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

Abstract

SUMMARYUbiquitination 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 P2Y1 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.

G protein–coupled receptors activate p38 MAPK via a non-canonical TAB1–TAB2– and TAB1–TAB3–dependent pathway in endothelial cells

“…However, in most cases it remains unclear how NEDD4 E3 ligase activity is regulated to facilitate GPCR ubiquitination. In recent work, we showed that thrombin activation of PAR1 stimulates c-Src-mediated tyrosine phosphorylation and activation of NEDD4-2 to promote p38 signaling and endothelial barrier disruption (43). Moreover, the P2Y 1 also required c-Src and NEDD4-2 tyrosine phosphorylation for p38 activation in endothelial cells (43).…”

“…In recent work, we showed that thrombin activation of PAR1 stimulates c-Src-mediated tyrosine phosphorylation and activation of NEDD4-2 to promote p38 signaling and endothelial barrier disruption (43). Moreover, the P2Y 1 also required c-Src and NEDD4-2 tyrosine phosphorylation for p38 activation in endothelial cells (43). However, it remains to be determined whether other endothelial GPCR agonists utilize a conserved c-Src-dependent mechanism to control NEDD4 E3 ligase activity similarly to promote p38 MAPK-induced inflammatory responses in endothelial cells.…”

YAP/TAZ: Drivers of Tumor Growth, Metastasis, and Resistance to Therapy

“…[ 228 ] As noted above, how Rho activation leads to inhibition of LATS1/2 is still unclear, but could involve increased tension on Integrin adhesions and thus FAK‐SRC signaling. In addition, classic GPCR signaling studies implicate SRC family kinases as key downstream effectors, particularly in cross‐talk between GPCR and growth factor signaling [ 220,221,229‐242 ] and cytokine signaling. [ 196,229 ] Consistent with this view, in uveal melanoma, mutations in G q require FAK activation to activate YAP.…”

Endo‐lysosomal sorting of G‐protein‐coupled receptors by ubiquitin: Diverse pathways for G‐protein‐coupled receptor destruction and beyond

“…We previously showed that phosphorylation of PAR1 is required for agonist-stimulated ubiquitination. 34 In recent work, we further demonstrated that NEDD4-2 E3 ligase is rapidly recruited to and ubiquitinates PAR1 following ligand stimulation 28,63 (Figure 2C). However, it is not known if phosphorylation of PAR1 mediates recruitment of "active" NEDD4-2 to the receptor or enables PAR1 internalization and localization to endocytic vesicles containing "active" NEDD4-2 to facilitate substrate-enzyme interaction and ubiquitination.…”

“…It is also possible that GPCR signaling triggers NEDD4‐2 catalytic activity and was recently examined for PAR1 and the P2Y 1 receptor. Unexpectedly, we discovered that PAR1 stimulates c‐Src‐mediated tyrosine phosphorylation and activation of NEDD4‐2, which promotes p38 inflammatory signaling (Figure C). Intriguingly, c‐Src is activated by PAR1 through a G q and G 12/13 ‐dependent pathway.…”