title: Phosphorylation of the M3/6 dual-specificity phosphatase enhances the activation of JNK by arsenite.
abstract: Specific outcomes upon activation of the c-Jun N-terminal kinase (JNK) pathway critically depend on the intensity and duration of signal transmission. Dual-specificity phosphatases (DUSPs) play a very important role in these events by modulating the extent of JNK phosphorylation and activation and thus regulating cellular responses to stress. M3/6 (DUSP8) is one of the dual-specificity protein phosphatases with distinct specificity towards JNK. It has been shown that M3/6 itself is phosphorylated by JNK upon stimulation with arsenite, but the role of this phosphorylation has not been investigated. In this study, we mapped JNK-induced phosphorylation sites on M3/6 using mass spectrometry. Phosphorylated residues Ser 515, Thr 518 and Ser 520 were identified and site-directed mutagenesis was employed to investigate their role. Upon arsenite stimulation, M3/6 mutated at these sites exhibited decreased phosphorylation compared to the wild-type protein. No difference was observed in terms of the enzyme's in vitro phosphatase activity, its substrate specificity towards JNK isoforms, its interactions with JNK and the scaffold family of JNK-interacting proteins (JIPs), its stability or its subcellular localization. Interestingly, expression of M3/6 phosphorylation mutants delayed the time-course of JNK phosphorylation and activation by arsenite. We propose that phosphorylation of the M3/6 phosphatase by JNK in response to stress stimuli results in attenuation of phosphatase activity and acceleration of JNK activation.
authors.affiliation: Institute of Molecular Oncology, Biomedical Sciences Research Center Alexander Fleming, Vari, Greece.
authors.forename: Marina
authors.initials: M
authors.lastname: Cotsiki
authors.forename: Wolf
authors.initials: W
authors.lastname: Oehrl
authors.forename: Martina
authors.initials: M
authors.lastname: Samiotaki
authors.forename: Aspasia
authors.initials: A
authors.lastname: Theodosiou
authors.forename: George
authors.initials: G
authors.lastname: Panayotou
mesh_terms: D000595:Amino Acid Sequence; D018053:Arsenites / Q000494:pharmacology*; D002460:Cell Line; D054637:Dual-Specificity Phosphatases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D013723:Teratogens / Q000494:pharmacology
chemical_list: D018053:Arsenites; D010748:Phosphopeptides; D020033:Protein Isoforms; D013723:Teratogens; D048031:JNK Mitogen-Activated Protein Kinases; C517038:DUSP8 protein, human; D054637:Dual-Specificity Phosphatases; C015001:arsenite