Functional subcellular relocalization of the hsp90-binding immunophilins FKBP51 and FKBP52

GALLO, L.I.; GUBER, S.; BELLO, E., PIWIEN PILIPUK, G.; GALIGNIANA, M.D.

Concepción, Chile

Workshop; 5th International Workshop on the Molecular Biology of Stress Responses; 2006

Cell Stress Society International

In their mature form, the members of the steroid receptor (SR) family form heterocomplexes with hsp90, hsp70, p23 and a co-chaperone that possesses tetratricopeptide repeat (TPR) domains. To date, five high molecular weight immunophilins (MMs) with TPR domains have been discovered as components of SR•hsp90 complexes: FKBP52, FKBP51, CyP40, XAP2 and PP5, the latter of which also shows Ser/Thr-phosphatase activity. Recent evidence shows that the hsp90•FKBP52 complex is required for the retrograde movement of both the glucocorticoid receptor (GR) (Galigniana et al., J Biol Chem (2001) 276:14884) and the proapoptotic nuclear factor, p53 (Galigniana et al., J Biol Chem (2004) 279:22483). Such movement towards the nucleus is powered by dynein/dynactin, a microtubule-associated motor protein complex that binds to cargo via the peptidyl-prolyl isomerase domain of the hsp90-bound IMM. Clearly, this model for protein movement suggests that the hsp90•IMM-based heterocomplex should be associated to cargoes during the time they are transported throughout the cytoplasm towards the nucleus. As such, the novel model collides with the classical dogma for SR transformation that was posited twenty years ago. Therefore, if the hsp90-binding IMMs are bound to cargoes during trafficking, it would also be possible that IMMs may participate in the subnuclear redistribution of nuclear factors when they reach the nuclear compartment. In this work we analyze the subcellular distribution of IMMs and the GR upon steroid binding. Dexamethasone (Dex) binding promotes the displacement of FKBP51 from the GR•hsp90 complex and the recruitment of FKBP52, PP5 and dynein. Confocal microscopy imaging of 3T3-L1 cells grown in a steroid-free medium shows that FKBP51 is concentrated in the soluble cytoplasmic fraction and, surprisingly, in mitochondria. This was confirmed by cell fractionation. GR becomes nuclear with Dex and some FKBP51 is also accumulated in the nucleus. Interestingly, the IMM also concentrates in a perinuclear rim that is excluded from the nuclear envelope as it is evidenced by co-staining with an anti-lamin B antibody. On the other hand, both the IMMsFKBP52 and PP5 localizes in the cytoplasm and nuclei of 3T3-L1 cells. Dex promotes nuclear acumulation of the GR, and hundred percent of the nuclear GR colocalizes with both IMMs in specific nuclear speckles. Interestingly, the nuclear matrix protein NuMA is also observed in those nuclear speckles, suggesting that the GR is anchored to nuclear matrix structures via IMMs. The hsp90 co-chaperone, p23, is also recruited to nuclear speckles. Cell treatment with the hsp90-disrupting agent radicicol disassembles these structures and also favors GR nuclear export, suggesting that these structures require hsp90. Similar nuclear speckles were also observed for the nuclear factor p53. Transcriptional assays performed by using the MMTV-Luc reporter gene demonstrate that FKBP51, but not FKBP52, exerts an inhibitory effect on the Dex-dependent transactivation of GR. Interestingly, when 3T3-L1 cells are induced to differentiate into adipocytes, there is an early strong induction of FKBP51, which moves from cytoplasm to nuclei. After 48 h of differentiation, FKBP51 cycles back to cytoplasm and relocates in cytosol and mitochondria. FKBP51 migration towards the nucleus is not observed when 3T3-L1 cells are induced to differenciate in a steroid-free medium. We conclude that IMMs are implicated in the subcellular distribution of SRs. We postulate that GR anchoring to the nuclear matrix is not direct but depends on the hsp90•FKBP52/PP5 complex. Thus, GR could be immobilized on staging regulatory nuclear areas where it lays as a reserve or undergoes a maturation process (e.g., phosphorylation) before becoming transcriptionally active. GR complexes with FKBP51 inhibit glucococrticoid biological effects, which may be a requirement for the efficient differentiation of fibroblasts into adipocytes after the GR is activated by Dex at the very early steps of this event.