Signal Transduction of Vascular Endothelial Cell-specific Growth Factor Receptors.

Flt-1 (VEGFR-1) 和 KDR/Flk-1 (VEGFR-2) 是血管内皮生长因子 (VEGF) 的酪氨酸激酶受体，在生理和病理条件下的血管生成中发挥重要作用。然而，人们对这些受体的信号转导知之甚少。特别是，Flt-1 的激酶活性极低是进一步分析的主要问题。在本研究项目中，我们试图通过引入杆状病毒系统来克服这个问题，并比较了Flt-1和KDR/Flk-1之间的信号传导。(1)。我们发现使用杆状病毒系统在昆虫细胞中过度表达 Flt-1 对于检查自磷酸化位点以及与 Flt-1 的接头分子的关联非常有用。 Flt-1 上的 Tyr-1169 和 Tyr-1213 被发现是自磷酸化的，但只有 Tyr-1169 的苯丙氨酸突变体强烈抑制其与 PLCgamma 的关联。在过表达 Flt-1 的 NIH3T3 细胞中，VEGF 诱导 Flt-1 的自身磷酸化、PLCgamma 的酪氨酸磷酸化以及蛋白激酶 C 依赖性的 MAP 激酶激活。这些结果强烈表明，Flt-1 上的 Tyr-1169 是 PLCgamma 的主要结合位点，对于细胞内的 Flt-1 信号转导非常重要。(2)。在过表达KDR/Flk-1的NIN3T3细胞中，我们发现KDR/Flk-1激酶的主要靶分子是PLC-gamma，并且VEGF诱导MAP激酶的激活，主要由蛋白激酶C（PKC）介导。在显示严格 VEGF 依赖性生长的原代窦内皮细胞中，我们发现 VEGF 刺激 PLCgamma 和 Raf-1-MEK-MAP 激酶级联的激活。令我们惊讶的是，重要的调节因子 Ras 并没有因 VEGF 的反应而有效激活至显着水平。另一方面，PLC 特异性抑制剂严重降低了 VEGF 依赖性 MEK 磷酸化、MAP 激酶的激活以及随后的 DNA 合成。PI_3 激酶抑制剂无法抑制其中任何一个。这些结果表明，在原代内皮细胞中，VEGF诱导的Raf-MEK-MAP激酶激活和DNA合成主要由PKC依赖性途径介导。较少的

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 … More 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