Global site-specific neddylation profiling reveals that NEDDylated cofilin regulates actin dynamics

Nat Struct Mol Biol. 2020 Feb;27(2):210-220. doi: 10.1038/s41594-019-0370-3. Epub 2020 Feb 3.


Neddylation is the post-translational protein modification most closely related to ubiquitination. Whereas the ubiquitin-like protein NEDD8 is well studied for its role in activating cullin-RING E3 ubiquitin ligases, little is known about other substrates. We developed serial NEDD8-ubiquitin substrate profiling (sNUSP), a method that employs NEDD8 R74K knock-in HEK293 cells, allowing discrimination of endogenous NEDD8- and ubiquitin-modification sites by MS after Lys-C digestion and K-εGG-peptide enrichment. Using sNUSP, we identified 607 neddylation sites dynamically regulated by the neddylation inhibitor MLN4924 and the de-neddylating enzyme NEDP1, implying that many non-cullin proteins are neddylated. Among the candidates, we characterized lysine 112 of the actin regulator cofilin as a novel neddylation event. Global inhibition of neddylation in developing neurons leads to cytoskeletal defects, altered actin dynamics and neurite growth impairments, whereas site-specific neddylation of cofilin at K112 regulates neurite outgrowth, suggesting that cofilin neddylation contributes to the regulation of neuronal actin organization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism*
  • Animals
  • Cell Line
  • Cells, Cultured
  • Cofilin 1 / metabolism*
  • Gene Knock-In Techniques
  • HEK293 Cells
  • Humans
  • Mice
  • Mice, Inbred C57BL
  • NEDD8 Protein / genetics
  • NEDD8 Protein / metabolism*
  • Neurons / cytology
  • Neurons / metabolism*
  • Point Mutation
  • Rats
  • Rats, Sprague-Dawley
  • Ubiquitin / metabolism
  • Ubiquitination


  • Actins
  • Cofilin 1
  • NEDD8 Protein
  • Ubiquitin