Medical Research Council Centre for Synaptic Plasticity, University of Bristol, Bristol BS8 1TD, United Kingdom.
Proc Natl Acad Sci U S A. 2011 Dec 6;108(49):19772-7. doi: 10.1073/pnas.1111575108. Epub 2011 Nov 16.
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.
红藻氨酸（KA）受体（KAR）的表面表达和受调控的内吞作用对神经元功能至关重要。蛋白激酶 C（PKC）可以调节 KAR 的运输，但作用部位和分子后果尚未完全阐明。小泛素样修饰物（SUMO）对 KAR 亚基 GluK2 的修饰介导激动剂诱导的内吞作用，但 KAR 激活如何导致 GluK2 SUMO 化尚不清楚。本文中，我们发现 KA 刺激可导致 PKC 对 GluK2 的快速磷酸化，且 PKC 激活可增加体外和神经元中 GluK2 的 SUMO 化。GluK2 的胞内 C 末端结构域包含两个预测的 PKC 磷酸化位点，S846 和 S868，KA 刺激均可使其磷酸化。S868 的磷酸模拟突变可增加 GluK2 的 SUMO 化，而 S868 突变为不可磷酸化的丙氨酸则可防止 KA 诱导的神经元内 SUMO 化和内吞作用。SUMO-1 的输注可显著降低表达 WT GluK2 或不可磷酸化 S846A 突变体的 HEK293 细胞中 KAR 介导的电流，但对 S868A 突变体介导的电流没有影响。这些数据表明，激动剂激活 GluK2 可促进 PKC 依赖性 S846 和 S868 的磷酸化，但只有 S868 磷酸化才是增强 GluK2 SUMO 化并促进内吞作用所必需的。因此，PKC 的直接磷酸化和 GluK2 的 SUMO 化在调节含 GluK2 的 KAR 的表面表达和功能方面密切相关。
