A Tyrosine Switch on NEDD4-2 E3 Ligase Transmits GPCR Inflammatory Signaling

Ubiquitination is essential for protein degradation and signaling and pivotal to many physiological processes. Ubiquitination of a subset of G-protein-coupled receptors (GPCRs) by the E3 ligase NEDD4–2 is required for p38 activation, but how GPCRs activate NEDD4–2 to promote ubiquitinmediated signaling is not known. Here, we report that the GPCR protease-activated receptor-1 (PAR1) stimulates c-Src-mediated tyrosine phosphorylation and activation of NEDD4–2 to promote p38 signaling and endothelial barrier disruption. Using mass spectrometry, we identified a unique phosphorylated tyrosine (Y)-485 within the 2,3-linker peptide between WW domain 2 and 3 of NEDD4–2 in agonist-stimulated cells. Mutation of NEDD4–2 Y485 impaired E3 ligase activity and failed to rescue PAR1-stimulated p38 activation and endothelial barrier permeability. The purinergic P2Y(1) receptor also required c-Src and NEDD4–2 tyrosine phosphorylation for p38 activation. These studies reveal a novel role for c-Src in GPCR-induced NEDD4–2 activation, which is critical for driving ubiquitin-mediated p38 inflammatory signaling.