Fibroblast Growth Factor‐2‐Induced Endothelial Cell Migration Is Regulated by Junctional Adhesion Molecule‐A Through Its Tyrosine Phosphorylation and Interaction With Afadin

Abstract

Neovascularization, or angiogenesis, plays an important role in tumor growth and ischemic diseases. Fibroblast growth factor‐2 (FGF‐2) is a major angiogenic growth factor. We have previously shown that FGF‐2‐induced angiogenesis requires junctional adhesion molecule‐A (JAM‐A). However, the signaling mechanism through which JAM‐A regulates the FGF‐2‐induced signaling pathway is currently unknown. To assess the role of JAM‐A in FGF‐2 signaling, we generated various JAM‐A mutants as well as Jam‐A null ECs and performed cell adhesion and migration assays, western blot analysis, co‐immunoprecipitation assays, and proximity ligation assays. Here we show that FGF‐2‐induced endothelial cell (EC) adhesion and migration on vitronectin is dependent on JAM‐A. FGF‐2‐induced ERK1/2 activation and Rap1 activation in EC also requires the presence of JAM‐A. We show JAM‐A to be phosphorylated on serine 284 residue in its cytoplasmic tail and that it associates with EC integrin avβ3 and CD9 in unstimulated conditions. However, upon FGF‐2 stimulation, JAM‐A gets phosphorylated on tyrosine 280, which is accompanied by dephosphorylation of phosphoserine 284 and dissociation from the integrin avβ3/CD9 complex. We also found that JAM‐A associates with PDZ domain‐containing protein zona occludens‐1 (ZO‐1) in resting conditions but dissociates from ZO‐1 and associates with afadin (AF‐6) through its PDZ‐domain binding motif present in its cytoplasmic tail upon FGF‐2 treatment. To test the role of specific residues of JAM‐A involved in regulating FGF‐2‐induced cell migration, we ectopically expressed various mutants of JAM‐A in human umbilical vein endothelial cells (HUVECs). Expression of PDZ domain‐binding motif‐deleted mutant JAM‐A showed significantly attenuated FGF‐2‐induced Rap1 activation, ERK1/2 activation, as well as cell migration. We found that tyrosine 280 phosphorylated JAM‐A associates with C‐terminal Src kinase (CSK) upon FGF‐2 stimulation and this association is found to be through the SH2 domain of CSK. Ectopic expression of JAM‐A tyrosine 280 to phenylalanine substitution mutant resulted in abrogation of FGF‐2 induced cell migration. Our results suggest that FGF‐2‐induced signaling in ECs is dependent on the tyrosine 280 residue and the PDZ domain‐binding motif of JAM‐A. This regulates its interaction with signaling proteins such as AF6 to regulate Rap1 and ERK1/2 activation induced by FGF‐2 which subsequently leads to EC migration and angiogenesis.Support or Funding InformationNHLBI/NIH 2R01 HL119374‐06 to UPN.