Rac1 acts in conjunction with Nedd4 and Dishevelled-1 to promote maturation of cell-cell contacts

Abstract

SummaryThe Rho-GTPase Rac1 promotes actin polymerization and membrane protrusion that mediate initial contact and subsequent maturation of cell-cell junctions. Here we report that Rac1 associates with the ubiquitin-protein ligase neural precursor cell expressed developmentally down-regulated 4 (Nedd4). This interaction requires the hypervariable C-terminal domain of Rac1 and the WW domains of Nedd4. Activated Rac1 colocalises with endogenous Nedd4 at epithelial cell-cell contacts. Reduction of Nedd4 expression by shRNA results in reduced transepithelial electrical resistance (TER) and concomitant changes in the distribution of adherens and tight junction markers. Conversely, expression of Nedd4 promotes TER, suggesting that Nedd4 cooperates with Rac1 in the induction of junctional maturation. We found that Nedd4, but not Nedd4-2, mediates the ubiquitylation and degradation of the adapter protein dishevelled-1 (Dvl1), the expression of which negatively regulates cell-cell contact. Nedd4-mediated ubiquitylation requires its binding to the C-terminal domain of Dvl1, comprising the DEP domain, and targets an N-terminal lysine-rich region upstream of the Dvl1 DIX domain. We found that endogenous Rac1 colocalises with endogenous Dvl1 in intracellular puncta as well as on cell-cell junctions. Finally, activated Rac1 was found to stimulate Nedd4 activity, resulting in increased ubiquitylation of Dvl1. Together, these data reveal a novel Rac1-dependent signalling pathway that, through Nedd4-mediated ubiquitylation of Dvl1, stimulates the maturation of epithelial cell-cell contacts.

The Rac1 hypervariable region in targeting and signaling

“…Expression of an inactive Rac1N17 mutant promoted tubule formation, whereas an activated Rac1V12 mutant reduced tubule formation. These findings are identical to results from our ubiquitylation and degradation of the adaptor protein Dishevelled (Dvl1) 75 (Fig. 4).…”

“…74 Notably, efficient interaction of K-Ras4B with calmodulin was nucleotidedependent and required the catalytic region of the GTPase. This co-operativity between the hypervariable C-terminus and the N-terminal effector domains in K-Ras4B is again similar to the interactions described for Rac1 and PRK, 15 for Rac1 and Nedd4 75 and may also apply to the interaction Rac1 and calmodulin. The functional consequences of Rac1 interacting with calmodulin were analyzed for the Rac1-PIP5K interaction.…”

“…103 In line with this latter finding, our lab showed that Nedd4 is required for the formation of stable cell-cell contacts in epithelial HeLa cells, which is a Rac1-dependent process. 75 Nedd4 binds, via its WW domains, to the HVR of Rac1 and this interaction is largely nucleotide-independent, although we detected increased interaction of Nedd4 with an activated mutant of Rac1.…”

“…However, Rac1 is not ubiquitylated by Nedd4. 75,93 Nedd4 has been implicated in various physiological processes including heart development, 101 adaptive immunity 102 and development of the neuromuscular junction, a specialized form of cell-cell contact. 103 In line with this latter finding, our lab showed that Nedd4 is required for the formation of stable cell-cell contacts in epithelial HeLa cells, which is a Rac1-dependent process.…”

Peptide and small molecule inhibitors of HECT-type ubiquitin ligases

“…We next tested it in an assay for the ubiquitination and degradation of the Dishevelled protein (Dvl). Dvl contains a PY motif and is a substrate for several members of the Nedd4 family (22)(23)(24)(25), which bind PY motifs via their WW domains. This was readily demonstrated by coexpression of Dvl2 with Smurf2 and other ligases.…”