Objective: Serum-and glucocorticoid-inducible kinase 1 (SGK1) inhibits the ubiquitin ligase neuronal cell expressed developmentally downregulated 4-2 (Nedd4-2), which retards the retrieval of the epithelial Na ϩ channel ENaC. Accordingly, SGK1 enhances ENaC abundance in the cell membrane. The significance of this effect is shown by an association of an E8CC/CT;I6CC polymorphism in the SGK1 gene with increased blood pressure. However, strong expression of SGK1 in enterocytes not expressing ENaC points to further functions of SGK1. This study was performed to test for regulation of Na ϩ -coupled glucose transporter 1 (SGLT1) by Nedd4-2, SGK1, and/or the related kinases SGK3 and PKB. Additional studies searched for an association of the SGK1 gene with BMI. Research Methods and Procedures: mRNA encoding SGLT1, wild-type Nedd4-2, inactive C938S Nedd4-2, wild type SGK1, constitutively active S422D SGK1 or inactive K127N SGK1, wild-type SGK3, and constitutively active T308DS473D PKB or inactive T308AS473A PKB were injected into Xenopus oocytes, and glucose transport was quantified from glucose-induced current (I glc ). BMI was determined in individuals with or without the E8CC/CT;I6CC polymorphism. Results: I glc was significantly decreased by coexpression of Nedd4-2 but not of C938S Nedd4-2. Coexpression of SGK1, S422D SGK1, SGK3, or T308DS473D PKB, but not of K127N SGK1 or T308AS473A PKB, enhanced I glc and reversed the effect of Nedd4-2. SGK1 and SGK3 phosphorylated Nedd4-2. Deletion of the SGK/PKB phosphorylation sites in Nedd4-2 blunted the kinase effects. BMI was significantly (p Ͻ 0.008) greater in individuals with the E8CC/CT;I6CC polymorphism than in individuals without. Discussion: Overactivity of SGK1 may lead not only to excessive ENaC activity and hypertension but also to enhanced SGLT1 activity and obesity.
The inwardly rectifying Cl(-) current in C. elegans oocytes is due to the activity of a ClC channel encoded by clh-3. Functional and structural similarities suggest that CLH-3 and mammalian ClC-2 are orthologs. CLH-3 is activated during oocyte meiotic maturation and functions in part to modulate ovulatory contractions of gap junction-coupled gonadal sheath cells.
Surface expression of the glial glutamate transporter EAAT1 is stimulated by insulin-like growth factor 1 through activation of phosphatidylinositol-3-kinase. Downstream targets include serum and glucocorticoid-sensitive kinase isoforms SGK1, SGK2 and SGK3, and protein kinase B. SGK1 regulates Nedd4-2, a ubiquitin ligase that prepares cell membrane proteins for degradation. The glial exitatory amino acid transporter (EAAT)1 is a Na Address correspondence and reprint requests to Professor Florian Lang, Physiologisches Institut, der Universität Tübingen, Gmelinstrasse 5, D-72076 Tübingen, Germany. E-mail: florian.lang@uni-tuebingen.deAbbreviations used: EAAT, excitatory amino acid transporter; ENaC, epithelial Na + channel; IGF, insulin-like growth factor; I GLU , glutamateinduced inward current; Nedd4-2, neuronal developmentally downregulated gene 4, isoform 2; PKB, protein kinase B; SGK, serum and glucocorticoid-inducible kinase.
Abstract. Mineralocorticoids stimulate Na ϩ reabsorption and K ϩ secretion in principal cells of connecting tubule and collecting duct. The involved ion channels are ENaC and ROMK1, respectively. In Xenopus oocytes, the serum and glucocorticoid-sensitive kinase SGK1 has been shown to increase ENaC activity by enhancing its abundance in the plasma membrane. With the same method, ROMK1 appeared to be insensitive to regulation by SGK1. On the other hand, ROMK1 has been shown to colocalize with NHERF2, a protein mediating targeting and trafficking of transport proteins into the cell membrane. The present study has been performed to test whether NHERF2 is required for regulation of ROMK1 by SGK1. Coexpression of neither NHERF2 nor SGK1 with ROMK1 increases ROMK1 activity. However, coexpression of NHERF2 and SGK1 together with ROMK1 markedly increases K ϩ channel activity. The combined effect of SGK1 and NHERF2 does not significantly alter the I/V relation of the channel but increases the abundance of the channel in the membrane and decreases the decay of channel activity after inhibition of vesicle insertion with brefeldin. Coexpression of NHERF2 and SGK1 does not modify cytosolic pH but leads to a slight shift of pK a of ROMK1 to more acidic values. In conclusion, NHERF2 and SGK1 interact to enhance ROMK1 activity in large part by enhancing the abundance of channel protein within the cell membrane. This interaction allows the integration of genomic regulation and activation of SGK1 and NHERF2 in the control of ROMK1 activity and renal K ϩ excretion.
The human excitatory amino acid transporter (EAAT)2 is the major glutamate carrier in the mammalian CNS. Defective expression of the transporter results in neuroexcitotoxicity that may contribute to neuronal disorders such as amyotrophic lateral sclerosis (ALS). The serum and glucocorticoid inducible kinase (SGK) 1 is expressed in the brain and is known to interact with the ubiquitin ligase Nedd4-2 to modulate membrane transporters and ion channels. The present study aimed to investigate whether SGK isoforms and the related kinase, protein kinase B (PKB), regulate EAAT2. Expression studies in Xenopus oocytes demonstrated that glutamate-induced inward current (I GLU ) was stimulated by co-expression of SGK1, SGK2, SGK3 or PKB. I GLU is virtually abolished by Nedd4-2, an effect abrogated by additional co-expression of either kinase. The kinases diminish the effect through Nedd4-2 phosphorylation without altering Nedd4-2 protein abundance. SGKs increase the transporter maximal velocity without significantly affecting substrate affinity. Tanaka et al. 1997;Vorwerk et al. 2000). Consistent with this idea, a reduction in EAAT2 function has been reported in amyotrophic lateral sclerosis (ALS) . Despite the fundamental role of EAAT2 in CNS glutamate shuttling, only a few studies have addressed the regulation of trafficking and function of this carrier, which has been shown to be regulated by protein kinase C (Casado et al. 1993;Kalandadze et al. 2002;Kalandadze et al. 2004). Regulation of the closely related glutamate transporter subtype, EAAT1, has recently been shown to involve members of the serum and glucocorticoid inducible kinase (SGK) protein kinase family (Boehmer et al. 2003a). The effect of those kinases on EAAT1 is mediated, in part, by direct phosphorylation of the SGK phosphorylation site on EAAT1, and by interference with the down-regulating effect of the ubiquitin ligase Address correspondence and reprint requests to Prof. Dr Florian Lang, Physiologisches Institut der Universität Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany. E-mail: florian.lang@uni-tuebingen.deAbbreviations used: ALS, amyotrophic lateral sclerosis; EAAT, excitatory amino acid transporters; ENaC, epithelial sodium channel; HA, hemagglutinin; NAT, neutral amino acid transporter; PBS, phosphatebuffered saline; PKB, protein kinase B; RLU, relative light unit; SGK, serum and glucocorticoid inducible kinase; wt, wild-type.
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