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 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.
Persaud
Avinash
A
Programs in Cell Biology and Molecular Structure and Function, The Hospital for Sick Children, Toronto, Ontario, Canada.
Alberts
Philipp
P
Amsen
Eva M
EM
Xiong
Xuejian
X
Wasmuth
James
J
Saadon
Zachary
Z
Fladd
Chris
C
Parkinson
John
J
Rotin
Daniela
D
D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D006801:Humans; D000075702:Nedd4 Ubiquitin Protein Ligases; D040081:Protein Array Analysis; D011485:Protein Binding; D020543:Proteome; D013379:Substrate Specificity; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
D056827:Endosomal Sorting Complexes Required for Transport; D020543:Proteome; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases