Neural precursor cells expressed developmentally downregulated protein 4-2 (Nedd4-2), a homologous to the E6-AP carboxyl terminus (HECT) ubiquitin ligase, triggers the endocytosis and degradation of its downstream target molecules by regulating signal transduction through interactions with other targets, including 14-3-3 proteins. In our previous study, we found that 14-3-3 binding induces a structural rearrangement of Nedd4-2 by inhibiting interactions between its structured domains. Here, we used time-resolved fluorescence intensity and anisotropy decay measurements, together with fluorescence quenching and mass spectrometry, to further characterize interactions between Nedd4-2 and 14-3-3 proteins. The results showed that 14-3-3 binding affects the emission properties of AEDANS-labeled WW3, WW4, and, to a lesser extent, WW2 domains, and reduces their mobility, but not those of the WW1 domain, which remains mobile. In contrast, 14-3-3 binding has the opposite effect on the active site of the HECT domain, which is more solvent exposed and mobile in the complexed form than in the apo form of Nedd4-2. Overall, our results suggest that steric hindrance of the WW3 and WW4 domains combined with conformational changes in the catalytic domain may account for the 14-3-3 binding-mediated regulation of Nedd4-2.

Department of Structural Biology of Signaling Proteins Division BIOCEV Institute of Physiology of the Czech Academy of Sciences Czech Republic

Department of Structural Biology of Signaling Proteins Division BIOCEV Institute of Physiology of the Czech Academy of Sciences Czech Republic; Department of Physical and Macromolecular Chemistry Faculty of Science Charles University Prague Czech Republic

Institute of Physics Faculty of Mathematics and Physics Charles University Prague Czech Republic

Komentář v

Biophys J. 2022 Apr 5;121(7):1115-1116. doi: 10.1016/j.bpj.2022.03.007. PubMed

Zobrazit více v PubMed

Yang B., Kumar S. Nedd4 and Nedd4-2: closely related ubiquitin-protein ligases with distinct physiological functions. Cell Death Differ. 2010;17:68–77. PubMed PMC

Itani O.A., Campbell J.R., et al. Thomas C.P. Alternate promoters and variable splicing lead to hNedd4-2 isoforms with a C2 domain and varying number of WW domains. Am. J. Physiol. Ren. Physiol. 2003;285:F916–F929. PubMed

Fotia A.B., Ekberg J., et al. Kumar S. Regulation of neuronal voltage-gated sodium channels by the ubiquitin-protein ligases Nedd4 and Nedd4-2. J. Biol. Chem. 2004;279:28930–28935. PubMed

Arroyo J.P., Lagnaz D., et al. Staub O. Nedd4-2 modulates renal Na+-Cl- cotransporter via the aldosterone-SGK1-Nedd4-2 pathway. J. Am. Soc. Nephrol. 2011;22:1707–1719. PubMed PMC

Kamynina E., Debonneville C., Bens M., Vandewalle A., Staub O. A novel mouse Nedd4 protein suppresses the activity of the epithelial Na+ channel. FASEB J. 2001;15:204–214. PubMed

Ekberg J., Schuetz F., et al. Adams D.J. Regulation of the voltage-gated K(+) channels KCNQ2/3 and KCNQ3/5 by ubiquitination. Novel role for Nedd4-2. J. Biol. Chem. 2007;282:12135–12142. PubMed

Zhu J., Lee K.Y., et al. Tsai N.P. Epilepsy-associated gene Nedd4-2 mediates neuronal activity and seizure susceptibility through AMPA receptors. PLoS Genet. 2017;13:e1006634. PubMed PMC

Broix L., Jagline H., et al. Chelly J. Mutations in the HECT domain of NEDD4L lead to AKT-mTOR pathway deregulation and cause periventricular nodular heterotopia. Nat. Genet. 2016;48:1349–1358. PubMed PMC

Popovic D., Vucic D., Dikic I. Ubiquitination in disease pathogenesis and treatment. Nat. Med. 2014;20:1242–1253. PubMed

Rizzo F., Staub O. NEDD4-2 and salt-sensitive hypertension. Curr. Opin. Nephrol. Hypertens. 2015;24:111–116. PubMed

Vanli-Yavuz E.N., Ozdemir O., et al. Baykan B. Investigation of the possible association of NEDD4-2 (NEDD4L) gene with idiopathic photosensitive epilepsy. Acta Neurol. Belg. 2015;115:241–245. PubMed

Corbalan-Garcia S., Gomez-Fernandez J.C. Signaling through C2 domains: more than one lipid target. Biochim. Biophys. Acta. 2014;1838:1536–1547. PubMed

Bork P., Sudol M. The WW domain: a signalling site in dystrophin? Trends Biochem. Sci. 1994;19:531–533. PubMed

Chen H.I., Sudol M. The WW domain of Yes-associated protein binds a proline-rich ligand that differs from the consensus established for Src homology 3-binding modules. Proc. Natl. Acad. Sci. U S A. 1995;92:7819–7823. PubMed PMC

Todaro D.R., Augustus-Wallace A.C., et al. Haas A.L. The mechanism of neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2)/NEDD4L-catalyzed polyubiquitin chain assembly. J. Biol. Chem. 2017;292:19521–19536. PubMed PMC

French M.E., Klosowiak J.L., et al. Hunter T. Mechanism of ubiquitin chain synthesis employed by a HECT domain ubiquitin ligase. J. Biol. Chem. 2017;292:10398–10413. PubMed PMC

Fotia A.B., Dinudom A., et al. Kumar S. The role of individual Nedd4-2 (KIAA0439) WW domains in binding and regulating epithelial sodium channels. FASEB J. 2003;17:70–72. PubMed

Harvey K.F., Dinudom A., et al. Kumar S. All three WW domains of murine Nedd4 are involved in the regulation of epithelial sodium channels by intracellular Na+ J. Biol. Chem. 1999;274:12525–12530. PubMed

Snyder P.M., Olson D.R., et al. Bucher D.B. Multiple WW domains, but not the C2 domain, are required for inhibition of the epithelial Na+ channel by human Nedd4. J. Biol. Chem. 2001;276:28321–28326. PubMed

Bruce M.C., Kanelis V., et al. Rotin D. Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain. Biochem. J. 2008;415:155–163. PubMed

Zhang W., Na T., et al. Peng J.B. Down-regulation of intestinal apical calcium entry channel TRPV6 by ubiquitin E3 ligase Nedd4-2. J. Biol. Chem. 2010;285:36586–36596. PubMed PMC

Ichimura T., Yamamura H., et al. Isobe T. 14-3-3 proteins modulate the expression of epithelial Na+ channels by phosphorylation-dependent interaction with Nedd4-2 ubiquitin ligase. J. Biol. Chem. 2005;280:13187–13194. PubMed

Snyder P.M., Olson D.R., et al. Steines J.C. cAMP and serum and glucocorticoid-inducible kinase (SGK) regulate the epithelial Na(+) channel through convergent phosphorylation of Nedd4-2. J. Biol. Chem. 2004;279:45753–45758. PubMed

Bhalla V., Daidie D., et al. Pearce D. Serum- and glucocorticoid-regulated kinase 1 regulates ubiquitin ligase neural precursor cell-expressed, developmentally down-regulated protein 4-2 by inducing interaction with 14-3-3. Mol. Endocrinol. 2005;19:3073–3084. PubMed

Lee I.H., Dinudom A., et al. Cook D.I. Akt mediates the effect of insulin on epithelial sodium channels by inhibiting Nedd4-2. J. Biol. Chem. 2007;282:29866–29873. PubMed

Hallows K.R., Bhalla V., et al. Pearce D. Phosphopeptide screen uncovers novel phosphorylation sites of Nedd4-2 that potentiate its inhibition of the epithelial Na+ channel. J. Biol. Chem. 2010;285:21671–21678. PubMed PMC

Edinger R.S., Lebowitz J., et al. Hallows K.R. Functional regulation of the epithelial Na+ channel by IkappaB kinase-beta occurs via phosphorylation of the ubiquitin ligase Nedd4-2. J. Biol. Chem. 2009;284:150–157. PubMed PMC

Nagaki K., Yamamura H., et al. Ichimura T. 14-3-3 Mediates phosphorylation-dependent inhibition of the interaction between the ubiquitin E3 ligase Nedd4-2 and epithelial Na+ channels. Biochemistry. 2006;45:6733–6740. PubMed

Chandran S., Li H., et al. Bhalla V. Neural precursor cell-expressed developmentally down-regulated protein 4-2 (Nedd4-2) regulation by 14-3-3 protein binding at canonical serum and glucocorticoid kinase 1 (SGK1) phosphorylation sites. J. Biol. Chem. 2011;286:37830–37840. PubMed PMC

Pohl P., Joshi R., et al. Obsilova V. 14-3-3-protein regulates Nedd4-2 by modulating interactions between HECT and WW domains. Commun. Biol. 2021;4:899. PubMed PMC

Mackintosh C. Dynamic interactions between 14-3-3 proteins and phosphoproteins regulate diverse cellular processes. Biochem. J. 2004;381(Pt 2):329–342. PubMed PMC

Obsilova V., Obsil T. The 14-3-3 proteins as important allosteric regulators of protein kinases. Int. J. Mol. Sci. 2020;21:8824. PubMed PMC

Sluchanko N.N. Association of multiple phosphorylated proteins with the 14-3-3 regulatory hubs: problems and perspectives. J. Mol. Biol. 2018;430:20–26. PubMed

Gogl G., Tugaeva K.V., et al. Sluchanko N.N. Hierarchized phosphotarget binding by the seven human 14-3-3 isoforms. Nat. Commun. 2021;12:1677. PubMed PMC

Sluchanko N.N., Bustos D.M. Intrinsic disorder associated with 14-3-3 proteins and their partners. Prog. Mol. Biol. Transl Sci. 2019;166:19–61. PubMed

Bustos D.M. The role of protein disorder in the 14-3-3 interaction network. Mol. Biosyst. 2012;8:178–184. PubMed

Horvath M., Petrvalska O., et al. Obsil T. 14-3-3 proteins inactivate DAPK2 by promoting its dimerization and protecting key regulatory phosphosites. Commun. Biol. 2021;4:986. PubMed PMC

Rotin D., Staub O. Nedd4-2 and the regulation of epithelial sodium transport. Front Physiol. 2012;3:212. PubMed PMC

Obsil T., Ghirlando R., et al. Dyda F. Crystal structure of the 14-3-3zeta:serotonin N-acetyltransferase complex. a role for scaffolding in enzyme regulation. Cell. 2001;105:257–267. PubMed

Obsilova V., Herman P., et al. Obsil T. 14-3-3zeta C-terminal stretch changes its conformation upon ligand binding and phosphorylation at Thr232. J. Biol. Chem. 2004;279:4531–4540. PubMed

Boura E., Silhan J., et al. Obsil T. Both the N-terminal loop and wing W2 of the forkhead domain of transcription factor Foxo4 are important for DNA binding. J. Biol. Chem. 2007;282:8265–8275. PubMed

Niesen F.H., Berglund H., Vedadi M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat. Protoc. 2007;2:2212–2221. PubMed

Ballone A., Centorrino F., et al. Ottmann C. Protein X-ray crystallography of the 14-3-3zeta/SOS1 complex. Data Brief. 2018;19:1683–1687. PubMed PMC

Kacirova M., Kosek D., et al. Obsil T. Structural characterization of phosducin and its complex with the 14-3-3 protein. J. Biol. Chem. 2015;290:16246–16260. PubMed PMC

Vecer J., Herman P. Maximum entropy analysis of analytically simulated complex fluorescence decays. J. Fluorescence. 2011;21:873–881. PubMed

Lakowicz J.R. Springer; 2006. Principles of Fluorescence Spectroscopy.

Lakowicz J.R. Plenum Press; 1983. Principles of Fluorescence Spectroscopy.

Lehrer S.S. Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion. Biochemistry. 1971;10:3254–3263. PubMed

Yong W., Tsukasa I., et al. Fujio T. Dansyl-β-cyclodextrins as fluorescent sensors responsive to organic compounds. Bull. Chem. Soc. Jpn. 1994;67:1598–1607.

Sudol M., Chen H.I., et al. Bork P. Characterization of a novel protein-binding module--the WW domain. FEBS Lett. 1995;369:67–71. PubMed

Staub O., Rotin D. WW domains. Structure. 1996;4:495–499. PubMed

Snyder P.M., Olson D.R., Thomas B.C. Serum and glucocorticoid-regulated kinase modulates Nedd4-2-mediated inhibition of the epithelial Na+ channel. J. Biol. Chem. 2002;277:5–8. PubMed

Chan W., Tian R., et al. Manser E. Down-regulation of active ACK1 is mediated by association with the E3 ubiquitin ligase Nedd4-2. J. Biol. Chem. 2009;284:8185–8194. PubMed PMC

Roy A., Al-Qusairi L., et al. Subramanya A.R. Alternatively spliced proline-rich cassettes link WNK1 to aldosterone action. J. Clin. Invest. 2015;125:3433–3448. PubMed PMC

Asher C., Sinha I., Garty H. Characterization of the interactions between Nedd4-2, ENaC, and sgk-1 using surface plasmon resonance. Biochim. Biophys. Acta. 2003;1612:59–64. PubMed

Wiemuth D., Lott J.S., et al. McDonald F.J. Interaction of serum- and glucocorticoid regulated kinase 1 (SGK1) with the WW-domains of Nedd4-2 is required for epithelial sodium channel regulation. PLoS One. 2010;5:e12163. PubMed PMC

Bhalla V., Hallows K.R. Mechanisms of ENaC regulation and clinical implications. J. Am. Soc. Nephrol. 2008;19:1845–1854. PubMed

Snyder P.M. Down-regulating destruction: phosphorylation regulates the E3 ubiquitin ligase Nedd4-2. Sci. Signal. 2009;2:pe41. PubMed

Manning J.A., Kumar S. Physiological functions of Nedd4-2: lessons from knockout mouse models. Trends Biochem. Sci. 2018;43:635–647. PubMed

Wu P.Y., Hanlon M., et al. Pickart C.M. A conserved catalytic residue in the ubiquitin-conjugating enzyme family. EMBO J. 2003;22:5241–5250. PubMed PMC

Soini L., Redhead M., et al. Ottmann C. Identification of molecular glues of the SLP76/14-3-3 protein-protein interaction. RSC Med. Chem. 2021;12:1555–1564. PubMed PMC

Wolter M., Valenti D., et al. Ottmann C. An exploration of chemical properties required for cooperative stabilization of the 14-3-3 interaction with NF-kappaB-Utilizing a reversible covalent tethering approach. J. Med. Chem. 2021;64:8423–8436. PubMed PMC

Bryan R.K. Maximum entropy analysis of oversampled data problems. Eur. Biophys. J. 1990;18:165–174.

Marquardt D.W. An algorithm for least-squares estimation of Nonlinear parameters. J. Soc. Ind. Appl. Math. 1963;11:431–441.

Look for the Scaffold: Multifaceted Regulation of Enzyme Activity by 14-3-3 Proteins

Structural insights into the functional roles of 14-3-3 proteins