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 ubiquitinlike 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 PKCdependent 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.

Exciting Times: New Advances Towards Understanding the Regulation and Roles of Kainate Receptors

“…Infusion of the catalytic domain of the deSUMOylating enzyme SENP1 increases KAR currents at MF-CA3 synapses, highlighting SUMOylaton as an endogenous regulator of the number of synaptic KARs [67]. Subsequent studies have demonstrated that SUMOylation of GluK2 is enhanced by prior PKCmediated phosphorylation of serine 868 and is required for LTD of kainate receptors at MF-CA3 synapses [15,69].…”

“…Although there is a strong base of knowledge about the activity-dependent regulation of KAR endocytosis and recycling [13,15,[67][68][69][70][71], compared to AMPARs [72] and tsVSVG cargo [73], little is known about the activitydependence of secretory pathway trafficking of KARs. A very recent study reported that secretory pathway KAR trafficking is indeed highly regulated in multiple different cellular activity contexts [48].…”

“…Phosphorylation of S846 promotes basal internalisation of GluK2-containing KARs in both HeLa cells and neurons [75], potentially via regulating the interaction between GluK2 and 4.1 proteins [71]. Furthermore, phosphorylation of both sites occurs in response to kainate stimulation of cultured neurons, and phosphorylation at S868 is required for agonist-induced endocytosis of GluK2, by promoting SUMOylation at lysine 886 [15,69,107] (see below). It should be noted, however, that PKC phosphorylation of S868 also appears to be involved in recycling of endocytosed GluK2 back to the plasma membrane [15], suggesting that whether phosphorylation of S868 promotes insertion or removal of GluK2 from the plasma membrane is likely to be context-dependent.…”

“…Thus, the current inconsistencies may reflect a complex relationship between Netos and KARs, which can be affected differential subunit expression and the availability of cell type-specific interacting proteins. Moreover, GluK2 is subject to multiple, coordinated post-translational modifications (PTMs) including phosphorylation [75], SUMOylation [67,69], ubiquitination [104,105] and palmitoylation [71,106]…”

SUMOylation alters CRMP2 regulation of calcium influx in sensory neurons

“…The Flag antibody detected an ~67 kilodalton (kD) CRMP2 band in immunoprecipitates from cells expressing HA-tagged SUMOs of its targeted proteins 19 and is emerging as a novel regulator of neuronal function especially in control of ion channels (e.g., voltage-gated potassium channel Kv1. 5,20 Kv2.1, 21 the K + leak channel K2P1 22,23 ) and receptors (e.g., the kainate receptor subunit GluR6, 24 and the ionotropic glutamate receptor subunits GluR7a/b 16 ). We are focusing on protein-protein interactions that regulate CaV2.2-channels that are essential mediators of the neurotransmitter release pathway in nerve terminals, 25,26 including those involved in pain networks.…”