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

• Applicant: Professorin Dr. Juping Yuan
• Subject Area: Cell Biology
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 406036362

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.

• DFG Programme: Research Grants