Comparison of Substrate Specificity of the Ubiquitin Ligases Nedd4 and Nedd4-2 Using Proteome Arrays

Overview

Journal Mol Syst Biol

Specialty Molecular Biology

Date 2009 Dec 3

PMID 19953087

Citations 86

Authors

Avinash Persaud

Philipp Alberts

Eva M Amsen

Xuejian Xiong

James Wasmuth

Zachary Saadon

Chris Fladd

John Parkinson

Daniela Rotin

Affiliations

Soon will be listed here.

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.

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