Calcium Activates Nedd4 E3 Ubiquitin Ligases by Releasing the C2 Domain-mediated Auto-inhibition

Nedd4 E3 ligases are members of the HECT E3 ubiquitin ligase family and regulate ubiquitination-mediated protein degradation. In this report, we demonstrate that calcium releases the C2 domain-mediated auto-inhibition in both Nedd4-1 and Nedd4-2. Calcium disrupts binding of the C2 domain to the HECT domain. Consistent with this, calcium activates the E3 ubiquitin ligase activity of Nedd4. Elevation of intracellular calcium by ionomycin treatment, or activation of acetylcholine receptor or epidermal growth factor receptor by carbachol or epidermal growth factor stimulation induced activation of endogenous Nedd4 in vivo evaluated by assays of either Nedd4 E3 ligase activity or ubiquitination of Nedd4 substrate ENaC-β. The activation effect of calcium on Nedd4 E3 ligase activity was dramatically enhanced by a membrane-rich fraction, suggesting that calcium-mediated membrane translocation through the C2 domain might be an activation mechanism of Nedd4 in vivo. Our studies have revealed an activation mechanism of Nedd4 E3 ubiquitin ligases and established a connection of intracellular calcium signaling to regulation of protein ubiquitination. Nedd4 E3 ligases are members of the HECT E3 ubiquitin ligase family and regulate ubiquitination-mediated protein degradation. In this report, we demonstrate that calcium releases the C2 domain-mediated auto-inhibition in both Nedd4-1 and Nedd4-2. Calcium disrupts binding of the C2 domain to the HECT domain. Consistent with this, calcium activates the E3 ubiquitin ligase activity of Nedd4. Elevation of intracellular calcium by ionomycin treatment, or activation of acetylcholine receptor or epidermal growth factor receptor by carbachol or epidermal growth factor stimulation induced activation of endogenous Nedd4 in vivo evaluated by assays of either Nedd4 E3 ligase activity or ubiquitination of Nedd4 substrate ENaC-β. The activation effect of calcium on Nedd4 E3 ligase activity was dramatically enhanced by a membrane-rich fraction, suggesting that calcium-mediated membrane translocation through the C2 domain might be an activation mechanism of Nedd4 in vivo. Our studies have revealed an activation mechanism of Nedd4 E3 ubiquitin ligases and established a connection of intracellular calcium signaling to regulation of protein ubiquitination. IntroductionProtein ubiquitination is a major intracellular signaling event. E3 ubiquitin ligase (E3), 3The abbreviations used are: E3ubiquitin ligaseE2ubiquitin-conjugating enzymeE1ubiquitin-activating enzymeSGK1serum- and glucocorticoid-inducible kinase 1ENaCepithelial sodium channelGFPgreen fluorescent proteinULRubiquitin ligation reactionGSTglutathione S-transferaseHAhemagglutininEGFepidermal growth factorEGFREGF receptorJNKc-Jun amino-terminal kinase. including the HECT (homologous to E6-AP carboxyl terminus) domain containing and the RING (the really interesting new gene) domain containing E3 ligases, is the key enzyme that catalyzes ubiquitination and confers specificity of ubiquitination substrates (1Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2887) Google Scholar, 2Jackson P.K. Eldridge A.G. Freed E. Furstenthal L. Hsu J.Y. Kaiser B.K. Reimann J.D. Trends Cell Biol. 2000; 10: 429-439Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, 3Passmore L.A. Barford D. Biochem. J. 2004; 379: 513-525Crossref PubMed Scopus (229) Google Scholar). Nedd4 E3 ubiquitin ligases are members of the WW domain-containing HECT E3 ubiquitin ligase subfamily (4Shearwin-Whyatt L. Dalton H.E. Foot N. Kumar S. BioEssays. 2006; 28: 617-628Crossref PubMed Scopus (132) Google Scholar). There are two Nedd4 E3 ligases, Nedd4-1 and Nedd4-2, in mammalian cells (5Chen H. Ross C.A. Wang N. Huo Y. MacKinnon D.F. Potash J.B. Simpson S.G. McMahon F.J. DePaulo Jr., J.R. McInnis M.G. Eur. J. Hum. Genet. 2001; 9: 922-930Crossref PubMed Scopus (50) Google Scholar). Human Nedd4-1 gene (Nedd4) is localized on chromosome 15, and Nedd4-2 gene (Nedd4L) is on chromosome 18 (5Chen H. Ross C.A. Wang N. Huo Y. MacKinnon D.F. Potash J.B. Simpson S.G. McMahon F.J. DePaulo Jr., J.R. McInnis M.G. Eur. J. Hum. Genet. 2001; 9: 922-930Crossref PubMed Scopus (50) Google Scholar). Both Nedd4-1 and Nedd4-2 have the same domain structure, with the C2 domain at the N terminus, followed by four WW domains, and the HECT domain at the C terminus. The primary peptide sequences of human Nedd4-1 and Nedd4-2 are ∼65% identical. The most unconserved regions are located between the WW1 and the WW3 domains.Four groups of Nedd4 substrates have been identified: 1) ion channels and membrane transporters; 2) membrane receptors; 3) tumor suppressors; and 4) endocytic regulation proteins. Accordingly, Nedd4 is involved in regulation of hypertension (6Russo C.J. Melista E. Cui J. DeStefano A.L. Bakris G.L. Manolis A.J. Gavras H. Baldwin C.T. Hypertension. 2005; 46: 488-491Crossref PubMed Scopus (68) Google Scholar), neuronal signal transmission (7Arévalo J.C. Waite J. Rajagopal R. Beyna M. Chen Z.Y. Lee F.S. Chao M.V. Neuron. 2006; 50: 549-559Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 8Fotia A.B. Ekberg J. Adams D.J. Cook D.I. Poronnik P. Kumar S. J. Biol. Chem. 2004; 279: 28930-28935Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), tumorigenesis and cell growth (9Xu L.L. Shi Y. Petrovics G. Sun C. Makarem M. Zhang W. Sesterhenn I.A. McLeod D.G. Sun L. Moul J.W. Srivastava S. Cancer Res. 2003; 63: 4299-4304PubMed Google Scholar), cellular metabolism (10Lang F. Böhmer C. Palmada M. Seebohm G. Strutz-Seebohm N. Vallon V. Physiol. Rev. 2006; 86: 1151-1178Crossref PubMed Scopus (553) Google Scholar), receptor endocytosis and degradation (11Marmor M.D. Yarden Y. Oncogene. 2004; 23: 2057-2070Crossref PubMed Scopus (318) Google Scholar), and viral endocytosis and budding (12Vana M.L. Tang Y. Chen A. Medina G. Carter C. Leis J. J. Virol. 2004; 78: 13943-13953Crossref PubMed Scopus (51) Google Scholar, 13Segura-Morales C. Pescia C. Chatellard-Causse C. Sadoul R. Bertrand E. Basyuk E. J. Biol. Chem. 2005; 280: 27004-27012Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Although Nedd4-1 and Nedd4-2 show partially redundant cellular functions (14Henry P.C. Kanelis V. O'Brien M.C. Kim B. Gautschi I. Forman-Kay J. Schild L. Rotin D. J. Biol. Chem. 2003; 278: 20019-20028Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), there seems to be a distinct preference for substrates between Nedd4-1 and Nedd4-2. For example, Nedd4-1 preferentially ubiquitinates tumor suppressors and endocytic proteins (15Magnifico A. Ettenberg S. Yang C. Mariano J. Tiwari S. Fang S. Lipkowitz S. Weissman A.M. J. Biol. Chem. 2003; 278: 43169-43177Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 16Woelk T. Oldrini B. Maspero E. Confalonieri S. Cavallaro E. Di Fiore P.P. Polo S. Nat. Cell Biol. 2006; 8: 1246-1254Crossref PubMed Scopus (158) Google Scholar, 17Katz M. Shtiegman K. Tal-Or P. Yakir L. Mosesson Y. Harari D. Machluf Y. Asao H. Jovin T. Sugamura K. Yarden Y. Traffic. 2002; 3: 740-751Crossref PubMed Scopus (120) Google Scholar, 18Aoh Q.L. Castle A.M. Hubbard C.H. Katsumata O. Castle J.D. Mol. Biol. Cell. 2009; 20: 1816-1832Crossref PubMed Scopus (52) Google Scholar), whereas Nedd4-2 ubiquitinates ion channels and membrane transporters (19Snyder P.M. Steines J.C. Olson D.R. J. Biol. Chem. 2004; 279: 5042-5046Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 20van Bemmelen M.X. Rougier J.S. Gavillet B. Apothéloz F. Daidié D. Tateyama M. Rivolta I. Thomas M.A. Kass R.S. Staub O. Abriel H. Circ. Res. 2004; 95: 284-291Crossref PubMed Scopus (173) Google Scholar, 21Zhou R. Patel S.V. Snyder P.M. J. Biol. Chem. 2007; 282: 20207-20212Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 22Ekberg J. Schuetz F. Boase N.A. Conroy S.J. Manning J. Kumar S. Poronnik P. Adams D.J. J. Biol. Chem. 2007; 282: 12135-12142Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 23Sorkina T. Miranda M. Dionne K.R. Hoover B.R. Zahniser N.R. Sorkin A. J. Neurosci. 2006; 26: 8195-8205Crossref PubMed Scopus (152) Google Scholar). It is not known how substrate preferences of Nedd4-1 and Nedd4-2 are determined. In addition, both Nedd4-1 and Nedd4-2 have multiple cellular substrates. How Nedd4 discriminates substrates in response to cellular signaling to execute corresponding cellular function is entirely unknown.Regulation of Nedd4 activity in cells is poorly of Nedd4-2 by and glucocorticoid-inducible kinase is an major in regulation of Nedd4-2 activity to C. E. C. Thomas M.A. C. A. J.D. D. J. Staub O. J. 2001; 20: PubMed Scopus Google Scholar). was a kinase hypertension through activation of sodium A. A. R. C. S. T. Y. S. A.M. F. Hypertension. 2002; PubMed Scopus Google Scholar, D. Trends 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Nedd4-2 on V. Daidié D. H. Wang J. A. J.D. Staub O. D. Mol. 2005; PubMed Scopus (148) Google Scholar), is for of Nedd4-2 with in ubiquitination of and enhanced activity D. Trends 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In to is involved in regulation of and sodium through and of Nedd4-2 M. Palmada M. J. A. A. C. F. Res. 2004; PubMed Scopus Google Scholar, C. G. R. Palmada M. J.D. S. F. J. 2003; 86: PubMed Scopus Google Scholar, C. F. I. S. F. Biochem. Res. 2003; PubMed Scopus Google Scholar, C. V. Palmada M. S. G. H. P. B. F. Res. 2003; PubMed Scopus (68) Google Scholar). is by a kinase is a known for hypertension C. 2003; PubMed Scopus Google Scholar). activates Nedd4-2 and in hypertension S. A. Lee B. 2005; PubMed Scopus Google Scholar). of Nedd4-2 by of was V. M.A. D. D. K.R. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). of Nedd4 by kinase be an for regulation of the E3 ligase activity or of the that Nedd4 is by receptor kinase A. Kumar S. Cook D.I. Biochem. Res. 2007; PubMed Scopus Google Scholar). The effect of by receptor kinase on Nedd4 activity is not C2 domain was a domain in protein kinase is for activation of the kinase activity J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). proteins are known to the C2 domain PubMed Scopus Google Scholar). The C2 domain of Nedd4 been a H. Staub O. P. Rotin D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (158) Google Scholar, R. L. J. Cell Biol. 2004; PubMed Scopus Google Scholar). studies that the C2 domain the translocation of Nedd4 to membrane by binding to H. Staub O. P. Rotin D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (158) Google Scholar). was a for the C2 domain of Nedd4 to to in cells F. S. P. K. Rotin D. J. Cell Biol. 2000; PubMed Scopus Google Scholar). In the C2 domain of the of is for of and ubiquitination of substrate R. L. J. Cell Biol. 2004; PubMed Scopus Google Scholar). In mammalian cells the WW domains, not the C2 are for Nedd4 to P.M. Olson D.R. F.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). It was that the C2 domain of Nedd4 of activity Nedd4 P.M. Olson D.R. F.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), suggesting that the C2 domain an in Nedd4 that the C2 domain of to the HECT domain and functions an domain for E3 ligase activity S. Wang Rotin D. F. Forman-Kay J.D. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google this report, we that the C2 domain of Nedd4 is the domain for E3 ubiquitin ligase of the C2 domain activation of Nedd4. Calcium activates Nedd4 E3 ubiquitin ligase activity both in and in vivo by the that intracellular calcium activates Nedd4 E3 ubiquitin ligase we that of the C2 domain-mediated auto-inhibition by calcium or cellular is a key mechanism for activation of Nedd4 E3 ubiquitin ligases in response to cellular have by and domain assays that the C2 domain of Nedd4 functions an domain through binding to the HECT domain. that the C2 domain of to the HECT domain and functions an auto-inhibition to the E3 ligase activity S. Wang Rotin D. F. Forman-Kay J.D. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). that Nedd4 a to and that of the C2 domain activates Nedd4 E3 ubiquitin ligase activity in an in ubiquitin ligase activity S. Wang Rotin D. F. Forman-Kay J.D. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). the auto-inhibition of the E3 ligase activity by the C2 domain is a key of Nedd4. we not the auto-inhibition is through the or the between the C2 and the HECT that the both the that activation of Nedd4 E3 ligase activity is to the Our show that this mechanism is by Calcium activates Nedd4 by binding to the C2 domain and the C2 domain-mediated The membrane-rich calcium to to the C2 calcium and the membrane-rich a activation of Nedd4. show activation of endogenous Nedd4 in we show that calcium-mediated activation of Nedd4 is enhanced by a membrane on we a for activation of Nedd4 E3 ubiquitin ligase activity in In this Nedd4 is by either the or (the in between the C2 and the HECT in or calcium signaling intracellular calcium and in binding of calcium to the C2 domain of Nedd4. The calcium binding the of the C2 domain to the and Nedd4 translocation to the membrane fraction, releases the auto-inhibition and activates Nedd4 E3 ubiquitin ligase that binding of calcium to the C2 domain partially disrupts of the C2 domain with the HECT domain and the C2 domain to is on that calcium partially activates Nedd4 E3 ubiquitin ligase activity to the the of C2 domain of Nedd4 been to to and an H. Staub O. P. Rotin D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (158) Google Scholar, R. L. J. Cell Biol. 2004; PubMed Scopus Google Scholar). The C2 domain to in a H. Staub O. P. Rotin D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (158) Google Scholar). studies have that translocation of Nedd4 to membrane in response to of intracellular calcium in cells H. Staub O. P. Rotin D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (158) Google Scholar, F. S. P. K. Rotin D. J. Cell Biol. 2000; PubMed Scopus Google Scholar). that membrane translocation of Nedd4 is a for activation in response to intracellular a in calcium-mediated activation of Nedd4 by a was by the for at suggesting that the in the that with calcium to Nedd4 might be a be an endogenous of Nedd4 that activates Nedd4 by the activation effect of calcium by the C2 domain-mediated of this to the mechanism of Nedd4 ligase activation and signaling of Nedd4 to the C2 domain-mediated for regulation of the E3 ubiquitin ligase and the two members of the HECT E3 ubiquitin ligase family that are to not to have a activation effect with the C2 domain of and not It been that an in activation of E. M. M. 2006; PubMed Scopus Google Scholar). The was located between the C2 domain and the WW1 domain. It was that the auto-inhibition by with the HECT releases the auto-inhibition E. M. M. 2006; PubMed Scopus Google Scholar). be involved in activation of Nedd4. It was that Nedd4-2, and the enhanced binding of Nedd4-2 to substrate of activity V. M.A. D. D. K.R. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google the mechanism of Nedd4 E3 ubiquitin ligases been (1Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2887) Google Scholar, 2Jackson P.K. Eldridge A.G. Freed E. Furstenthal L. Hsu J.Y. Kaiser B.K. Reimann J.D. Trends Cell Biol. 2000; 10: 429-439Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, 3Passmore L.A. Barford D. Biochem. J. 2004; 379: 513-525Crossref PubMed Scopus (229) Google Scholar), activation of the E3 ubiquitin ligase activity of Nedd4 in response to cellular signaling is poorly we show that the C2 domain of Nedd4 E3 ubiquitin ligases functions an domain to the ligase and that the auto-inhibition is by mechanism a for of Nedd4 in cells and cellular and acetylcholine to protein and It been that Nedd4-1 ubiquitinates tumor proteins and be (15Magnifico A. Ettenberg S. Yang C. Mariano J. Tiwari S. Fang S. Lipkowitz S. Weissman A.M. J. Biol. Chem. 2003; 278: 43169-43177Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 16Woelk T. Oldrini B. Maspero E. Confalonieri S. Cavallaro E. Di Fiore P.P. Polo S. Nat. Cell Biol. 2006; 8: 1246-1254Crossref PubMed Scopus (158) Google Scholar, 17Katz M. Shtiegman K. Tal-Or P. Yakir L. Mosesson Y. Harari D. Machluf Y. Asao H. Jovin T. Sugamura K. Yarden Y. Traffic. 2002; 3: 740-751Crossref PubMed Scopus (120) Google Scholar, 18Aoh Q.L. Castle A.M. Hubbard C.H. Katsumata O. Castle J.D. Mol. Biol. Cell. 2009; 20: 1816-1832Crossref PubMed Scopus (52) Google Scholar). Nedd4-2 ubiquitinates ion channels and membrane transporters and degradation (19Snyder P.M. Steines J.C. Olson D.R. J. Biol. Chem. 2004; 279: 5042-5046Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 20van Bemmelen M.X. Rougier J.S. Gavillet B. Apothéloz F. Daidié D. Tateyama M. Rivolta I. Thomas M.A. Kass R.S. Staub O. Abriel H. Circ. Res. 2004; 95: 284-291Crossref PubMed Scopus (173) Google Scholar, 21Zhou R. Patel S.V. Snyder P.M. J. Biol. Chem. 2007; 282: 20207-20212Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 22Ekberg J. Schuetz F. Boase N.A. Conroy S.J. Manning J. Kumar S. Poronnik P. Adams D.J. J. Biol. Chem. 2007; 282: 12135-12142Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 23Sorkina T. Miranda M. Dionne K.R. Hoover B.R. Zahniser N.R. Sorkin A. J. Neurosci. 2006; 26: 8195-8205Crossref PubMed Scopus (152) Google Scholar). Nedd4 in cell and The for the ligase activity how Nedd4 and in response to cellular IntroductionProtein ubiquitination is a major intracellular signaling event. E3 ubiquitin ligase (E3), 3The abbreviations used are: E3ubiquitin ligaseE2ubiquitin-conjugating enzymeE1ubiquitin-activating enzymeSGK1serum- and glucocorticoid-inducible kinase 1ENaCepithelial sodium channelGFPgreen fluorescent proteinULRubiquitin ligation reactionGSTglutathione S-transferaseHAhemagglutininEGFepidermal growth factorEGFREGF receptorJNKc-Jun amino-terminal kinase. including the HECT (homologous to E6-AP carboxyl terminus) domain containing and the RING (the really interesting new gene) domain containing E3 ligases, is the key enzyme that catalyzes ubiquitination and confers specificity of ubiquitination substrates (1Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2887) Google Scholar, 2Jackson P.K. Eldridge A.G. Freed E. Furstenthal L. Hsu J.Y. Kaiser B.K. Reimann J.D. Trends Cell Biol. 2000; 10: 429-439Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, 3Passmore L.A. Barford D. Biochem. J. 2004; 379: 513-525Crossref PubMed Scopus (229) Google Scholar). Nedd4 E3 ubiquitin ligases are members of the WW domain-containing HECT E3 ubiquitin ligase subfamily (4Shearwin-Whyatt L. Dalton H.E. Foot N. Kumar S. BioEssays. 2006; 28: 617-628Crossref PubMed Scopus (132) Google Scholar). There are two Nedd4 E3 ligases, Nedd4-1 and Nedd4-2, in mammalian cells (5Chen H. Ross C.A. Wang N. Huo Y. MacKinnon D.F. Potash J.B. Simpson S.G. McMahon F.J. DePaulo Jr., J.R. McInnis M.G. Eur. J. Hum. Genet. 2001; 9: 922-930Crossref PubMed Scopus (50) Google Scholar). Human Nedd4-1 gene (Nedd4) is localized on chromosome 15, and Nedd4-2 gene (Nedd4L) is on chromosome 18 (5Chen H. Ross C.A. Wang N. Huo Y. MacKinnon D.F. Potash J.B. Simpson S.G. McMahon F.J. DePaulo Jr., J.R. McInnis M.G. Eur. J. Hum. Genet. 2001; 9: 922-930Crossref PubMed Scopus (50) Google Scholar). Both Nedd4-1 and Nedd4-2 have the same domain structure, with the C2 domain at the N terminus, followed by four WW domains, and the HECT domain at the C terminus. The primary peptide sequences of human Nedd4-1 and Nedd4-2 are ∼65% identical. The most unconserved regions are located between the WW1 and the WW3 domains.Four groups of Nedd4 substrates have been identified: 1) ion channels and membrane transporters; 2) membrane receptors; 3) tumor suppressors; and 4) endocytic regulation proteins. Accordingly, Nedd4 is involved in regulation of hypertension (6Russo C.J. Melista E. Cui J. DeStefano A.L. Bakris G.L. Manolis A.J. Gavras H. Baldwin C.T. Hypertension. 2005; 46: 488-491Crossref PubMed Scopus (68) Google Scholar), neuronal signal transmission (7Arévalo J.C. Waite J. Rajagopal R. Beyna M. Chen Z.Y. Lee F.S. Chao M.V. Neuron. 2006; 50: 549-559Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 8Fotia A.B. Ekberg J. Adams D.J. Cook D.I. Poronnik P. Kumar S. J. Biol. Chem. 2004; 279: 28930-28935Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), tumorigenesis and cell growth (9Xu L.L. Shi Y. Petrovics G. Sun C. Makarem M. Zhang W. Sesterhenn I.A. McLeod D.G. Sun L. Moul J.W. Srivastava S. Cancer Res. 2003; 63: 4299-4304PubMed Google Scholar), cellular metabolism (10Lang F. Böhmer C. Palmada M. Seebohm G. Strutz-Seebohm N. Vallon V. Physiol. Rev. 2006; 86: 1151-1178Crossref PubMed Scopus (553) Google Scholar), receptor endocytosis and degradation (11Marmor M.D. Yarden Y. Oncogene. 2004; 23: 2057-2070Crossref PubMed Scopus (318) Google Scholar), and viral endocytosis and budding (12Vana M.L. Tang Y. Chen A. Medina G. Carter C. Leis J. J. Virol. 2004; 78: 13943-13953Crossref PubMed Scopus (51) Google Scholar, 13Segura-Morales C. Pescia C. Chatellard-Causse C. Sadoul R. Bertrand E. Basyuk E. J. Biol. Chem. 2005; 280: 27004-27012Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Although Nedd4-1 and Nedd4-2 show partially redundant cellular functions (14Henry P.C. Kanelis V. O'Brien M.C. Kim B. Gautschi I. Forman-Kay J. Schild L. Rotin D. J. Biol. Chem. 2003; 278: 20019-20028Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), there seems to be a distinct preference for substrates between Nedd4-1 and Nedd4-2. For example, Nedd4-1 preferentially ubiquitinates tumor suppressors and endocytic proteins (15Magnifico A. Ettenberg S. Yang C. Mariano J. Tiwari S. Fang S. Lipkowitz S. Weissman A.M. J. Biol. Chem. 2003; 278: 43169-43177Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 16Woelk T. Oldrini B. Maspero E. Confalonieri S. Cavallaro E. Di Fiore P.P. Polo S. Nat. Cell Biol. 2006; 8: 1246-1254Crossref PubMed Scopus (158) Google Scholar, 17Katz M. Shtiegman K. Tal-Or P. Yakir L. Mosesson Y. Harari D. Machluf Y. Asao H. Jovin T. Sugamura K. Yarden Y. Traffic. 2002; 3: 740-751Crossref PubMed Scopus (120) Google Scholar, 18Aoh Q.L. Castle A.M. Hubbard C.H. Katsumata O. Castle J.D. Mol. Biol. Cell. 2009; 20: 1816-1832Crossref PubMed Scopus (52) Google Scholar), whereas Nedd4-2 ubiquitinates ion channels and membrane transporters (19Snyder P.M. Steines J.C. Olson D.R. J. Biol. Chem. 2004; 279: 5042-5046Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 20van Bemmelen M.X. Rougier J.S. Gavillet B. Apothéloz F. Daidié D. Tateyama M. Rivolta I. Thomas M.A. Kass R.S. Staub O. Abriel H. Circ. Res. 2004; 95: 284-291Crossref PubMed Scopus (173) Google Scholar, 21Zhou R. Patel S.V. Snyder P.M. J. Biol. Chem. 2007; 282: 20207-20212Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 22Ekberg J. Schuetz F. Boase N.A. Conroy S.J. Manning J. Kumar S. Poronnik P. Adams D.J. J. Biol. Chem. 2007; 282: 12135-12142Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 23Sorkina T. Miranda M. Dionne K.R. Hoover B.R. Zahniser N.R. Sorkin A. J. Neurosci. 2006; 26: 8195-8205Crossref PubMed Scopus (152) Google Scholar). It is not known how substrate preferences of Nedd4-1 and Nedd4-2 are determined. In addition, both Nedd4-1 and Nedd4-2 have multiple cellular substrates. How Nedd4 discriminates substrates in response to cellular signaling to execute corresponding cellular function is entirely unknown.Regulation of Nedd4 activity in cells is poorly of Nedd4-2 by and glucocorticoid-inducible kinase is an major in regulation of Nedd4-2 activity to C. E. C. Thomas M.A. C. A. J.D. D. J. Staub O. J. 2001; 20: PubMed Scopus Google Scholar). was a kinase hypertension through activation of sodium A. A. R. C. S. T. Y. S. A.M. F. Hypertension. 2002; PubMed Scopus Google Scholar, D. Trends 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Nedd4-2 on V. Daidié D. H. Wang J. A. J.D. Staub O. D. Mol. 2005; PubMed Scopus (148) Google Scholar), is for of Nedd4-2 with in ubiquitination of and enhanced activity D. Trends 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In to is involved in regulation of and sodium through and of Nedd4-2 M. Palmada M. J. A. A. C. F. Res. 2004; PubMed Scopus Google Scholar, C. G. R. Palmada M. J.D. S. F. J. 2003; 86: PubMed Scopus Google Scholar, C. F. I. S. F. Biochem. Res. 2003; PubMed Scopus Google Scholar, C. V. Palmada M. S. G. H. P. B. F. Res. 2003; PubMed Scopus (68) Google Scholar). is by a kinase is a known for hypertension C. 2003; PubMed Scopus Google Scholar). activates Nedd4-2 and in hypertension S. A. Lee B. 2005; PubMed Scopus Google Scholar). of Nedd4-2 by of was V. M.A. D. D. K.R. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). of Nedd4 by kinase be an for regulation of the E3 ligase activity or of the that Nedd4 is by receptor kinase A. Kumar S. Cook D.I. Biochem. Res. 2007; PubMed Scopus Google Scholar). The effect of by receptor kinase on Nedd4 activity is not C2 domain was a domain in protein kinase is for activation of the kinase activity J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). proteins are known to the C2 domain PubMed Scopus Google Scholar). The C2 domain of Nedd4 been a H. Staub O. P. Rotin D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (158) Google Scholar, R. L. J. Cell Biol. 2004; PubMed Scopus Google Scholar). studies that the C2 domain the translocation of Nedd4 to membrane by binding to H. Staub O. P. Rotin D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (158) Google Scholar). was a for the C2 domain of Nedd4 to to in cells F. S. P. K. Rotin D. J. Cell Biol. 2000; PubMed Scopus Google Scholar). In the C2 domain of the of is for of and ubiquitination of substrate R. L. J. Cell Biol. 2004; PubMed Scopus Google Scholar). 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