极性蛋白hDLG多位点磷酸化以及在胃癌生长和转移中作用的研究

Loss of cell polarity is a hallmark and a precondition for the development and progression of human carcinoma. Scribble polarity protein complex participates in the establishment and maintenance of different modes of cell polarity, being considered as a group of tumor suppressors. Human DLG (hDLG), a polarity protein in scribble complex, also implicated in regulating of the process of cell cycle, has been demonstrated to have both tumor suppressing and oncogenic activity. The behavior of hDLG protein is regulated by multisite phosphorylation, and our current results showed that the multi-phosphorylated hDLG promoted the growth and metastasis of gastric cancers in the nude mice. By mutagenesis analysis, we identified two switch-like phosphorylation sites within a phosphorylation clustering region at the N-terminus. Next, we will identify the primary kinases and phosphatases responsible for the phosphorylation level of those two sites, and analyze their expression and activity state in gastric cancers. Furthermore, to understand the effects and regulatory mechanisms of hDLG multi-phosphorylation, we will study the behavior of whole phosphorylated isoforms rather than one of its certain phosphorylation state in cells, and investigate the changes of the molecular structure and its protein interactome after multi-phosphorylation. Finally, we will design and identify small molecular compounds to specifically block the key phosphorylation sites in hDLG and related changes in signaling pathways for the treatment of gastric cancers.

失去细胞极性是上皮肿瘤发生发展的重要标志和前提条件，Scribble极性蛋白复合体参与细胞极性及细胞间粘附连接的生物学发生和维持，被认为是一组肿瘤抑制因子。这一极性复合体中的hDLG蛋白同时也是细胞周期进程的重要调控分子，具有抑癌和致癌的神秘双重特性。hDLG蛋白的功能行为受多位点高度磷酸化的调控，我们的初步试验结果显示，其多位点磷酸化的蛋白分子具有促进胃癌细胞的生长和迁移能力。同时，我们的点突变研究确定了其氨基端磷酸化聚集区内具有开关作用的两个磷酸化位点。我们接下来的研究将确定调控这两个关键位点磷酸化水平的蛋白激酶和磷酸酯酶，以及它们在胃癌细胞内的活性状态。我们将以全分子多位点不同磷酸化的hDLG蛋白同源体群作为研究对象，分析其多位点磷酸化导致的整体分子构象和相互作用蛋白熵的改变，以揭示其多位点磷酸化促进胃癌生长和转移的效应机制。我们还将筛选出阻断关键磷酸化位点的治疗性靶向小分子。

细胞有丝分裂末期的纺锤丝迅速和完全的解聚是细胞成活所必须的，然而这一过程如何发生在哺乳类细胞中完全不清楚。我们使用实时荧光显微镜技术和生化分析的方法发现，一个原发性矮小症关联的富含半胱氨酸的CRIPT蛋白通过氧化还原的方式决定人类细胞纺锤丝的解聚。CRIPT一直被认为是胞浆蛋白，然而我们的研究发现它在静止的细胞中是一个细胞核内蛋白并主要表达在核仁，但在细胞分裂期间定位到纺锤丝并被氧化还原修饰形成CXXC对内的二硫键。CRIPT直接结合纺锤丝中微管蛋白，通过半胱氨酸间多层次的交换传递氧化还原反应到微管蛋白导致纺锤丝微管的解聚。转染任一半胱氨酸替代的CRIPT突变体都完全阻断纺锤丝的解聚导致细胞固定在分裂中期或纺锤丝残留。实时荧光显微镜记录一个疾病相关的CRIPT突变体(CRIPTC3Y) 揭示CRIPT介导的氧化还原同时负责有丝分裂后期（anaphase)纺锤丝末端的微管解聚和有丝分裂末期（telophase)纺锤丝的完全溶解。进一步，我们确定了一个微管蛋白特异的分子伴侣TBCB与CRIPT相互作用并协助纺锤丝的解聚。