Abstract
NUB1 (Nedd8 ultimate buster 1) is an adaptor protein which negatively regulates the ubiquitin-like protein Nedd8 as well as neddylated proteins levels through proteasomal degradation. However, molecular mechanisms underlying this function are not completely understood. Here, we report that the oncogenic E3 ubiquitin ligase Mdm2 is a new NUB1 interacting protein which induces its ubiquitination. Interestingly, we found that Mdm2-mediated ubiquitination of NUB1 is not a proteolytic signal. Instead of promoting the conjugation of polyubiquitin chains and the subsequent proteasomal degradation of NUB1, Mdm2 rather induces its di-ubiquitination on lysine 159. Importantly, mutation of lysine 159 into arginine inhibits NUB1 activity by impairing its negative regulation of Nedd8 and of neddylated proteins. We conclude that Mdm2 acts as a positive regulator of NUB1 function, by modulating NUB1 ubiquitination on lysine 159.
Highlights
Post-translational modifications (PTMs) by ubiquitin and ubiquitin-like proteins (Ubls) have emerged as major regulators of cellular functions [1,2]
This suggests that the shifted NUB1 was a substrate for deubiquitinating enzymes (DUBs) activity in cellulo and that it corresponded to ubiquitinated NUB1
In order to discriminate between NUB1 poly- or multi-ubiquitination, we have studied the effect of Mdm2 on NUB1 ubiquitination using either WT ubiquitin or a K0 mutant ubiquitin in which all lysine residues are mutated into arginine residues to prevent the formation of polyubiquitin chains
Summary
Post-translational modifications (PTMs) by ubiquitin and ubiquitin-like proteins (Ubls) have emerged as major regulators of cellular functions [1,2]. E3s modify substrate proteins with a single ubiquitin molecule (mono-ubiquitination), several single ubiquitin molecules (multi-ubiquitination) or by eight different types of poly-ubiquitin chains (poly-ubiquitination), obtained through the sequential attachment of ubiquitin molecules using either one of the seven lysine residues or their N-terminal methionine. These ubiquitination patterns dictate specific outcomes for the substrate proteins such as degradation, protein interactions, sub-cellular localization and/or activity. Ubiquitination can be reversed through the action of specific deubiquitinating enzymes (DUBs) Ubls proteins, such as Nedd (neural precursor cell expressed developmentally down-regulated 8), share a very similar ternary structure and are conjugated to their substrates by specific sets of E1 / E2 / E3 enzymes. Neddylation primarily occurs on cullins thereby enabling the PLOS ONE | DOI:10.1371/journal.pone.0169988 January 18, 2017
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