Ribosomal protein S6 kinases (S6Ks) belong to the AGC family of Ser/Thr kinases and are important players in cellular PI3K/mTOR signalling network, implicated in the regulation of cell size, growth and metabolism. Previous studies using yeast two hybrid screening with S6K1 as bait allowed to identify a novel binding partner of S6K1 - TDRD7 (Tudor Domain Containing Protein 7), a scaffold protein involved in regulation of cytoskeleton dynamics, mRNA transport, protein translation, piRNAs processing. In H. sapiens, the full-length TDRD7 protein represents a 1098 amino acids polypeptide and contains three so-called LOTUS domains at the N-terminal part and Tudor domains more close to C-part of TDRD7 molecule. LOTUS domains are necessary for RNA binding, as far as Tudor domains are unique structures which allow to interact with proteins methylated Arg and Lys as well as nucleic acids. Tudor module are common for scaffold proteins, which are involved in regulation of chromosomes stability in cell nucleus through interaction with methylated histones. First of all, we have performed the bioinformatical analysis of TDRD7 primary structure. Next, six different peptides of TDRD7 were cloned, overexpressed and purified from bacteria cells. These recombinant proteins were used in a set of pull-down experiments with full-length S6K1. Direct interaction between C-terminal Tudor domain containing fragment of TDRD7 and S6K1 has been shown. This interaction was further confirmed in Far-western blot on recombinant S6K1 and TDRD7 fragments. Moreover, purified domains of TDRD7 were used as antigens for mouse immunizations and generation of monoclonal antibodies. The generated antibodies were used for studying S6K1/TDRD7 interaction in mammalian cells in vivo. We succeed to detect a complex formation between TDRD7 and S6K1 in HEK293 lysates. The application of previously generated anti-TDRD7 and anti-S6K2 antibodies, allowed us to detect the existence of S6K2-TDRD7 complexes in HEK293. Moreover, we have detected that C-terminal synthetic peptides of S6K2 with methylated Arg directly interact with recombinant TDRD7 fragments, as far as peptide with methylated Arg475 precipitated endogenous TDRD7 from HEPG2 lysates. We have found that S6K1 and S6K2 phosphorylate three from five fragments of TDRD7 in the in vitro kinase reaction, indicating that TDRD7 could be a new substrate for S6K1 as well as for S6K2. We suppose that the phosphorylation of TDRD7 by S6K1 or/and S6K2 may influence TDRD7 association with other proteins/RNAs and its subcellular localization. Taking into account that S6K1 and S6K2 possess NLS-motifs in their structure, interaction with TDRD7 also may be important for the regulation of S6K1 and/or S6K2 nucleocytoplsmic shuttling. The application of generated anti-TDRD7 antibodies in immunofluorescent microscopy allowed us to detect TDRD7 not only in cytoplasm, but also in the nucleus of HEK293 cells. This observation was further supported with Western blot analysis of subcellular fractionation. Hence we suppose that several new TDRD7 isoforms were identified in HEK293. And finally, confocal microscopy studies showed co-localization of S6K1/TDRD7 and S6K2/TDRD7 within perinuclear region in HEK293, HEPG2 cells and in soma of primary rat hippocampal neurons. We should point out the possible involvement of TDRD7 in the regulation of S6K1 and/or S6K2 nuclear-cytoplasmic transport. We assume that the localization TDRD7 complexes with both kinases in the perinuclear region indicate exactly the same role of TDRD7. Another cellular compartment for S6K1/2 and TDRD7 interaction may be the cell nucleus. TDRD7 is involved in chromatin remodelling process, including the interaction with methylated histones, and also transcription regulation. Since S6K is involved in regulation of transcription factor CREM activity, these processes are closely linked, it is possible that TDRD7-S6K interaction have a regulatory role in signal transduction during the spermatogenetic cycle, as well as other complex processes. Moreover, recent studies have shown the formation of T-cells active complex involving mTOR, S6K, 4E-BP1, Aurora kinase B and protein survivin is involved in cell cycle regulation, particulary in the control of cells transition from G1 to S phase. Our data revealed that under physiological conditions, a certain fraction of TDRD7 is co-localized with S6K kinases in the perinuclear region. As far as this co-localization in cells of different tissue origin suggests a specificity of TDRD7 and S6K complexes formation and, in addition, may indicate for its possible physiological importance. Thus, taking into account the obtained results, we can conclude that TDRD7-S6K interaction may be a regulatory element in many cellular processes, including splicing, mRNA translation and transport.
