Nck and Crk mediate distinct VEGF-induced signaling pathways that serve overlapping functions in focal adhesion turnover and integrin activation.

We have asked whether the Nck and Crk adaptor proteins play important roles in the vascular endothelial growth factor (VEGF)-induced signaling pathways that lead to an enhancement in cell migration. The introduction into human umbilical vein endothelial cells of a dominant-negative inhibitor for either Nck or Crk blocked the recruitment of both endogenous proteins to the KDR VEGF receptor subtype indicating that both proteins are recruited to the same docking site. The Nck and Crk dominant-negatives led to the formation of abnormally large focal adhesion, blocked VEGF-induced integrin activation, and blocked VEGF-induced actin dynamics. The dominant-negatives had no effects on these properties in cells expressing constitutively active Rac1 or RhoA. Since a DN to either Nck or Crk blocks the cellular responses mediated by both proteins, we performed experiments directed at clarifying signaling pathways specifically mediated by each protein. Inhibition of the interaction between Nck with its downstream effector PAK led to abnormally large focal adhesions, but had no effect on integrin activation or cell adhesiveness. Evidence is presented that Crk complexes with C3G in control cells, and VEGF treatment leads to the recruitment of the complex to the cell surface. Inhibition of the C3G downstream effector Rap1 leads to enlarged focal adhesions and blocks VEGF-induced integrin activation. We conclude that Nck and Crk mediate distinct VEGF-induced signaling pathways that serve overlapping functions in cell migration.