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 (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

N-Cadherin Is an In Vivo Substrate for Protein Tyrosine Phosphatase Sigma (PTPσ) and Participates in PTPσ-Mediated Inhibition of Axon Growth

Protein tyrosine phosphatase sigma (PTPσ) belongs to the LAR family of receptor tyrosine phosphatases and was previously shown to negatively regulate axon growth. The substrate for PTPσ and the effector(s) mediating this inhibitory effect were unknown. Here we report the identification of N-cadherin as an in vivo substrate for PTPσ. Using brain lysates from PTPσ knockout mice, in combination with substrate trapping, we identified a hyper-tyrosine-phosphorylated protein of ∼120 kDa in the knockout animals (relative to sibling controls), which was identified by mass spectrometry and immunoblotting as N-cadherin. β-Catenin also precipitated in the complex and was also a substrate for PTPσ. Dorsal root ganglion (DRG) neurons, which highly express endogenous N-cadherin and PTPσ, exhibited a faster growth rate in the knockout mice than in the sibling controls when grown on laminin or N-cadherin substrata. However, when N-cadherin function was disrupted by an inhibitory peptide or lowering calcium concentrations, the differential growth rate between the knockout and sibling control mice was greatly diminished. These results suggest that the elevated tyrosine phosphorylation of N-cadherin in the PTPσ(−/−) mice likely disrupted N-cadherin function, resulting in accelerated DRG nerve growth. We conclude that N-cadherin is a physiological substrate for PTPσ and that N-cadherin (and likely β-catenin) participates in PTPσ-mediated inhibition of axon growth