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

血管内皮生长因子（VEGF）酪氨酸激酶受体Flt-1 （VEGFR-1）和KDR/Flk-1 （VEGFR-2）在生理和病理条件下的血管生成中起重要作用。然而，人们对这些受体的信号转导知之甚少。特别是，非常低的Flt-1激酶活性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一问题，并比较了Flt-1和KDR/Flk-1之间的信号传导(1)。我们发现，利用杆状病毒系统在昆虫细胞中过表达Flt-1，对于检测Flt-1的自磷酸化位点及其与适配器分子的关联非常有用。Flt-1上的tyr1 -1169和tyr1 -1213被发现是自动磷酸化的，但只有tyr1 -1169的苯丙氨酸突变体强烈抑制其与PLCgamma的关联。在Flt-1过表达的NIH3T3细胞中，VEGF诱导Flt-1的自磷酸化、PLCgamma的酪氨酸磷酸化和mapk的蛋白激酶c依赖性活化。这些结果强烈表明，Flt-1上的tyr1 -1169是PLCgamma的主要结合位点，对细胞内Flt-1信号转导很重要(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 - γ， VEGF诱导MAP激酶的激活，主要由蛋白激酶C （PKC）介导。在严格依赖VEGF生长的原代窦状内皮细胞中，我们发现VEGF刺激了PLCgamma和Raf-1-MEK-MAP激酶级联的激活。令我们惊讶的是，一个重要的调节因子Ras在VEGF反应中并没有被有效地激活到显著水平。另一方面，plc特异性抑制剂严重降低了vegf依赖性的MEK磷酸化、MAP激酶的激活和随后的DNA合成。PI_3激酶抑制剂对二者均无抑制作用。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合成主要通过pkc依赖途径介导。少

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