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Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4-2 using proteome arrays

Target recognition by the ubiquitin system is mediated by E3 ubiquitin ligases. Nedd4 family members are E3 ligases comprised of a C2 domain, 2–4 WW domains that bind PY motifs (L/PPxY) and a ubiquitin ligase HECT domain. The nine Nedd4 family proteins in mammals include two close relatives: Nedd4 (...

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Autores principales: Persaud, Avinash, Alberts, Philipp, Amsen, Eva M, Xiong, Xuejian, Wasmuth, James, Saadon, Zachary, Fladd, Chris, Parkinson, John, Rotin, Daniela
Formato: Texto
Lenguaje:English
Publicado: Nature Publishing Group 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2824488/
https://www.ncbi.nlm.nih.gov/pubmed/19953087
http://dx.doi.org/10.1038/msb.2009.85
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author Persaud, Avinash
Alberts, Philipp
Amsen, Eva M
Xiong, Xuejian
Wasmuth, James
Saadon, Zachary
Fladd, Chris
Parkinson, John
Rotin, Daniela
author_facet Persaud, Avinash
Alberts, Philipp
Amsen, Eva M
Xiong, Xuejian
Wasmuth, James
Saadon, Zachary
Fladd, Chris
Parkinson, John
Rotin, Daniela
author_sort Persaud, Avinash
collection PubMed
description Target recognition by the ubiquitin system is mediated by E3 ubiquitin ligases. Nedd4 family members are E3 ligases comprised of a C2 domain, 2–4 WW domains that bind PY motifs (L/PPxY) and a ubiquitin ligase HECT domain. The nine Nedd4 family proteins in mammals include two close relatives: Nedd4 (Nedd4-1) and Nedd4L (Nedd4-2), but their global substrate recognition or differences in substrate specificity are unknown. We performed in vitro ubiquitylation and binding assays of human Nedd4-1 and Nedd4-2, and rat-Nedd4-1, using protein microarrays spotted with ∼8200 human proteins. Top hits (substrates) for the ubiquitylation and binding assays mostly contain PY motifs. Although several substrates were recognized by both Nedd4-1 and Nedd4-2, others were specific to only one, with several Tyr kinases preferred by Nedd4-1 and some ion channels by Nedd4-2; this was subsequently validated in vivo. Accordingly, Nedd4-1 knockdown or knockout in cells led to sustained signalling via some of its substrate Tyr kinases (e.g. FGFR), suggesting Nedd4-1 suppresses their signalling. These results demonstrate the feasibility of identifying substrates and deciphering substrate specificity of mammalian E3 ligases.
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spelling pubmed-28244882010-02-18 Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4-2 using proteome arrays Persaud, Avinash Alberts, Philipp Amsen, Eva M Xiong, Xuejian Wasmuth, James Saadon, Zachary Fladd, Chris Parkinson, John Rotin, Daniela Mol Syst Biol Article Target recognition by the ubiquitin system is mediated by E3 ubiquitin ligases. Nedd4 family members are E3 ligases comprised of a C2 domain, 2–4 WW domains that bind PY motifs (L/PPxY) and a ubiquitin ligase HECT domain. The nine Nedd4 family proteins in mammals include two close relatives: Nedd4 (Nedd4-1) and Nedd4L (Nedd4-2), but their global substrate recognition or differences in substrate specificity are unknown. We performed in vitro ubiquitylation and binding assays of human Nedd4-1 and Nedd4-2, and rat-Nedd4-1, using protein microarrays spotted with ∼8200 human proteins. Top hits (substrates) for the ubiquitylation and binding assays mostly contain PY motifs. Although several substrates were recognized by both Nedd4-1 and Nedd4-2, others were specific to only one, with several Tyr kinases preferred by Nedd4-1 and some ion channels by Nedd4-2; this was subsequently validated in vivo. Accordingly, Nedd4-1 knockdown or knockout in cells led to sustained signalling via some of its substrate Tyr kinases (e.g. FGFR), suggesting Nedd4-1 suppresses their signalling. These results demonstrate the feasibility of identifying substrates and deciphering substrate specificity of mammalian E3 ligases. Nature Publishing Group 2009-12-01 /pmc/articles/PMC2824488/ /pubmed/19953087 http://dx.doi.org/10.1038/msb.2009.85 Text en Copyright © 2009, EMBO and Nature Publishing Group http://creativecommons.org/licenses/by-nc-sa/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits distribution and reproduction in any medium, provided the original author and source are credited. Creation of derivative works is permitted but the resulting work may be distributed only under the same or similar licence to this one. This licence does not permit commercial exploitation without specific permission.
spellingShingle Article
Persaud, Avinash
Alberts, Philipp
Amsen, Eva M
Xiong, Xuejian
Wasmuth, James
Saadon, Zachary
Fladd, Chris
Parkinson, John
Rotin, Daniela
Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4-2 using proteome arrays
title Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4-2 using proteome arrays
title_full Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4-2 using proteome arrays
title_fullStr Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4-2 using proteome arrays
title_full_unstemmed Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4-2 using proteome arrays
title_short Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4-2 using proteome arrays
title_sort comparison of substrate specificity of the ubiquitin ligases nedd4 and nedd4-2 using proteome arrays
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2824488/
https://www.ncbi.nlm.nih.gov/pubmed/19953087
http://dx.doi.org/10.1038/msb.2009.85
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