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-β-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 [α-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-2α.
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-Δ785-1108 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 rB− mB−; 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 β-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-2α 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.
Expression of PEK in TOP10 E. coli cells (Invitrogen) was carried out by using plasmid pBK-RK3, which was obtained by in vivo excision from the lambda phages from the antiphosphothreonine screen. Plasmid pBK-RK3 carries the full-length coding region for PEK along with 150 bp of 5′-untranslated sequence subcloned into the EcoRI and XhoI sites of expression vector pBK-CMV (Stratagene). Expression of PEK was driven by the lac promoter induced by IPTG. In addition to the coding region, both the 5′-untranslated sequences and part of the polylinker sequences upstream from the EcoRI site were included in the recombinant PEK gene. E. coli cells expressing PEK were collected by centrifugation, washed, and resuspended in a solution of 20 mM Tris-HCl (pH 7.9), 50 mM NaCl, and 10 mM MgCl2 and lysed with a French press. Lysates were clarified by centrifugation at 10,000 × g.
For baculoviral expression of PEK in Sf-9 cells, a 4.5-kb DNA fragment containing the entire coding region and a portion of the 5′-UTR of the PEK cDNA was subcloned from plasmid pBK-RK3 into the EcoRI and XhoI sites of the baculoviral expression vector pFastBac (Gibco-BRL). The selection of recombinant virus and the expression of PEK in Sf-9 cells were carried out according to a protocol provided by Gibco-BRL. Sf-9 cells expressing PEK were resuspended in cell lysis buffer (10 mM HEPES [pH 7.4], 1 mM EGTA, 1 mM MgCl2, 1 mM 2-aminoethylisothiouronium bromide, 1% Triton X-114, 1× Complete protease inhibitor cocktail [Boehringer Mannheim, Indianapolis, Ind.]), followed by centrifugation at 10,000 × g for 10 min to eliminate insoluble material. Human eIF-2α was similarly expressed in Sf-9 cells. The coding region of human eIF-2α was amplified by PCR with anchored primers and Marathon Ready human testis cDNAs (Clontech). Primers GCTAGAGCTCATGCCGGGTCTAAGTTGTAGATT and AGTCGAATTCAAATTGGACTCTGTTTCCCACAA contained a XhoI site or an EcoRI site to facilitate direct cloning into the respective sites in expression vector pTrcHis A (Invitrogen), generating plasmid pTrcHis-hIF2α. The human eIF-2α cDNA sequences were removed from plasmid pTrcHis-hIF2α and inserted between the BamHI and HindIII sites of the baculoviral expression vector pFastBacHTb (Gibco-BRL). The eIF-2α with six fused histidines at the N terminus was expressed in Sf-9 cells, and the recombinant protein was purified with a ProBond column (Invitrogen) containing nickel chelation resin. The column was washed, and human eIF-2α was eluted with native wash buffer containing 200 mM imidazole. Combined fractions containing the fusion protein were concentrated and desalted on a Centricon concentrator (Amicon) with 10,000-molecular-weight cutoff, washed once with kinase buffer (20 mM HEPES [pH 7.5], 50 mM NaCl, 10% [vol/vol] glycerol), and stored at −80°C. Protein concentrations were determined with bicinchoninic acid protein assay reagents from Pierce.
Yeast eIF-2α used in the in vitro kinase assays was a modified form lacking residues 200 to 304 and containing polyhistidine sequences for rapid purification (52
). Deletion of the carboxy-terminal residues of eIF-2α removed phosphorylation sites for casein kinase II (12
). Modified versions of yeast eIF-2α possessing or lacking the serine-51 phosphorylation site were expressed and purified from E. coli
as previously described (52
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-2α 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-2α was carried out by the addition of 30 μl of reaction mixture containing 1, 2, or 4 μg of purified human eIF-2α and 20 μCi of [γ-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.
The in vitro kinase assays using recombinant yeast eIF-2α substrate were carried out as described previously (52
). In a final reaction volume of 25 μl, 2 μg of recombinant PEK or PEK-Δ785-1108 was added, along with 1 μg of yeast eIF-2α and 10 μCi of [γ-32
P]ATP in a final concentration of 60 μM ATP. After incubation for 6 min at 30°C, the phosphorylated proteins were analyzed by electrophoresis in an SDS–12.5% polyacrylamide gel, followed by autoradiography. Phosphoamino acid analysis of PEK radiolabeled by in vitro autophosphorylation was carried out by first transferring 32
P-labeled PEK from the SDS-polyacrylamide gel to an Immobilon-P membrane (Millipore). The portion of the membrane containing PEK was excised, and the protein was hydrolyzed in 5.7 N HCl at 110°C for 60 min. Hydrolyzed samples were applied to cellulose-coated sheets (Kodak) and separated by one-dimensional thin-layer electrophoresis as described previously (53
). Radiolabeled amino acids were detected by autoradiography.
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 β-actin, α-tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were radiolabeled with [α-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-2α 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 Sac
I DNA fragment containing the PEK cDNA was removed from pBK-RK3 and inserted between the Sac
I and Sal
I 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 Δgcn2
), H1816 (MATa ura3-52 leu2-3 leu2-112 Δgcn2 Δsui2 GCN4-lacZ
p1097 [SUI2 LEU2
]), and H1817 (MATa ura3-52 leu2-3 leu2-112 Δgcn2 Δsui2 GCN4-lacZ
p1098 [SUI2S51A LEU2
). Strains H1817 and H1816 are isogenic and differ only in their SUI2
alleles, which encode eIF-2α. 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-Δ785-1008 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.