Malanchuk O. Identification of binding partners of tuberous sclerosis complex TSC1/2 and characterization of identified interaction

3.The object - molecular mechanisms of cell signal system function. The aim of the work was the search of novel functional interaction of TSC1/2 complex and elucidation of regulatory role of identified interactions in mammalian cells. Applied methods - yeast two-hybrid screen, cDNA fragment cloning, transient transfection of mammalian cell lines of cDNA fragments, expression and affinity purification of recombinant proteins, immunobloting, immunoprecipitation and immunocytochemestry, generation and characterization of polyclonal and monoclonal antibodies (hybridoma technique), analysis of protein-protein interactions by reciprocal co-immunoprecipitation, in vitro kinase reaction, in vitro phosphatase reaction. Results and novelty - 10 TSC2-binding partners have been identified using yeast two-hybrid system. Among the isolated clones are an adaptor proteins of 14.3.3 family, the proteins of proteasomal complex, and serine/threonine protein phosphatase 5 (PP5). The monoclonal anti- TSC2 antibodies have been generated. The possibility of TSC2/PP5 complex formation in vivo has been shown and efficiency of such interaction depends on physiological status of cells. In addition, PP5 is capable of dephosphorylating TSC2 in vitro at AMPK potential sites. The hypothetic model of regulation of TSC2 activity by PP5 dephosphorylation has been proposed.