Identification and Characterization of Pancreatic Eukaryotic Initiation Factor 2 α-Subunit Kinase, PEK, Involved in Translational Control

Author:

Shi Yuguang1,Vattem Krishna M.2,Sood Ruchira2,An Jie1,Liang Jingdong1,Stramm Lawrence1,Wek Ronald C.2

Affiliation:

1. Diabetes Research, Endocrine Division, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285 1 and

2. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 462022

Abstract

ABSTRACT In response to various environmental stresses, eukaryotic cells down-regulate protein synthesis by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF-2α). In mammals, the phosphorylation was shown to be carried out by eIF-2α 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-2α kinase, or PEK. In addition to a catalytic domain with sequence and structural features conserved among eIF-2α 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-2α 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-2α kinase, GCN2, by a process requiring the serine-51 phosphorylation site in eIF-2α. We also identified PEK homologs from both Caenorhabditis elegans and the puffer fish Fugu rubripes , suggesting that this eIF-2α kinase plays an important role in translational control from nematodes to mammals.

Publisher

American Society for Microbiology

Subject

Cell Biology,Molecular Biology

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