Dopamine transporter phosphorylation site threonine 53 regulates substrate reuptake and amphetamine-stimulated efflux.
Dopamine transporter phosphorylation site threonine 53 regulates substrate reuptake and amphetamine-stimulated efflux.
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DOI：
10.1074/jbc.m112.367706
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发表时间：
2012-08-24
期刊：
影响因子：
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通讯作者：
Vaughan RA
中科院分区：
文献类型：
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作者：
Foster JD;Yang JW;Moritz AE;Challasivakanaka S;Smith MA;Holy M;Wilebski K;Sitte HH;Vaughan RA
背景：DAT 活性受蛋白激酶调节。结果：我们通过质谱和磷酸化特异性抗体将 Thr53 鉴定为大鼠纹状体中的 DAT 磷酸化位点； Thr53 突变减少了多巴胺流入并消除了转运蛋白介导的流出。结论：DAT Thr53 的磷酸化参与转运活性。意义：这些结果鉴定了 DAT 体内 Thr53 磷酸化并阐明了相关的功能特性。在中枢神经系统中，神经元外多巴胺的水平主要由多巴胺转运蛋白（DAT）的作用控制。多种信号通路通过目前未知的机制调节转运活性、底物流出和其他 DAT 功能。 DAT 被细胞质 N 末端远端丝氨酸簇内的蛋白激酶 C 磷酸化，而最近在模型细胞中的研究揭示了大鼠 DAT 在膜近端残基 Thr53 处的脯氨酸定向磷酸化。在本报告中，我们使用质谱法和新开发的磷酸化特异性抗体来阳性鉴定啮齿动物纹状体组织和异源表达系统中 Thr53 处的 DAT 磷酸化。 Thr53 的基础磷酸化以约 50% 的化学计量发生，并且通过佛波酯和蛋白磷酸酶抑制剂强烈增加，证明通过影响 DAT 活性的信号通路对该位点进行调节。 Thr53 突变阻止磷酸化，导致多巴胺转运 Vmax 降低和安非他明刺激的底物流出明显丧失，这支持了该残基在转运动力学机制中的主要作用。
Background: DAT activity is regulated by protein kinases. Results: We identify Thr53 as a DAT phosphorylation site in rat striatum by mass spectrometry and a phospho-specific antibody; Thr53 mutation reduced dopamine influx and ablated transporter-mediated efflux. Conclusion: Phosphorylation of DAT Thr53 is involved in transport activity. Significance: These results identify Thr53 phosphorylation of DAT in vivo and elucidate associated functional properties. In the central nervous system, levels of extraneuronal dopamine are controlled primarily by the action of the dopamine transporter (DAT). Multiple signaling pathways regulate transport activity, substrate efflux, and other DAT functions through currently unknown mechanisms. DAT is phosphorylated by protein kinase C within a serine cluster at the distal end of the cytoplasmic N terminus, whereas recent work in model cells revealed proline-directed phosphorylation of rat DAT at membrane-proximal residue Thr53. In this report, we use mass spectrometry and a newly developed phospho-specific antibody to positively identify DAT phosphorylation at Thr53 in rodent striatal tissue and heterologous expression systems. Basal phosphorylation of Thr53 occurred with a stoichiometry of ∼50% and was strongly increased by phorbol esters and protein phosphatase inhibitors, demonstrating modulation of the site by signaling pathways that impact DAT activity. Mutations of Thr53 to prevent phosphorylation led to reduced dopamine transport Vmax and total apparent loss of amphetamine-stimulated substrate efflux, supporting a major role for this residue in the transport kinetic mechanism.