NCK and PAK Participate in the Signaling Pathway by Which Vascular Endothelial Growth Factor Stimulates the Assembly of Focal Adhesions

Bruce I. Terman,Simone C. Spring,Konstantin V. Stoletov,Kirsty E. Ratcliffe

doi:10.1074/jbc.m009720200

Bruce I. Terman, Simone C. Spring + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m009720200

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Abstract

Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is a key step in the angiogenic response and is mediated, in part, by an accelerated rate of focal adhesion complex assembly and disassembly. We investigated the signaling pathway by which VEGF regulates focal adhesion complex assembly by examining the signaling proteins involved. VEGF stimulated the tyrosine phosphorylation of the SH2 domain-containing signaling proteins NCK and CRK in human umbilical vein endothelial cells. The signaling pathways that couple the kinase insert domain-containing receptor to NCK and CRK is most likely mediated by another cellular protein, as NCK and CRK were tyrosine-phosphorylated in response to VEGF in cells expressing receptors mutated at each of several candidate SH2 domain-interacting cytosolic tyrosines. In the absence of VEGF treatment, NCK (but not CRK) associated with the p21 GTPase-activated kinase PAK. PAK catalytic activity was augmented after VEGF treatment; an association of PAK with 60- and 90-kDa tyrosine-phosphorylated proteins accompanied this. VEGF stimulated the recruitment of PAK to focal adhesions, and FAK immunoprecipitated with both NCK and PAK in VEGF-treated (but not untreated) human umbilical vein endothelial cells. Inhibition of NCK protein expression using antisense oligonucleotides led to the inhibition of both VEGF-induced focal adhesion assembly and VEGF-induced cell migration, demonstrating a necessary role of NCK in these cellular responses.

Highlights

Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is a key step in the angiogenic response and is mediated, in part, by an accelerated rate of focal adhesion complex assembly and disassembly
Both receptors possess insert sequences within their catalytic domains and seven immunoglobulin-like domains in the extracellular regions and are related to the platelet-derived growth factor family of receptor tyrosine kinases. Expression of both VEGF receptor types occurs in adult endothelial cells, including human umbilical vein endothelial (HUVE) cells, recent findings suggest that kinase insert domain-containing receptor (KDR), and not Flt-1, is able to mediate the mitogenic and chemotactic effects of VEGF in endothelial cells [36]
We describe a signal transduction pathway that couples VEGF binding to its receptor with focal adhesion formation and cell migration

Summary

Introduction

Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is a key step in the angiogenic response and is mediated, in part, by an accelerated rate of focal adhesion complex assembly and disassembly. VEGF stimulated the tyrosine phosphorylation of the SH2 domain-containing signaling proteins NCK and CRK in human umbilical vein endothelial cells. Both receptors possess insert sequences within their catalytic domains and seven immunoglobulin-like domains in the extracellular regions and are related to the platelet-derived growth factor family of receptor tyrosine kinases Expression of both VEGF receptor types occurs in adult endothelial cells, including human umbilical vein endothelial (HUVE) cells, recent findings suggest that KDR, and not Flt-1, is able to mediate the mitogenic and chemotactic effects of VEGF in endothelial cells [36]. In porcine aortic endothelial cells transfected with KDR and Flt-1, VEGF has no effect on phosphatidylinositol 3-kinase activity and only a weak effect on p120GAP tyrosine phosphorylation [39]

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