucose levels by modulating secretion of both insulin and glucagon. In contrast to transcriptional regulation over time intervals of hours, the glucose-stimulated biosynthesis of insulin occurs within minutes at the level of protein
synthesis (10, 58). A number of other membrane and secretory proteins
in the pancreas are also believed to be regulated at the translational
level (54-56), although little is known about the mechanisms involved
in the processes. Our demonstration that PEK is an islet delta
cell-specific eIF-2
kinase will have important repercussions on
future studies of translational control in pancreatic islets, a tissue
that plays important roles in health and disease, such as diabetes.