INTRODUCTION

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Mammalian protein synthesis is promptly adjusted in response to variety of different cellular stresses including nutrient starvation, iron deficiency, heat shock, and viral infection (27). One of the best-studied mechanisms regulating translation involves phosphorylation of the  subunit of eukaryotic initiation factor 2 (eIF-2) (6, 7, 18, 39, 45). eIF-2 associates with initiator Met-tRNA and GTP and participates in the ribosomal selection of the start codon. During the process of translation initiation, GTP complexed with eIF-2 is hydrolyzed to GDP and eIF-2-GDP is released from the ribosomal machinery (28). To facilitate subsequent rounds of translation initiation, the GDP-bound eIF-2 has to be converted to active eIF-2-GTP. This guanine exchange reaction is carried out by eIF-2B. Phosphorylation of the  subunit of eIF-2 at residue serine-51 leads to inhibition of eIF-2B activity, reducing guanine nucleotide exchange and thus the rate of translation.

A family of protein kinases phosphorylate eIF-2 in response to different cellular stress conditions. While each member of the eIF-2 kinase family shares sequence and structural features distinguishable from those of other families of serine/threonine kinases, there is little similarity in their flanking regulatory regions, which facilitate the different stress signals controlling each eIF-2 kinase. Included in this family are two mammalian eIF-2 kinases: the double-stranded RNA (ds-RNA)-dependent kinase (PKR) (34) and the heme-regulated inhibitor kinase (HRI) (5). PKR participates in an antiviral defense mechanism that is mediated by interferon. It has been proposed that when cells are infected by viruses, ds-RNA that is synthesized during viral replication stimulates PKR activity by interacting with two ds-RNA-binding domains located in the amino terminus of this kinase (6, 26, 34). Phosphorylation of eIF-2 by PKR blocks total protein synthesis, preventing viral replication and infection of neighboring cells. Interestingly, RNA and protein products encoded by viruses have been shown to thwart the antiviral pathway by binding to PKR and inhibiting its kinase activity. Recent studies indicate that PKR also plays an important role in the regulation of cell growth and division and in the control of apoptosis (2, 22, 24, 25, 29, 42).

The second well-characterized mammalian eIF-2 kinase, HRI, is expressed in erythroid tissues and couples the synthesis of globin, the predominant protein product in these cells, to hemin and iron availability (5). In addition to being modulated by hemin, the activity of HRI is proposed to be modulated by association with heat shock proteins (5, 49). Another eIF-2 kinase that is regulated by hemin, PfPK4, was recently identified from the malarial parasite Plasmodium falciparum (30). PfPK4 is expressed during each stage of parasite development, and it is proposed that PfPK4 allows the parasite to sense its environment during the invasion process. Although PfPK4 and HRI are both inhibited by hemin, these two kinases do not have similar sequences flanking their kinase catalytic domains.

In contrast to mammalian kinases PKR and HRI, which inhibit global protein synthesis in response to stress signals, the eIF-2 kinase in Saccharomyces cerevisiae, GCN2, controls translation of a single species of mRNA encoding GCN4 (19, 45). GCN4 is a transcriptional activator of over 30 genes involved in amino acid biosynthesis. Control of GCN4 translation is mediated by four short upstream open reading frames (ORFs) located in the 5'-untranslated portion of the GCN4 mRNA. When cells are grown under conditions limiting for amino acids, the ORFs inhibit translation of the GCN4 coding region. In response to amino acid limitation, phosphorylation of eIF-2 by GCN2 kinase leads to reduced eIF-2-GTP levels that overcome the inhibitory effects of the ORFs, allowing for increased translation of GCN4 (1, 9, 19, 45). Activation of GCN2 kinase during starvation conditions involves sequences homologous to those of histidyl-tRNA synthetases, which bind uncharged tRNAs that accumulate when amino acids are limiting (46-48, 52). Recently, GCN2 kinase was characterized from Drosophila melanogaster (33, 41). Expression of Drosophila GCN2 is developmentally regulated and at later stages becomes restricted to the central nervous system. The physiological role of GCN2 kinase in Drosophila is currently unclear. Furthermore, it is not certain whether Drosophila GCN2 mediates total protein synthesis or controls gene-specific translation.

In the present study, we identified and characterized a new eIF-2 kinase from a rat pancreatic islet. Like the members of the eIF-2 kinase family, this new kinase, which we refer to as PEK, pancreatic eIF-2 kinase, phosphorylates the  subunit of eIF-2 at residue serine-51. While the kinase domain of PEK is similar to those of eIF-2 kinases, including the characteristic large insert between subdomains IV and V, its flanking 550-residue amino-terminal sequences are distinct. Our Northern analysis indicates that PEK is expressed in many different rat tissues, with the greatest levels in the pancreas. In agreement with pancreatic expression for this kinase, PEK was predominantly detected by an immunoprecipitation kinase assay of pancreas and pancreatic islet cells. PEK was found to function in translation regulation in both the yeast and reticulocyte lysate model systems. Results from these studies indicate that PEK is a new mammalian eIF-2 kinase important for mediating translational control.

	MATERIALS AND METHODS

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Isolation of cDNA clones encoding PEK. cDNAs encoding proteins immunoreactive with antiphosphothreonine antibodies were isolated from a lambda Zap-Express library generated from rat pancreatic islet poly(A)-selected RNA. The library was screened with a picoBlue immunoscreening kit from Stratagene according to the manufacturer's instructions. A total of 5 × 10⁵ plaques were screened by infecting the XL1-Blue MRF' bacterial strain with the phage library. Following incubation at 42°C for 4 to 5 h, plates were overlaid with filters presoaked with 10 mM isopropylthio--D-galactoside (IPTG) and incubated for an additional 3.5 h. Upon removal of the first membrane, a duplicate nitrocellulose membrane presoaked with 10 mM IPTG was overlaid, and the plates were incubated overnight at 37°C. The membranes were incubated with blocking solutions containing rabbit antiphosphothreonine antibody (Zymed), rinsed three times with washing solution, and treated with alkaline phosphatase conjugated to goat anti-rabbit secondary antibody (Zymed). Positive plaques were detected with Nitro Blue Tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (Sigma). Following purification by two subsequent rounds of screening, the cDNA inserts from positive plaques were subcloned into plasmid pBK-CMV by in vivo excision from the lambda phages as described by Stratagene. Additional rounds of screening were carried out to isolate full-length cDNA clones by using an [-³²P]dCTP-labeled DNA insert from the subcloned plasmid as a probe to rescreen the library, according to a protocol recommended by Stratagene for plaque hybridization and purification.

Bacterial and baculoviral expression of PEK and eIF-2. PEK was expressed in Escherichia coli by using the T7 promoter system. A 3.4-kb EcoRI DNA fragment encoding the entire PEK cDNA was inserted into the EcoRI site of pET28a. An EcoRI site was engineered immediately 5' to the predicted start codon of the PEK gene to facilitate direct subcloning of the cDNA without the 5' untranslated region (UTR). The resulting plasmid, p259, contained the PEK ORF fused to an amino-terminal sequence containing a polyhistidine tag. To express a mutant version of PEK with residues 785 to 1108 deleted, a 2.4-kb EcoRI-to-HindIII fragment was inserted into the EcoRI-to-HindIII sites of pET28a, generating p260. The encoded PEK-785-1108 was fused to amino terminal polyhistidine sequences in pET28a. Expression plasmid p259 or p260 or vector pET28a was introduced into E. coli BL21 (DE3) (F ompT r_B m_B; containing lysogen DE3), and the strain was grown at 30°C in Luria-Bertani medium supplemented with 100 µg of ampicillin per ml until mid-logarithmic phase. Then, 1 mM IPTG was added to the culture, and the culture was incubated overnight at room temperature. The cell pellets were collected by centrifugation, washed, and then resuspended in solution A (20 mM Tris-HCl [pH 7.9], 500 mM NaCl, 10% glycerol, 1 mM -mercaptoethanol, 0.1% Triton X-100, 1 µM pepstatin A, 1 µM leupeptin, 0.15 µM aprotinin, 0.1 mM phenylmethylsulfonyl fluoride [PMSF]) with 5 mM imidazole and lysed with a French press. Lysates were clarified by centrifugation at 39,000 × g and subjected to an immunoblot assay using a polyclonal antibody against the polyhistidine tag (Pierce) or used in kinase reaction mixtures containing the eIF-2 substrate. No immunoreactive protein was detected in the lysate prepared from E. coli containing only vector pET28a. To purify the PEK fusion proteins, the clarified lysates were loaded onto a column containing nickel chelation resin (Qiagen) that binds to the polyhistidine tag of the fusion proteins, and PEK was partially purified by elution with solution A containing 200 mM imidazole.

In vitro kinase assays. The activity of recombinant rat PEK from Sf-9 cell lysate was assessed in immune-complex kinase assays using recombinant eIF-2 as a substrate. The supernatants were precleared with protein A-Sepharose, followed by immunoprecipitation with the polyclonal anti-PEK peptide antibody (PITK-289) at 4°C for 90 min. The PITK-289 antibody was developed by immunizing rabbits with synthetic peptides (ENAVFENLEFPGKTVLRQRS) derived from the C-terminal sequence of rat PEK. After incubation with protein A-Sepharose at 4°C for 1 h with rocking, the immune complexes were rinsed twice with wash buffer (10 mM HEPES [pH 7.4], 10 mM benzamidine, 150 mM NaCl, 0.5 mM methionine, 0.1 mg/ml bovine serum albumin [BSA], 5 mM EDTA) and twice with kinase buffer supplemented with 100 µM PMSF, 0.1 mM ATP, and 1 mM dithiothreitol. The kinase assay using human eIF-2 was carried out by the addition of 30 µl of reaction mixture containing 1, 2, or 4 µg of purified human eIF-2 and 20 µCi of [-³²P]ATP in a final concentration of 0.1 mM ATP to the bead-bound PEK. After the reaction mixtures were incubated at 37°C for 30 min, the assays were terminated by boiling the mixtures with an equal volume of 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer for 3 min, followed by characterization by SDS-PAGE. The gels were dried and subjected to autoradiography at 70°C.

Northern blot analysis. A Northern blot containing 2 µg of poly(A)⁺ RNA from different rat tissues per lane was purchased from Clontech. To measure PEK mRNA levels in pancreas cells, which were not included in the multiple-tissue Northern blot, a separate blot was prepared with mRNA from pancreas, skeletal muscle, kidney, and testis cells. DNA probes for PEK and the internal controls, which include -actin, -tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were radiolabeled with [-³²P]dCTP by random prime labeling with a kit from Gibco-BRL. Hybridization was carried out in 2× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate) with 0.5% SDS, 0.1% BSA, 0.1% polyvinylpyrolidone, 0.1% Ficoll, 100 µg of heparin per ml, and 1 mM EDTA at 60°C overnight, followed by three washings at 60°C in 2× SSC buffer with 0.1% SDS. The relative levels of PEK and control mRNAs were measured with a Molecular Dynamics PhosphorImager.

Preparation of tissue lysates. Tissues were freshly isolated from 8-week-old male Sprague-Dawley rats and were immediately frozen in liquid nitrogen. Frozen tissues were ground to a fine powder in liquid nitrogen and then resuspended in ice-cold lysis buffer containing 50 mM HEPES (pH 7.4), 2 mM EDTA, 1% Triton X-100, 10% glycerol, 10 mM NaF, 150 mM NaCl, and inhibitors (2 mM Na₃VO₄, 5 µg of leupeptin per ml, 1.5 mg of benzamidine per ml, 0.5 mg of pepstatin A per ml, 2 µg of aprotinin per ml, 1 mM PMSF, 10 µg of antipain per ml) at a final concentration of 100 mg of tissue/ml of buffer. The tissues were homogenized with a polytron for 30 s, and the homogenate was incubated on ice for 30 min, followed by centrifugation for 20 min at 10,000 × g. The supernatants were aliquotted and stored at 80°C. Isolated canine islets were lysed in cell lysis buffer (10 mM HEPES [pH 7.4], 1 mM EGTA, 1 mM MgCl₂, 1 mM 2-aminoethylisothiouronium bromide, 1× Complete medium (Boehringer Mannheim) and precleared by centrifugation for 10 min at 10,000 × g. Immunoprecipitation kinase assays were carried out with human or yeast eIF-2 substrate as described above. As a control, similar assays were carried out with preimmune serum in the immunoprecipitations.

Expression of PEK in yeast. To express PEK in yeast, a 3.5-kb SacI-to-XhoI DNA fragment containing the PEK cDNA was removed from pBK-RK3 and inserted between the SacI and SalI sites of yeast expression vector pEMBLyex4 (4), generating plasmid p504. p504 is a URA3-marked high-copy-number plasmid that contains the PEK cDNA downstream of the galactose-inducible GAL-CYC1 hybrid promoter. Plasmids p504, pEMBLyex4, and pC102-2 (47) encoding GCN2 were transformed into yeast strains H1894 (MATa ura3-52 leu2-3 leu 2-112 trp1 gcn2), H1816 (MATa ura3-52 leu2-3 leu2-112 gcn2 sui2 GCN4-lacZ p1097 [SUI2 LEU2]), and H1817 (MATa ura3-52 leu2-3 leu2-112 gcn2 sui2 GCN4-lacZ p1098 [SUI2S51A LEU2]) (19). Strains H1817 and H1816 are isogenic and differ only in their SUI2 alleles, which encode eIF-2. Plasmid-containing strains were selected for by uracil prototrophy. Yeast transformants were grown in patches on agar plates containing synthetic medium supplemented with 10% galactose-2% raffinose (SGal) (21), 2 mM leucine, and 1 mM tryptophan. After the plates were incubated for 1 day at 30°C, cell patches were replica printed onto agar plates containing SGal medium supplemented with leucine, tryptophan, 0.5 µg of sulfometuron methyl (SM) per ml or 30 mM 3-aminotriazole (3-AT) (48), and all amino acids except histidine. Agar plates were incubated for the indicated times at 30°C and photographed.

Protein synthesis in rabbit reticulate lysate. In vitro translation assays were carried out in a 20-µl reaction volume containing 50% untreated rabbit reticulocyte lysate (Promega), 20 mM HEPES-KOH (pH 6.8), 20 mM KCl, 1 mM Mg(OAc)₂, 10 mM creatine phosphate, 50 µg of creatine phosphokinase per ml, 0.8 mM ATP, 0.2 mM GTP, a 25 µM concentration of each amino acid except methionine, and 1 µCi of [³⁵S]methionine (1,200 Ci/mmol). Full-length PEK and PEK-785-1008 were purified by using their amino-terminal polyhistidine sequences and nickel chelation resin. The partially purified PEK and the mutant were added to the in vitro translation reaction mixtures at the indicated concentrations, and the reaction mixtures were incubated at 30°C for 30 min. Proteins from 5-µl aliquots were precipitated with trichloroacetic acid, and the incorporation of ³⁵S was quantified by scintillation counting.

Nucleotide sequence accession number. The nucleotide sequences determined in this study have been submitted to the GenBank/EMBL data bank under accession no. AF096835.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Isolation of cDNA encoding new protein kinase from rat pancreatic islet cells. In an effort to identify new threonine kinases in pancreatic tissue, we used antiphosphothreonine antibodies to screen a lambda Zap-Express cDNA library prepared from mRNA isolated from rat pancreatic islets. Since there was no detectable threonine kinase activity in the host strain, E. coli XL1-Blue MRF', on the basis of the antiphosphothreonine antibodies, it was expected that any positive signal would come from the expression of introduced cDNA clones. After screening 5 × 10⁵ recombinant plaques, we identified distinct clones encoding fusion proteins which reacted with the polyclonal antiphosphothreonine antibodies. The cDNA inserts were subcloned into plasmid pBK-CMV by in vivo excision from the lambda phages and characterized by sequence analysis. While the majority of the inserts encoded known threonine kinases, such as those encoded by lyn and pim-1, one cDNA clone with a 4.5-kb insert that did not match any previously characterized protein kinase coding sequence entered in the GenBank/EMBL database was identified. As will be described below, the corresponding new pancreatic kinase is related to the eIF-2 kinase family, and we will refer to it as pancreatic eIF-2 kinase, or PEK.

View larger version (81K): [in this window] [in a new window]   FIG. 1.   The predicted sequence of pancreatic eIF-2 kinase, PEK. PEK is 1,108 residues in length. The kinase catalytic sequences are underlined. A predicted signal sequence and a hydrophobic region are boxed, and possible amino-terminal myristoylation sites are in boldface.

New pancreatic protein kinase is related to eIF-2 kinases. The catalytic domain of PEK spans 540 amino acid residues and contains sequences corresponding to those of the 12 subdomains described by Hanks and Hunter (17) (Fig. 1 and 2). By using the sequence of PEK as a query in a BLAST search of the GenBank/EMBL database, we found that it is most closely related to a family of protein kinases that regulate protein synthesis by phosphorylation of eIF-2. The sum probability of random correspondence of the pairwise segments with the pancreatic kinase (i.e., the BLAST score) ranged from 6.0e⁴² and 4.3e⁴⁰ with rat HRI and human PKR, respectively, to 2.4e²⁵ with yeast GCN2. A multiple alignment of the catalytic domain of PEK and the eIF-2 kinases was generated with the program Pileup and is illustrated in Fig. 2. In this alignment PEK is 31% identical to HRI, 31% identical to PKR, and 24% identical to GCN2. A distinguishing sequence proposed to be important for eIF-2 kinase function is LFIQME(Y/F)C(D/E), which is present in subdomain V of PEK (Fig. 2). Additionally, 11 residue positions dispersed among the catalytic domains of the eIF-2 kinases were observed to be conserved among the family members but absent in the majority of other protein kinases (36). PEK contains the same residues at 10 of these positions. The position that is the sole exception is also not conserved in the rat HRI kinase. A final feature shared among the eIF-2 kinases is an insert between subdomains IV and V (36, 45). The inserts of the different eIF-2 kinases are quite variable in sequence and in length, ranging from 35 residues in PKR to 550 residues in PfPK4 from P. falciparum. For PEK, this insert is 220 residues long and has only weak sequence identity to other members of the eIF-2 kinase family (Fig. 2). Together, the above-described sequence and structural similarities are highly suggestive that the pancreatic kinase is a new member of the eIF-2 kinase family.

View larger version (62K): [in this window] [in a new window]   FIG. 2.   The sequences of PEK are related to eIF-2 kinases. Shown is a sequence alignment of the kinase catalytic domains of rat PEK, human PKR, rat HRI, and yeast GCN2 generated by the program Pileup. Identical amino acid residues of the eIF-2 kinases are designated by black boxes, and gaps in the alignment are indicated by dashes. Kinase subdomains are highlighted by bars above the multisequence alignment.

PEK phosphorylates eIF-2 on residue serine-51. To verify the predicted size of PEK and address whether it phosphorylates eIF-2, we expressed this protein kinase in E. coli and in Sf-9 cells. PEK sequences fused to an amino-terminal polyhistidine tag were expressed in E. coli by using the T7 promoter system. The recombinant protein was visualized by immunoblotting with a polyclonal antibody that recognizes the polyhistidine sequences (Fig. 3). This recombinant PEK had a molecular weight of 140,000, which is larger than the 128,000 molecular weight predicted for the fusion protein. This protein was absent in lysates prepared from E. coli containing only the parent vector, pET28a (Fig. 3). The reason why the recombinant PEK migrated more slowly by SDS-PAGE than the predicted molecular weight does not appear to be possible self-phosphorylation, because a similar preparation of a truncated version of PEK, lacking 324 residues in its carboxy catalytic domain, also revealed a higher molecular weight, calculated by SDS-PAGE, than that predicted on the basis of the deduced coding sequences (Fig. 3). Furthermore, we also expressed PEK in E. coli fused to a more-extended amino-terminal sequence and in Sf-9 cells by using the baculovirus expression system. In both cases, using immunoblot analysis and an antiphosphothreonine antibody, we detected recombinant PEK with a larger molecular weight than that predicted on the basis of the DNA sequence. No PEK protein was detected in lysates prepared from E. coli or Sf-9 cells containing the parent vector or expressing a heterologous control protein. It is noted that expression of PEK in the Sf-9 insect cells yielded very little protein, possibly due to the toxic effect of the expressed kinase or due to its instability.

View larger version (26K): [in this window] [in a new window]   FIG. 3.   Expression of PEK in E. coli as measured by immunoblotting. PEK or PEK-785-1108 fused to amino-terminal polyhistidine tags was expressed in E. coli by using the T7 promoter system, as described in Materials and Methods. Equal amounts of total cell lysates were analyzed by SDS-PAGE, and PEK was detected by immunoblotting with a polyclonal antibody that recognizes the polyhistidine sequences. Vector, lysates prepared from E. coli containing only the parent vector, pET28a. Molecular weight markers, in kilodaltons, are on the left.

View larger version (32K): [in this window] [in a new window]   FIG. 4.   PEK in an in vitro kinase assay is autophosphorylated on both serine and threonine residues. (A) Lysates prepared from E. coli expressing PEK or vector alone were incubated with [-32P]ATP and analyzed by SDS-PAGE, followed by autoradiography. The radiolabeled PEK is indicated by an arrow on the right. Sizes of protein standards in kilodaltons are on the left. (B) Phosphoamino acid analysis of 32P-labeled PEK from the in vitro assay. Radiolabeled PEK was hydrolyzed with HCl, applied to cellulose-coated sheets, and separated by one-dimensional thin-layer electrophoresis. In parallel, eIF-2 that was phosphorylated by PEK in an in vitro assay was similarly analyzed. The 32P-labeled amino acids were detected by autoradiography. The positions of serine, threonine, and tyrosine are indicated by one-letter codes.

View larger version (52K): [in this window] [in a new window]   FIG. 5.   Immunoprecipitated PEK phosphorylates human eIF-2. PEK or an unrelated bacterial protein was expressed in Sf-9 cells and the PEK was immunoprecipitated with polyclonal antisera prepared against a synthetic peptide derived from the carboxy terminus of the kinase. Immunocomplexes prepared from lysates expressing an unrelated bacterial protein (lanes 1 to 3) or PEK (lanes 4 to 6) were incubated with [-32P]ATP and increasing amounts of human eIF-2 as described in Materials and Methods. Radiolabeled proteins were separated by electrophoresis with an SDS-polyacrylamide gel and visualized by autoradiography. Kinase assay mixtures contained 1 (lane 1 and 4), 2 (lanes 2 and 5), or 4 (lanes 3 and 6) µg of purified human eIF-2 protein. The arrowhead indicates the position of the phosphorylated eIF-2.

View larger version (26K): [in this window] [in a new window]   FIG. 6.   PEK phosphorylation of eIF-2 is dependent on residue serine-51. Full-length PEK and truncated PEK-785-1108 were partially purified and added to kinase reaction mixtures containing [-32P]ATP and a modified form of yeast eIF-2 containing wild-type serine-51 (WT) or alanine substituted for serine at this phosphorylation site (S51A). Radiolabeled proteins were analyzed by SDS-PAGE, followed by autoradiography. The reason full-length PEK is a doublet appears to be in vitro proteolysis. In other preparations, as determined by autophosphorylation or immunoblotting, a single species of PEK was detected. Phosphorylated PEK and eIF-2 are indicated to the right. The sizes of protein standards in kilodaltons are on the left.

PEK is expressed in many different rat tissues, with the greatest level in the pancreas. The expression of PEK mRNA in various rat tissues was examined by Northern blot analysis of poly(A)⁺ RNA using a cDNA probe containing the entire coding region of PEK. A major 5.2-kb transcript was detected in all tissues examined (Fig. 7). A higher-molecular-weight band of lower intensity was also detected in RNA samples from some of the tissues. This larger transcript was not reproducibly seen in our Northern blot analyses using different RNA preparations, suggesting an unspliced variant rather than different isoforms of PEK. PEK mRNA was readily detected in rat liver, kidney, spleen, brain, lung, and heart cells. Much lower levels of PEK mRNA were detected in testis and skeletal muscle cells. Given that the PEK cDNA was originally isolated from a pancreatic islet library, we carried out a separate Northern blot analysis using poly(A)⁺ RNA derived from rat pancreas cells. We found that the PEK transcript was greatly elevated in pancreas cells, with about 10 times the levels found in kidney cells. To ensure that similar amounts of RNA from each kind of tissue were used in the Northern analyses, we carried out a similar experiment using a DNA probe encoding -actin. While -actin mRNA levels were similar among most of the different tissue samples, the level was actually reduced in pancreas cells. As expected, the mRNA level of -actin was elevated in skeletal muscle cells. We also carried out similar Northern analyses using DNA probes encoding frequently used standards -tubulin and GAPDH and again detected lower mRNA levels in pancreas cells (data not shown). Based on these studies, we conclude that PEK mRNA is expressed at the highest level in the pancreas. The PEK mRNA level was 10-fold higher than those seen in other tissues, and, if the level was adjusted for differences in the amounts of RNA loaded between lanes by using the -actin standard, this relative difference would be even greater.

View larger version (44K): [in this window] [in a new window]   FIG. 7.   Northern blot analysis of tissue distributions of the PEK mRNA. A Northern blot containing 2 µg of poly(A)+ RNA purified from the different rat tissues indicated (A) and a separate blot containing ~2 µg of mRNA from rat skeletal muscle, kidney, testis, and pancreas tissue (B) were hybridized with an -32P-labeled cDNA probe encoding PEK. Following autoradiography to visualize the ~5.2-kb PEK mRNA (top panel), the membranes were rehybridized with a radiolabeled rat -actin probe, and the resulting autoradiogram is shown in the bottom panel. The PEK and -actin mRNAs are indicated by arrowheads.

View larger version (41K): [in this window] [in a new window]   FIG. 8.   PEK immunoprecipitated from mammalian tissues phosphorylates eIF-2 at serine-51. (A) PEK was immunoprecipitated from the indicated tissues by using polyclonal PEK antibody and incubated with [-32P]ATP and human eIF-2 as described in Materials and Methods. As a control, a similar immunoprecipitation assay was carried out with preimmune serum. Radiolabeled proteins were separated by SDS-PAGE and were visualized by autoradiography. The arrowhead indicates the position of phosphorylated eIF-2. All tissues used in this study are from rats, with the exception of islets, which were isolated from dogs. (B) PEK was immunoprecipitated from pancreas, islets, and Sf-9 cells. As a control, similar immunoprecipitations were carried out with lysates prepared from Sf-9 insect cells containing only the vector. Immunoprecipitates were added to kinase reaction mixtures containing [-32P]ATP and a modified form of yeast eIF-2 containing wild-type serine-51 (WT) or alanine substituted for serine at this phosphorylation site (S51A). Radiolabeled proteins were analyzed by SDS-PAGE, followed by autoradiography. Sizes of protein standards in kilodaltons on the right sides of both panels.

PEK mediates translational control in yeast and reticulocyte model system. Yeast is a useful model system for studying the in vivo role of eIF-2 kinases in translational control. Several previous studies have shown that the expression of mammalian PKR or HRI or Drosophila GCN2 kinase in yeast can complement a deletion of the endogenous yeast eIF-2 kinase encoded by GCN2 (8, 33, 38, 51). GCN2 kinase in yeast participates in the general amino acid control response, and deletion of the GCN2 gene renders the cell hypersensitive to chemical inhibitors of amino acid biosynthesis, such as 3-AT and SM (19, 48). To address whether PEK can functionally substitute for the GCN2 kinase in strain H1894 (gcn2), we expressed PEK from a high-copy-number expression vector by using a galactose-inducible promoter. Yeast cells expressing PEK were compared to cells containing plasmid-encoded yeast GCN2 or only expression vector pEMBLyex4. While all three versions of H1894 grew on galactose-inducing medium, only the GCN2- and PEK-expressing cells were growth resistant to the same medium supplemented with 3-AT or SM (Fig. 9A). Minimal growth of cells containing only the expression vector was detected in the presence of either chemical inhibitor. This experiment indicates that PEK can function as an eIF-2 kinase in the yeast model system and replace GCN2 activity in this translational control pathway.

View larger version (45K): [in this window] [in a new window]   View larger version (38K): [in this window] [in a new window]   FIG. 9.   PEK functionally substitutes for the eIF-2 kinase GCN2 in a yeast model system. (A) Strain H1894 (gcn2) was transformed with plasmid pC102-2, encoding GCN2 kinase (GCN2), p504, expressing PEK from a galactose-inducible promoter (PEK), or only vector pEBMLyex4 (Vector). Patches of transformed cells were replica printed onto agar plates containing SGal, SGal supplemented with 3-AT, or SGal supplemented with SM. GCN2-deficient strains are hypersensitive to the amino acid inhibitors 3-AT and SM. Replicated plates were grown for 4 days at 30°C and photographed. (B) PEK was similarly expressed in strains H1816 (gcn2 SUI2) and H1817 (gcn2 SUI2-S51A), which are related to H1894, and cells were replica printed onto SGal or galactose-inducing medium containing SM. Strain H1817 contains a mutant version of eIF-2 that has an alanine substituted for the phosphorylated residue serine-51. While expression of either GCN2 or PEK in H1816 facilitates growth of this strain in SGal medium containing SM, neither eIF-2 kinase mediated growth when expressed in H1817 cells.

gcn2 SUI2

gcn2 SUI2-S51A

View larger version (19K): [in this window] [in a new window]   FIG. 10.   Addition of recombinant PEK to reticulocyte lysates reduced protein synthesis. Partially purified full-length PEK or truncated PEK-785-1108 was added at the indicated concentrations to reticulocyte lysates. [35S]methionine was added to the cell-free translation system concomitant with recombinant PEK, and synthesized polypeptides were precipitated with tricarboxylic acid. The incorporation of [35S]methionine into the protein samples was measured by scintillation counting. The effect of PEK on protein synthesis in the cell-free system is expressed as a percentage of inhibition.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

In this report, we describe the isolation and characterization of a new mammalian kinase, PEK, that phosphorylates the  subunit of eIF-2 at residue serine-51. Consistent with this observed kinase activity, the sequence of the catalytic domain of PEK is similar to those of members of the eIF-2 kinase family, including that of the characteristic large insert between subdomains IV and V. PEK contains a 550-residue flanking sequence that is quite divergent from those of previously characterized eIF-2 kinases, suggesting that PEK is regulated by different physiological conditions. Northern blot analysis suggests that while PEK is expressed in many different rat tissues, the highest levels are found in the pancreas. The eIF-2 kinase assays using PEK immunoprecipitated from different rat tissues further supported the idea that PEK is predominately expressed in pancreas and pancreatic islet tissues. PEK functionally substitutes for eIF-2 kinase GCN2, demonstrating that PEK can mediate translational control in the yeast model system. Furthermore, the addition of PEK to reticulocyte lysates reduced protein synthesis. Taken together, this study strongly suggests that PEK regulates translation initiation predominately in pancreatic cells by phosphorylation of eIF-2.

Roles of eIF-2 kinases in cellular stress and proliferation. A variety of cellular stresses have been observed to elicit the phosphorylation of mammalian eIF-2, resulting in altered protein synthesis. In addition to viral infection and iron deficiency, known activators of PKR and HRI, respectively, identified stress conditions that increase eIF-2 phosphorylation include starvation for amino acids, glucose, or serum; growth factor deprivation; heat shock; ischemia; and altered calcium levels (6). Many of these conditions do not appear to be mediated by PKR and HRI. Additionally, as HRI is predominantly expressed in erythroid tissues, its role in translational control would appear to be restricted to certain cell types (5). This suggests that additional mammalian eIF-2 kinases, such as PEK, may mediate translation control in response to many of these stress conditions. Previous biochemical studies have partially characterized eIF-2 kinases, although it was unclear whether these enzymes were distinct from PKR and HRI and whether their activity was specific for serine-51 (7, 32, 35). Such new mammalian kinases may act individually or in combination with other eIF-2 kinase family members to increase the levels of eIF-2 phosphorylation. In support of a different physiological role for PEK, our preliminary studies show that PEK kinase activity in vitro was not affected by treatment with poly(I)·poly(C) or heparin, known activators of PKR.

Translational control by eIF-2 kinases in the yeast model system. In mammalian cells, eIF-2 phosphorylation in response to viral infection or heme deprivation inhibits general protein synthesis. In contrast, GCN2 phosphorylation of eIF-2 in yeast does not reduce total protein synthesis but rather stimulates the translation of GCN4 mRNA. The GCN4 protein increases the transcription of over 30 genes involved in amino acid biosynthesis, including those that contribute to growth resistance in medium containing 3-AT or SM. It is thought that the level of eIF-2 phosphorylation during amino acid starvation in yeast leads to a modest decrease in the activity of this initiation factor that does not affect primary translation (45). By comparison, translation reinitiation that occurs during ribosome scanning of the 5' leader of the GCN4 mRNA may be particularly sensitive to reductions in the levels of the eIF-2 ternary complex (19). It is this delay in reinitiation, which occurs during modest reductions in eIF-2-GTP levels, that is proposed to allow leaky scanning through the negative-acting ORFs, increasing the expression of the GCN4 coding sequence. Consistent with the idea that the levels of eIF-2 phosphorylation can be used to discriminate between general and gene-specific translation, cells expressing constitutively active mutants of GCN2 were found to suffer a slow-growth phenotype due to elevated eIF-2 phosphorylation that exceeded the levels measured in wild-type strains grown under amino acid starvation conditions (36, 44).

Regulation of eIF-2 kinases by autophosphorylation and cellular targeting. Autophosphorylation is an important step for the activation of the eIF-2 kinases (5, 34, 37, 39, 43). Romano et al. (37) presented evidence that autophosphorylation at two threonine residues in the predicted activation loops of PKR and GCN2 facilitates the function of these two eIF-2 kinases. Many protein kinases are known to be activated by the autophosphorylation of residues in this loop region (17). Such autophosphorylation may elicit a protein conformation that enhances the binding of ATP or peptide substrates or that facilitates the phosphoryl transfer reaction itself (17, 37). Interestingly, the autophosphorylated residues in subdomain VIII of PKR and GCN2 align with Thr-974 and Thr-979 of PEK. Given the observed PEK autophosphorylation at threonine residue(s) (Fig. 4B), it is inviting to speculate that a similar mode of PEK activation occurs by phosphorylation of these residues in its subdomain VIII.

New eIF-2 kinase predominately expressed in pancreas. We have identified a new mammalian eIF-2 kinase that differs from PKR and HRI in tissue distribution. While our Northern analyses suggest that PEK is widely distributed among rat tissues, it is most highly expressed in the pancreas (Fig. 7 and 8). This was confirmed by our kinase assays that showed PEK activity in pancreas and islet cells (Fig. 8). The pancreas has both exocrine and endocrine functions that require regulated protein synthesis and secretion. The majority of the pancreas consists of exocrine acinar cells, which produce and secrete digestive enzymes. Scattered among the vast majority of exocrine tissue are the islets of Langerhans. Whereas these pancreatic islets occupy less than 5% of the total pancreatic mass, they play a pivotal role in regulating glucose homeostasis in mammals by secreting different hormones including insulin, glucagon, somatostatin, and pancreatic polypeptide (3). The synthesis and secretion of the hormones are coordinated and are regulated by changes in metabolic and nutritional conditions such as hyperglycemia or hypoglycemia. 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 (13, 31). A number of other membrane and secretory proteins in the pancreas are also believed to be regulated at the translational level (14-16). Our identification and characterization of PEK may facilitate future studies on translational control of proteins secreted from pancreatic tissues.