Requirement of protein kinase D tyrosine phosphorylation for VEGF-A165-induced angiogenesis through its interaction and regulation of phospholipase Cγ phosphorylation

Vascular endothelial cell growth factor-A(165)(VEGF-A(165)) is critical for angiogenesis. Although protein kinase C-mediated protein kinase D(PKD) activation was implicated in the response, the detailed mechanism remains unclear. In this study, we found that VEGF-A(165)-stimulated tyrosine phosphorylation of PKD and the dominant negative mutant of PKD, PKD(Y463F), inhibited VEGF-A(165)-induced human umbilical vein endothelial cell ( HUVEC) proliferation. In addition, PKD(S738A/S742A) overexpression inhibited VEGF-induced HUVEC migration. Furthermore, knockdown of PKD by its specific small interfering RNA inhibited VEGF-induced HUVEC proliferation and migration. Moreover transfection of PKD(Y463F), PKD(S738A/S742A), or PKD-small interfering RNA blocked VEGF-induced angiogenesis in vivo. Our signaling experiments show that KDR not Flt-1 mediated PKD tyrosine phosphorylation and KDR tyrosine residues 951 and 1059 were required for VEGF-A(165)-stimulated PKD serine and tyrosine phosphorylation, respectively. Whereas G protein G beta gamma subunits were required for both PKD serine phosphorylation and tyrosine phosphorylation, intracellular Ca2+ mobilization was required for VEGF-A(165)-stimulated PKD tyrosine phosphorylation and phospholipase C (PLC) activity was required for PKD serine phosphorylation. Surprisingly, the PLC inhibitor did not inhibit PKD tyrosine phosphorylation. Instead, PKD tyrosine 463 was required for VEGF-A(165)-stimulated PLC gamma tyrosine phosphorylation. Moreover, PKD interacted with PLC gamma even in unstimulated cells, and PKD tyrosine 463 phosphorylation was not required for this interaction. Together, we demonstrate that PKD interacts with PLC gamma and becomes tyrosine phosphorylated upon VEGF stimulation, leading to PLC gamma activation and angiogenic response of VEGF-A(165).