title: Functional characterization of Drosophila melanogaster PERK eukaryotic initiation factor 2alpha (eIF2alpha) kinase.
abstract: Four distinct eukaryotic initiation factor 2alpha (eIF2alpha) kinases phosphorylate eIF2alpha at S51 and regulate protein synthesis in response to various environmental stresses. These are the hemin-regulated inhibitor (HRI), the interferon-inducible dsRNA-dependent kinase (PKR), the endoplasmic reticulum (ER)-resident kinase (PERK) and the GCN2 protein kinase. Whereas HRI and PKR appear to be restricted to mammalian cells, GCN2 and PERK seem to be widely distributed in eukaryotes. In this study, we have characterized the second eIF2alpha kinase found in Drosophila, a PERK homologue (DPERK). Expression of DPERK is developmentally regulated. During embryogenesis, DPERK expression becomes concentrated in the endodermal cells of the gut and in the germ line precursor cells. Recombinant wild-type DPERK, but not the inactive DPERK-K671R mutant, exhibited an autokinase activity, specifically phosphorylated Drosophila eIF2alpha at S50, and functionally replaced the endogenous Saccharomyces cerevisiae GCN2. The full length protein, when expressed in 293T cells, located in the ER-enriched fraction, and its subcellular localization changed with deletion of different N-terminal fragments. Kinase activity assays with these DPERK deletion mutants suggested that DPERK localization facilitates its in vivo function. Similar to mammalian PERK, DPERK forms oligomers in vivo and DPERK activity appears to be regulated by ER stress. Furthermore, the stable complexes between wild-type DPERK and DPERK-K671R mutant were mediated through the N terminus of the proteins and exhibited an in vitro eIF2alpha kinase activity.
authors.affiliation: Centro de Biología Molecular 'Severo Ochoa', Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.
authors.forename: Natalia
authors.initials: N
authors.lastname: Pomar
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Berlanga
authors.forename: Sonsoles
authors.initials: S
authors.lastname: Campuzano
authors.forename: Greco
authors.initials: G
authors.lastname: Hernández
authors.forename: Mónica
authors.initials: M
authors.lastname: Elías
authors.forename: César
authors.initials: C
authors.lastname: de Haro
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D020869:Gene Expression Profiling; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*
chemical_list: D015852:Eukaryotic Initiation Factor-2; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D029701:Saccharomyces cerevisiae Proteins; D019892:eIF-2 Kinase; C512912:GCN2 protein, S cerevisiae