Global ubiquitinome profiling identifies NEDD4 as a regulator of Profilin 1 and actin remodeling in neural crest cells

Neural crest cell (NCC) development occurs through the regulation of key transcription factors and its downstream effector genes.  However, recent studies suggest additional regulatory mechanisms such as post-translational modifications also have important roles; specifically, Schwarz and colleagues identified the ubiquitin ligase, NEDD4, as an important factor that NCCs utilize to control their development.  In this recent article, the group deciphered the mechanisms by which NEDD4 ubiquitination controls NCC development.  Preliminary ubiquitinome studies were performed and they found 276 proteins had altered levels of ubiquitination events (259 decreased, 17 increased) when NEDD4 was depleted.   Interestingly, 19 of these NEDD4 targets have established roles in regulating the actin cytoskeleton.  Through validation studies, they identified profilin 1 as a key target that is ubiquitinated by NEDD4. In NEDD4 CRISPR knockout cells they saw a modest but significant decrease in profilin 1 ubiquitination and a subsequent increase in profilin 1 levels. Cycloheximide chase assays and proteosome and lysosome inhibitors were used to confirm that the changes in profilin were due to conventional ubiquitination mechanisms.  Quantitative imaging studies were performed in vitro and in vivo to show NEDD4 regulates actin levels in NCCs.  A mild knockdown of profilin 1 was used to show that it was responsible for the NEDD4 knockout-induced changes in actin.  Collectively, these data identify critical players that mediate NEDD4’s ability to control morphological changes observed in NCCs. Cytoskeleton Inc’s G-Actin/F-Actin In Vivo Assay Biochem Kit (Cat. #BK037) was essential to measure levels of G-actin and F-actin in response to profilin 1 in NEDD4 knockout cells.