Phosphorylation and regulation of antidepressant-sensitive serotonin transporters
Phosphorylation and regulation of antidepressant-sensitive serotonin transporters
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DOI：
10.1074/jbc.273.4.2458
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发表时间：
1998-01-23
影响因子：
4.8
通讯作者：
Blakely, RD
中科院分区：
文献类型：
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作者：
Ramamoorthy, S;Giovanetti, E;Blakely, RD
抗抑郁药敏感的5-羟色胺（5-hydroxytryptamine，5 HT）转运蛋白（SERT）负责细胞外5 HT的有效突触清除。以前（Qian，Y.，Galli，A.，Ramamoorthy，S.，Risso，S.，德费利斯湖J.，和Blakely，R. D.等人（1997）J. Neurosci. 17，45-47），我们证明了转染的HEK-293细胞中的蛋白激酶（PKC）连接的途径导致细胞表面人（h）SERT蛋白的内化和5 HT摄取能力的降低。在本研究中，我们报告说，PKC激活剂迅速，并以浓度依赖性的方式，提高基础水平的hSERT磷酸化5-6倍。类似地，蛋白磷酸酶（PP 1/PP 2A）抑制剂下调5 HT转运并显著提高hSERT P-32掺入，其作用与PKC激活剂的作用相加。此外，由β-佛波醇12-肉豆蔻酸酯13-乙酸酯诱导的hSERT磷酸化被PKC抑制剂星形孢菌素和双吲哚马来酰亚胺I选择性地消除，而由磷酸酶抑制剂诱导的hSERT磷酸化在可比浓度下对这些试剂不敏感。蛋白激酶A和蛋白激酶G激活剂不能急性下调5 HT摄取，但显着增强hSERT磷酸化。基础hSERT和冈田酸诱导的磷酸化对细胞内钙离子和Ca 2 +/钙调素依赖性蛋白激酶抑制剂的螯合不敏感。总之，这些结果表明hSERT是一种磷蛋白，其磷酸化状态可能受到多种激酶和磷酸酶途径的严格控制，这些途径也可能影响转运蛋白的调节运输。
Antidepressant-sensitive serotonin (5-hydroxytryptamine, 5HT) transporters (SERTs) are responsible for efficient synaptic clearance of extracellular 5HT. Previously (Qian, Y., Galli, A., Ramamoorthy, S., Risso, S., DeFelice, L. J., and Blakely, R. D. (1997) J. Neurosci. 17, 45-47), we demonstrated that protein kinase (PKC)-linked pathways in transfected HEK-293 cells lead to the internalization of cell-surface human (h) SERT protein and a reduction in 5HT uptake capacity. In the present study, we report that PKC activators rapidly, and in a concentration-dependent manner, elevate the basal level of hSERT phosphorylation 5-6-fold. Similarly, protein phosphatase (PP1/PP2A) inhibitors down-regulate 5HT transport and significantly elevate hSERT P-32 incorporation, effects that are additive with those of PKC activators. Moreover, hSERT phosphorylation induced by beta-phorbol 12-myristate 13-acetate is abolished selectively by the PKC inhibitors staurosporine and bisindolymaleimide I, whereas hSERT phosphorylation induced by phosphatase inhibitors is insensitive to these agents at comparable concentrations. Protein kinase A and protein kinase G activators fail to acutely down-regulate 5HT uptake but significantly enhance hSERT phosphorylation. Basal hSERT and okadaic acid-induced phosphorylation were insensitive to chelation of intracellular calcium and Ca2+/calmodulin-dependent protein kinase inhibitors. Together these results reveal hSERT to be a phosphoprotein whose phosphorylation state is likely to be tightly controlled by multiple kinase and phosphatase pathways that may also influence the transporter's regulated trafficking.