Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4‐2 using proteome arrays
Abstract 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...
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Springer Nature
2009-12-01
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| Series: | Molecular Systems Biology |
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| Online Access: | https://doi.org/10.1038/msb.2009.85 |
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| author | Avinash Persaud Philipp Alberts Eva M Amsen Xuejian Xiong James Wasmuth Zachary Saadon Chris Fladd John Parkinson Daniela Rotin |
| author_facet | Avinash Persaud Philipp Alberts Eva M Amsen Xuejian Xiong James Wasmuth Zachary Saadon Chris Fladd John Parkinson Daniela Rotin |
| author_sort | Avinash Persaud |
| collection | DOAJ |
| description | Abstract 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. |
| format | Article |
| id | doaj-art-e83f2a1f6a294aa1a3ac55ce704caf2f |
| institution | Kabale University |
| issn | 1744-4292 |
| language | English |
| publishDate | 2009-12-01 |
| publisher | Springer Nature |
| record_format | Article |
| series | Molecular Systems Biology |
| spelling | doaj-art-e83f2a1f6a294aa1a3ac55ce704caf2f2025-08-24T11:59:32ZengSpringer NatureMolecular Systems Biology1744-42922009-12-015111810.1038/msb.2009.85Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4‐2 using proteome arraysAvinash Persaud0Philipp Alberts1Eva M Amsen2Xuejian Xiong3James Wasmuth4Zachary Saadon5Chris Fladd6John Parkinson7Daniela Rotin8Programs in Cell Biology and Molecular Structure and Function, The Hospital for Sick ChildrenPrograms in Cell Biology and Molecular Structure and Function, The Hospital for Sick ChildrenPrograms in Cell Biology and Molecular Structure and Function, The Hospital for Sick ChildrenPrograms in Cell Biology and Molecular Structure and Function, The Hospital for Sick ChildrenPrograms in Cell Biology and Molecular Structure and Function, The Hospital for Sick ChildrenPrograms in Cell Biology and Molecular Structure and Function, The Hospital for Sick ChildrenPrograms in Cell Biology and Molecular Structure and Function, The Hospital for Sick ChildrenPrograms in Cell Biology and Molecular Structure and Function, The Hospital for Sick ChildrenPrograms in Cell Biology and Molecular Structure and Function, The Hospital for Sick ChildrenAbstract 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.https://doi.org/10.1038/msb.2009.85E3 ubiquitin ligaseHECT domainNedd4proteome arrayubiquitin |
| spellingShingle | Avinash Persaud Philipp Alberts Eva M Amsen Xuejian Xiong James Wasmuth Zachary Saadon Chris Fladd John Parkinson Daniela Rotin Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4‐2 using proteome arrays Molecular Systems Biology E3 ubiquitin ligase HECT domain Nedd4 proteome array ubiquitin |
| 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 | E3 ubiquitin ligase HECT domain Nedd4 proteome array ubiquitin |
| url | https://doi.org/10.1038/msb.2009.85 |
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