Functional Study Of Lysines On Ubiquitin-like Protein NEDD8 And Identification Of NEDD8 Substrates

Background and purpose: Neural-precursor-cell-expressed developmentally down-regulated 8(NEDD8) is one of the most important ubiquitin-like modifiers. The process that NEDD8 forms covalent conjugates on lysines of its substrates is called neddylation. Neddylation plays important roles in tumor cell proliferation, regulation of transcription factor and so on. Ubiquitin can form diverse polyubiquitin chains on its seven lysines, which play important functions in various biological processes. However, the roles of lysines in NEDD8 have not been explored systematically. Here, we generated nine NEDD8 point mutants, each with one lysine replaced by an arginine, to study the putative function of lysines in NEDD8. The first identified neddylated proteins are cullin family proteins, which are components of ubiquitin E3 ligases. Cullins neddylation enhances the activity of cullin-RING E3 ligases(CRLs). There are few studies on NEDD8-modified proteins comparing with ubiquitinated proteins. In this project, we also isolated NEDD8-modified proteins through the immobilized metal affinity chromatography and identified them by mass spectrometry.Methods: We generated nine NEDD8 point mutants, each with one lysine replaced by an arginine. The effect of the lysine mutation on neddylation was examined by immunoblotting. We coexpressed wild-type(wt) and mutant NEDD8 with two subunits of the NEDD8-activating enzyme complex(NAE1 and Uba3) and NEDD8-conjugating enzyme UBE2 M. The effect of the lysine residues on the activation and conjugation of NEDD8 was detected by immunoblotting. We coexpressed wt and mutant NEDD8 with two cullin family members and the effect of the lysine mutation on neddylation and ubiqutination of the cullins was detected by immunoblotting after the purification of cullins. We coexpressed wt or mutant NEDD8 with cullin family members, a CRL E3 ligase substrate, and studied the effect of the lysine mutation on the function of the CRL E3 substrates through affinity purification and immunoblotting. We overexpressed His6-NEDD8, isolated NEDD8-modified proteins through the immobilized metal affinity chromatography and then digested them for proteomic identification.Results:(1) K27 R NEDD8 mutant almost completely abolishes proteins neddylation.(2) The NEDD8 activation is not significantly altered by the NEDD8 mutation.(3) K27 R NEDD8 mutant impairs the NEDD8 conjugation by forming stable covalent bond(s) with the E2 enzyme.(4) K27 R mutant prevents cullins from neddylation.(5) K27 R mutant prevents cullins from ubiquitination.(6) K27 R NEDD8 mutant reduces ubiquitination of a CRL E3 ligase substrate, IkBa.(7) In total 84 proteins were identified as potential NEDD8-modified proteins by mass spectrometry. Among them 67 are new potential NEDD8-modified proteins such as regulator of chromosome condensation 2.Conclusions: The critical lysine residue K27 on NEDD8 significantly alters the neddylation cascades. The K27 R NEDD8 mutant is unable to form a thioester bond with the active cysteine of NEDD8 E2 for subsequent neddylation. Instead, this mutant forms stable covalent bond(s) with NEDD8 E2. Expression of the K27 R NEDD8 mutant almost completely hampers cullins neddylation and ubiquitination. The reduction of cullins neddylation by this mutant significantly decreases the activity of cullin-associated E3 ligases. K27 R NEDD8 could serve as a useful dominant negative mutant in investigating the biological functions of protein neddylation. Identification of potential NEDD8-modified proteins indicates that NEDD8 may be involved in some important physiological processes, such as cell cycle.