Role of Nck and Crk adapter proteins in the VEGF promoted endothelial cell migration

Abstract

Vascular Endothelial Growth Factor (VEGF) is an important regulator of angiogenesis in both normal and pathological processes. The signaling mechanisms by which VEGF exerts its effects on vascular endothelial cells are poorly understood. Here we describe mechanisms by which KDR mediates endothelial cell migration.;We found that in HUVE cells the Nck and Crk adapter proteins associate with KDR after VEGF treatment. Nck is constitutively associated with p21-activated kinase PAK. VEGF augments PAK catalytic activity and induces the translocation of PAK to focal adhesions where it co-localizes with focal adhesion kinase FAK. Both Nck and PAK co-immunoprecipitate with FAK in VEGF stimulated cells. HUVE cells transfected with anti-Nck oligonucleotides display reduced motility and focal adhesion formation in response to VEGF.;In HUVE cells Crk is constitutively associated with the Rap1 exchanging factor C3G and VEGF induces redistribution of C3G to the cell membrane. Introduction of the Crk or Nck SH2 domain fusions with Drosophila antennapedia peptide into HUVE cells resulted in the formation of enlarged focal adhesions and cell detachment. Moreover, Nck and Crk AP fusions prevented VEGF mediated integrin activation. HUVE cells transfection with an AP fusion of a PAK-derived peptide responsible for Nck binding promoted formation of enlarged focal adhesions but had no effect on integrin activation. Expression in HUVE cells of a dominant negative Rap1 mutant led to both inhibition of VEGF induced integrin activation and the formation of enlarged focal adhesions.;Mutation of the KDR phosphorylation sites did not abolish Crk or Nck phosphorylation or association with receptor. However, an adapter protein FRS2 was found to immunoprecipitate with Nck, Crk and PAK in response to VEGF stimulation. FRS2 is constitutively associated with KDR and undergoes rapid tyrosine phosphorylation in response to VEGF.;Based on our results we conclude that both Nck and Crk participate in the signaling pathways, which are necessary for VEGF promoted endothelial cell migration. Particularly, they are responsible for the regulation of integrins activation and focal adhesion turnover. Their interaction with KDR is indirect and mediated by another adapter protein-FRS2.