Promotion of PDGF-induced endothelial cell migration by phosphorylated VASP depends on PKA anchoring via AKAP

【摘要】：正Vasodilator-stimulated phosphoprotein(VASP),an important substrate of PKA,plays a critical role in VASP remodeling of actin cytoskeleton and actin-based cell motility.However,how PKA accurately transfers extracellular signals to VASP and then how phosphorylation of VASP regulates endothelial cell migration have not been clearly defined.Protein kinase A anchoring proteins(AKAPs) are considered to regulate intracellular-specific signal targeting of PKA via AKAP-mediated PKA anchoring.Thus,our study investi-gated the relationship among AKAP anchoring of PKA,PKA activity,and VASP phosphorylation,which is to clarify the exact role of VASP and its upstream regulatory mechanism in PKA-dependent migration.Our results show that chemotactic factor PDGF activated PKA,increased phosphorylation of VASP at Serl57,and enhanced ECV304 endothelial cell migration.However,phosphorylation site-directed mutation of VASP at Ser157 attenuated the chemotactic effect of PDGF on endothelial cells,suggesting phosphorylation of VASP at Serl57 promotes PKA-mediated endothelial cell migratioa Furthermore,disrupting PKA anchoring to AKAP or PKA activity significantly attenuated the PKA activity,VASP phosphorylation,and subsequent cell migratioa Meanwhile,disrupting PKA anchoring to AKAP abolished PDGF-induced lamellipodia formation and special VASP accumu-lation at leading edge of lamellipodia. These results indicate that PKA activation and PKA-mediated substrate responses in VASP phosphorylation and localization depend on PKA anchoring via AKAP in PDGF-induced endothelial cell migratioa In conclusion, AKAP anchoring of PKA is an essential upstream event in regulation of PKA-mediated VASP phosphorylation and subsequent endothelial cell migration,which contributes to explore new methods for controlling endothelial cell migration related diseases and angiogenesis.