Bmx is a downstream Rap1 effector in VEGF-induced endothelial cell activation.

We had previously shown that Rap1 mediates certain of the signaling pathways involved in VEGF-induced endothelial cell migration, although the downstream Rap1 effectors are not known. Towards the goal of identifying those effectors, we utilized a commercially available antibody array filter to identify proteins that either directly interact with Rap1 or interact indirectly through a multi-protein complex. The protocol identified 10 possible Rap1-interacting proteins, including the Bmx non-receptor tyrosine kinase. The conclusion that VEGF treatment leads to a Rap1/Bmx complex was confirmed by an experiment in which cell lysates from VEGF and control cells were immunoprecipitated with Bmx antibodies and Western blotting was done using anti-Rap1 antibodies. VEGF treatment led to the recruitment of Bmx to the CAS scaffolding protein, and inhibition of the Bmx kinase blocked VEGF-induced cell migration. Formation of a Rap1/Bmx complex was not observed in cells transfected with an expression vector for a dominant-negative Rap1, indicating that Bmx is a downstream Rap1 effector in VEGF-induced endothelial cell activation.

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.

VEGF treatment induces signaling pathways that regulate both actin polymerization and depolymerization.

The angiogenic growth factor vascular endothelial growth factor (VEGF) enhances endothelial cell migration through the activation of multiple signaling transduction pathways. Actin reorganization is an important component in VEGF-induced migration, yet the signaling pathways mediating this process remain unclear. Actin reorganization involves both actin polymerization and depolymerization, and in this study we demonstrate that VEGF-treatment regulates both of these activities. With respect to actin polymerization, our results indicate that the actin nucleation promoting factors (NPF) neural Wiskott-Aldrich syndrome protein (N-WASP) binds the SH2- plus SH3-domain containing adaptor protein Nck in both control and VEGF-treated cells. We had previously showed that VEGF treatment leads to the recruitment of Nck to activated receptor, and our current results indicate a VEGF-dependent redistribution of N-WASP to the cell surface. A Nck dominant-negative blocked Nck recruitment to receptor, blocked N-WASP cellular redistribution and attenuated actin stress fiber formation. With respect to actin depolymerization, VEGF-treatment led to the rapid phosphorylation of the actin depolymerization factor cofilin, and its upstream regulator, LIM-kinase (LIMK). Unlike what is observed in certain other cell types, the p21-activated kinase (PAK), a Nck binding protein, does not mediate VEGF-induced LIMK phosphorylation, as a PAK dominant-negative had no effect on this activity. The PAK dominant-negative also did not affect VEGF-induced actin reorganization. Pharmacological inhibitors of phosphoinositide-3 kinase (PI3-K) and the rho-activated kinase (ROCK) attenuated VEGF-induced LIMK phosphorylation, indicating a role for (PI3-K) and ROCK in the signaling pathways leading to regulation of LIMK activity.

Sck is expressed in endothelial cells and participates in vascular endothelial growth factor-induced signaling.

Sck, a member of the Shc family of cell signaling proteins, has only been studied in neuronal cells, though previous studies have demonstrated its expression in tissues other than brain. Using RT-PCR and RNase protection assays, we detected Sck mRNA expression in endothelial cells, and Sck protein was detected by Western blotting using polyclonal and monoclonal antibodies targeting the Sck CH1 domain. Immunohistochemistry protocols demonstrate that Sck is expressed in KDR and PECAM positive cells found in the mouse retina, mouse heart and human umbilical chord. Treatment of human umbilical vein endothelial (HUVE) cells with vascular endothelial growth factor (VEGF) leads to the recruitment of Sck to the KDR VEGF receptor and an enhanced Sck tyrosine phosphorylation. Sck is recruited to KDR tyrosine 1175, as co-immunoprecipitation of KDR and Sck is not observed in VEGF-treated porcine aortic endothelial cells expressing a receptor mutated at this autophosphorylation site. The Sck and Shc SH2 domains, and not the PTB domain, mediates its interactions with KDR, as recombinant Sck SH2 domain binds to a tyrosine phosphorylated KDR 1175-derived synthetic peptide, but not to a peptide synthesized without tyrosine phosphate. Recombinant PLCgamma SH2 domain also interacts with the phosphotyrosine 1175 containing peptide. VEGF-induced MAPK activation is dependent upon PLCgamma activity, and chimeric proteins consisting of the Shc or Sck SH2 domains fused with a cellular internalization sequence attenuated this activation. Taken together, these results demonstrate that Sck is expressed in vascular endothelial cells, and participates in VEGF-induced signal transduction.

Fibroblast growth factor receptor substrate 2 participates in vascular endothelial growth factor-induced signaling.

Vascular endothelial growth factor (VEGF) activates endothelial cells, in part, by interacting with the kinase insert domain-containing receptor (KDR) receptor tyrosine kinase. Although progress has been made in the identification of cell-signaling proteins that participate in the VEGF-induced response, questions remain concerning the molecular interactions that allow coupling of receptor activation with an increased cellular response. Evidence is provided in this manuscript that indicates a role for the fibroblast growth factor receptor substrate 2 (FRS2) in VEGF-induced signal transduction. VEGF treatment of human umbilical vein endothelial cells (HUVECs) and KDR-transfected porcine aortic endothelial cells leads to the rapid tyrosine phosphorylation of FRS2. FRS2 is associated constitutively with KDR, and VEGF treatment has no effect on this interaction. VEGF treatment of KDR-expressing cells leads to the recruitment of Nck, p21-activated kinase, Crk, Grb2, and protein kinase C l to FRS2. The ability of FRS2 to recruit cell-signaling proteins to the cell is significant because it provides a mechanism for enhancing the repertoire of VEGF-induced signaling pathways.