Interactome of S65 phosphorylated NEDD8 and Ub

In this project we present for the first time a proteome-wide interaction study of S65 phosphorylated NEDD8 and ubiquitin (Ub). By using cell extracts and affinity enrichment coupled to mass spectrometry, we show that pNEDD8 and pUb have different interactomes that are also distinct from those of the non-modified forms. Among the preferential pNEDD8 interactors are HSP70 family members and, indeed, pNEDD8 stimulates HSP70 ATPase activity more pronouncedly than non-modified NEDD8. Our findings provide evidence that phosphorylation of Ub and NEDD8 at S65 is involved in stress response signaling and underscore the importance of studying non-covalent interactions of NEDD8 in particular and post-translational modifications of Ubls in general.