Ubiquitin Modification of Serum and Glucocorticoid-induced Protein Kinase-1 (SGK-1)

Abstract

The serum and glucocorticoid-induced protein kinase gene (sgk-1) encodes a multifunctional kinase that can be phosphorylated and activated through a phosphatidylinositol 3-kinase-dependent signaling pathway. In many cell types, endogenous SGK-1 steady-state protein levels are very low but can be acutely up-regulated after glucocorticoid receptor-mediated transcriptional activation; in breast epithelial and cancer cell lines, this up-regulation is associated with promotion of cell survival. We and others have noted that ectopically introduced full-length SGK-1 is poorly expressed, although SGK-1 lacking the first 60 amino acids (Δ60SGK-1) is expressed at much higher-fold protein levels than wild-type SGK-1 in both human embryonic kidney 293T and MCF10A mammary epithelial cells. In this report, we demonstrate for the first time that the low steady-state expression level of SGK-1 is due to polyubiquitination and subsequent degradation by the 26S proteasome. Deletion of the amino-terminal 60 amino acids of SGK-1 results in a mutant SGK-1 protein that is neither efficiently polyubiquitinated nor degraded by the 26S proteasome, accounting for the higher steady-state levels of the truncated protein. We also demonstrate that a subset of SGK-1 localizes to the plasma membrane and that the polyubiquitin-modified SGK-1 localizes to a membrane-associated fraction of the cell. Taken together, these data suggest that a significant fraction of SGK-1 is membrane-associated and ubiquitinated. These findings are consistent with the recently described role of SGK-1 in phosphorylating the membrane-associated protein Nedd4-2 and the integral membrane Na+/H+ exchanger isoform 3 (NHE3) and suggest a novel mechanism of regulation of SGK-1. The serum and glucocorticoid-induced protein kinase gene (sgk-1) encodes a multifunctional kinase that can be phosphorylated and activated through a phosphatidylinositol 3-kinase-dependent signaling pathway. In many cell types, endogenous SGK-1 steady-state protein levels are very low but can be acutely up-regulated after glucocorticoid receptor-mediated transcriptional activation; in breast epithelial and cancer cell lines, this up-regulation is associated with promotion of cell survival. We and others have noted that ectopically introduced full-length SGK-1 is poorly expressed, although SGK-1 lacking the first 60 amino acids (Δ60SGK-1) is expressed at much higher-fold protein levels than wild-type SGK-1 in both human embryonic kidney 293T and MCF10A mammary epithelial cells. In this report, we demonstrate for the first time that the low steady-state expression level of SGK-1 is due to polyubiquitination and subsequent degradation by the 26S proteasome. Deletion of the amino-terminal 60 amino acids of SGK-1 results in a mutant SGK-1 protein that is neither efficiently polyubiquitinated nor degraded by the 26S proteasome, accounting for the higher steady-state levels of the truncated protein. We also demonstrate that a subset of SGK-1 localizes to the plasma membrane and that the polyubiquitin-modified SGK-1 localizes to a membrane-associated fraction of the cell. Taken together, these data suggest that a significant fraction of SGK-1 is membrane-associated and ubiquitinated. These findings are consistent with the recently described role of SGK-1 in phosphorylating the membrane-associated protein Nedd4-2 and the integral membrane Na+/H+ exchanger isoform 3 (NHE3) and suggest a novel mechanism of regulation of SGK-1. ubiquitin-protein isopeptide ligase green fluorescent protein hemagglutinin fetal calf serum N-acetyl-Leu-Leu-norleucinal N-acetyl-Leu-Leu-normethional horseradish peroxidase Glucocorticoid receptor activation in mammary epithelial cells (1Moran T.J. Gray S. Mikosz C.A. Conzen S.D. Cancer Res. 2000; 60: 867-872PubMed Google Scholar) and hepatocytes (2Evans-Storms R.B. Cidlowski J.A. Endocrinology. 2000; 141: 1854-1862Crossref PubMed Scopus (83) Google Scholar) initiates a potent antiapoptotic signaling pathway. Activation of the glucocorticoid receptor by ligand binding directly regulates the transcription of several potential downstream mediators of this survival pathway, including SGK-1 (3Webster M.K. Goya L., Ge, Y. Maiyar A.C. Firestone G.L. Mol. Cell. Biol. 1993; 13: 2031-2040Crossref PubMed Scopus (492) Google Scholar, 4Mikosz C.A. Brickley D.R. Sharkey M.S. Moran T.W. Conzen S.D. J. Biol. Chem. 2001; 276: 16649-16654Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). SGK-1 is a member of an important subfamily of protein kinases known as the “AGC” subfamily that includes protein kinase A, protein kinase B, protein kinase G, and protein kinase C isoforms. SGK-1 is 54% identical in its catalytic domain to protein kinase Bα, also known as AKT-1. Two other isoforms of sgk, sgk-2 and sgk-3 (or cytokine-independent survival kinase, cisk), have been identified, and all three products can be phosphorylated and activated following phosphatidylinositol 3-kinase pathway signaling to downstream phosphatidylinositol-dependent kinases including phosphatidylinositol-dependent kinase-1 and -2 (5Kobayashi T. Deak M. Morrice N. Cohen P. Biochem. J. 1999; 344: 189-197Crossref PubMed Scopus (333) Google Scholar). Furthermore, phosphatidylinositol-dependent kinase-1 and -2 activation of AKT, SGK-1, and SGK-3/CISK (6Xu J. Liu D. Gill G. Songyang Z. J. Cell Biol. 2001; 154: 699-705Crossref PubMed Scopus (94) Google Scholar) can promote cell survival under a variety of conditions that normally favor apoptosis (for review, see Ref. 7Kandel E.S. Hay N. Exp. Cell Res. 1999; 253: 210-229Crossref PubMed Scopus (788) Google Scholar).In addition to SGK-1 regulation via reversible phosphorylation,sgk-1 transcription is acutely up-regulated after glucocorticoid receptor activation, serum stimulation, and cell stress (8Lang F. Cohen P. Science's STKE. 2001; (http://www.stke.org/cgi/content/full/oc_sigtrans;2001/108/re17)PubMed Google Scholar). The role of transcriptional activation in modulating a serine-threonine kinase is unusual and to date has not been implicated in the regulation of either sgk-2 or sgk-3expression (5Kobayashi T. Deak M. Morrice N. Cohen P. Biochem. J. 1999; 344: 189-197Crossref PubMed Scopus (333) Google Scholar).Another increasingly recognized mechanism of regulation of signaling proteins is through posttranslational modification via covalent addition of one or more ubiquitin molecules (reviewed in Ref. 9Pickart C.M. Mol. Cell. 2001; 8: 499-504Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Covalent attachment of ubiquitin, either as a single molecule or as a polyubiquitin chain, can regulate a variety of processes including protein degradation and subcellular trafficking of the substrate protein. For example, activity of the non-receptor tyrosine kinase c-Abl has been recently shown to be down-regulated by a ubiquitin-dependent degradation pathway (10Echarri A. Pendergast A.M. Curr. Biol. 2001; 11: 1759-1765Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Similarly, protein kinase C-λ is ubiquitinated and down-regulated by the Von Hippel Lindau-Cul 2 E31ubiquitin ligase (11Okuda H. Saitoh K. Hirai S. Iwai K. Takaki Y. Baba M. Minato N. Ohno S. Shuin T. J. Biol. Chem. 2001; 276: 43611-43617Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Polyubiquitination also directly regulates the ability of TAK1 to phosphorylate MKK6, which in turn activates the c-Jun NH2-terminal kinase-p38 kinase pathway (12Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1612) Google Scholar). Finally, the PHD domain of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase-1 was recently found to act as an E3 ubiquitin ligase and to thereby mediate ubiquitination and degradation of extracellular signal-regulated kinase 1/2 (13Lu Z., Xu, S. Joazeiro C. Cobb M.H. Hunter T. Mol. Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar).In the present study we demonstrate that the serine-threonine kinase SGK-1 is modified by polyubiquitination and ultimately degraded by the 26S proteasome. The rapid degradation of SGK-1 suggests that in addition to transcriptional up-regulation and reversible phosphorylation, ubiquitin modification plays an important role in determining the availability of SGK-1 as a kinase. We also demonstrate that the amino terminus of SGK-1 contains a domain that regulates both ubiquitin/proteasome-mediated degradation and efficient association of SGK-1 with the plasma membrane. Furthermore, fractionation studies reveal that although SGK-1 is found in both membrane and cytosolic fractions, the ubiquitin-modified SGK-1 is predominantly membrane-associated. Taken together, these findings suggest that SGK-1, a serine-threonine kinase that phosphorylates Nedd4-2 (14Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Munster C. Chraibi A. Prat J.H. Horisberger J.D. Pearce D. Loffing J. Staub O. EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (574) Google Scholar, 15Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar) and the Na+/H+ exchanger 3 (NHE3) (16Yun C.C. Chen Y. Lang F. J. Biol. Chem. 2002; 277: 7676-7683Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), is negatively regulated by ubiquitin modification and proteasome degradation.DISCUSSIONAlthough it is well-established that sgk-1 mRNA is up-regulated as an immediate early response to serum and glucocorticoid stimulation, the regulation of SGK-1 kinase activity is only partly understood. It has been previously established that SGK-1 is phosphorylated and activated by a phosphatidylinositol 3-kinase-dependent pathway on conserved serine and threonine residues homologous to those found in AKT-1. However, in most cell types, steady-state levels of SGK-1 are far less abundant than AKT-1 (8Lang F. Cohen P. Science's STKE. 2001; (http://www.stke.org/cgi/content/full/oc_sigtrans;2001/108/re17)PubMed Google Scholar). In this report, we demonstrate for the first time that in addition to the transcriptional regulation of sgk-1, SGK-1 protein levels are regulated by the ubiquitin-proteasome pathway, adding another dimension to the mechanism of regulation of this unusual serine-threonine kinase. We also show that polyubiquitin-modified SGK-1 is predominantly localized to the membrane-associated fraction of the cell. These observations suggest that SGK-1 is ubiquitinated at or near the membrane rather than in the cytosol. Alternatively, deubiquitinating enzymes may preferentially act on SGK-1 in the cytosolic compartment, thereby resulting in accumulation of polyubiquitinated SGK-1 in the membrane fraction.While these studies were ongoing, SGK-1 was shown to directly phosphorylate and inactivate Nedd4-2, a known membrane-associated protein that is the E3 ligase responsible for ubiquitination and degradation of the epithelial sodium channel (ENaC) (14Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Munster C. Chraibi A. Prat J.H. Horisberger J.D. Pearce D. Loffing J. Staub O. EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (574) Google Scholar, 15Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). This raises the interesting possibility that SGK-1 may both phosphorylate and act as an E3 substrate of Nedd4-2 (or another E3 ligase), thereby achieving a tight negative feedback of SGK-1 activity. Our finding that the majority of ubiquitinated SGK-1 is associated with the plasma membrane is consistent with this proposed model (see Fig.6).Unlike SGK-3 (CISK), which contains a complete phosphatidylinositol phosphate-binding PX domain that is required to localize SGK-3 to early endosomes (24Virbasius J.V. Song X. Pomerleau D.P. Zhan Y. Zhou G.W. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12908-12913Crossref PubMed Scopus (75) Google Scholar), SGK-1 contains only the α-helix-2 of this domain and does not co-localize with early endosomes. 2C. A. Mikosz and S. D. Conzen, unpublished data. Furthermore, mutation of the α-helix-2 domain arginine residues that are predicted to be required for binding phosphatidylinositols (25Bravo J. Karathanassis D. Pacold C.M. Pacold M.E. Ellson C.D. Anderson K.E. Butler P.J. Lavenir I. Perisic O. Hawkins P.T. Stephens L. Williams R.L. Mol. Cell. 2001; 8: 829-839Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar) does not alter SGK-1 membrane localization or kinase activity. 3D. R. Brickley, C. A. Mikosz, and S. D. Conzen, unpublished data. Therefore, SGK-1 appears to use an independent mechanism, perhaps via a protein-protein interaction requiring the amino terminus, for translocation to the cell membrane.In summary, we have demonstrated that the steady-state protein levels and kinase activity of SGK-1 are down-regulated via polyubiquitin modification of the membrane-associated fraction of SGK-1. Regulation of serine-threonine kinases via ubiquitin modification underscores the potential importance of phosphorylation-independent mechanisms in the regulation of signaling molecules. In cell types or disease conditions in which phosphatidylinositol 3-kinase activity is constitutively high (e.g. with HER-2/neu amplification, Ras mutations, or PTEN deletions), the availability of downstream targets is likely to be of critical importance to the activity of these pathways. Glucocorticoid receptor activation in mammary epithelial cells (1Moran T.J. Gray S. Mikosz C.A. Conzen S.D. Cancer Res. 2000; 60: 867-872PubMed Google Scholar) and hepatocytes (2Evans-Storms R.B. Cidlowski J.A. Endocrinology. 2000; 141: 1854-1862Crossref PubMed Scopus (83) Google Scholar) initiates a potent antiapoptotic signaling pathway. Activation of the glucocorticoid receptor by ligand binding directly regulates the transcription of several potential downstream mediators of this survival pathway, including SGK-1 (3Webster M.K. Goya L., Ge, Y. Maiyar A.C. Firestone G.L. Mol. Cell. Biol. 1993; 13: 2031-2040Crossref PubMed Scopus (492) Google Scholar, 4Mikosz C.A. Brickley D.R. Sharkey M.S. Moran T.W. Conzen S.D. J. Biol. Chem. 2001; 276: 16649-16654Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). SGK-1 is a member of an important subfamily of protein kinases known as the “AGC” subfamily that includes protein kinase A, protein kinase B, protein kinase G, and protein kinase C isoforms. SGK-1 is 54% identical in its catalytic domain to protein kinase Bα, also known as AKT-1. Two other isoforms of sgk, sgk-2 and sgk-3 (or cytokine-independent survival kinase, cisk), have been identified, and all three products can be phosphorylated and activated following phosphatidylinositol 3-kinase pathway signaling to downstream phosphatidylinositol-dependent kinases including phosphatidylinositol-dependent kinase-1 and -2 (5Kobayashi T. Deak M. Morrice N. Cohen P. Biochem. J. 1999; 344: 189-197Crossref PubMed Scopus (333) Google Scholar). Furthermore, phosphatidylinositol-dependent kinase-1 and -2 activation of AKT, SGK-1, and SGK-3/CISK (6Xu J. Liu D. Gill G. Songyang Z. J. Cell Biol. 2001; 154: 699-705Crossref PubMed Scopus (94) Google Scholar) can promote cell survival under a variety of conditions that normally favor apoptosis (for review, see Ref. 7Kandel E.S. Hay N. Exp. Cell Res. 1999; 253: 210-229Crossref PubMed Scopus (788) Google Scholar). In addition to SGK-1 regulation via reversible phosphorylation,sgk-1 transcription is acutely up-regulated after glucocorticoid receptor activation, serum stimulation, and cell stress (8Lang F. Cohen P. Science's STKE. 2001; (http://www.stke.org/cgi/content/full/oc_sigtrans;2001/108/re17)PubMed Google Scholar). The role of transcriptional activation in modulating a serine-threonine kinase is unusual and to date has not been implicated in the regulation of either sgk-2 or sgk-3expression (5Kobayashi T. Deak M. Morrice N. Cohen P. Biochem. J. 1999; 344: 189-197Crossref PubMed Scopus (333) Google Scholar). Another increasingly recognized mechanism of regulation of signaling proteins is through posttranslational modification via covalent addition of one or more ubiquitin molecules (reviewed in Ref. 9Pickart C.M. Mol. Cell. 2001; 8: 499-504Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Covalent attachment of ubiquitin, either as a single molecule or as a polyubiquitin chain, can regulate a variety of processes including protein degradation and subcellular trafficking of the substrate protein. For example, activity of the non-receptor tyrosine kinase c-Abl has been recently shown to be down-regulated by a ubiquitin-dependent degradation pathway (10Echarri A. Pendergast A.M. Curr. Biol. 2001; 11: 1759-1765Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Similarly, protein kinase C-λ is ubiquitinated and down-regulated by the Von Hippel Lindau-Cul 2 E31ubiquitin ligase (11Okuda H. Saitoh K. Hirai S. Iwai K. Takaki Y. Baba M. Minato N. Ohno S. Shuin T. J. Biol. Chem. 2001; 276: 43611-43617Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Polyubiquitination also directly regulates the ability of TAK1 to phosphorylate MKK6, which in turn activates the c-Jun NH2-terminal kinase-p38 kinase pathway (12Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1612) Google Scholar). Finally, the PHD domain of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase-1 was recently found to act as an E3 ubiquitin ligase and to thereby mediate ubiquitination and degradation of extracellular signal-regulated kinase 1/2 (13Lu Z., Xu, S. Joazeiro C. Cobb M.H. Hunter T. Mol. Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). In the present study we demonstrate that the serine-threonine kinase SGK-1 is modified by polyubiquitination and ultimately degraded by the 26S proteasome. The rapid degradation of SGK-1 suggests that in addition to transcriptional up-regulation and reversible phosphorylation, ubiquitin modification plays an important role in determining the availability of SGK-1 as a kinase. We also demonstrate that the amino terminus of SGK-1 contains a domain that regulates both ubiquitin/proteasome-mediated degradation and efficient association of SGK-1 with the plasma membrane. Furthermore, fractionation studies reveal that although SGK-1 is found in both membrane and cytosolic fractions, the ubiquitin-modified SGK-1 is predominantly membrane-associated. Taken together, these findings suggest that SGK-1, a serine-threonine kinase that phosphorylates Nedd4-2 (14Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Munster C. Chraibi A. Prat J.H. Horisberger J.D. Pearce D. Loffing J. Staub O. EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (574) Google Scholar, 15Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar) and the Na+/H+ exchanger 3 (NHE3) (16Yun C.C. Chen Y. Lang F. J. Biol. Chem. 2002; 277: 7676-7683Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), is negatively regulated by ubiquitin modification and proteasome degradation. DISCUSSIONAlthough it is well-established that sgk-1 mRNA is up-regulated as an immediate early response to serum and glucocorticoid stimulation, the regulation of SGK-1 kinase activity is only partly understood. It has been previously established that SGK-1 is phosphorylated and activated by a phosphatidylinositol 3-kinase-dependent pathway on conserved serine and threonine residues homologous to those found in AKT-1. However, in most cell types, steady-state levels of SGK-1 are far less abundant than AKT-1 (8Lang F. Cohen P. Science's STKE. 2001; (http://www.stke.org/cgi/content/full/oc_sigtrans;2001/108/re17)PubMed Google Scholar). In this report, we demonstrate for the first time that in addition to the transcriptional regulation of sgk-1, SGK-1 protein levels are regulated by the ubiquitin-proteasome pathway, adding another dimension to the mechanism of regulation of this unusual serine-threonine kinase. We also show that polyubiquitin-modified SGK-1 is predominantly localized to the membrane-associated fraction of the cell. These observations suggest that SGK-1 is ubiquitinated at or near the membrane rather than in the cytosol. Alternatively, deubiquitinating enzymes may preferentially act on SGK-1 in the cytosolic compartment, thereby resulting in accumulation of polyubiquitinated SGK-1 in the membrane fraction.While these studies were ongoing, SGK-1 was shown to directly phosphorylate and inactivate Nedd4-2, a known membrane-associated protein that is the E3 ligase responsible for ubiquitination and degradation of the epithelial sodium channel (ENaC) (14Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Munster C. Chraibi A. Prat J.H. Horisberger J.D. Pearce D. Loffing J. Staub O. EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (574) Google Scholar, 15Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). This raises the interesting possibility that SGK-1 may both phosphorylate and act as an E3 substrate of Nedd4-2 (or another E3 ligase), thereby achieving a tight negative feedback of SGK-1 activity. Our finding that the majority of ubiquitinated SGK-1 is associated with the plasma membrane is consistent with this proposed model (see Fig.6).Unlike SGK-3 (CISK), which contains a complete phosphatidylinositol phosphate-binding PX domain that is required to localize SGK-3 to early endosomes (24Virbasius J.V. Song X. Pomerleau D.P. Zhan Y. Zhou G.W. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12908-12913Crossref PubMed Scopus (75) Google Scholar), SGK-1 contains only the α-helix-2 of this domain and does not co-localize with early endosomes. 2C. A. Mikosz and S. D. Conzen, unpublished data. Furthermore, mutation of the α-helix-2 domain arginine residues that are predicted to be required for binding phosphatidylinositols (25Bravo J. Karathanassis D. Pacold C.M. Pacold M.E. Ellson C.D. Anderson K.E. Butler P.J. Lavenir I. Perisic O. Hawkins P.T. Stephens L. Williams R.L. Mol. Cell. 2001; 8: 829-839Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar) does not alter SGK-1 membrane localization or kinase activity. 3D. R. Brickley, C. A. Mikosz, and S. D. Conzen, unpublished data. Therefore, SGK-1 appears to use an independent mechanism, perhaps via a protein-protein interaction requiring the amino terminus, for translocation to the cell membrane.In summary, we have demonstrated that the steady-state protein levels and kinase activity of SGK-1 are down-regulated via polyubiquitin modification of the membrane-associated fraction of SGK-1. Regulation of serine-threonine kinases via ubiquitin modification underscores the potential importance of phosphorylation-independent mechanisms in the regulation of signaling molecules. In cell types or disease conditions in which phosphatidylinositol 3-kinase activity is constitutively high (e.g. with HER-2/neu amplification, Ras mutations, or PTEN deletions), the availability of downstream targets is likely to be of critical importance to the activity of these pathways. Although it is well-established that sgk-1 mRNA is up-regulated as an immediate early response to serum and glucocorticoid stimulation, the regulation of SGK-1 kinase activity is only partly understood. It has been previously established that SGK-1 is phosphorylated and activated by a phosphatidylinositol 3-kinase-dependent pathway on conserved serine and threonine residues homologous to those found in AKT-1. However, in most cell types, steady-state levels of SGK-1 are far less abundant than AKT-1 (8Lang F. Cohen P. Science's STKE. 2001; (http://www.stke.org/cgi/content/full/oc_sigtrans;2001/108/re17)PubMed Google Scholar). In this report, we demonstrate for the first time that in addition to the transcriptional regulation of sgk-1, SGK-1 protein levels are regulated by the ubiquitin-proteasome pathway, adding another dimension to the mechanism of regulation of this unusual serine-threonine kinase. We also show that polyubiquitin-modified SGK-1 is predominantly localized to the membrane-associated fraction of the cell. These observations suggest that SGK-1 is ubiquitinated at or near the membrane rather than in the cytosol. Alternatively, deubiquitinating enzymes may preferentially act on SGK-1 in the cytosolic compartment, thereby resulting in accumulation of polyubiquitinated SGK-1 in the membrane fraction. While these studies were ongoing, SGK-1 was shown to directly phosphorylate and inactivate Nedd4-2, a known membrane-associated protein that is the E3 ligase responsible for ubiquitination and degradation of the epithelial sodium channel (ENaC) (14Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Munster C. Chraibi A. Prat J.H. Horisberger J.D. Pearce D. Loffing J. Staub O. EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (574) Google Scholar, 15Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). This raises the interesting possibility that SGK-1 may both phosphorylate and act as an E3 substrate of Nedd4-2 (or another E3 ligase), thereby achieving a tight negative feedback of SGK-1 activity. Our finding that the majority of ubiquitinated SGK-1 is associated with the plasma membrane is consistent with this proposed model (see Fig.6). Unlike SGK-3 (CISK), which contains a complete phosphatidylinositol phosphate-binding PX domain that is required to localize SGK-3 to early endosomes (24Virbasius J.V. Song X. Pomerleau D.P. Zhan Y. Zhou G.W. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12908-12913Crossref PubMed Scopus (75) Google Scholar), SGK-1 contains only the α-helix-2 of this domain and does not co-localize with early endosomes. 2C. A. Mikosz and S. D. Conzen, unpublished data. Furthermore, mutation of the α-helix-2 domain arginine residues that are predicted to be required for binding phosphatidylinositols (25Bravo J. Karathanassis D. Pacold C.M. Pacold M.E. Ellson C.D. Anderson K.E. Butler P.J. Lavenir I. Perisic O. Hawkins P.T. Stephens L. Williams R.L. Mol. Cell. 2001; 8: 829-839Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar) does not alter SGK-1 membrane localization or kinase activity. 3D. R. Brickley, C. A. Mikosz, and S. D. Conzen, unpublished data. Therefore, SGK-1 appears to use an independent mechanism, perhaps via a protein-protein interaction requiring the amino terminus, for translocation to the cell membrane. In summary, we have demonstrated that the steady-state protein levels and kinase activity of SGK-1 are down-regulated via polyubiquitin modification of the membrane-associated fraction of SGK-1. Regulation of serine-threonine kinases via ubiquitin modification underscores the potential importance of phosphorylation-independent mechanisms in the regulation of signaling molecules. In cell types or disease conditions in which phosphatidylinositol 3-kinase activity is constitutively high (e.g. with HER-2/neu amplification, Ras mutations, or PTEN deletions), the availability of downstream targets is likely to be of critical importance to the activity of these pathways. We thank members of our laboratory and Yair Argon, Jan Burkhardt, Geoffrey Greene, Clive Palfrey, and Marsha Rosner for reagents and useful discussions. We also thank Jerrold Turner and Vytas Bindokas for assistance with confocal microscopy.