Ragolia L, Cherpalis B, Srinivasan M, Begum N
The Diabetes Research Laboratory, Winthrop University Hospitol, Mineola, New York 11501, USA.
J Biol Chem. 1997 Sep 19;272(38):23653-8. doi: 10.1074/jbc.272.38.23653.
In this study, we examined the potential role of serine/threonine protein phosphatase-1 (PP-1) and PP-2A in the mechanism of Na+/K+-ATPase activation by insulin in the rat skeletal muscle cell line L6. Incubation of L6 cells with insulin caused a time- and dose-dependent stimulation of ouabain-sensitive plasma membrane Na+/K+-ATPase activity. Pretreatment with okadaic acid (OA; 0.1-1 microM) or calyculin A (1 microM) blocked insulin's effect on Na+/K+-ATPase activation. Low concentrations of OA that specifically inhibit PP-2A were ineffective. Immunoprecipitation of the enzyme from 32P-labeled cells with an antibody directed against the alpha-1 subunit of the enzyme revealed a 60% decrease in 110-kDa protein phosphorylation in insulin-treated cells. The presence of calyculin A blocked insulin-mediated dephosphorylation of Na+/K+-ATPase, whereas low concentrations of OA were ineffective. To further confirm the role of PP-1, we used L6 cell lines that overexpress the glycogen/SR-associated regulatory subunit of PP-1, PP-1G. Overexpression of PP-1G resulted in a 3-fold increase in insulin-stimulated PP-1 catalytic activity. This was accompanied by a 30% increase in basal Na+/K+-ATPase activity and a >2-fold increase in insulin's effect on pump activity. Inhibition of phosphatidylinositol-3 kinase with wortmannin blocked insulin-stimulated PP-1 activation as well as the dephosphorylation and activation of Na+/K+-ATPase. We conclude that insulin regulates the activity of Na+/K+-ATPase by promoting dephosphorylation of the alpha subunit via an insulin-stimulated PP-1 and that phosphatidylinositol-3 kinase-generated signals may mediate insulin activation of PP-1 and Na+/K+-ATPase.
在本研究中，我们检测了丝氨酸/苏氨酸蛋白磷酸酶-1（PP-1）和PP-2A在胰岛素激活大鼠骨骼肌细胞系L6中Na⁺/K⁺-ATP酶机制中的潜在作用。用胰岛素孵育L6细胞会引起哇巴因敏感的质膜Na⁺/K⁺-ATP酶活性呈时间和剂量依赖性刺激。用冈田酸（OA；0.1 - 1微摩尔）或花萼海绵诱癌素A（1微摩尔）预处理可阻断胰岛素对Na⁺/K⁺-ATP酶激活的作用。特异性抑制PP-2A的低浓度OA无效。用针对该酶α-1亚基的抗体从³²P标记的细胞中免疫沉淀该酶，结果显示胰岛素处理的细胞中110-kDa蛋白磷酸化减少了60%。花萼海绵诱癌素A的存在可阻断胰岛素介导的Na⁺/K⁺-ATP酶去磷酸化，而低浓度的OA无效。为进一步证实PP-1的作用，我们使用了过表达PP-1的糖原/肌浆网相关调节亚基PP-1G的L6细胞系。PP-1G的过表达导致胰岛素刺激的PP-1催化活性增加了3倍。这伴随着基础Na⁺/K⁺-ATP酶活性增加30%以及胰岛素对泵活性的作用增加2倍以上。用渥曼青霉素抑制磷脂酰肌醇-3激酶可阻断胰岛素刺激的PP-1激活以及Na⁺/K⁺-ATP酶的去磷酸化和激活。我们得出结论，胰岛素通过促进α亚基经胰岛素刺激的PP-1去磷酸化来调节Na⁺/K⁺-ATP酶的活性，并且磷脂酰肌醇-3激酶产生的信号可能介导胰岛素对PP-1和Na⁺/K⁺-ATP酶的激活。
