Identification and characterization of phosphorylation in RITA, a new player in oncogenesis

微管动力学的微调时空调节对于有丝分裂和细胞运动等各种关键细胞活动至关重要。其失调常常导致染色体不稳定、侵袭和化疗耐药。然而，其分子机制尚未完全了解。新发现的RITA，RBP-J相互作用和微管蛋白相关蛋白，是Notch通路的负作用者。我们最近确定RITA作为一个重要的调制器在微管动力学。RITA在体外和体内包覆微管并影响其结构。缺乏RITA的肿瘤细胞系显示乙酰化α-微管蛋白增加，微管稳定性增强和微管动力学降低。RITA的敲除/敲除导致多种有丝分裂缺陷，包括染色体错配和分离错误，这是肿瘤细胞中染色体不稳定的主要原因。重要的是，RITA水平与肿瘤细胞的侵袭能力密切相关。更有趣的是，抑制RITA诱导的微管稳定性增加增强了Aurora A的活性，Aurora A是一种经常在不同肿瘤实体中过表达的激酶。这些数据高度表明，RITA出现作为一个新的演员在肿瘤发生。事实上，RITA在多种恶性实体中是失调的，并且RITA敲除小鼠易于发生淋巴瘤。因此，了解RITA本身在正常细胞中是如何调节的以及它在肿瘤细胞中是如何解除调节的是最重要的。初步数据表明，磷酸化是RITA的翻译后修饰之一。在本提案中，使用磷酸化质谱、CRISPR/CAS9细胞系、敲除/敲除小鼠成纤维细胞、特异性小分子抑制剂、活细胞成像以及其他分子/生物化学方法，我们希望解决以下问题：1. RITA磷酸化位点的体内外鉴定2.磷酸化后RITA的表征3. RITA磷酸化在肿瘤和正常细胞中的功能分析鉴于RITA在调节微管动力学中的关键作用及其在肿瘤发生中的参与，本研究将为RITA自身调节及其失调促进恶性进展的分子机制提供新的线索。此外，这项工作将证实失调激酶对癌症发展和化疗耐药性的贡献。

The fine-tuned spatiotemporal regulation of microtubule dynamics is vital for various crucial cellular activities like mitosis and cell motility. Its deregulation leads very often to chromosome instability, invasion and chemotherapy resistance. The molecular mechanisms are however not totally understood. The newly discovered RITA, the RBP-J interacting and tubulin associated protein, is a negative player of the Notch pathway. We have recently identified RITA as an important modulator in microtubule dynamics. RITA coats microtubules and affects their structures in vitro as well as in vivo. Tumor cell lines deficient of RITA display increased acetylated α-tubulin, enhanced microtubule stability and reduced microtubule dynamics. Knockdown/knockout of RITA results in multiple mitotic defects including chromosome misalignment and segregation errors, the major reasons for chromosome instability in tumor cells. Importantly, the RITA level is well correlated with invasive capability of tumor cells. More interestingly, increased microtubule stability induced by suppression of RITA enhances the activity of Aurora A, a kinase frequently overexpressed in diverse tumor entities. These data highly suggest that RITA emerges as a novel actor in oncogenesis. Indeed, RITA is deregulated in a variety of malignant entities and RITA knockout mice are prone to lymphoma. It is thus of most importance to understand how RITA itself is regulated in normal cells and how it is deregulated in tumor cells. The preliminary data suggest that phosphorylation is one of the post-translational modifications in RITA. In this proposal, using phospho-mass spectrometry, CRISPR/CAS9 cell lines, knockdown/knockout mouse fibroblasts, specific small molecule inhibitors, living cell imaging as well as other molecular/biochemical methodology, we would like to address following issues:1. Identification of the phosphorylation sites in RITA in vitro and in vivo 2. Characterization of RITA upon phosphorylation 3. Functional analysis of RITA phosphorylation in tumor and normal cellsGiven the crucial roles of RITA in regulating microtubule dynamics and its involvement in oncogenesis, this study will shed new light on the molecular mechanisms whereby RITA itself is regulated and its deregulation facilitates malignant progression. Moreover, this work will substantiate the contribution of deregulated kinases to the cancer development and chemotherapy resistance.