tailieunhanh - Báo cáo khoa học: Functional characterization of Drosophila melanogaster PERK eukaryotic initiation factor 2a (eIF2a) kinase

Four distinct eukaryotic initiation factor 2a(eIF2a)kinases phosphorylate eIF2aat S51and regulateprotein synthesis in response to various environmental stresses. These are the hemin-regulated inhibitor (HRI), the interferon-inducible dsRNA-dependent kinase (PKR), the endoplasmic reticu-lum (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. | Eur. J. Biochem. 270 293-306 2003 FEBS 2003 doi Functional characterization of Drosophila melanogaster PERK eukaryotic initiation factor 2a eIF2a kinase Natalia Pomar Juan J. Berlanga Sonsoles Campuzano Greco Hernandezf Monica Elias and Cesar de Haro Centro de Biologia Molecular Severo Ochoa Consejo Superior de Investigaciones Cientificas Universidad Autonoma de Madrid Cantoblanco Madrid Spain Four distinct eukaryotic initiation factor 2a eIF2a kinases phosphorylate eIF2a 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 eIF2a 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 eIF2a 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 eIF2a kinase .