Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control.
In response to various environmental stresses, eukaryotic cells down-regulate protein synthesis by phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). In mammals, the phosphorylation was shown to be carried out by eIF-2alpha kinases PKR and HRI. We report the identification and characterization of a cDNA from rat pancreatic islet cells that encodes a new related kinase, which we term pancreatic eIF-2alpha kinase, or PEK. In addition to a catalytic domain with sequence and structural features conserved among eIF-2alpha kinases, PEK contains a distinctive amino-terminal region 550 residues in length. Using recombinant PEK produced in Escherichia coli or Sf-9 insect cells, we demonstrate that PEK is autophosphorylated on both serine and threonine residues and that the recombinant enzyme can specifically phosphorylate eIF-2alpha on serine-51. Northern blot analyses indicate that PEK mRNA is expressed in all tissues examined, with highest levels in pancreas cells. Consistent with our mRNA assays, PEK activity was predominantly detected in pancreas and pancreatic islet cells. The regulatory role of PEK in protein synthesis was demonstrated both in vitro and in vivo. The addition of recombinant PEK to reticulocyte lysates caused a dose-dependent inhibition of translation. In the Saccharomyces model system, PEK functionally substituted for the endogenous yeast eIF-2alpha kinase, GCN2, by a process requiring the serine-51 phosphorylation site in eIF-2alpha. We also identified PEK homologs from both Caenorhabditis elegans and the puffer fish Fugu rubripes, suggesting that this eIF-2alpha kinase plays an important role in translational control from nematodes to mammals.
Shi
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Diabetes Research, Endocrine Division, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA. Shi_Yuguang@Lilly.com
Vattem
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Sood
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An
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Liang
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Stramm
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Wek
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D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D007515:Islets of Langerhans / Q000201:enzymology*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D014176:Protein Biosynthesis / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics; D012156:Reticulocytes / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D019892:eIF-2 Kinase / Q000737:chemistry / Q000235:genetics*
D010750:Phosphoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D010768:Phosphoserine; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase