Object: the molecular mechanisms of the NADPH-oxidase complex functional activity control and redox-dependent signaling. Aim: to determine the potential role Ruk/CIN85 adapter protein in the regulation of the functional activity of NADPH-oxidase and intracellular signaling in tumor cells. Methods: cell transfection, chemiluminescent assay, migration assay using modified Boyden chambers, PAGE protein electrophoresis, Western blot analysis, immunoprecipitation, GST in vitro pull down assay, confocal microscopy. The results of the study demonstrated the existence of the regulatory relationship between the level of Ruk/CIN85 expression in MCF-7 cells and the intensity of ROS production dependent on NADPH oxidase functional activity. Genetic constructs, which encode the intracellular H2O2 biosensor, Hyper, and the fusion protein Ruk/CIN85-HyPer were obtained. Using intravital fluorescence microscopy, colocalization of H2O2 production and adaptor protein Ruk/CIN85 was demonstrated in «dot»-like vesicular structures of different sizes in transiently transfected MCF-7 cells. According to our data, pretreatment of G10 MCF-7 cells with inhibitor of NADPH-oxidase complex assembly apocynin and trap for ROS N-acetylcysteine led to the reversion from sustained Akt kinase activation to transient with simultaneous increase of EGF receptor autophosphorylation and inhibition of cell migration. The data obtained can suggest that ROS produced by NADPH oxidases are signaling components, upstream to Akt kinase, that mediate the increased migratory potential of Ruk/CIN85-overexpressing MCF-7 cells. The system multidirectional changes in mRNA levels for NOX1, NOX2, NOX5, DUOX2 and p22Phox were revealed in Ruk/CIN85 overexpressing cells in comparison to control cells. Knocking down of Ruk/CIN85 using technology of RNA-interference resulted in the reversion of these changes. Further studies are necessary to elucidate, by which molecular mechanisms Ruk/CIN85 could affect transcriptional regulation of NOXs genes. Using GST in vitro pull-down assay, we determined that SH3A domain of adaptor protein Ruk/CIN85 precipitated full-length form of adaptor protein Tks4 (Mr 120 kDa) from lysates of human breast (MCF-7, MDA-MB-231), melanoma (MM-4), colon (HT-29, DLD-1) tumor cells as well as from lysates of mouse Lewis lung carcinoma cells (LLC) and mouse fibroblasts (NIH 3T3). It has been also revealed that all Ruk/CIN85 SH3 domains (A, B and C) with high efficiency precipitated the additional forms of Tks4 with Mr 75, 90 and 160 kDa from lysates of human colon carcinoma cells and mouse fibroblasts. The molecular nature of new multiple forms of Tks4 has not been determined to date. The data obtained suggest that interaction between Ruk/CIN85 SH3 domains with Tks4 endogenous forms is determined by cellular context while a level of this interaction can be regulated in the course of physiological cellular responses. Increased interaction between the endogenous forms of both adaptor proteins in MCF-7 cells overexpressing Ruk/CIN85 was demonstrated in the course of EGF treatment.
