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

Mol Syst Biol. 2009:5:333. doi: 10.1038/msb.2009.85. Epub 2009 Dec 1.

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 approximately 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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Endosomal Sorting Complexes Required for Transport / metabolism*
  • Humans
  • Nedd4 Ubiquitin Protein Ligases
  • Protein Array Analysis
  • Protein Binding
  • Proteome
  • Substrate Specificity*
  • Ubiquitin-Protein Ligases / metabolism*

Substances

  • Endosomal Sorting Complexes Required for Transport
  • Proteome
  • NEDD4L protein, rat
  • Nedd4 Ubiquitin Protein Ligases
  • Nedd4 protein, human
  • Nedd4 protein, rat
  • Nedd4L protein, human
  • Ubiquitin-Protein Ligases