Differential regulation by protein kinases of activity and cell surface expression of glutamate transporters in neuron-enriched cultures
Differential regulation by protein kinases of activity and cell surface expression of glutamate transporters in neuron-enriched cultures
复制标题
DOI：
10.1016/j.neuint.2004.10.006
复制
发表时间：
2005-03-01
影响因子：
4.2
通讯作者：
Pisano, P
中科院分区：
文献类型：
--
作者：
Guillet, BA;Velly, LJ;Pisano, P
本研究描述了短期PKA、PKC或PI3K磷酸化介导的过程参与调控兴奋性氨基酸转运体EAAC 1、GLAST和GLT-1在富含神经元的培养物中内源性表达的活性和运输。蛋白激酶A （PKA）、[N-[2-（对溴肉桂胺）-乙基]-5（异喹啉磺酰胺）]（H89）或磷脂酰肌醇3-激酶（PI3K） （wortmannin）抑制剂可剂量依赖性地降低谷氨酸摄取，但在蛋白激酶C （PKC）抑制（staurosporine）或激活phorol -12-肉豆蔻酸-13-乙酸（PMA）后不发生改变。生物素化和免疫印迹结果（占对照组的%）显示，H89（71.9 +/- 4.7%）和wortmannin（63.3 +/- 20.0%）显著降低EAAC1膜表达，PMA（137.7 +/- 15.5%）显著升高EAAC1膜表达。H89和PMA显著降低了GLAST的细胞表面含量（分别为54.0 +/- 34.1%和73.3 +/- 14.3%），而wortmannin显著提高了GLAST的细胞表面含量（119.8 +/- 9.3%）。经H89治疗后，GLT-1膜水平升高2倍（179.4 +/- 19.7%）。相反，PMA和wortmannin诱导GLT-1细胞表面表达显著降低（分别为49.0 +/- 1 15.4%和40.7 +/- 33.7%）。共聚焦显微镜显示wortmannin诱导的EAAC1聚集在细胞内。这些数据表明，谷氨酸转运体的运输可以通过PKA-， PKC-和pi3k依赖的信号通路进行不同的调节，因此可以控制谷氨酸的总摄取活性。这些过程可能代表了对细胞环境变化的快速适应性反应，这对EAA的传递有重要的调节作用，并进一步防止可能的兴奋性毒性事件。(C) 2004 Elsevier Ltd.版权所有。
This study described the involvement of short-term PKA, PKC or PI3K phosphorylation-mediated processes in the regulation of activity and trafficking of the excitatory amino acid transporters EAAC 1, GLAST and GLT-1 endogenously expressed in neuron-enriched cultures. Glutamate uptake was dose-dependently decreased by inhibitors of protein kinase A (PKA), [N-[2-(p-bromocinnamylamino)-ethyl]-5(isoquinolinesulfonamide)] (H89) or phosphatidylinositol 3-kinase (PI3K) (wortmannin), but not altered after protein kinase C (PKC) inhibition (staurosporine) or activation phorbol-12-myristate-13-acetate (PMA). Biotinylation and immunoblotting results (% of controls) showed that EAAC1 membrane expression was significantly decreased by H89 (71.9 +/- 4.7%) and wortmannin (63.3 +/- 20.0%) and increased by PMA (137.7 +/- 15.5%). H89 and PMA induced a significant decrease of the cell surface fraction of GLAST (54.0 +/- 34.1% and 73.3 +/- 14.3%, respectively) whereas wortmannin significantly increased this fraction (119.8 +/- 9.3%). After treatment with H89, the GLT-1 membrane level showed a two-fold increase (179.4 +/- 19.7%). Conversely, PMA and wortmannin induced a significant decrease of the cell surface expression of GLT-1 (49.0 +/- 1 15.4% and 40.7 +/- 33.7%, respectively). Confocal microscopy revealed a wortmannin-induced clustering of EAAC1 in the intracellular compartment. These data suggest that trafficking of glutamate transporters can be differentially regulated by PKA-, PKC- and PI3K-dependent signaling pathways and could therefore control total glutamate uptake activity. These processes may represent rapid adaptive responses to changes in the cellular environment, which significantly contribute to regulation of EAA transmission and further prevent possible excitotoxic events. (C) 2004 Elsevier Ltd. All rights reserved.