Subcellular rearrangement of hsp90‐binding immunophilins accompanies neuronal differentiation and neurite outgrowth

Abstract

Immunophilins (IMMs) are a family of proteins that bind immunosuppressive drugs and possess cis/trans-peptidylprolyl-isomerase activity. They are classified as FKBPs (FK506-binding protein) when they bind FK506, and cyclophilins when they bind cyclosporine A (CsA) . FKBP51 and FKBP52 (gene names FKBP5 and FKBP4, respectively) are highly homologous tetratricopeptide-domain immunophilins whose sequences share 60% identity and 75% similarity. The best characterized interactions of FKBP52 and FKBP51 are with hsp90 and steroid receptor complexes. It has been shown that FKBP52 is required for dynein/dynactin-dependent retrograde movement of glucocorticoid receptor (GR) Address correspondence and reprint requests to Dr Mario D. Galigniana, Departamento de Química Bioló gica, Pabellón II, Facultad de Ciencias Exactas y Naturales, Ciudad Universitaria (C1428EGA), Buenos Aires, Argentina. E-mail: mgali@qb.fcen.uba.arAbbreviations used: AIF, apoptosis-inducing factor; CHX, cycloheximide; CREB, cAMP-response element binding protein; CsA, cyclosporine A; DMEM, Dulbecco's modified Eagle's medium; ERK1/2, extracellular signal-regulated kinases 1 and 2; FKBP, FK506-binding protein; GR, glucocorticoid receptor; IBMX, 3-isobutyl-1-methylxanthine; IMM, immunophilin; MEK, mitogen-activated protein kinase/ ERK kinase; PKA, protein kinase A; RAC3, receptor-associated coactivator 3; RAD, radicicol; TRPC, transient receptor potential channel. Abstract FKBP51 and FKBP52 (FK506-binding protein 51 and 52) are tetratricopeptide repeat-domain immunophilins belonging to the tetratricopeptide-protein•hsp90•hsp70•p23 heterocomplex bound to steroid receptors. Immunophilins are related to receptor folding, subcellular localization, and hormonedependent transcription. Also, they bind the immunosuppressant macrolide FK506, which shows neuroregenerative and neuroprotective actions by a still unknown mechanism. In this study, we demonstrate that in both, undifferentiated neuroblastoma cells and embryonic hippocampal neurons, the FKBP52•hsp90•p23 heterocomplex concentrates in a perinuclear structure. Upon cell stimulation with FK506, this structure disassembles and this perinuclear area becomes transcriptionally active. The acquisition of a neuronal phenotype is accompanied by increased expression of bIII-tubulin, Map-2, Tau-1, but also hsp90, hsp70, p23, and FKBP52. During the early differentiation steps, the perinuclear heterocomplex redistributes along the cytoplasm and nascent neurites, p23 binds to intermediate filaments and microtubules acquired higher filamentary organization. While FKBP52 moves towards neurites and concentrates in arborization bodies and terminal axons, FKBP51, whose expression remains constant, replaces FKBP52 in the perinuclear structure. Importantly, neurite outgrowth is favored by FKBP52 over-expression or FKBP51 knock-down, and is impaired by FKBP52 knock-down or FKBP51 over-expression, indicating that the balance between these FK506-binding proteins plays a key role during the early mechanism of neuronal differentiation.

The 90-kDa Heat-shock Protein (Hsp90)-binding Immunophilin FKBP51 Is a Mitochondrial Protein That Translocates to the Nucleus to Protect Cells against Oxidative Stress

“…Recent studies demonstrated that in both undifferentiated neuroblastoma cells and embryonic hippocampal neurons, the early subcellular relocalization of FKBP51 and FKBP52 relates to neuronal differentiation and neurite outgrowth (28). During these studies on the subcellular redistribution of immunophilins, we noted that FKBP51 shows a peculiar cytoplasmic pattern compatible with mitochondrial localization.…”

“…Whether FKBP51 may use the Hsp90/Hsp70-dependent mechanism for mitochondrial import is still uncertain, although it is possible in view of the requirement of the TPR domain of FKBP51. Recently, it was shown that neuronal FKBP51 also forms complexes with Hsc70 (28). Previous studies have already demonstrated that Hsp70 selectively targets the TPR domains in SGT1 (41) and heat-shock organizing protein (Hop) (42), whereas CyP40 (43) and carboxy terminus of Hsc-70 interacting protein (CHIP) (44) are less discriminatory and can bind either Hsp90 or Hsc70.…”

NF-κB Transcriptional Activity Is Modulated by FK506-binding Proteins FKBP51 and FKBP52

“…Indirect Immunofluorescence Assays-Cells grown on polylysine-coated coverslips were fixed with 3% p-formaldehyde for 30 min and permeabilized with 0.1% Triton X-100 as described (31). Indirect immunofluorescence was performed using 1/100 dilutions of the following primary antibodies: rabbit monoclo-nal IgG anti-FKBP51 (Affinity BioReagents, Golden, CO), MG19 mouse monoclonal IgG anti-FKBP51 (32), UP30 rabbit antiserum against FKBP52 (33), and anti-HA or anti-RelA mouse monoclonal antibodies (Santa Cruz Biotechnology, Dallas, TX) to detect transfected RelA protein or endogenous RelA protein, respectively.…”

Glycan-dependent binding of galectin-1 to neuropilin-1 promotes axonal regeneration after spinal cord injury

“…With the same settings, the confocal images were obtained to develop 3D reconstruction images to perform the 3D colocalization analysis as previously described. 57 Assessment of the spinal cord scar and regenerated axons. Quantification of the spinal cord scar, lesion and neurite-regenerated areas were performed using the Fiji program (v.1.45) (NIH; Bethesda, MA, USA) with an area measurement and a calibration/set scale plug-in for the pre-calibration of each image.…”