Rzhepetskyy Y. Phosphorylation of adaptor protein Ruk/CIN85 in the control of biological cell responses

Object of the research: molecular mechanisms of adaptor/scaffold proteins functioning . The aim of the work: study the possibility of post-translational modification of adaptor protein Ruk/CIN85 through phosphorylation using models of different tumour cells treated with various regulatory factors as well as to search for protein kinases able to interact with Ruk/CIN85 and modify it. Methods: approaches of cellular and molecular biology, biochemical. It was demonstrated that Ruk/CIN85 is phosphorylated in cellulo and changes in the level of its modification were revealed in cells in the process of biological responses realization. In particular, increase of full-length Ruk/CIN85 isoform phosphorylation has been shown in human embryonic kidney cells HEK293 stimulated by serum and insulin. On the contrary, under the same conditions phosphorylation of Rukm isoform without first SH3AB domains is decreased. In addition, differential changes in the level of Ruk/CIN85 phosphorylation on Tyr, Ser and Thr residues in rat pheochromocytoma PC12 cells induced to proliferation and differentiation with EGF and NGF, respectively, were revealed. It was shown for the first time that Ser/Thr-specific PKD is a binding partner of Ruk/CIN85 and molecular mechanisms of intermolecular interaction as well as potential sites of its phosphorylation were identified. It has been also demonstrated the suppression of Ruk/CIN85-PKD2 interaction in B lymphocytes isolated from human tonsils and Burkitt's lymphoma Ramos cells in the process of BCR stimulation. Importantly, colocalization of Ruk/CIN85 and PKD2 was revealed in human breast adenocarcinoma MCF-7 cells, which stably overexpress GFP-fused full length Ruk form, in lamellipodia and membrane ruffles, structures recponsible for cell migration. It was also established that these cells are characterized by increased motility in comparison to mock-transfected cells. The interaction of Ruk/CIN85 with cortactin, being also the binding partner of PKD and the component of machinery involved in the control of cell motility and invasion, was identified. Together, the data obtained can suggest the functional role of of Ruk/CIN85-PKD interaction in regulation of cell migration and invasion. Area of use: cellular biology, molecular biology.