|Budget Amount *help
¥5,400,000 (Direct Cost: ¥5,400,000)
Fiscal Year 1997: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1996: ¥3,000,000 (Direct Cost: ¥3,000,000)
Flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2), tyrosine kinase receptors for vascular endothelial growth factor (VEGF), play important roles in the angiogenesis at physiological and pathological conditions. However, the signal transduction of these receptors is poorly understood. Particularly, a very low kinase activity of Flt-1 was a major problem for further analysis. In this research project, we attempted to overcome this problem by introducting Baculovirus sysem, and compared the signaling between Flt-1 and KDR/Flk-1.
(1). we found that overexpression of Flt-1 in insect cells using the Baculovirus system was very useful to examine the autophosphorylation sites and association with adapter molecules with Flt-1. Tyr-1169 and Tyr-1213 on Flt-1 were found to be auto-phosphorylated, but only a phenylalanine mutant of Tyr-1169 strongly suppressed its association with PLCgamma. In Flt-1 overexpressing NIH3T3 cells, VEGF induced autophosphorylation of Flt-1, tyrosine-phosphorylation of PLCgamma a
nd protein kinase C-dependent activation of MAP kinase. These results strongly suggest that Tyr-1169 on Flt-1 is a major binding site for PLCgamma and important for Flt-1 signal transduction within the cell.
(2). In the NIN3T3 cells overexpressing KDR/Flk-1, we found that a major target molecule of KDR/Flk-1 kinase is PLC-gamma, and that VEGF induces activation of MAP kinase, mainly mediated by Protein kinase C (PKC). In primary sinusoidal endothelial cells which showed strictly VEGF-dependent growth, we found that VEGF stimulated the activation of PLCgamma, and Raf-1-MEK-MAP kinase cascade. To our surprise, an important regulator, Ras was not efficiently activated to a significant level in response to VEGF.On the other hand, PLC-specific inhibitors severely reduced VEGF-dependent phosphorylation of MEK,activation of MAP kinase and subsequent DNA synthesis.PI_3 kinase inhibitor could not inhibit either of them. These results suggest that in primary endothelial cells, VEGF-induced activation of Raf-MEK-MAP kinase and DNA synthesis are mainly mediated by PKC-dependent pathway. Less