The epithelial Na+ channel (ENaC) forms the pathway for Na+ absorption across epithelia, including the kidney collecting duct, where it plays a critical role in Na+ homeostasis and blood pressure control. Na+ absorption is regulated in part by mechanisms that control the expression of ENaC at the apical cell surface. Nedd4 family members (e.g. Nedd4, Nedd4-2) bind to the channel and decrease its surface expression by catalyzing its ubiquitination and degradation. Conversely, serum and glucocorticoid-regulated kinase (SGK), a downstream mediator of aldosterone, increases the expression of ENaC at the cell surface. Here we show that SGK and human Nedd4-2 (hNedd4-2) converge in a common pathway to regulate epithelial Na+ absorption. Consistent with this model, we found that SGK bound to hNedd4-2 and hNedd4. A PY motif in SGK mediated the interaction and was required for SGK to stimulate ENaC. SGK phosphorylated hNedd4-2 (but not hNedd4), altering hNedd4-2 function; phosphorylation reduced the binding of hNedd4-2 to αENaC, and hence, the hNedd4-2-mediated inhibition of Na+ absorption. These data suggest that SGK regulates epithelial Na+ absorption in part by modulating the function of hNedd4-2. The epithelial Na+ channel (ENaC) forms the pathway for Na+ absorption across epithelia, including the kidney collecting duct, where it plays a critical role in Na+ homeostasis and blood pressure control. Na+ absorption is regulated in part by mechanisms that control the expression of ENaC at the apical cell surface. Nedd4 family members (e.g. Nedd4, Nedd4-2) bind to the channel and decrease its surface expression by catalyzing its ubiquitination and degradation. Conversely, serum and glucocorticoid-regulated kinase (SGK), a downstream mediator of aldosterone, increases the expression of ENaC at the cell surface. Here we show that SGK and human Nedd4-2 (hNedd4-2) converge in a common pathway to regulate epithelial Na+ absorption. Consistent with this model, we found that SGK bound to hNedd4-2 and hNedd4. A PY motif in SGK mediated the interaction and was required for SGK to stimulate ENaC. SGK phosphorylated hNedd4-2 (but not hNedd4), altering hNedd4-2 function; phosphorylation reduced the binding of hNedd4-2 to αENaC, and hence, the hNedd4-2-mediated inhibition of Na+ absorption. These data suggest that SGK regulates epithelial Na+ absorption in part by modulating the function of hNedd4-2. epithelial Na+ channel human Nedd4 human Nedd4-2 serum and glucocorticoid-regulated kinase Fischer rat thyroid green fluorescent protein 4-morpholinepropanesulfonic acid The epithelial Na+ channel (ENaC)1 forms the pathway for Na+ absorption across a variety of epithelia, including the kidney collecting duct, airway, and distal colon (reviewed in Refs. 1Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1025) Google Scholarand 2Stokes J.B. Kidney Int. 1999; 56: 2318-2333Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Three homologous subunits (α-, β-, and γENaC) form the channel complex (3Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1739) Google Scholar, 4McDonald F.J. Price M.P. Snyder P.M. Welsh M.J. Am. J. Physiol. 1995; 268: C1157-C1163Crossref PubMed Google Scholar). Dominant gain of function mutations in ENaC cause Liddle's syndrome, an inherited form of hypertension (5Lifton R.P. Science. 1996; 272: 676-680Crossref PubMed Scopus (560) Google Scholar). Conversely, loss of function mutations cause salt wasting and hypotension (pseudohypoaldosteronism type I) (5Lifton R.P. Science. 1996; 272: 676-680Crossref PubMed Scopus (560) Google Scholar). Thus, the regulation of ENaC is critical for the maintenance of Na+ homeostasis and for blood pressure control. In the kidney collecting duct, Na+ absorption must vary over a wide range in response to conditions of Na+depletion or Na+ excess. This occurs in large part by mechanisms that modulate the expression of ENaC at the apical cell surface (reviewed in Ref. 6Snyder P.M. Endocr. Rev. 2001; (in press)Google Scholar). For example, the Nedd4 family of ubiquitin protein-ligases (including Nedd4 and Nedd4-2) reduce ENaC surface expression (7Goulet C.C. Volk K.A. Adams C.M. Prince L.S. Stokes J.B. Snyder P.M. J. Biol. Chem. 1998; 273: 30012-30017Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 8Abriel H. Loffing J. Rebhun J.F. Pratt J.H. Schild L. Horisberger J.D. Rotin D. Staub O. J. Clin. Invest. 1999; 103: 667-673Crossref PubMed Scopus (318) Google Scholar, 9Kamynina E. Debonneville C. Bens M. Vandewalle A. Staub O. FASEB J. 2001; 15: 204-214Crossref PubMed Scopus (247) Google Scholar). They contain multiple WW domains that bind to PY motifs in the C termini of α-, β-, and γENaC (10Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (721) Google Scholar, 11Farr T.J. Coddington-Lawson S.J. Snyder P.M. McDonald F.J. Biochem. J. 2000; 345: 503-509Crossref PubMed Google Scholar, 12Snyder P.M. Olson D.R. McDonald F.J. Bucher D.B. J. Biol. Chem. 2001; 276: 28321-28326Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). This interaction facilitates the ubiquitination of ENaC, catalyzed by a ubiquitin ligase domain at the C terminus of Nedd4 family members (7Goulet C.C. Volk K.A. Adams C.M. Prince L.S. Stokes J.B. Snyder P.M. J. Biol. Chem. 1998; 273: 30012-30017Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar,8Abriel H. Loffing J. Rebhun J.F. Pratt J.H. Schild L. Horisberger J.D. Rotin D. Staub O. J. Clin. Invest. 1999; 103: 667-673Crossref PubMed Scopus (318) Google Scholar). Ubiquitination reduces ENaC at the cell surface by increasing the rate of channel degradation (7Goulet C.C. Volk K.A. Adams C.M. Prince L.S. Stokes J.B. Snyder P.M. J. Biol. Chem. 1998; 273: 30012-30017Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Liddle's syndrome is caused by defects in this regulatory pathway; mutations in the ENaC PY motifs disrupt their interaction with Nedd4 family members, resulting in increased expression of ENaC at the cell surface, and hence, excessive Na+ absorption (10Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (721) Google Scholar, 13Snyder P.M. Price M.P. McDonald F.J. Adams C.M. Volk K.A. Zeiher B.G. Stokes J.B. Welsh M.J. Cell. 1995; 83: 969-978Abstract Full Text PDF PubMed Scopus (390) Google Scholar, 14Schild L. Lu Y. Gautschi I. Schneeberger E. Lifton R.P. Rossier B.C. EMBO J. 1996; 15: 2381-2387Crossref PubMed Scopus (356) Google Scholar). Thus, the Nedd4 family of proteins is critically important in reducing Na+absorption. Conversely, the renin-angiotensin-aldosterone pathway increases renal Na+ absorption, in part by increasing the expression of ENaC at the cell surface (15Masilamani S. Kim G.H. Mitchell C. Wade J.B. Knepper M.A. J. Clin. Invest. 1999; 104: R19-R23Crossref PubMed Scopus (612) Google Scholar). This pathway plays a key role in responding to Na+ depletion and hypovolemia. Moreover, disruption of this pathway underlies several acquired and genetic disorders of blood pressure control, including primary aldosteronism and glucocorticoid-remediable aldosteronism (5Lifton R.P. Science. 1996; 272: 676-680Crossref PubMed Scopus (560) Google Scholar). An important downstream mediator of aldosterone is serum and glucocorticoid-regulated kinase (SGK), (16Chen S.Y. Bhargava A. Mastroberardino L. Meijer O.C. Wang J. Buse P. Firestone G.L. Verrey F. Pearce D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2514-2519Crossref PubMed Scopus (629) Google Scholar, 17Naray-Fejes-Toth A. Canessa C. Cleaveland E.S. Aldrich G. Fejes-Toth G. J. Biol. Chem. 1999; 274: 16973-16978Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). SGK transcription is induced by aldosterone over a very rapid time course (30–60 min) (16Chen S.Y. Bhargava A. Mastroberardino L. Meijer O.C. Wang J. Buse P. Firestone G.L. Verrey F. Pearce D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2514-2519Crossref PubMed Scopus (629) Google Scholar,17Naray-Fejes-Toth A. Canessa C. Cleaveland E.S. Aldrich G. Fejes-Toth G. J. Biol. Chem. 1999; 274: 16973-16978Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar), and it is post-translationally activated in response to insulin and other stimuli by phosphorylation through the phosphoinositide 3-kinase pathway (18Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Crossref PubMed Scopus (479) Google Scholar). Thus, it has been proposed that SGK integrates a variety of signals that modulate renal Na+ absorption (19Wang J. Barbry P. Maiyar A.C. Rozansky D.J. Bhargava A. Leong M. Firestone G.L. Pearce D. Am. J. Physiol. Renal Physiol. 2001; 280: F303-F313Crossref PubMed Google Scholar). SGK increases the expression of ENaC at the cell surface (20Alvarez de la Rosa D. Zhang P. Naray-Fejes-Toth A. Fejes-Toth G. Canessa C.M. J. Biol. Chem. 1999; 274: 37834-37839Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar), but little is known about the mechanisms involved. Previous work reported that SGK phosphorylates Ser/Thr residues within the sequence RXRXXS/T (18Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Crossref PubMed Scopus (479) Google Scholar, 21Kobayashi T. Deak M. Morrice N. Cohen P. Biochem. J. 1999; 344: 189-197Crossref PubMed Scopus (332) Google Scholar). However, ENaC subunits are not phosphorylated by SGK (19Wang J. Barbry P. Maiyar A.C. Rozansky D.J. Bhargava A. Leong M. Firestone G.L. Pearce D. Am. J. Physiol. Renal Physiol. 2001; 280: F303-F313Crossref PubMed Google Scholar). Thus, it seems likely that SGK phosphorylates one or more proteins involved in controlling ENaC surface expression, although such SGK substrates have not yet been identified. Two observations suggest the possibility that the Nedd4 family and SGK might converge in a common pathway to regulate ENaC surface expression. First, SGK contains a PY motif (see Fig. 1A), suggesting that it might bind directly to WW domains in Nedd4 or Nedd4-2. Second, Nedd4-2 (but not hNedd4) contains three sequences that fit the consensus for phosphorylation by SGK, suggesting it might be a SGK substrate. The goal of this work was to test the hypothesis that SGK modulates ENaC surface expression in part through the phosphorylation of Nedd4-2. hNedd4 was cloned as described previously (12Snyder P.M. Olson D.R. McDonald F.J. Bucher D.B. J. Biol. Chem. 2001; 276: 28321-28326Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). hNedd4-2 and human SGK were cloned by PCR of cDNA reverse transcribed from kidney poly(A)+ RNA (CLONTECH). Mutations were created using QuikChange Kit (Stratagene) and each cDNA was sequenced in the University of Iowa DNA Sequencing Core. Human α-, β-, and γENaC in pMT3 or pcDNA3 were cloned as described previously (4McDonald F.J. Price M.P. Snyder P.M. Welsh M.J. Am. J. Physiol. 1995; 268: C1157-C1163Crossref PubMed Google Scholar). The PY motifs of each subunit were mutated to PLP motifs (P7LP6) to disrupt binding to the hNedd4-2 WW domains. A FLAG epitope (DYKDDDDK) was introduced at the C terminus of SGK to allow immunodetection. This epitope did not alter ENaC stimulation by SGK. cDNA encoding wild-type or mutant (Y298A or K127M) SGK or GFP (negative control) was expressed in COS-7 cells by electroporation, as described previously (13Snyder P.M. Price M.P. McDonald F.J. Adams C.M. Volk K.A. Zeiher B.G. Stokes J.B. Welsh M.J. Cell. 1995; 83: 969-978Abstract Full Text PDF PubMed Scopus (390) Google Scholar). The cells were lysed and protein solubilized in TBS (150 mm NaCl, 50 mm Tris, pH 7.4) containing 1% Triton X-100 and protease inhibitors (0.4 mmphenylmethylsulfonyl fluoride, 20 μg/ml aprotonin, 20 μg/ml leupeptin, and 10 μg/ml pepstatin A). SGK was immunoprecipitated from 100 μl of lysate (1 μg/μl total protein) with anti-FLAG M2 monoclonal antibody (1:1000, Eastman Kodak Co.) and protein A beads (Pierce). hNedd4-2 and hNedd4 (20 μl) were generated and [35S]methionine-labeled by in vitrotranscription and translation (TNT kit, Promega) then incubated with immunoprecipitated SGK or SGKY298A (or GFP) for 16 h. The beads were washed three times with TBS/1% Triton X-100, separated by SDS-PAGE, and imaged by fluorography. To detect total SGK, SGKY298A, or SGKK127M, 30 μg of lysate was separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and blocked overnight with 5% dry milk in TBS containing 0.1% Triton X-100. The membrane was incubated for 2 h with anti-FLAG M2 antibody (1:1000), 1 h with horseradish peroxidase-coupled sheep anti-mouse IgG (1:50,000, Amersham Biosciences, Inc.), and imaged by chemiluminescence (ECL Plus, Amersham Biosciences, Inc.). FRT cells were grown on permeable filter supports as described (22Snyder P.M. J. Clin. Invest. 2000; 105: 45-53Crossref PubMed Scopus (135) Google Scholar). One day after seeding, cells were cotransfected with α-, β-, and γENaC (0.07 μg each) and hNedd4-2, SGK, or GFP as a negative control (0.02–0.8 μg using TFX 50 (22)). The total DNA was held constant by varying the ratio of hNedd4-2 or SGK to GFP. Expression of GFP did not alter ENaC Na+ currents. Na+ transport was measured 2–3 days after transfection in modified Ussing chambers (Warner Instrument Corporation). The apical and basolateral surfaces were bathed in 135 mm NaCl, 1.2 mm CaCl2, 1.2 mm MgCl2, 2.4 mm K2HPO4, 0.6 mmKH2PO4, 10 mm dextrose, 10 mm HEPES, pH 7.4, at 37 °C and bubbled with O2. Amiloride-sensitive short-circuit current was determined as the difference in current with and in the apical encoding hNedd4-2, or GFP (negative control) were expressed in COS-7 solubilized in TBS containing 1% Triton X-100, and μg of the were immunoprecipitated with sheep and GFP) or sheep (12Snyder P.M. Olson D.R. McDonald F.J. Bucher D.B. J. Biol. Chem. 2001; 276: 28321-28326Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). protein was in 100 20 mm pH mm mm 1 mm Na+ 1 2 protein kinase A and 10 of The were incubated with or activated SGK of for at 30 The beads were washed three times with μl of TBS containing 1% Triton X-100 and proteins by and fluorography. To detect total hNedd4-2 and hNedd4 COS-7 cells were for 1 h with and cell were immunoprecipitated as described hNedd4-2, or GFP expressed in COS-7 cells were incubated with or activated SGK (1 mm for and incubated for 16 h with 20 μl of and [35S]methionine-labeled in The beads were washed three times with TBS 1% Triton X-100, separated by SDS-PAGE, and imaged by fluorography. The PY motif is a sequence that protein through its binding to type WW domains M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). SGK contains a sequence that the PY motif consensus Fig. of the Nedd4 family contain multiple WW bind to PY motifs in ENaC. the hypothesis that their WW domains might bind to SGK. SGK a FLAG epitope at the C was expressed in COS-7 SGK protein by anti-FLAG M2 in cells SGK, but not in cells GFP To test for we incubated immunoprecipitated SGK with hNedd4-2 or hNedd4 and [35S]methionine-labeled by in hNedd4-2 and hNedd4 bound to SGK, but not to immunoprecipitated protein from cells GFP The PY motif of SGK mediated of a critical within the motif SGK binding to hNedd4-2 and hNedd4 This did not from protein the mutant and wild-type generated of SGK protein To the role of this we the SGK PY motif was required to stimulate ENaC. Expression of α-, β-, and γENaC in FRT epithelial cells generated short-circuit Na+ that were blocked by (22Snyder P.M. J. Clin. Invest. 2000; 105: 45-53Crossref PubMed Scopus (135) Google Scholar). of ENaC with SGK increased Na+ current with ENaC expressed with GFP) In SGKY298A did not stimulate ENaC suggesting that the SGK PY motif is required for SGK kinase is required for it to stimulate ENaC in In a of a in the binding the of SGK to a (18Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Crossref PubMed Scopus (479) Google Scholar). found that this SGK from ENaC expressed in FRT cells but it did not alter of SGK protein This that SGK ENaC by one or more SGK phosphorylates or residues in the of the sequence RXRXXS/T (18Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Crossref PubMed Scopus (479) Google Scholar, 21Kobayashi T. Deak M. Morrice N. Cohen P. Biochem. J. 1999; 344: 189-197Crossref PubMed Scopus (332) Google Scholar). hNedd4-2 contains three sequences that fit this consensus and but are not in hNedd4 the hypothesis that hNedd4-2 is a for SGK hNedd4-2, or GFP (negative control) were expressed in COS-7 and incubated with with or an activated form of SGK found that SGK phosphorylated hNedd4-2 but not hNedd4 a control for expression, we cells with and immunoprecipitated with the WW domains. of the were in cells with hNedd4-2 and but not in cells with GFP the hypothesis that phosphorylation hNedd4-2 ENaC was with SGK or GFP (negative control) in FRT with increasing of hNedd4-2 ENaC was expressed with hNedd4-2 Na+ current in a In hNedd4-2 reduced current to a expressed with SGK However, a kinase mutant did not decrease inhibition suggesting that phosphorylation was Thus, phosphorylation modulates hNedd4-2 its to ENaC. For hNedd4-2 and hNedd4 to Na+ their WW domains must bind to PY motifs in ENaC (7Goulet C.C. Volk K.A. Adams C.M. Prince L.S. Stokes J.B. Snyder P.M. J. Biol. Chem. 1998; 273: 30012-30017Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 8Abriel H. Loffing J. Rebhun J.F. Pratt J.H. Schild L. Horisberger J.D. Rotin D. Staub O. J. Clin. Invest. 1999; 103: 667-673Crossref PubMed Scopus (318) Google Scholar, 9Kamynina E. Debonneville C. Bens M. Vandewalle A. Staub O. FASEB J. 2001; 15: 204-214Crossref PubMed Scopus (247) Google Scholar). the SGK consensus are the WW domains of hNedd4-2, suggesting that phosphorylation might alter its binding to ENaC. To test this we immunoprecipitated hNedd4-2 or hNedd4 from COS-7 by with one of the ENaC subunits in and with to the work that the three ENaC subunits are in their binding to WW domains E. Debonneville C. Bens M. Vandewalle A. Staub O. FASEB J. 2001; 15: 204-214Crossref PubMed Scopus (247) Google Scholar, 11Farr T.J. Coddington-Lawson S.J. Snyder P.M. McDonald F.J. Biochem. J. 2000; 345: 503-509Crossref PubMed Google Scholar, 12Snyder P.M. Olson D.R. McDonald F.J. Bucher D.B. J. Biol. Chem. 2001; 276: 28321-28326Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, A. S. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). bound to hNedd4-2 and but not to immunoprecipitated from cells GFP of hNedd4-2 by SGK the binding of hNedd4-2 to In SGK caused in the binding of hNedd4 to with that SGK did not hNedd4. The data suggest that SGK might Na+ current in part by the binding of hNedd4-2 to ENaC. a be by of the ENaC PY are required for this To test this we expressed ENaC or PY motif with SGK (or GFP as negative control) in FRT cells and measured short-circuit Na+ currents. SGK increased in cells wild-type ENaC but not the PY motifs were mutated in α-, β-, and γENaC Thus, in epithelial stimulation by SGK was on the PY motifs of ENaC. The regulation of ENaC is critically important to Na+ of this regulation genetic and acquired forms of hypertension and hypotension (5Lifton R.P. Science. 1996; 272: 676-680Crossref PubMed Scopus (560) Google Scholar). Two important of ENaC are aldosterone and its downstream mediator SGK, and the Nedd4 family of ubiquitin protein modulate ENaC surface expression in a data suggest that ENaC in part through a common The data a in SGK regulates in part by modulating the inhibition of ENaC by hNedd4-2 conditions hNedd4-2 is and epithelial Na+ its WW domains bind to ENaC PY resulting in and degradation of the channel a hNedd4-2 Na+ current by reducing the expression of ENaC at the cell surface. In response to salt or in aldosterone this of Na+absorption. the transcription of SGK (16Chen S.Y. Bhargava A. Mastroberardino L. Meijer O.C. Wang J. Buse P. Firestone G.L. Verrey F. Pearce D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2514-2519Crossref PubMed Scopus (629) Google Scholar, 17Naray-Fejes-Toth A. Canessa C. Cleaveland E.S. Aldrich G. Fejes-Toth G. J. Biol. Chem. 1999; 274: 16973-16978Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar), to hNedd4-2 and phosphorylates one or more Ser/Thr consensus The binding of SGK to hNedd4-2 to be of the SGK PY motif it from ENaC. of hNedd4-2 reduces its binding to ENaC, resulting in increased ENaC at the cell surface, and hence, increased Na+ absorption. might phosphorylation alter the binding of hNedd4-2 to we not yet have to a it is to that the phosphorylation are in WW domains. For example, are WW domains 1 and and is WW domains 2 and phosphorylation a in hNedd4-2, altering the of the WW domains to bind ENaC. contains at three hNedd4-2 in the of SGK phosphorylation The form we known as to Nedd4-2 and has three phosphorylation A a acid WW domains 1 and one of the SGK A the and SGK as as WW domain each form to be expressed in collecting epithelia, P. and three ENaC and Ref. E. C. Staub O. Am. J. Physiol. Renal Physiol. 2001; PubMed Google Scholar). Thus, it seems that the forms be and regulated by SGK. the in three forms is phosphorylated is the function of the interaction SGK and A is that to their to through such as protein kinase A proteins D. J.D. J. Sci. 2001; PubMed Google Scholar). be phosphorylation of acid by and protein kinase is mediated by a interaction Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). Thus, SGK binding its kinase to hNedd4-2. be required to a acid binding and protein kinase a to be important for and P. H. Nature. 2001; PubMed Scopus Google Scholar). SGK might be activated through its binding to hNedd4-2. In to hNedd4-2, the function of ENaC be regulated by other ubiquitin Nedd4 is expressed in the renal collecting O. H. Kim H. Rotin D. Am. J. Physiol. 1997; 272: PubMed Google and ENaC (7Goulet C.C. Volk K.A. Adams C.M. Prince L.S. Stokes J.B. Snyder P.M. J. Biol. Chem. 1998; 273: 30012-30017Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 8Abriel H. Loffing J. Rebhun J.F. Pratt J.H. Schild L. Horisberger J.D. Rotin D. Staub O. J. Clin. Invest. 1999; 103: 667-673Crossref PubMed Scopus (318) Google Scholar, A. P. S. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). However, SGK consensus phosphorylation are not in Nedd4, suggesting that SGK not modulate the of this ubiquitin Consistent with such a we found that SGK did not hNedd4 or alter its binding to This several Nedd4 ENaC in a the inhibition of ENaC by Nedd4 might be regulated by In this it has been reported that the of Nedd4, resulting in its to the cell surface H. Staub O. P. Rotin D. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). Nedd4 might be phosphorylated by a kinase or might be it is that Nedd4 not regulate Na+ transport in epithelia, its to ENaC expressed in SGK modulate ENaC function by observations this For example, it is that the SGK PY motif with ENaC for binding to the WW domains of hNedd4-2 (or other ubiquitin In this SGK Na+ current by directly the binding of hNedd4-2 to ENaC. However, for hNedd4-2 binding not to be the SGK kinase was required for ENaC stimulation and for SGK to modulate hNedd4-2 In SGK might stimulate ENaC through mechanisms of hNedd4-2. In of this of the ENaC PY motifs did not SGK from increasing Na+ current (20Alvarez de la Rosa D. Zhang P. Naray-Fejes-Toth A. Fejes-Toth G. Canessa C.M. J. Biol. Chem. 1999; 274: 37834-37839Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar), in to data in was reported that SGK bind directly to the C terminus of and (19Wang J. Barbry P. Maiyar A.C. Rozansky D.J. Bhargava A. Leong M. Firestone G.L. Pearce D. Am. J. Physiol. Renal Physiol. 2001; 280: F303-F313Crossref PubMed Google Scholar). However, SGK did not ENaC, and the role and the sequences that this interaction are not yet data suggest that aldosterone, SGK, and hNedd4-2 converge a common pathway to regulate Na+ absorption. this hNedd4-2 and members of the Nedd4 family might form a of to regulate ENaC surface expression, and hence, to Na+ and other for and and for