Characterisation of the novel hsp90 interacting protein FKBPL and its potential role in steroid receptor complexes

The FKBPL protein was recently identified as an hsp90 interacting protein and shares significant homology to the immunophilins FKBP52 and cyclophilin 40. The FKBPs are involved in many cellular processes, one of the most documented being their role in hsp90-steroid 'receptor complexes. It was therefore thought logical to investigate a potential role for FKBPL in these complexes. The main aims were to evaluate potential interactions between FKBPL the glucocorticoid receptor (GR) and dynamitin. The functional role played by FKBPL would be addressed in overexpression studies. Since a single nucleotide polymorphic variant and a 12 bp insertion mutant of FKBPL had been identified, the ability of these variants to interact and function within steroid receptor complexes was also evaluated. Finally. a small study was carried out to determine if a correlation existed between the frequency of the FKBPL SNP and the incidence of cancer. Co-localisation studies revealed that FKBPL co-Iocalisea with the glucocorticoid receptor, dynamitin and tubulin. Interactions of FKBPL and its variants with dynamitin was verified using the biomolecular fluorescence complementation assay. furthermore, the cytoplasmic localisation of the interaction with hsp90 was determined in this assay. This assay also confirmed interactions of the FKBPL variants with hsp90. Interactions between endogenous FKBPL and polymorphic FKBPL with GR/dynamitin were confirmed by co-immunoprecipitation. These data suggested a role for FKBPL in steroid hormone receptor complexes. Co-localisation of GR and FKBPL was examined after treatment with the GR ligand dexamethasone. Both proteins translocated from the cytoplasm to the nucleus of DU145 cells within 10 min, indicative of retrograde transport. FKBPL overexpression was found to decrease GR protein levels and transactivity in L132 cells, and increase GR protein levels in DU145 cells. FKBPL overexpression also caused a dexamethasone . dose dependent increase in GR transactivity in DU145 cells. These data suggest FKBPL is involved in steroid rec~ptor complexes and modulates GR protein levels and signalling. Finally, we demonstrated that although the homozygous SNP variant was more prevalent in DNA from endometrial cancer and HNPCC patients, compared to controls, it \Vas not significant.EThOS - Electronic Theses Online ServiceGBUnited Kingdo