title: Functional analyses of phosphorylation events in human Argonaute 2.
abstract: Argonaute 2 (Ago2) protein is a central effector of RNA interference (RNAi) pathways and regulates mammalian genes on a global level. The mechanisms of Ago2-mediated silencing are well understood, but less is known about its regulation. Recent reports indicate that phosphorylation significantly affects Ago2 activity. Here, we investigated the effect of mutating all known phospho-residues within Ago2 on its localization and activity. Ago2 associates with two different cytoplasmic RNA granules known as processing bodies (P-bodies) and stress granules, but the nature of this phenomenon is controversial. We report that replacing serine with a phospho-mimetic aspartic acid at position 798 completely abrogates association of Ago2 with P-bodies and stress granules. The effect of this mutation on its activity in gene silencing was modest, which was surprising because association of Ago2 with cytoplasmic RNA granules is thought to be a consequence of its role in RNAi. As such, our data indicate that targeting of Ago2 to P-bodies and stress granules is separable from its role in RNAi and likely requires dynamic phosphorylation of serine 798.
authors.affiliation: Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Joaquin
authors.initials: J
authors.lastname: Lopez-Orozco
authors.affiliation: Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Justin M
authors.initials: JM
authors.lastname: Pare
authors.affiliation: Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Andrea L
authors.initials: AL
authors.lastname: Holme
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Steven G
authors.initials: SG
authors.lastname: Chaulk
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Richard P
authors.initials: RP
authors.lastname: Fahlman
authors.affiliation: Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Tom C
authors.initials: TC
authors.lastname: Hobman
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D060565:Argonaute Proteins / Q000235:genetics / Q000378:metabolism*; D002268:Carboxypeptidases / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D043244:Ribonuclease III / Q000378:metabolism
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D018841:HSP90 Heat-Shock Proteins; D035683:MicroRNAs; C408087:DICER1 protein, human; D043244:Ribonuclease III; D002268:Carboxypeptidases; C436327:SCPEP1 protein, human; D053487:DEAD-box RNA Helicases
keywords: Argonaute; P bodies; RNAi; phosphorylation; stress granules