The adaptor protein shb binds to tyrosine 1175 in vascular endothelial growth factor (VEGF) receptor-2 and regulates VEGF-dependent cellular migration
The adaptor protein shb binds to tyrosine 1175 in vascular endothelial growth factor (VEGF) receptor-2 and regulates VEGF-dependent cellular migration
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DOI：
10.1074/jbc.m312729200
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发表时间：
2004-05-21
影响因子：
4.8
通讯作者：
Welsh, M
中科院分区：
文献类型：
--
作者：
Holmqvist, K;Cross, MJ;Welsh, M
先前的研究表明，衔接蛋白Shb参与受体酪氨酸激酶信号传导。在这项研究中，我们使用表达人血管内皮生长因子受体2（VEGFR-2）（KDR）的猪主动脉内皮细胞证明，在血管内皮生长因子（VEGF）刺激后，Shb以Src依赖性方式磷酸化。在免疫共沉淀实验中，我们可以检测到Shb和VEGFR-2在人端粒酶永生化微血管内皮细胞之间的相互作用。此外，在谷胱甘肽S-转移酶下拉试验中，显示Shb的Src同源2结构域与VEGFR-2的C-末端尾部的磷酸化酪氨酸1175相互作用。在表达嵌合鼠VEGFR-2（Flk-1）的猪主动脉内皮细胞中，VEGF诱导的Shb磷酸化在相应位置发生突变。Shb表达特异性降低了80%，在一个短暂的方式，通过使用短干扰RNA技术。Shb表达减少导致磷脂酰肌醇3-激酶的刺激丧失，粘着斑激酶在酪氨酸576处磷酸化，粘着斑的产生，以及响应VEGF的应力纤维形成。此外，我们表明，VEGF诱导的迁移抑制Shb短干扰RNA处理的细胞。我们的数据表明Shb对于内皮细胞中的VEGF信号传导很重要。这是通过Shb与VEGFR-2中的酪氨酸1175结合来实现的，VEGFR-2调节VEGF诱导的粘着斑形成和细胞迁移，后者以磷脂酰肌醇3-激酶依赖性方式发生。
Previous studies have shown that the adaptor protein Shb is involved in receptor tyrosine kinase signaling. In this study, we demonstrate that Shb is phosphorylated in an Src-dependent manner upon vascular endothelial growth factor ( VEGF) stimulation using porcine aortic endothelial cells expressing the human VEGF receptor 2 (VEGFR-2) (KDR). In co-immunoprecipitation experiments, we could detect an interaction between Shb and the VEGFR-2 in human telomerase-immortalized microvascular endothelial cells. Furthermore, in a glutathione S-transferase pull-down assay, the Src homology 2 domain of Shb was shown to interact with phosphorylated tyrosine 1175 in the C-terminal tail of VEGFR-2. VEGF-induced Shb phosphorylation was lost in porcine aortic endothelial cells expressing a chimeric murine VEGFR-2 (Flk-1) with a mutation at the corresponding position. Shb expression was specifically decreased by 80%, in a transient manner, by using the short interfering RNA technique. Reduced Shb expression led to a loss of stimulation of phosphatidylinositol 3-kinase, phosphorylation of focal adhesion kinase at tyrosine 576, the generation of focal adhesions, and stress fiber formation in response to VEGF. Furthermore, we show that VEGF-induced migration is inhibited in Shb short interfering RNA-treated cells. Our data demonstrate that Shb is important for VEGF signaling in endothelial cells. This is achieved by Shb binding to tyrosine 1175 in the VEGFR-2, which regulates VEGF-induced formation of focal adhesions and cell migration, of which the latter occurs in a phosphatidylinositol 3-kinase-dependent manner.