Insulin-induced stimulation of Na+,K+-ATPase activity in kidney proximal tubule cells depends on phosphorylation of the α-subunit at Tyr-10
Insulin-induced stimulation of Na+,K+-ATPase activity in kidney proximal tubule cells depends on phosphorylation of the α-subunit at Tyr-10
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DOI：
10.1091/mbc.10.9.2847
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发表时间：
1999-09-01
影响因子：
3.3
通讯作者：
Favre, H
中科院分区：
文献类型：
--
作者：
Féraille, E;Carranza, ML;Favre, H
Na+，K+-ATP酶α亚基的磷酸化在该泵的调节中起重要作用。最近的研究表明，已知增加哺乳动物近曲小管（PCT）中溶质和液体重吸收的胰岛素通过酪氨酸磷酸化过程刺激Na+，K+-ATP酶活性。因此，本研究进行了评估的作用，酪氨酸磷酸化的Na+，K+-ATP酶α亚基在胰岛素的行动。在大鼠PCT中，胰岛素和原钒酸盐（一种酪氨酸磷酸酶抑制剂）使α亚基的酪氨酸磷酸化水平增加两倍以上。它们的作用不是累加的，表明它们有共同的作用机制。胰岛素诱导的酪氨酸磷酸化被酪氨酸激酶抑制剂染料木黄酮阻止。酪氨酸磷酸化位点通过大鼠PCT中的受控胰蛋白酶解和用大鼠α-亚基转染的负鼠肾细胞中的定点突变在Tyr-10上鉴定。通过以下方式评估Tyr-10磷酸化的功能相关性：1）在表达突变型大鼠α 1-亚基（其中酪氨酸被丙氨酸或谷氨酰胺取代）的负鼠肾细胞中消除胰岛素诱导的对哇巴因敏感性Rb-86摄取的刺激;和2）胰岛素诱导的哇巴因敏感性Rb-86摄取和酪氨酸磷酸化增加的时间过程和剂量依赖性的相似性。这些结果表明，磷酸化的Na+，K+-ATP酶α-亚基在Tyr-10可能参与生理控制的钠重吸收在PCT。
Phosphorylation of the alpha-subunit of Na+,K+-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na+,K+-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na+,K+-ATPase alpha-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the alpha-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat alpha-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive Rb-86 uptake in opossum kidney cells expressing mutant rat alpha 1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive Rb-86 uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na+,K+-ATPase alpha-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.