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

RITA 磷酸化的鉴定和表征，RITA 是肿瘤发生的新参与者

• 批准号: 406036362
• 负责人: Professorin Dr. Juping Yuan
• 金额: --
• 依托单位: Klinik für Frauenheilkunde und Geburtshilfe
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/406036362?language=en
• 关键词: Identification; characterization; phosphorylation; RITA; new

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.

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。它的失调常常导致染色体不稳定、侵袭和化疗耐药。然而，其分子机制尚不完全清楚。新发现的 RITA（RBP-J 相互作用和微管蛋白相关蛋白）是 Notch 通路的负向参与者。我们最近发现 RITA 是微管动力学的重要调节剂。 RITA 覆盖微管并在体外和体内影响其结构。缺乏 RITA 的肿瘤细胞系表现出乙酰化 α-微管蛋白增加、微管稳定性增强和微管动力学降低。 RITA 的敲低/敲除会导致多种有丝分裂缺陷，包括染色体错位和分离错误，这是肿瘤细胞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，通过抑制 RITA 诱导的微管稳定性增加增强了 Aurora A 的活性，Aurora A 是一种在多种肿瘤实体中经常过度表达的激酶。这些数据强烈表明 RITA 成为肿瘤发生中的一个新角色。事实上，RITA 在多种恶性实体中失调，并且 RITA 基因敲除小鼠容易患淋巴瘤。因此，了解 RITA 本身如何在正常细胞中受到调节以及它在肿瘤细胞中如何解除调节是最重要的。初步数据表明磷酸化是 RITA 的翻译后修饰之一。在本提案中，我们希望利用磷质谱、CRISPR/CAS9细胞系、敲低/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/生化方法来解决以下问题：1．体外和体内 RITA 磷酸化位点的鉴定 2. RITA 磷酸化的表征 3. 肿瘤和正常细胞中 RITA 磷酸化的功能分析 鉴于 RITA 在调节微管动力学及其参与肿瘤发生中的关键作用，本研究将为 RITA 自身调节的分子机制及其作用提供新的线索。 放松管制会促进恶性进展。此外，这项工作将证实失调的激酶对癌症发展和化疗耐药性的贡献。