Evidence That the Peptidylprolyl Isomerase Domain of the hsp90-binding Immunophilin FKBP52 Is Involved in Both Dynein Interaction and Glucocorticoid Receptor Movement to the Nucleus

Abstract

Here, we also show that native GR⅐hsp90 heterocomplexes immunoadsorbed from L cell cytosol contain dynein and that GR⅐hsp90 heterocomplexes assembled in reticulocyte lysate contain cytoplasmic dynein in a manner that is competed by the PPIase domain of FKBP52.Steroid receptors move continuously into and out of the nucleus (Refs. 1-4; for review, see Ref. 5), and, depending upon the receptor, the hormone-free, untransformed receptor may have a predominantly nuclear or cytoplasmic localization. The hormone-free glucocorticoid receptor (GR) 1 is localized to the cytoplasm of most cells, and after steroid binding and transformation, it translocates to the nucleus (6 -8). Several studies with inhibitors suggest that the multiprotein hsp90-based chaperone system and the hsp90-binding immunophilin FKBP52 are involved in movement of the GR along microtubular tracks to the nucleus (for review, see Ref. 9). Assembly of receptors into heterocomplexes with hsp90 is a dynamic process (10), and it has been shown that the GR and hsp90 can move together from the cytoplasm to the nucleus (11). A couple of observations suggest that the role of hsp90 in receptor movement is likely to involve dynamic assembly and disassembly of GR⅐hsp90 heterocomplexes. For example, Yang and DeFranco (12) showed that molybdate, which binds to hsp90 and stabilizes GR⅐hsp90 heterocomplexes in vivo (13), traps the GR in the cytoplasm of cells continuously exposed to hormone. Molybdate in this case was thought to inhibit reimport of the GR into the nucleus by inhibiting the dynamic cycling of receptors into and out of their complexes with the hsp90 chaperone. Also, geldanamycin, an antibiotic that binds to the nucleotide binding site on hsp90 (14) and prevents formation of normal receptor⅐hsp90 heterocomplexes (15), impedes steroid-induced movement of the GR from the cytoplasm to the nucleus (16, 17).Some localization studies have shown the untransformed GR to colocalize with microtubules (for review, see Ref. 18), but the evidence supporting movement along microtubular tracks is indirect. Although microtubule disrupting agents, such as colcemid, do not affect the overall rate of steroid-dependent receptor translocation to the nucleus (8, 19), they eliminate the hsp90-dependent mode of receptor movement (17). Using a fusion protein of murine GR with Aequorea green fluorescent protein (GFP), we found that steroid-dependent GFP-GR translocation to the nucleus is rapid (t1 ⁄2 ϭ ϳ5 min) both in cells with intact cytoskeleton and in cells with disrupted cytoskeletal networks (17). However, in cells with normal cytoskeleton, the hsp90 inhibitor geldanamycin slowed translocation of the GFP-GR by an order of magnitude (t1 ⁄2 ϭ ϳ45 min), whereas in cells with colcemid-disrupted microtubules, geldanamycin had no effect on the translocation rate (t1 ⁄2 ϭ ϳ5 min). This suggests two mechanisms of GR movement. Under physiological conditions where the cytoskeleton is intact, diffusion is limited, and the GFP-GR utilizes a movement machinery in which the hsp90 heter...

Importin 7 and Importin α/Importin β Are Nuclear Import Receptors for the Glucocorticoid Receptor

“…Pratt and colleagues have demonstrated that components of the apoGR complex bind cytoskeletal factors (Galigniana et al, 2001). Alternatively, Kino et al (2003) have suggested that binding to the 14-3-3 protein helps tether apoGR to the cytoplasmic compartment.…”

Hsp90-binding Immunophilins Link p53 to Dynein During p53 Transport to the Nucleus

“…The pGEX1 T plasmid encoding the GST-rabbit FKBP52 Gly 32 -Lys 138 expression vector that comprises the PPIase core domain I (provided by Drs. Michel Renoir and Christine Radanyi, UMR8612 CNRS, Paris, France) and the purification of the PPIase core domain I protein were described previously (23,34). The mammalian expression plasmid pSG5PL-PPIase core domain I was described in a previous work (23).…”

“…GR⅐hsp90 heterocomplexes immunoadsorbed from cell lysates contain cytoplasmic dynein (23,24), a molecular motor that processes along microtubular tracks toward the nucleus (25). These heterocomplexes also contain one of several immunophilins with tetratricopeptide repeat (TPR) domains that interact with a TPR acceptor site on hsp90 (13).…”

Independent Functions of hsp90 in Neurotransmitter Release and in the Continuous Synaptic Cycling of AMPA Receptors

“…hsp90 was proposed to be a mediator of protein trafficking over a decade ago (Pratt, 1992(Pratt, , 1993. Since then, ample evidence has accumulated indicating that hsp90 is required for the subcellular targeting of a variety of proteins, including the glucocorticoid receptor (Owens-Grillo et al, 1996;Czar et al, 1997;Silverstein et al, 1999;Galigniana et al, 2001), the dioxin receptor (Kazlauskas et al, 2001), the receptor tyrosine kinase ErbB2 (Xu et al, 2002), the epidermal growth factor receptor (Supino-Rosin et al, 2000), the CFTR (cystic fibrosis transmembrane regulator) chloride channel (Loo et al, 1998) and the G-protein G␣ 12 (Waheed and Jones, 2002). hsp90 is also required for protein translocation into mitochondria (Young et al, 2003) and peroxisomes (Crookes and Olsen, 1998).…”