Klaus F, Palmada M, Lindner R, Laufer J, Jeyaraj S, Lang F, Boehmer C
Physiologisches Institut der Universität Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany.
J Physiol. 2008 Mar 15;586(6):1539-47. doi: 10.1113/jphysiol.2007.146191. Epub 2008 Jan 17.
Mechanisms of regulatory cell volume increase following cell shrinkage include accumulation of organic osmolytes such as betaine, taurine, sorbitol, glycerophosphorylcholine (GPC) and myo-inositol. Myo-inositol is taken up by the sodium-myo-inositol-transporter SMIT1 (SLC5A3) expressed in a wide variety of cell types. Hypertonicity induces the transcription of the SMIT1 gene upon binding of the transcription factor tonicity enhancer binding protein (TonEBP) to tonicity responsive enhancers (TonE) in the SMIT1 promoter region. However, little is known about post-translational regulation of the carrier protein. In this study we show that SMIT1 is modulated by the serum- and glucocorticoid-inducible kinase SGK1, a protein genomically up-regulated by hypertonicity. As demonstrated by two-electrode voltage-clamp in the Xenopus oocyte expression system, SMIT1-mediated myo-inositol-induced currents are up-regulated by coexpression of wild type SGK1 and constitutively active (S422D)SGK1 but not by inactive (K127N)SGK1. The increase in SMIT1 activity is due to an elevated cell surface expression of the carrier while its kinetic properties remain unaffected. According to the decay of SMIT1 activity in the presence of brefeldin A, SGK1 stabilizes the SMIT1 protein in the plasma membrane. The SGK isoforms SGK2, SGK3 and the closely related protein kinase B (PKB) are similarly capable of activating SMIT1 activity. SMIT1-mediated currents are decreased by coexpression of the ubiquitin-ligase Nedd4-2, an effect counteracted by additional coexpression of SGK1. In conclusion, the present observations disclose SGK isoforms and protein kinase B as novel regulators of SMIT1 activity.
细胞皱缩后调节性细胞容积增加的机制包括有机渗透溶质的蓄积，如甜菜碱、牛磺酸、山梨醇、甘油磷酰胆碱（GPC）和肌醇。肌醇通过在多种细胞类型中表达的钠 - 肌醇转运体SMIT1（SLC5A3）被摄取。高渗状态通过转录因子张力增强子结合蛋白（TonEBP）与SMIT1启动子区域的张力反应增强子（TonE）结合，诱导SMIT1基因的转录。然而，关于载体蛋白的翻译后调节知之甚少。在本研究中，我们表明SMIT1受血清和糖皮质激素诱导激酶SGK1的调节，SGK1是一种在基因组水平上因高渗而上调的蛋白质。正如在非洲爪蟾卵母细胞表达系统中通过双电极电压钳所证明的，野生型SGK1和组成型活性（S422D）SGK1的共表达上调了SMIT1介导的肌醇诱导电流，但无活性的（K127N）SGK1则没有。SMIT1活性的增加是由于载体的细胞表面表达升高，而其动力学特性未受影响。根据布雷菲德菌素A存在下SMIT1活性的衰减情况，SGK1使SMIT1蛋白在质膜中稳定。SGK同工型SGK2、SGK3以及密切相关的蛋白激酶B（PKB）同样能够激活SMIT1活性。泛素连接酶Nedd4 - 2的共表达降低了SMIT1介导的电流，而SGK1的额外共表达可抵消这种作用。总之，目前的观察结果揭示了SGK同工型和蛋白激酶B是SMIT1活性的新型调节因子。
