title: Agonist-induced PKC phosphorylation regulates GluK2 SUMOylation and kainate receptor endocytosis.
abstract: The surface expression and regulated endocytosis of kainate (KA) receptors (KARs) plays a critical role in neuronal function. PKC can modulate KAR trafficking, but the sites of action and molecular consequences have not been fully characterized. Small ubiquitin-like modifier (SUMO) modification of the KAR subunit GluK2 mediates agonist-evoked internalization, but how KAR activation leads to GluK2 SUMOylation is unclear. Here we show that KA stimulation causes rapid phosphorylation of GluK2 by PKC, and that PKC activation increases GluK2 SUMOylation both in vitro and in neurons. The intracellular C-terminal domain of GluK2 contains two predicted PKC phosphorylation sites, S846 and S868, both of which are phosphorylated in response to KA. Phosphomimetic mutagenesis of S868 increased GluK2 SUMOylation, and mutation of S868 to a nonphosphorylatable alanine prevented KA-induced SUMOylation and endocytosis in neurons. Infusion of SUMO-1 dramatically reduced KAR-mediated currents in HEK293 cells expressing WT GluK2 or nonphosphorylatable S846A mutant, but had no effect on currents mediated by the S868A mutant. These data demonstrate that agonist activation of GluK2 promotes PKC-dependent phosphorylation of S846 and S868, but that only S868 phosphorylation is required to enhance GluK2 SUMOylation and promote endocytosis. Thus, direct phosphorylation by PKC and GluK2 SUMOylation are intimately linked in regulating the surface expression and function of GluK2-containing KARs.
authors.affiliation: Medical Research Council Centre for Synaptic Plasticity, University of Bristol, Bristol BS8 1TD, United Kingdom.
authors.forename: Filip A
authors.initials: FA
authors.lastname: Konopacki
authors.forename: Nadia
authors.initials: N
authors.lastname: Jaafari
authors.forename: Dan L
authors.initials: DL
authors.lastname: Rocca
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Wilkinson
authors.forename: Sophie
authors.initials: S
authors.lastname: Chamberlain
authors.forename: Philip
authors.initials: P
authors.lastname: Rubin
authors.forename: Sriharsha
authors.initials: S
authors.lastname: Kantamneni
authors.forename: Jack R
authors.initials: JR
authors.lastname: Mellor
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Henley
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004705:Endocytosis; D057809:HEK293 Cells; D006801:Humans; D007608:Kainic Acid / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D018092:Kainic Acid Receptors / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D025842:SUMO-1 Protein / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D058207:Sumoylation / Q000187:drug effects; D000097806:GluK2 Kainate Receptor
chemical_list: D000409:Alanine; D007608:Kainic Acid; D008164:Luminescent Proteins; D011493:Protein Kinase C; D018092:Kainic Acid Receptors; D025842:SUMO-1 Protein; D012694:Serine; D000097806:GluK2 Kainate Receptor
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