Identification and characterization of phosphorylation in RITA, a new player in oncogenesis
Final Report Abstract
The finely-tuned spatiotemporal regulation of microtubule dynamics is vital for various crucial cellular activities such as mitosis and cell motility. Its deregulation leads very often to chromosome instability, invasion and therapy resistance. The molecular mechanisms are however not totally understood. RITA, the RBP-J interacting and tubulin-associated protein, is a new microtubule-associated protein. We have reported that RITA modulates microtubule dynamics and the activity of Aurora A. Its depletion causes severe chromosome defects in mitosis. Overexpression or down-regulation of RITA has been observed in various tumor entities. These data suggest that RITA may emerge as a novel player in oncogenesis. It is thus of importance to understand how RITA itself is regulated in tumor cells. To examine this issue, various cellular and molecular techniques were used, including gene deletion and mutation, kinase assay in vitro, in vivo labelling, confocal microscopy, indirect immunofluorescence staining, immunoprecipitation and Western blot analysis. We show here that RITA is phosphorylated in vitro by several kinases including Polo-like kinase 1 (Plk1), mitogen-activated protein kinase (MAPK) and cyclin-dependent kinase 1 (Cdk1). Among multiple serine or threonine residues, Cdk1 phosphorylates T254 of RITA in vitro as well in vivo. While HeLa CRISPRi RITA T254D cells, where RITA is stably depleted and transiently transfected with the phosphorylation mimicking RITA T254D construct, displays defective central spindle, HeLa CRISPRi RITA T254A demonstrates stabilized mitotic microtubules in mitosis. Neither of them is able to fully rescue the mitotic defects induced by depletion of RITA. Moreover, the loss of the C-terminus (aa 157-269), where T254 locates, renders RITA instable. Transfection of this construct into HeLa CRISPRi cells induces an enlarged area of PRC1 staining associated with disorganized central spindle. These findings suggest that RITA must be precisely regulated and Cdk1 is a critical kinase for its regulation. Highly activated mitotic kinases may be responsible for deregulated RITA, contributing to abnormal chromosomal movement and leading to chromosome instability, a hallmark of malignant cells. Further studies are required to investigate the phosphorylation status of RITA in tumor tissues, whether this is correlated with therapy resistance and if kinase inhibitors could re-activate the therapy response in tumor cells.
Publications
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Adipose Tissue-Derived Mesenchymal Stromal/Stem Cells, Obesity and the Tumor Microenvironment of Breast Cancer. Cancers, 14(16), 3908.
Ritter, Andreas; Kreis, Nina-Naomi; Hoock, Samira Catharina; Solbach, Christine; Louwen, Frank & Yuan, Juping
