FLJ22318 - a novel binding partner of NKX3.1 in prostate cancer cells Free

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NKX3.1 is a prostate-restricted homeodomain protein and prostatic tumour suppressor that controls proliferation of epithelial cells in the adult prostate. Yeast two-hybrid analysis, performed to isolate candidate NKX3.1 binding proteins, resulted in the identification of FLJ22318 (RMND5B). FLJ22318 is a novel hypothetical gene located at 5q35, a chromosomal region frequently disrupted in a variety of tumours and recently implicated in familial prostate cancer. FLJ22318 mRNA is found in cDNA libraries derived from adult and embryonic tissues, suggesting that it is ubiquitously expressed. Bioinformatic analyses have predicted multiple FLJ22318 mRNA transcripts and northern blotting of LNCaP, DU145 and PC-3 prostate cancer cells has identified ~4kb and ~2kb mRNA’s in each cell line that correspond to predicted mRNA species. All of the proposed mRNA’s encode proteins with a novel domain structure that includes three protein-protein interaction domains, a Lissencephaly type-1-like homology (LisH), a C-terminal to LisH (CTLH) and a CT11-RanBPM (CRA) domain. This domain composition is found in only seven human proteins, including an FLJ22318 orthologue, designated FLJ13190 (RMND5A). FLJ22318 orthologues have been identified in other mammalian species and exhibit an extraordinary degree of amino acid conservation, with human and mouse FLJ22318 each containing 393 amino acids and differing by only 9 amino acids scattered throughout the protein. Remarkably, of the 44 single nucleotide polymorphisms identified in the FLJ22318 gene, none are located in the proposed coding region, findings which suggest that FLJ22318 structure and therefore function are essential in the cell and consequently invariant. FLJ22318 interaction with NKX3.1 was confirmed using reverse yeast two-hybrid analysis, GST-pulldown and co-immunoprecipitation assays. Following transfection of LNCaP cells, perinuclear and nuclear co-localisation of Myc-FLJ22318 and NKX3.1-V5 was detected using confocal microscopy. Co-localisation was enhanced by DHT treatment of transfected cells, which increased nuclear localisation of NKX3.1. FLJ22318 exhibited transcriptional repressor activity from a NKX3.1 responsive luciferase reporter plasmid and increased the transcriptional repressor activity of NKX3.1 from this plasmid. These studies have therefore identified NKX3.1 interaction with the novel protein, FLJ22318. Further characterisation of the functional consequences of this interaction and elucidation of the biological activity of FLJ22318 will identify the contribution of this novel protein in normal cellular physiology and in prostate and other tumours.