Azuma Kotaro, Tanaka Masamitsu, Uekita Takamasa, Inoue Satoshi, Yokota Jun, Ouchi Yasuyoshi, Sakai Ryuichi
Growth Factor Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
Oncogene. 2005 Jul 14;24(30):4754-64. doi: 10.1038/sj.onc.1208654.
To acquire information on signal alteration corresponding to the changes in metastatic potential, we analysed protein tyrosine phosphorylation of low- and high-metastatic human osteosarcoma HuO9 sublines, which were recently established as the first metastatic model of human osteosarcoma. Tyrosine phosphorylation of proteins around 60, 70, and 120-130 kDa was enhanced in high-metastatic sublines. Among these proteins, the protein around 70 kDa, which was most remarkably phosphorylated, was identified as paxillin, a scaffold protein in integrin signaling. Activity of Src family kinase correlated well with metastatic potential, and a Src family kinase inhibitor, PP2, not only abolished tyrosine phosphorylation of paxillin but also impaired the motility of high-metastatic sublines. The expression of paxillin was also elevated in high-metastatic sublines, and knocking down of paxillin expression by RNAi method resulted in attenuated motility of high-metastatic cells. We also demonstrated that the phosphorylated form of paxillin is essential for the migration-promoting effect in human osteosarcoma. These findings suggest that enhanced activity of Src family kinases and overexpression of paxillin synergistically contribute to the high metastatic potential of human osteosarcoma through the hyperphosphorylation of paxillin.
为获取与转移潜能变化相对应的信号改变信息，我们分析了低转移和高转移人骨肉瘤HuO9亚系的蛋白质酪氨酸磷酸化情况，这两个亚系最近被确立为首个骨肉瘤转移模型。在高转移亚系中，60、70以及120 - 130 kDa左右的蛋白质酪氨酸磷酸化增强。在这些蛋白质中，70 kDa左右磷酸化最为显著的蛋白质被鉴定为桩蛋白，它是整合素信号通路中的一种支架蛋白。Src家族激酶的活性与转移潜能密切相关，一种Src家族激酶抑制剂PP2不仅消除了桩蛋白的酪氨酸磷酸化，还损害了高转移亚系的迁移能力。高转移亚系中桩蛋白的表达也有所升高，通过RNAi方法敲低桩蛋白的表达会导致高转移细胞的迁移能力减弱。我们还证明，桩蛋白的磷酸化形式对于人骨肉瘤的迁移促进作用至关重要。这些发现表明，Src家族激酶活性增强和桩蛋白过表达通过桩蛋白的过度磷酸化协同促进了人骨肉瘤的高转移潜能。
