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      Activation of Na +/K +-ATPase by the Serum and Glucocorticoid-Dependent Kinase Isoforms

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          Abstract

          Background/Aim: Expression of the constitutively active form of serum and glucocorticoid-dependent kinase (<sup>S422D</sup>SGK1) in Xenopus oocytes has recently been shown to upregulate endogenous Na<sup>+</sup>/K<sup>+</sup>-ATPase activity, an effect presumably participating in the regulation of cellular K<sup>+</sup> uptake and transepithelial Na<sup>+</sup> transport. SGK1 and the two isoforms SGK2 and SGK3 are stimulated by insulin and insulin-like growth factor-1 (IGF-1), which have been shown to enhance Na<sup>+</sup>/K<sup>+</sup>-ATPase activity in a variety of cells. The present experiments have been performed to elucidate whether or not wild-type SGK1, SGK2 and SGK3 are similar to <sup>S422D</sup>SGK1 in being effective regulators of Na<sup>+</sup>/K<sup>+</sup>-ATPase. Methods: To this end, dual-electrode voltage clamp experiments were performed in Xenopus oocytes injected either with water or with mRNA of constitutively active <sup>S422D</sup>SGK1 and wild-type SGK1, SGK2 or SGK3. Na<sup>+</sup>/K<sup>+</sup>-ATPase activity was estimated from the outward-directed current created by readdition of extracellular K<sup>+</sup> in the presence of K<sup>+</sup> channel blocker Ba<sup>2+</sup> following a 10-min exposure to K<sup>+</sup>-free extracellular fluid. Results: The outward-directed current was fully abolished by incubation with 1 m M ouabain and was significantly larger in oocytes expressing <sup>S422D</sup>SGK1, SGK1, SGK2 or SGK3, as compared to those injected with water. Conclusion: The stimulating effect of SGK1 on the Xenopus oocyte Na<sup>+</sup>/K<sup>+</sup>-ATPase is mimicked by the isoforms SGK2 and SGK3. Thus, all three kinases may participate in the regulation of Na<sup>+</sup>/K<sup>+</sup>-ATPase activity by hormones such as insulin and IGF-1.

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          Most cited references 25

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          Epithelial sodium channel regulated by aldosterone-induced protein sgk.

           J. Wang,  P E Buse,  F Verrey (1999)
          Sodium homeostasis in terrestrial and freshwater vertebrates is controlled by the corticosteroid hormones, principally aldosterone, which stimulate electrogenic Na+ absorption in tight epithelia. Although aldosterone is known to increase apical membrane Na+ permeability in target cells through changes in gene transcription, the mechanistic basis of this effect remains poorly understood. The predominant early effect of aldosterone is to increase the activity of the epithelial sodium channel (ENaC), although ENaC mRNA and protein levels do not change initially. Rather, the open probability and/or number of channels in the apical membrane are greatly increased by unknown modulators. To identify hormone-stimulated gene products that modulate ENaC activity, a subtracted cDNA library was generated from A6 cells, a stable cell line of renal distal nephron origin, and the effect of candidates on ENaC activity was tested in a coexpression assay. We report here the identification of sgk (serum and glucocorticoid-regulated kinase), a member of the serine-threonine kinase family, as an aldosterone-induced regulator of ENaC activity. sgk mRNA and protein were strongly and rapidly hormone stimulated both in A6 cells and in rat kidney. Furthermore, sgk stimulated ENaC activity approximately 7-fold when they were coexpressed in Xenopus laevis oocytes. These data suggest that sgk plays a central role in aldosterone regulation of Na+ absorption and thus in the control of extracellular fluid volume, blood pressure, and sodium homeostasis.
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            Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2.

            The PtdIns(3,4,5)P3-dependent activation of protein kinase B (PKB) by 3-phosphoinositide-dependent protein kinases-1 and -2 (PDK1 and PDK2 respectively) is a key event in mediating the effects of signals that activate PtdIns 3-kinase. The catalytic domain of serum- and glucocorticoid-regulated protein kinase (SGK) is 54% identical with that of PKB and, although lacking the PtdIns(3,4, 5)P3-binding pleckstrin-homology domain, SGK retains the residues that are phosphorylated by PDK1 and PDK2, which are Thr256 and Ser422 in SGK. Here we show that PDK1 activates SGK in vitro by phosphorylating Thr256. We also show that, in response to insulin-like growth factor-1 (IGF-1) or hydrogen peroxide, transfected SGK is activated in 293 cells via a PtdIns 3-kinase-dependent pathway that involves the phosphorylation of Thr256 and Ser422. The activation of SGK by PDK1 in vitro is unaffected by PtdIns(3,4,5)P3, abolished by the mutation of Ser422 to Ala, and greatly potentiated by mutation of Ser422 to Asp (although this mutation does not activate SGK itself). Consistent with these findings, the Ser422Asp mutant of SGK is activated by phosphorylation (probably at Thr256) in unstimulated 293 cells, and activation is unaffected by inhibitors of PtdIns 3-kinase. Our results are consistent with a model in which activation of SGK by IGF-1 or hydrogen peroxide is initiated by a PtdIns(3,4, 5)P3-dependent activation of PDK2, which phosphorylates Ser422. This is followed by the PtdIns(3,4,5)P3-independent phosphorylation at Thr256 that activates SGK, and is catalysed by PDK1. Like PKB, SGK preferentially phosphorylates serine and threonine residues that lie in Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr motifs, and SGK and PKB inactivate glycogen synthase kinase-3 similarly in vitro and in co-transfection experiments. These findings raise the possibility that some physiological roles ascribed to PKB on the basis of the overexpression of constitutively active PKB mutants might be mediated by SGK.
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              Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume.

              Hepatic metabolism and gene expression are among other regulatory mechanisms controlled by the cellular hydration state, which changes rapidly in response to anisotonicity, concentrative substrate uptake, oxidative stress, and under the influence of hormones such as insulin and glucagon. Differential screening for cell volume sensitive transcripts in a human hepatoma cell line revealed a gene for a putative serine/threonine kinase, h-sgk, which has 98% sequence identity to a serum- and glucocorticoid regulated kinase, sgk, cloned from a rat mammary tumor cell line. h-sgk transcript levels were strongly altered during anisotonic and isotonic cell volume changes. Within 30 min h-sgk RNA was, independent of de novo protein synthesis, induced upon cell shrinkage and, due to a complete stop in h-sgk transcription, reduced upon cell swelling. Comparable changes of sgk transcript levels were observed in a renal epithelial cell line. h-sgk mRNA was detected in all human tissues tested, with the highest levels in pancreas, liver, and heart. The putative serine/threonine protein kinase h-sgk may provide a functional link between the cellular hydration state and metabolic control.
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                Author and article information

                Journal
                KBR
                Kidney Blood Press Res
                10.1159/issn.1420-4096
                Kidney and Blood Pressure Research
                S. Karger AG
                1420-4096
                1423-0143
                2002
                2002
                26 February 2003
                : 25
                : 6
                : 370-374
                Affiliations
                Department of Physiology, University of Tübingen, Tübingen, Germany
                Article
                68699 Kidney Blood Press Res 2002;25:370–374
                10.1159/000068699
                12590200
                © 2002 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 1, Tables: 1, References: 49, Pages: 5
                Product
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/68699
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