NCK and PAK participate in the signaling pathway by which vascular endothelial growth factor stimulates the assembly of focal adhesions

Vascular endothelial growth factor (VEGF)-induced endothelial cell migratio n 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 inv estigated the signaling pathway by which VEGF regulates focal adhesion comp lex assembly by examining the signaling proteins involved. VEGF stimulated the tyrosine phosphorylation of the SH2 domain containing signaling protein s NCK and CRK in human umbilical vein endothelial cells. The signaling path ways that couple the kinase insert domain-containing receptor to NCK and CR K is most likely mediated by another cellular protein, as NCK and CRK were tyrosine phosphorylated in response to VECF in cells expressing receptors m utated at each of several candidate SH2 domain-interacting cytosolic tyrosi nes, In the absence of VEGF treatment, NCK (but not CRK) associated with th e p21 GTPase-activated kinase PAK. PAK catalytic activity was augmented aft er VEGF treatment; an association of PAK with 60- and 90-kDa tyrosine-phosp horylated proteins accompanied this. VEGF stimulated the recruitment of PAK to focal adhesions, and FAK immunoprecipitated with both NCK and PAK in VE GF-treated (but not untreated) human umbilical vein endo thelial cells. Inh ibition of NCK protein expression using antisense oligonucleotides led to t he inhibition of both VEGF-induced focal adhesion assembly and VEGF-induced cell migration, demonstrating a necessary role of NCK in these cellular re sponses.