title: The Drosophila DUSP puckered is phosphorylated by JNK and p38 in response to arsenite-induced oxidative stress.
abstract: Dual-Specificity Phosphatases (DUSPs) are enzymes that remove phosphate groups from both phospho-tyrosine and phospho-serine/threonine residues. A subgroup of DUSPs specifically targets Mitogen-Activated Protein Kinases (MAPKs) and has been shown to participate in the regulation of differential cellular responses to the large variety of stimuli conveyed by MAPK-pathways. In Drosophila, Puckered has been identified as a DUSP, exhibiting specificity towards the c-Jun-N-terminal kinase (JNK). Recent studies have signified its role in regulating JNK-dependent processes, including immunity, stress tolerance and longevity. Puckered expression depends on the activation of the JNK pathway whereas it's degradation is mediated by the ubiquitin-proteasome system. In this study we show that Puckered is phosphorylated by JNK and p38 in response to arsenite-induced oxidative stress and that phosphorylation affects the interaction between Puckered and these MAPKs. In silico analysis of the Puckered amino acid sequence revealed several MAPK consensus phosphorylation motifs. Expression of Puckered in the heterologous system of HEK293 cells and subsequent stimulation with arsenite resulted in reduced mobility of Puckered in SDS-PAGE. Similar results were obtained when Puckered was co-expressed with the constitutively active forms of JNK and p38. This mobility shift was abolished by lambda-phosphatase treatment or by simultaneous inhibition of JNK and p38. Analysis by mass-spectrometry identified Puckered phosphorylation in Ser413, though phosphorylation on this site was found irrespective of stimulation. Finally, phosphorylation of Puckered enhanced its interaction both with JNK and p38. Our results suggest a possible functional role of Puckered phosphorylation by MAPKs.
authors.affiliation: Institute of Molecular Oncology, Biomedical Sciences Research Center Alexander Fleming, 34 Fleming Street, Vari 166 72, Greece.
authors.forename: Katerina
authors.initials: K
authors.lastname: Karkali
authors.forename: George
authors.initials: G
authors.lastname: Panayotou
mesh_terms: D000818:Animals; D018053:Arsenites / Q000494:pharmacology; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D054637:Dual-Specificity Phosphatases / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000378:metabolism*; D018384:Oxidative Stress; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
chemical_list: D018053:Arsenites; D029721:Drosophila Proteins; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; C110854:puc protein, Drosophila; D010749:Phosphoprotein Phosphatases; D054637:Dual-Specificity Phosphatases; C015001:arsenite