The 230 kDa mature form of KDR/Flk-1 (VEGF receptor-2) activates the PLC-γ pathway and partially induces mitotic signals in NIH3T3 fibroblasts

Abstract

KDR/Flk-1 tyrosine kinase, one of the two receptors for Vascular Endothelial Growth Factor (VEGF) has been shown to generate the major part of mitotic signals in endothelial cells, although the mechanisms are poorly understood. Here we examined the processing and signal transduction of KDR/Flk-1. Both in endothelial cells and in NIH3T3 cells expressing KDR/Flk-1, an immature form of KDR/Flk-1 with a molecular mass of about 150 kDa was glycosylated to create a 200 kDa intermediate, and after further glycosylation a mature 230 kDa was expressed on the cell surface. Only this 230 kDa form was rapidly and transiently phosphorylated on tyrosine residues in the presence of VEGF. As a major substrate of KDR/Flk-1, PLC-g was found to be rapidly tyrosine-phosphorylated and associated with KDR/Flk-1 both in endothelial cells and NIH3T3 cells. Interestingly, however, a prompt activation of MAP kinase and subsequent strong mitotic signaling were generated only in the endothelial cell background. Activation of MAP kinase in NIH3T3 cells overexpressing KDR/Flk-1 showed a slower response as maximum levels were only attained after 20 min compared to 5 min in sinusoidal endothelial cells. These results suggest that the KDR/Flk-1 utilizes cell typespeci®c signal transduction pathway(s) for MAP kinase activation and the mitotic response in endothelial cells.

Roles of two VEGF receptors, Flt-1 and KDR, in the signal transduction of VEGF effects in human vascular endothelial cells

“…However, ERK1/2 may not play a major role in this system, since speci®c inhibition of ERK1/2 by PD98059 exhibited only a marginal e ect on vinculin assembly and phosphorylation of FAK and paxillin. Previous reports indicate that VEGF, by binding to KDR, activates ERK1/2 via a phospholipase Cg (PLCg)-PKC-dependent pathway (Seetharam et al, 1995;Guo et al, 1995;Takahashi and Shibuya, 1997). We observed that speci®c inhibition of PKC by GF109203X completely abrogated vinculin assembly as well as DNA sysnthesis.…”

Angiogenesis in patients with craniopharyngiomas

“…Nonetheless, various lines of evidence in different tumor types suggest that the VEGF/VEGFR-2 system plays an important role in the regulation of tumor cell growth through the activation of the mitogen-activated protein kinase pathway and, subsequently, the mitogenic response. 56,57 It also is possible that, like endothelial cells, activity of the VEGF/VEGFR-2 system in craniopharyngiomas may be linked to the increasing permeability of epithelial cells and cyst formation. This signaling pathway provides an intriguing mechanism that may establish a direct correlation between VEGF expression and tumor cyst formation, as observed by Vaquero et al 22 in their study of craniopharyngiomas.…”

Vascular endothelial growth factor receptors in the regulation of angiogenesis and lymphangiogenesis

“…Activation of the p42/44 MAP kinase pathway by VEGF and subsequent cell proliferation has been documented for many types of endothelial cells (Seetharam et al, 1995;D'Angelo et al, 1995;Rousseau et al, 1997;Kroll and Waltenberger, 1997;Takahashi and Shibuya, 1997;Landgren et al, 1998;Takahashi et al, 1999). Association of VEGFR-2 with Grb2 suggests that activated Ras might be involved in the regulation of the VEGF-induced mitogenic response.…”