Journal of Biological Chemistry

The ubiquitin ligase Nedd4-2/NEDD4L, comprised of C2-WW(x4)-HECT domains, is known to regulate several ion transporters and channels. We recently showed that elevated intracellular [Na+] and osmolarity enhances Nedd4-2 enzymatic activity. To globally identify its interactome and substrates in cells under hyperosmotic stress, we performed a BioID screen using miniTurbo with Nedd4-2 as a bait under hyperosmotic (vs. isosmotic) conditions. One of the top hits identified that preferentially binds Nedd4-2 under hyperosmolarity was Dynamin Binding Protein (DNMBP)/Tuba, a known GEF for Cdc42. We then showed that DNMBP is a substrate for Nedd4-2, and that active Nedd4-2 targets DNMBP to P-body condensates under hyperosmotic stress. Moreover, DNMBP itself promotes P-body formation under hyperosmolarity. Both Nedd4-2 and DNMBP are required for the activation of Cdc42 following hyperosmotic treatment, and accordingly, knockout of DNMBP results in suppression of Cdc42 and its downstream effector p38-MAPK. We thus propose that Nedd4-2–mediated targeting of DNMBP to P-bodies under hyperosmotic stress facilitates the activation of Cdc42 by this GEF.

NEDD4L

dynamin-binding protein

condensates

osmolarity

4′,6-diamidino-2-phenylindole

mRNA-Decapping Enzyme1

Dulbecco's Modified Eagle Medium

dynamin binding protein (also known as Tuba)

Early Endosome Antigen 1

Epithelial Na+ Channel

Guanine nucleotide Exchange Factors

Homologous to the E6-AP Carboxyl Terminus

Human Embryonic Kidney cells

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

Neural precursor cell Expressed Developmentally Downregulated 4 - 2

Trans-Golgi Network protein 46

© 2025 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biologyé