Three ubiquitin conjugation sites in the amino terminus of the dopamine transporter mediate protein kinase C-dependent endocytosis of the transporter
Three ubiquitin conjugation sites in the amino terminus of the dopamine transporter mediate protein kinase C-dependent endocytosis of the transporter
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DOI：
10.1091/mbc.e06-08-0704
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发表时间：
2007-01-01
影响因子：
3.3
通讯作者：
Sorkin, Alexander
中科院分区：
文献类型：
--
作者：
Miranda, Manuel;Dionne, Kalen R.;Sorkin, Alexander
脑内多巴胺水平受质膜多巴胺转运蛋白（DAT）控制。细胞表面的数据量是由其内化和再循环的相对速率决定的。蛋白激酶C （PKC）的激活导致DAT内吞作用的加速。我们最近证明PKC的激活也会导致DAT的泛素化。为了直接解决DAT泛素化的作用，DAT中的赖氨酸残基发生突变。每个赖氨酸的突变不影响DAT的泛素化和内吞作用。相比之下，携带多个赖氨酸取代的突变体的泛素化在用佛博尔酯处理的细胞中降低到未刺激细胞中检测到的水平。总之，突变数据表明聚集在DAT氨基端的Lys19、Lys27和Lys35是主要的泛素结合位点。数据与模型是一致的，即在任何给定的时间只有一个赖氨酸在DAT与短泛素链共轭。重要的是，细胞表面生物素化、免疫荧光和下调实验表明，pkc依赖性多赖氨酸突变体的内化基本上被消除了。这些数据提供了第一个证据，证明与DAT结合的泛素片段可能作为转运蛋白与内吞机制相互作用的分子界面。
Dopamine levels in the brain are controlled by the plasma membrane dopamine transporter (DAT). The amount of DAT at the cell surface is determined by the relative rates of its internalization and recycling. Activation of protein kinase C (PKC) leads to acceleration of DAT endocytosis. We have recently demonstrated that PKC activation also results in ubiquitylation of DAT. To directly address the role of DAT ubiquitylation, lysine residues in DAT were mutated. Mutations of each lysine individually did not affect ubiquitylation and endocytosis of DAT. By contrast, ubiquitylation of mutants carrying multiple lysine substitutions was reduced in cells treated with phorbol ester to the levels detected in nonstimulated cells. Altogether, mutagenesis data suggested that Lys19, Lys27, and Lys35 clustered in the DAT aminoterminus are the major ubiquitin-conjugation sites. The data are consistent with the model whereby at any given time only one of the lysines in DAT is conjugated with a short ubiquitin chain. Importantly, cell surface biotinylation, immunofluorescence and down-regulation experiments revealed that PKC-dependent internalization of multilysine mutants was essentially abolished. These data provide the first evidence that the ubiquitin moieties conjugated to DAT may serve as a molecular interface of the transporter interaction with the endocytic machinery.