Regulation of intestinal phosphate cotransporter NaPi IIb by ubiquitin ligase Nedd4–2 and by serum- and glucocorticoid-dependent kinase 1

Abstract

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 ^C938SNedd4–2, significantly downregulates I_P, whereas the coinjection of ^S422DSGK1 markedly stimulates I_P and even fully reverses the effect of Nedd4–2 on I_P. The effect of ^S422DSGK1 on NaPi IIb is mimicked by wild-type SGK3 but not by wild-type SGK2, constitutively active ^T308D,S473DPKB, or inactive ^K127NSGK1. Moreover, ^S422DSGK1 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.