THE 59-KDA FK506-BINDING PROTEIN, A 90-KDA HEAT-SHOCK PROTEIN-BINDINGIMMUNOPHILIN (FKBP59-HBI), IS ASSOCIATED WITH THE NUCLEUS, THE CYTOSKELETON AND MITOTIC APPARATUS

FKBP59-HBI, a 59 kDa FK506 binding protein which binds the 90 kDa heat shock protein hsp90 and thus is a heat shock protein binding immunoph ilin (HBI), was originally discovered in association with unliganded s teroid receptors in their heat shock protein containing heterooligomer form. It belongs to a growing family including other FKBPs which bind the immunosuppressants FK506 and rapamycin, and cyclophilins which bi nd cyclosporin A, all having rotamase (peptidyl-prolyl cis-trans isome rase) activity which may be involved in protein folding. Targets for d rug-immunophilin complexes have been mostly studied in vivo in T lymph ocytes; however, immunophilins are present in all cell types, where th eir role and distribution are still unknown. Here we report the locali zation of FKBP59-HBI in various non lymphoid cells (mouse fibroblasts (L-929), monkey kidney cells (Cos-7), Madin-Darby canine kidney epithe lial cells (MDCK), and mouse neuronal cells (GT1)). Two polyclonal ant ipeptide antibodies directed against the C-terminal end (amino acids 4 41-458) (Ab 173) or the sequence 182-201 (Ab 790) of the FKBP59-HBI we re used in light and confocal laser immunofluorescence. FKBP59-HBI was found in the cytoplasm and nucleus of interphase cells. Specific immu nofluorescence was much stronger in the cytoplasm than in the nucleus when using Ab 173, and stronger in the nucleus than in the cytoplasm w ith Ab 790. Detailed observations of L-cells, which have a particularl y flat morphology, showed a punctate as well as a fibrous cytoskeletal staining in the cytoplasm using antibody 173, a result which suggests interactions of FKBP59-HBI with an organized network. Colocalization experiments (using antibodies against tubulin, vimentin or actin) and use of cytoskeletal-disrupting drugs revealed partial association of F KBP59-HBI with the microtubules. Western blot experiments confirmed th at the protein was present in the subcellular fractions containing eit her 'soluble' proteins released from cells exposed to NP40 detergent, or proteins released from the cytoskeleton exposed to calcium ions (i. e. in microtubule depolymerizing conditions). Exposure of cells to 1 m u M FK506 and rapamycin for 1 hour did not modify significantly the st aining, although rapamycin treatment rendered the network stained by 1 73 clearly visible. Interestingly, during mitosis FKBP59-HBI segregate d from the region of the chromosomes; it mainly localized with the mit otic apparatus (centrosome, spindle and interzone separating the chrom osomes), the cleavage furrow and the midbodies during cytokinesis. It appeared again as a fibrous network in the cytoplasm of the two daught ers cells. In conclusion, the present immunocytochemical analysis docu ments the presence of FKBP59-HBI in different cell compartments, where the differential labelling with Ab 173 and Ab 790 suggests different conformations of the protein or different interactions with other prot eins which need to be clarified using biochemical methods. The role of FKBP59-HBI, as well as the roles of other FKBPs present in the same c ells, needs further investigation. Preliminary immunofluorescence obse rvations showed that FKBP12, a FK506 binding protein of 12 kDa homolog ous to the N-terminal region of PKBP59 (59% identity with amino acids 18-134), was only present in the cytoplasm associated with elements of the cytoskeleton, not concentrated into the mitotic apparatus.