REGULATION OF BASIC BIOLOGICAL PROCESSES BY THE FKBP51 TO FKBP52 EXPRESSION RATIO

ERLEJMAN AG, MAZAIRA GI, DE LEO SG, CAMISAY MF, DANERI CR, FEDERICCI F, CAUERHFF A, GALIGNIANA MD

Heraklion

Congreso; EMBO Meeting on Molecular Chaperones: From Molecules to Cells and Misfolding Diseases; 2015

EMBO

The conserved MEEVD motif of the C-terminal domain ofHsp90 serves as the docking site for co-chaperones showing a tetratricopeptide-repeat(TPR) clamp. The general consensus is that for mature heterocomplexes, astoichiometry equal to one TPR protein bound per dimer of Hsp90 is the mostprobable association existing in the cell. This is also supported by thedrastic changes observed in biological processes when a functional exchange ofTPR-proteins takes place in a mutually exclusive fashion. Early studies showedthat upon hormone-binding to steroid receptors, the Hsp90-binding immunophilinFKBP51 is exchanged by FKBP52. Based on this model, we subsequently analyzedother Hsp90-interacting factors such as p53 and RAC3/AIF, and found similarantagonistic properties for both immunophilins. Here we report two novelimmunophilin-dependent regulatory mechanisms, one of them is linked to Hsp90complexes and the other is Hsp90-independent. When undifferentiated nervouscells are incubated with the macrolide FK506, they rapidly acquire a neuronalphenotype. Accordingly, neuronal biochemical markers are also induced. Both, undifferentiatedneuroblastoma cells and primary cultures of embryonic hippocampal neurons, form a perinuclear heterocomplex composed by FKBP52, Hsp90, and p23, which colocalizes with nuclear lamina. Upon celldifferentiation with FK506, this annular structuredisassembles in a MAPK-(ERK1/2)-dependent manner, the chaperones redistribute in the cytoplasm, and FKBP52 and Hsp90 concentrate in terminal axons.Interestingly, the perinuclear areathat had been occupied by these chaperones becomes transcriptionallyhyperactive. All theseeffects are prevented by the Hsp90-disrupting agent, geldanamycin. FKBP51, whose overall expression remains constant during cell differentiation, is weaklydetected in neurites and axons, increases the levels of a specificphosphorylated isoform, and relocalizes to those perinuclear areas where FKBP52was originally present. Analysis of isolated nuclear envelopes by biochemicalfractionation and the co-immunoprecipitation of both immunophilins and Hsp90with perinuclear lamin confirm that FKBP52 and FKBP51 are associated tochromatin filaments linked to the nuclear envelope. Importantly, theoverexpression of FKBP52 per se accelerates neurodifferentiation (even in theabsence of FK506), whereas the overexpression of FKBP51 shows antagonisticaction. Accordingly, the knock-down of FKBP52 impairs differentiation whereasthe knock-down of FKBP51 favors it. Taken together, these data clearlydemonstrate that both immunophilins play an antagonistic role duringneurodifferentiation at various levels of the cell organization. On the otherhand, the NF-êB signalling system is also a regulator of neuronal differentiation, such that permanent activation of NF-êB is detrimentaland favors the opposite phenomenon, neurodegeneration. Therefore, the putativeregulation of NF-êB action by immunophilins was also analyzed. In unstimulated cells,FKBP51 and not FKBP52 coimmunoprecipitates with RelA/p65, and the oppositeassociation pattern is seen after NF-êB activation. FKBP51 overexpression impairs both thenuclear translocation rate of NF-kB and its transcriptional activity. Neither thepeptidylprolyl-isomerase activity nor Hsp90-binding is required for thisinhibitory action, but the TPR domain. On the other hand, the recruitment of FKBP52favors the nuclear retention time of RelA/p65, its association to DNA consensusbinding sequences, and NF-kB transcriptional activity, the latter effect beingstrongly dependent on both the peptidylprolyl-isomerase activity and the TPR-domainof FKBP52, although Hsp90 is not required. Importantly, FKBP52 is functionally associatedto the promoter region of NF-kB target genes upon cell stimulation, whereas FKBP51is released from those sites. Protein-protein competition studies reveal thatboth immunophilins antagonize one another, and binding assays with purifiedproteins suggest that the association with RelA/p65 is direct. Taken together,all these observations demonstrate that the FKBP52/FKBP51 expression ratio in agiven cell type is a determining condition for shaping basic cellular processessuch as the rearrangement of nuclear and cytoplasmic architectures, celldifferentiation, cell cycle, gene expression, and the regulation of keysignalling cascades.