Role of protein kinase Czeta in Ras-mediated transcriptional activation of vascular permeability factor/vascular...

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), a multifunctional cytokine, is regulated by different factors including degree of cell differentiation, hypoxia, and certain oncogenes namely, ras and src. The up-regulation of VPF/VEGF expression by Ras has been found to be through both transcription and mRNA stability. The present study investigates a novel pathway whereby Ras promotes the transcription of VPF/VEGF by activating protein kinase Czeta (PKCzeta). The Ras-mediated overexpression of VPF/VEGF was also found to be inhibited by using the antisense or the dominant-negative mutant of PKCzeta. In co-transfection assays, by overexpressing oncogenic Ha-Ras (12 V) and PKCzeta, there was an additive effect up to 4-fold in activation of Sp1-mediated VPF/VEGF transcription. It has been shown through electrophoretic mobility shift assay that Ras promoted the PKCzeta-induced binding of Sp1 to the VPF/VEGF promoter. In the presence of PDK-1, a major activating kinase for PKC, the Ras-mediated activation of VPF/VEGF promoter through PKCzeta was further increased, suggesting that PKCzeta can serve as an effector for both Ras and PDK-1. In other experiments, with the use of a dominant-negative mutant of phosphatidylinositol 3-kinase, the activation of VPF/VEGF promoter through Ras, PDK-1, and PKCzeta was completely repressed, indicating phosphatidylinositol 3-kinase as an important component of this pathway. Taken together, these data elucidate the signaling mechanism of Ras-mediated VPF/VEGF transcriptional activation through PKCzeta and also provide insight into PKCzeta and Sp1-dependent transcriptional regulation of VPF/VEGF.