Dopamine transporters are phosphorylated on N-terminal serines in rat striatum
Dopamine transporters are phosphorylated on N-terminal serines in rat striatum
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DOI：
10.1074/jbc.m200294200
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发表时间：
2002-07-12
影响因子：
4.8
通讯作者：
Vaughan, RA
中科院分区：
文献类型：
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作者：
Foster, JD;Pananusorn, B;Vaughan, RA
多巴胺转运蛋白 (DAT) 是神经元磷酸蛋白，可清除突触间隙中的多巴胺。冈田酸 (OA) 激活蛋白激酶 C (PKC) 和抑制蛋白磷酸酶会增加 DAT 的磷酸化，并导致 DAT 活性和细胞表面表达同时降低。 DAT 上存在许多潜在的磷酸化位点，但所利用的位点及其与运输调节的关系目前尚不清楚。我们使用肽图谱和表位特异性免疫沉淀来鉴定大鼠纹状体组织中发生磷酸化的 DAT 区域。磷酸氨基酸分析显示基础样品和刺激样品主要在丝氨酸上被磷酸化。用胰蛋白酶消化 (PO4)-P-32 标记的 DAT 并用 N 端或 C 端特异性抗血清进行免疫沉淀，未能分离出与包含所有内部螺旋间环的光亲和标记片段相对应的磷酸肽片段。然而，用内蛋白酶 asp-N 消化 (PO4)-P-32 标记的 DAT 并用 N 端抗血清进行免疫沉淀，从基础样品和 PKC/OA 刺激的样品中提取了两个磷酸肽片段，证明 N 端胞质尾部是磷酸化的主要位点。氨肽酶处理 PKC 和/或 OA 刺激的 DAT 基本上裂解了所有 (PO4)-P-32 标记，而没有蛋白水解延伸超过跨膜结构域 1 和 2，这进一步证明大多数磷酸化位点位于 N 末端附近，而不是在细胞内环或 C 末端结构域中。 N 末端尾部的原位蛋白水解表明大多数受刺激的磷酸化位点位于抗体表位残基 42-59 的 N 末端。纯化蛋白的二维分析产生了可能由多个位点磷酸化产生的三种胰蛋白酶磷酸肽，但这些片段不与丝氨酸 2 和 4 处磷酸化的合成胰蛋白酶肽共迁移。这些结果表明纹状体组织中 DAT 的大部分或全部基础磷酸化和刺激磷酸化发生在聚集在远端的一组六个丝氨酸中的一个或多个残基上 细胞质N末端的末端。
Dopamine transporters (DATs) are neuronal phospho-proteins that clear dopamine from the synaptic cleft. Activation of protein kinase C (PKC) and inhibition of protein phosphatases by okadaic acid (OA) increase phosphorylation of DAT and lead to concomitant reduction in DAT activity and cell surface expression. Numerous potential sites for phosphorylation are present on DAT, but the sites utilized and their relationship to transport regulation are currently unknown. We used peptide mapping and epitope-specific immunoprecipitation to identify the region of DAT that undergoes phosphorylation in rat striatal tissue. Phosphoamino acid analysis revealed that basal and stimulated samples were phosphorylated primarily on serine. Digestion of (PO4)-P-32-labeled DAT with trypsin and immunoprecipitation with N- or C-terminal specific antisera failed to isolate phosphopeptide fragments corresponding to photoaffinity-labeled fragments that contain all internal interhelical loops. However, digestion of (PO4)-P-32-labeled DAT with endoproteinase asp-N and immunoprecipitation with an N-terminal antiserum extracted two phosphopeptide fragments from both basal and PKC/OA-stimulated samples, demonstrating that the N-terminal cytoplasmic tail is a major site of phosphorylation. Aminopeptidase treatment of PKC- and/or OA-stimulated DAT cleaved essentially all (PO4)-P-32 label without proteolysis extending past transmembrane domains 1 and 2, providing further evidence that most phosphorylation sites are near the N terminus and not in intracellular loops or C-terminal domains. In situ proteolysis of the N-terminal tail indicates that the majority of stimulated phosphorylation sites are N-terminal to an antibody epitope at residues 42-59. Two-dimensional analysis of purified protein produced three tryptic phosphopeptides that may result from phosphorylation of multiple sites, but the fragments did not co-migrate with synthetic tryptic peptides phosphorylated at serines 2 and 4. These results indicate that most or all of the basal and stimulated phosphorylation of DAT in striatal tissue occurs on one or more residues in a group of six serines clustered near the distal end of the cytoplasmic N terminus.