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

VOGL, ANNETTE M.; PHU, LILIAN; BECERRA, RAQUEL; GIUSTI, SEBASTIAN A.; VERSCHUEREN, ERIK; HINKLE, TRENT B.; BORDENAVE, MARTÍN D.; ADRIAN, MAX; HEIDERSBACH, AMY; YANKILEVICH, PATRICIO; STEFANI, FERNANDO D.; WURST, WOLFGANG; HOOGENRAAD, CASPER C.; KIRKPATRICK, DONALD S.; REFOJO, DAMIAN; SHENG, MORGAN

Nature Structural and Molecular Biology

Nature Research

Lugar: Londres; Año: 2020 vol. 27 p. 210 - 220

1545-9993

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.