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

Cell Rep. 2018 Sep 18;24(12):3312-3323.e5. doi: 10.1016/j.celrep.2018.08.061.


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 ubiquitin-mediated 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.

Keywords: GPCR; HECT; NEDD4; c-Src; endothelial barrier permeability; p38 MAPK; protease-activated receptor; thrombin; ubiquitin.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Capillary Permeability
  • Human Umbilical Vein Endothelial Cells / metabolism
  • Humans
  • Nedd4 Ubiquitin Protein Ligases / chemistry*
  • Nedd4 Ubiquitin Protein Ligases / genetics
  • Nedd4 Ubiquitin Protein Ligases / metabolism
  • Protein Domains
  • Receptor, PAR-1 / metabolism*
  • Receptors, Purinergic P2Y1 / metabolism
  • Signal Transduction*
  • Tyrosine / genetics
  • Tyrosine / metabolism
  • p38 Mitogen-Activated Protein Kinases / metabolism
  • src-Family Kinases / metabolism


  • Receptor, PAR-1
  • Receptors, Purinergic P2Y1
  • Tyrosine
  • Nedd4 Ubiquitin Protein Ligases
  • Nedd4L protein, human
  • src-Family Kinases
  • p38 Mitogen-Activated Protein Kinases