Palmada M, Dieter M, Speil A, Böhmer C, Mack A F, Wagner H J, Klingel K, Kandolf R, Murer H, Biber J, Closs E I, Lang F
Physiologisches Institut, Universitat Tubingen, Gmelinstrasse 5, D-72076 Tubingen, Germany.
Am J Physiol Gastrointest Liver Physiol. 2004 Jul;287(1):G143-50. doi: 10.1152/ajpgi.00121.2003. Epub 2004 Mar 25.
Serum and glucocorticoid-inducible kinase 1 (SGK1) is highly expressed in enterocytes. The significance of the kinase in regulation of intestinal function has, however, remained elusive. In Xenopus laevis oocytes, SGK1 stimulates the epithelial Na(+) channel by phosphorylating the ubiquitin ligase Nedd4-2, which regulates channels by ubiquitination leading to subsequent degradation of the channel protein. Thus the present study has been performed to explore whether SGK1 regulates transport systems expressed in intestinal epithelial cells, specifically type IIb sodium-phosphate (Na(+)-P(i)) cotransporter (NaPi IIb). Immunohistochemistry in human small intestine revealed SGK1 colocalization with Nedd4-2 in villus enterocytes. For functional analysis cRNA encoding NaPi IIb, the SGK isoforms and/or the Nedd4-2 were injected into X. laevis oocytes, and transport activity was quantified as the substrate-induced current (I(P)). Exposure to 3 mM phosphate induces an I(P) in NaPi IIb-expressing oocytes. Coinjection of Nedd4-2, but not the catalytically inactive mutant (C938S)Nedd4-2, significantly downregulates I(P), whereas the coinjection of (S422D)SGK1 markedly stimulates I(P) and even fully reverses the effect of Nedd4-2 on I(P). The effect of (S422D)SGK1 on NaPi IIb is mimicked by wild-type SGK3 but not by wild-type SGK2, constitutively active (T308D,S473D)PKB, or inactive (K127N)SGK1. Moreover, (S422D)SGK1 and SGK3 phosphorylate Nedd4-2. In conclusion, SGK1 stimulates the NaPi IIb, at least in part, by phosphorylating and thereby inhibiting Nedd4-2 binding to its target. Thus the present study reveals a novel signaling pathway in the regulation of intestinal phosphate transport, which may be important for regulation of phosphate balance.
血清和糖皮质激素诱导激酶1（SGK1）在肠上皮细胞中高表达。然而，该激酶在肠道功能调节中的意义仍不清楚。在非洲爪蟾卵母细胞中，SGK1通过磷酸化泛素连接酶Nedd4-2刺激上皮钠通道，Nedd4-2通过泛素化调节通道，导致通道蛋白随后降解。因此，本研究旨在探讨SGK1是否调节肠上皮细胞中表达的转运系统，特别是IIb型钠-磷酸盐（Na(+)-P(i)）共转运体（NaPi IIb）。人小肠的免疫组织化学显示SGK1与Nedd4-2在绒毛肠上皮细胞中共定位。为了进行功能分析，将编码NaPi IIb、SGK同工型和/或Nedd4-2的cRNA注射到非洲爪蟾卵母细胞中，转运活性被量化为底物诱导电流（I(P)）。暴露于3 mM磷酸盐会在表达NaPi IIb的卵母细胞中诱导I(P)。共注射Nedd4-2，但不是催化无活性的突变体（C938S）Nedd4-2，会显著下调I(P)，而共注射（S422D）SGK1会显著刺激I(P)，甚至完全逆转Nedd4-2对I(P)的影响。野生型SGK3模拟了（S422D）SGK1对NaPi IIb的作用，但野生型SGK2、组成型活性（T308D，S473D）PKB或无活性（K127N）SGK1则没有。此外，（S422D）SGK1和SGK3使Nedd4-2磷酸化。总之，SGK1至少部分地通过磷酸化从而抑制Nedd4-2与其靶点的结合来刺激NaPi IIb。因此，本研究揭示了肠道磷酸盐转运调节中的一条新信号通路，这可能对磷酸盐平衡的调节很重要。
