Sawano A, Takahashi T, Yamaguchi S, Shibuya M
Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108, Japan.
Biochem Biophys Res Commun. 1997 Sep 18;238(2):487-91. doi: 10.1006/bbrc.1997.7327.
Flt-1, a tyrosine kinase receptor for vascular endothelial growth factor (VEGF), plays important roles in the angiogenesis required for embryogenesis and in monocyte/macrophage migration. However, the signal transduction of Flt-1 is poorly understood due to its very weak tyrosine kinase activity. Therefore, we overexpressed Flt-1 in insect cells using the Baculovirus system in order to examine for autophosphorylation sites and association with adapter molecules such as phospholipase Cgamma-1 (PLCgamma). 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 and 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.
Flt-1是血管内皮生长因子（VEGF）的酪氨酸激酶受体，在胚胎发育所需的血管生成以及单核细胞/巨噬细胞迁移中发挥重要作用。然而，由于其酪氨酸激酶活性非常弱，Flt-1的信号转导机制尚不清楚。因此，我们利用杆状病毒系统在昆虫细胞中过表达Flt-1，以检测其自身磷酸化位点以及与诸如磷脂酶Cγ-1（PLCγ）等衔接分子的结合情况。发现Flt-1上的酪氨酸-1169（Tyr-1169）和酪氨酸-1213（Tyr-1213）可发生自身磷酸化，但只有Tyr-1169的苯丙氨酸突变体强烈抑制其与PLCγ的结合。在过表达Flt-1的NIH3T3细胞中，VEGF诱导Flt-1自身磷酸化、PLCγ酪氨酸磷酸化以及丝裂原活化蛋白激酶（MAP激酶）的蛋白激酶C依赖性激活。这些结果强烈表明，Flt-1上的Tyr-1169是PLCγ的主要结合位点，并且对细胞内Flt-1信号转导很重要。
