The 59 kDa FK506-binding protein, a 90 kDa heat shock protein binding immunophilin (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 immunophilin (HBI), was originally discovered in association with unliganded steroid 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 bind cyclosporin A, all having rotamase (peptidyl-prolyl cis-trans isomerase) activity which may be involved in protein folding. Targets for drug-immunophilin complexes have been mostly studied in vivo in T lymphocytes; however, immunophilins are present in all cell types, where their role and distribution are still unknown. Here we report the localization of FKBP59-HBI in various non lymphoid cells (mouse fibroblasts (L-929), monkey kidney cells (Cos-7), Madin-Darby canine kidney epithelial cells (MDCK), and mouse neuronal cells (GT1)). Two polyclonal antipeptide antibodies directed against the C-terminal end (amino acids 441-458) (Ab 173) or the sequence 182-201 (Ab 790) of the FKBP59-HBI were used in light and confocal laser immunofluorescence. FKBP59-HBI was found in the cytoplasm and nucleus of interphase cells. Specific immunofluorescence was much stronger in the cytoplasm than in the nucleus when using Ab 173, and stronger in the nucleus than in the cytoplasm with Ab 790. Detailed observations of L-cells, which have a particularly 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 FKBP59-HBI with the microtubules. Western blot experiments confirmed that the protein was present in the subcellular fractions containing either '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 microM FK506 and rapamycin for 1 hour did not modify significantly the staining, although rapamycin treatment rendered the network stained by 173 clearly visible. Interestingly, during mitosis FKBP59-HBI segregated from the region of the chromosomes; it mainly localized with the mitotic apparatus (centrosome, spindle and interzone separating the chromosomes), the cleavage furrow and the midbodies during cytokinesis. It appeared again as a fibrous network in the cytoplasm of the two daughters cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Perrot-Applanat
M
M
INSERM U135 Hormones et Reproduction, Faculté de Médecine de Bicêtre, Kremlin-Bicêtre, France.
Cibert
C
C
Géraud
G
G
Renoir
J M
JM
Baulieu
E E
EE
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D002451:Cell Compartmentation; D002460:Cell Line; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000737:chemistry*; D002522:Chlorocebus aethiops; D003599:Cytoskeleton / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D003902:Detergents / Q000494:pharmacology; D004285:Dogs; D004848:Epithelium; D005347:Fibroblasts / Q000737:chemistry / Q000648:ultrastructure; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D007399:Interphase; D007668:Kidney; D007739:L Cells / Q000737:chemistry / Q000648:ultrastructure; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D008938:Mitosis; D009474:Neurons / Q000737:chemistry / Q000648:ultrastructure; D010446:Peptide Fragments / Q000276:immunology; D011090:Polyenes / Q000494:pharmacology; D011487:Protein Conformation; D020123:Sirolimus; D008941:Spindle Apparatus / Q000737:chemistry*; D016559:Tacrolimus / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D003902:Detergents; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D011090:Polyenes; D022021:Tacrolimus Binding Proteins; D002118:Calcium; D020123:Sirolimus; D016559:Tacrolimus