Src在Y74、Y272磷酸化TOPK对结肠癌发生的作用及机制研究

我们以往的研究揭示了TOPK的功能类似自然界中并不存在的致瘤性组成激活型MEK, 促进结肠癌发生。但TOPK的激活机制不明，阻碍了对TOPK的进一步研究。我们近来的研究发现：1)Src上调TOPK的蛋白水平；TOPK蛋白序列中有Src磷酸化保守序列；2)蛋白激酶实验证实Src磷酸化TOPK的Y74和Y272；3)免疫共沉淀证实Src与TOPK结合。因此，我们提出"Src是TOPK直接上游蛋白激酶的假说"。为了在细胞内验证这一假说，我们拟进行如下研究：1)在细胞内证实Src磷酸化TOPK；2)制备Y74、Y272的磷酸化抗体，探索该磷酸化的临床意义；3)应用细胞转化模型研究该磷酸化对于细胞转化和结肠癌发生的影响；4)研究该磷酸化对于TOPK信号通路的影响以及Src信号通路的影响；5)探索Src通过磷酸化调控TOPK信号通路的机制。我们的研究将对深入理解TOPK在结肠癌发生中的作用提供依据。

Although cancer caused constitutively activated MEK does not exist in nature, our study indicated that TOPK, like constitutively activated MEK, can promote carcinogenesis of colorecral cancer. However, the mechanism of activation of TOPK is still unrevealed, which hinders the progress of research in TOPK field. Our recent study indicated: 1) TOPK level was up-regulated by Src; and there existed consensus phosphorylation sequence of Src in TOPK; 2) Src phosphorylated TOPK at Y74 and Y272 in protein kinase assay; 3) Src could bind with TOPK in co-immunoprecipitation. Therefore, we make hypothesis that "Src is the direct up-strean kinase of TOPK. To test the hypothesis, the following experiments are designed: 1) test the phosphorylation exists in cells; 2) prepare the specific phosphorylation antibodies of Y74 or Y272, and explore the meaning of these phosphorylation in clinic; 3) study the effect of this phosphorylaton to cell transformation and carcinogenesis in colorectal cancer; 4) test the impact of this phosphorylation to TOPK signal pathway and Src signal pathway; and 5) explore the mechanism of regulation of TOPK by Src. Our study will provide new evidence to understand the role of TOPK in carcinogenesis of colorectal cancer.