Regulation of basic biological processes by the FKBP51 to FKBP52 expression ratio

ALEJANDRA G. ERLEJMAN; GISELA I. MAZAIRA; SONIA DE LEO; MARÍA FERNANDA CAMISAY; CRISTINA R. DANERI; FERNANDO FEDERICCI; ANA CAUERHFF; MARIO D. GALIGNIANA

Heraklion

Congreso; EMBO Conference, Molecular Chaperones: From Molecules to Cells and Misfolding Diseases; 2015

EMBO

The conserved MEEVD motif of the C-terminal domain of Hsp90 serves as the docking site for co-chaperones showing a tetratricopeptide-repeat (TPR) clamp. The general consensus is that for mature heterocomplexes, a stoichiometry equal to one TPR protein bound per dimer of Hsp90 is the most probable association existing in the cell. This is also supported by the drastic changes observed in biological processes when a functional exchange of TPR-proteins takes place in a mutually exclusive fashion. Early studies showed that upon hormone-binding to steroid receptors, the Hsp90-binding immunophilin FKBP51 is exchanged by FKBP52. Based on this model, we subsequently analyzed other Hsp90-interacting factors such as p53 and RAC3/AIF, and found similar antagonistic properties for both immunophilins. Here we report two novel immunophilin-dependent regulatory mechanisms, one of them is linked to Hsp90 complexes and the other is Hsp90-independent. When undifferentiated nervous cells are incubated with the macrolide FK506, they rapidly acquire a neuronal phenotype. Accordingly, neuronal biochemical markers are also induced. Both, undifferentiated neuroblastoma cells and primary cultures of embryonic hippocampal neurons, form a perinuclear heterocomplex composed by FKBP52, Hsp90, and p23, which colocalizes with nuclear lamina. Upon cell differentiation with FK506, this annular structure disassembles in a MAPK-(ERK1/2)-dependent manner, the chaperones redistribute in the cytoplasm, and FKBP52 and Hsp90 concentrate in terminal axons. Interestingly, the perinuclear area that had been occupied by these chaperones becomes transcriptionally hyperactive. All these effects are prevented by the Hsp90-disrupting agent, geldanamycin. FKBP51, whose overall expression remains constant during cell differentiation, is weakly detected in neurites and axons, increases the levels of a specific phosphorylated isoform, and relocalizes to those perinuclear areas where FKBP52 was originally present. Analysis of isolated nuclear envelopes by biochemical fractionation and the co-immunoprecipitation of both immunophilins and Hsp90 with perinuclear lamin confirm that FKBP52 and FKBP51 are associated to chromatin filaments linked to the nuclear envelope. Importantly, the overexpression of FKBP52 per se accelerates neurodifferentiation (even in the absence of FK506), whereas the overexpression of FKBP51 shows antagonistic action. Accordingly, the knock-down of FKBP52 impairs differentiation whereas the knock-down of FKBP51 favors it. Taken together, these data clearly demonstrate that both immunophilins play an antagonistic role during neurodifferentiation at various levels of the cell organization. On the other hand, the NF-κB signalling system is also a regulator of neuronal differentiation, such that permanent activation of NF-κB is detrimental and favors the opposite phenomenon, neurodegeneration. Therefore, the putative regulation of NF-κB action by immunophilins was also analyzed. In unstimulated cells, FKBP51 and not FKBP52 coimmunoprecipitates with RelA/p65, and the opposite association pattern is seen after NF-κB activation. FKBP51 overexpression impairs both the nuclear translocation rate of NF-B and its transcriptional activity. Neither the peptidylprolyl-isomerase activity nor Hsp90-binding is required for this inhibitory action, but the TPR domain. On the other hand, the recruitment of FKBP52 favors the nuclear retention time of RelA/p65, its association to DNA consensus binding sequences, and NF-B transcriptional activity, the latter effect being strongly dependent on both the peptidylprolyl-isomerase activity and the TPR-domain of FKBP52, although Hsp90 is not required. Importantly, FKBP52 is functionally associated to the promoter region of NF-B target genes upon cell stimulation, whereas FKBP51 is released from those sites. Protein-protein competition studies reveal that both immunophilins antagonize one another, and binding assays with purified proteins suggest that the association with RelA/p65 is direct. Taken together, all these observations demonstrate that the FKBP52/FKBP51 expression ratio in a given cell type is a determining condition for shaping basic cellular processes such as the rearrangement of nuclear and cytoplasmic architectures, cell differentiation, cell cycle, gene expression, and the regulation of key signalling cascades.