Characterization of a Mutant Pancreatic eIF-2alpha Kinase, PEK, and Co-localization with Somatostatin in Islet Delta Cells*

Yuguang ShiDagger , Jie An, Jingdong Liang, Scott E. Hayes, George E. Sandusky, Lawrence E. Stramm, and Na N. Yang

From the Diabetes Research, DC 0545, Endocrine Division, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285

    ABSTRACT
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Abstract
Introduction
References

Phosphorylation of eukaryotic translation initiation factor-2alpha (eIF-2alpha ) is one of the key steps where protein synthesis is regulated in response to changes in environmental conditions. The phosphorylation is carried out in part by three distinct eIF-2alpha kinases including mammalian double-stranded RNA-dependent eIF-2alpha kinase (PKR) and heme-regulated inhibitor kinase (HRI), and yeast GCN2. We report the identification and characterization of a related kinase, PEK, which shares common features with other eIF-2alpha kinases including phosphorylation of eIF-2alpha in vitro. We show that human PEK is regulated by different mechanisms than PKR or HRI. In contrast to PKR or HRI, which are dependent on autophosphorylation for their kinase activity, a point mutation that replaced the conserved Lys-614 with an alanine completely abolished the eIF-2alpha kinase activity, whereas the mutant PEK was still autophosphorylated when expressed in Sf-9 cells. Northern blot analysis indicates that PEK mRNA was predominantly expressed in pancreas, though low expression was also present in several tissues. Consistent with the high levels of mRNA in pancreas, the PEK protein was only detected in human pancreatic islets, and the kinase co-localized with somatostatin, a pancreatic delta cell-specific hormone. Thus PEK is believed to play an important role in regulating protein synthesis in the pancreatic islet, especially in islet delta cells.

    INTRODUCTION
Top
Abstract
Introduction
References

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 alpha -subunit of eukaryotic initiation factor-2 (eIF-2alpha )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-2alpha 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-2alpha 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-2alpha . 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-2alpha kinases share extensive homology within the catalytic domains and each of the kinases has been demonstrated to phosphorylate eIF-2alpha 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-2alpha kinase allows for different physiological signals to regulate phosphorylation of eIF-2alpha . 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-2alpha 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-2alpha 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-2alpha phosphorylation in mammalian cells (29).

Because additional eIF-2alpha 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-2alpha kinase from rat pancreas, named PEK, as pancreatic eIF-2alpha kinase (35). In this study, we have isolated genomic and cDNA clones encoding human PEK. We demonstrate that the PEK eIF-2alpha 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 [alpha -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 [alpha -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 [gamma -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-2alpha 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-2alpha kinase assay was carried out by addition of 1, 2, and 4 µg of purified human eIF-2alpha and 20 µCi of [gamma -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-2alpha 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 [alpha -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 beta -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).


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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-2alpha 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.


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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-2alpha Kinase Activity-- The distinct homology shared between human PEK and other eIF-2alpha kinases suggest that PEK is a novel eIF-2alpha kinase. To verify whether human PEK exhibits eIF-2alpha 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-2alpha and [gamma -32P]ATP, respectively, to the immunoprecipitates of human PEK resulted in a dose-dependent phosphorylation of eIF-2alpha (Fig. 3, lanes 1-3, top panel, left arrow). In contrast, no phosphorylation of eIF-2alpha 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).


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Fig. 3.   Analysis of autophosphorylation and eIF-2alpha kinase activity of the wild type and a mutant PEK. Human eIF-2alpha kinase activity was measured from immunoprecipitated PEK (top panel). Kinase activities were assayed using purified eIF-2alpha 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-2alpha protein. The left arrows indicate the positions of autophosphorylated human PEK and phosphorylated human eIF-2alpha . 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-2alpha 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-2alpha phosphorylation was detected from immunoprecipitates from Sf-9 cells expressing K164A after addition of 1, 2, or 4 µg of purified human eIF-2alpha and [gamma -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 beta -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.


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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 beta -actin probe (bottom panel). The names of the tissues are indicated at the top. The positions of human PEK (~5.2 kb) and beta -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.


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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-2alpha 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-2alpha kinases from other families of serine/threonine kinases (20). The size of the insert varies between different eIF-2alpha kinases, and has been shown to be important in regulating eIF-2alpha kinase activity and substrate specificity (24, 39).

eIF-2alpha 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-2alpha kinase, which through a dominant-negative mechanism can inhibit activity of eIF-2alpha 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-2alpha 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-2alpha , 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-2alpha 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-2alpha 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-2alpha (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-2alpha kinases. An invariant lysine residue within the kinase domain II of eIF-2alpha 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-2alpha 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 beta  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-2alpha 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.

    ACKNOWLEDGEMENTS

We thank Bruce Glover for oligonucleotide synthesis and members in the Lilly sequencing laboratory for DNA sequence analysis. We also thank Drs. Andrew Geiser and Michael Statnick for critically reading the manuscript. We are especially grateful to Drs. Armen Tashjian, Jose Caro, and Hansen Hsiung for valuable suggestions to the work presented here.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF110146.

Dagger To whom correspondence should be addressed. Diabetes Research, DC 0545, Endocrine Div., Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, IN 46285. Tel.: 317-276-6753; Fax: 317-276-9574; E-mail: Shi_Yuguang{at}Lilly.com.

    ABBREVIATIONS

The abbreviations used are: eIF-2alpha , eukaryotic protein synthesis initiation factor-2alpha subunit; PEK, pancreatic eIF-2alpha kinase; PKR, the double-stranded RNA-dependent eIF-2alpha kinase; HRI, the hemeregulated inhibitor kinase; GCN2, the yeast general amino acid control eIF-2alpha kinase; kb, kilobase(s); PCR, polymerase chain reaction; bp, base pair(s); EST, expressed sequence tag; RACE, rapid amplification of cDNA ends.

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Abstract
Introduction
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