发掘新的线粒体蛋白激酶及其功能与机制研究

在人类基因组编码的538个蛋白激酶中，有18个已知具有线粒体定位，它们对线粒体的结构与功能发挥调控作用。然而由于缺乏系统性研究，是否存在其它的线粒体定位蛋白激酶以及它们的功能仍然是重要的科学问题。在前期研究中，我们绘制了蛋白激酶组亚细胞定位图谱，并发现了4个新的线粒体定位蛋白激酶。进一步研究发现其中的MOK激酶调控线粒体能量代谢活性。MOK的线粒体定位受到EGF信号的动态调控，并且MOK的表达在黑色素瘤细胞中明显上调。因此，本项目拟通过对MOK及线粒体转运体系的研究阐明MOK线粒体定位在EGF信号调控下受到动态调控的机制；通过多种方式发掘MOK的底物和结合蛋白，阐明MOK调控线粒体活性的功能与机制；并通过对MOK在黑色素瘤细胞与肿瘤样品中的研究阐明癌细胞逃逸生长因子依赖的新机制。本项目将为线粒体细胞器的动态调控开辟新的研究领域，为干预线粒体在疾病中的异常找到新的靶点。

Mitochondrion is an important cellular organelle that generates energy and controls multiple other cellular activities. Previous investigations have found that protein kinases regulate mitochondrion structure and function via phosphorylation of certain substrates. Among the 538 kinases encoded by the human genome, 18 have been shown to have mitochondrial localization. However, the complete picture of the mitochondria kinome is lacking, and functions of kinases in mediating mitochondria regulation by extracellular stimuli are also not thoroughly understood. In order to systematically investigate the human kinome, we constructed a plasmid library expressing individual kinases of the human kinome in 96-well format. Through expression of the library and immunofluorescence staining, we made a subcellular localization map of the human kinome and identified 4 new kinases with specific mitochondrial localization. We further found that one of the kinases, MOK, play a specific role in regulation of the energy metabolism function of mitochondria. Interestingly, EGF signaling significantly promotes the mitochondrial localization of MOK. Furthermore, the expression level of MOK is markedly elevated in several melanoma cell lines. Based on these preliminary data, we plan to investigate the mechanisms underlying dynamic regulation of MOK mitochondria localization by EGF signaling through techniques such as super-resolution microscopy, and CRISPR/Cas9-mediated in vivo tagging of endogenous proteins. Furthermore, we will elucidate the mechanism by which MOK regulates mitochondria activities through identification of MOK phosphorylation substrates using multiple approaches such as the chemical genetic method. In addition, we will determine the role of MOK in melanoma tumor growth and progression to test the hypothesis of cancer cell evading addition to growth factors through up-regulation of MOK and thus mitochondrial metabolism. The completion of this projection will identify new mechanisms of dynamic regulation of mitochondria activities through the shuttling of kinases. It will also uncover new links of kinase dysregulation to mitochondria abnormalities in cancer. In addition, it will provide a start point to a thorough investigation of novel kinase localizations found in the screen.