Annexin VII and annexin XI are tyrosine phosphorylated in peroxovanadate-treated dogs and in platelet-derived growth factor-treated rat vascular smooth muscle cells
Annexin VII and annexin XI are tyrosine phosphorylated in peroxovanadate-treated dogs and in platelet-derived growth factor-treated rat vascular smooth muscle cells
复制标题
DOI：
10.1074/jbc.274.47.33504
复制
发表时间：
1999-11-19
影响因子：
4.8
通讯作者：
Cohen, S
中科院分区：
文献类型：
--
作者：
Furge, LL;Chen, K;Cohen, S
腹腔内给予过氧钒酸盐会导致许多酪氨酸磷酸化蛋白在治疗动物的肝脏和肾脏中快速积累。来自正常组织的大量酪氨酸磷酸化蛋白的可用性有助于纯化和鉴定先前未知的细胞酪氨酸激酶靶标。通过这个程序，我们迄今为止已经在经过过氧钒酸盐治疗的狗的肝脏和肾脏中鉴定出了四种蛋白质。其中两种，膜联蛋白 VII 和膜联蛋白 XI，是新的，之前没有报道过它们是酪氨酸激酶的底物，而其余两种，埃兹蛋白和网格蛋白，据报道在一些细胞培养系统中被酪氨酸磷酸化。在本研究中，通过序列分析和免疫学方法鉴定了分离的蛋白质。膜联蛋白 VII 和膜联蛋白 XI 存在于培养的大鼠血管平滑肌细胞中，并且两者均因生理配体血小板衍生生长因子-BB (PDGF-BB) 而被酪氨酸磷酸化。此外，通过向细胞培养物中共同添加过氧钒酸盐，增强了对 PDGF-BB 的酪氨酸磷酸化程度。体外磷酸化测定表明，PDGF 受体、钙依赖性酪氨酸激酶 (CADTK/Pyk-2)、Src 激酶和表皮生长因子受体均能够在酪氨酸残基上磷酸化纯化的膜联蛋白 VII 和 XI。这些发现证实了过氧钒酸盐抑制磷酸酶作为识别细胞蛋白酪氨酸激酶先前未知的生理靶标的工具的有用性。
The intraperitoneal administration of peroxovanadate results in the rapid accumulation of many tyrosine-phosphorylated proteins in the liver and kidney of treated animals. The availability of large pools of tyrosine-phosphorylated proteins derived from normal tissues facilitates the purification and identification of previously unknown targets for cellular tyrosine kinases. Using this procedure, we have thus far identified four proteins in the liver and kidney of peroxovanadate-treated dogs. Two of these, annexin VII and annexin XI, were novel and had not been previously reported to be substrates of tyrosine kinases while the remaining two, ezrin and clathrin, have been reported to be tyrosine phosphorylated in some cell culture systems. In the present study, isolated proteins were identified both by sequence analysis and immunological methods. Annexin VII and annexin XI are present in cultured rat vascular smooth muscle cells and both were tyrosine phosphorylated in response to a physiological ligand, platelet-derived growth factor-BB (PDGF-BB). Furthermore, the extent of tyrosine phosphorylation in response to PDGF-BB was augmented by the co-addition of peroxovanadate to cell cultures. In vitro phosphorylation assays showed that PDGF receptor, calcium-dependent tyrosine kinase (CADTK/Pyk-2), Src kinase, and epidermal growth factor receptor all were able to phosphorylate purified annexin VII and XI on tyrosine residues. These findings confirm the usefulness of phosphatase inhibition by peroxovanadate as a tool for identifying previously unknown physiological targets for cellular protein tyrosine kinases.