Regulation of amino acid transporter ATA2 by ubiquitin ligase Nedd4-2
Regulation of amino acid transporter ATA2 by ubiquitin ligase Nedd4-2
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DOI：
10.1074/jbc.m606577200
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发表时间：
2006-11-24
影响因子：
4.8
通讯作者：
Setou, Mitsutoshi
中科院分区：
文献类型：
--
作者：
Hatanaka, Takahiro;Hatanaka, Yasue;Setou, Mitsutoshi
我们在这里报告，泛素连接酶Nedd4 - 2调节细胞表面的氨基酸转运蛋白ATA2活性。我们首先发现蛋白酶体抑制剂MG 132增加了3T3-L1脂肪细胞和前脂肪细胞中α-（甲氨基）异丁酸（氨基酸转运系统A的模型底物）的摄取。Nedd4 - 2在非洲爪蟾卵母细胞和中国仓鼠卵巢细胞中的瞬时表达分别下调注射cRNA和转染cDNA诱导的ATA 2转运活性。Nedd4 - 2催化结构域缺陷突变体和c-Cbl对ATA2活性均无显著影响。RNA介导的Nedd4 - 2干扰增加了细胞中的ATA2活性，这与细胞表面膜上ATA2的多聚泛素化减少有关。用增强型绿色荧光蛋白（EGFP）标记的ATA 2稳定转染的脂肪细胞中Nedd4 - 2的免疫荧光分析显示Nedd4 - 2和EGFP-ATA 2共定位于质膜中，但不在核周ATA 2储存位点中，支持ATA 2泛素化的主要位点是质膜的观点。这些数据表明，质膜上的ATA2受到Nedd4 - 2的多泛素化，随后发生内吞螯合和蛋白酶体降解，并且该过程是细胞表面上ATA2功能密度的重要决定因素。
We report here that ubiquitin ligase Nedd4-2 regulates amino acid transporter ATA2 activity on the cell surface. We first found that a proteasome inhibitor MG132 increased the uptake of alpha-(methylamino) isobutyric acid, a model substrate for amino acid transport system A, in 3T3-L1 adipocytes as well as the preadipocytes. Transient expression of Nedd4-2 in Xenopus oocytes and Chinese hamster ovary cells down-regulated the ATA2 transport activity induced by injected cRNA and transfected cDNA, respectively. Neither the Nedd4-2 mutant with defective catalytic domain nor c-Cbl affected the ATA2 activity significantly. RNA-mediated interference of Nedd4-2 increased the ATA2 activity in the cells, and this was associated with decreased polyubiquitination of ATA2 on the cell surface membrane. Immunofluorescent analysis of Nedd4-2 in the adipocytes stably transfected with the enhanced green fluorescent protein (EGFP)-tagged ATA2 showed the co-localization of Nedd4-2 and EGFP-ATA2 in the plasma membrane but not in the perinuclear ATA2 storage site, supporting the idea that the primary site for the ubiquitination of ATA2 is the plasma membrane. These data suggest that ATA2 on the plasma membrane is subject to polyubiquitination by Nedd4-2 with consequent endocytotic sequestration and proteasomal degradation and that this process is an important determinant of the density of ATA2 functioning on the cell surface.