HEAT-SHOCK-PROTEIN 90-DEPENDENT (GELDANAMYCIN-INHIBITED) MOVEMENT OF THE GLUCOCORTICOID RECEPTOR THROUGH THE CYTOPLASM TO THE NUCLEUS REQUIRES INTACT CYTOSKELETON

We use here a chimera of the green fluorescent protein (GFP) and the g lucocorticoid receptor (GR) to test the notion that the protein chaper one heat shock protein-90 (hsp90) is required for steroid-dependent tr anslocation of the receptor through the cytoplasm along cytoskeletal t racks. The GFP-GR fusion protein undergoes steroid-mediated translocat ion from the cytoplasm to the nucleus, where it is transcriptionally a ctive. Treatment of 3T3 cells containing steroid-bound GFP-GR with gel danamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts i ts function, inhibits dexamethasone-dependent translocation from the c ytoplasm to the nucleus. The t(1/2) for translocation in the absence o f geldanamycin is similar to 5 min, and the t(1/2) in the presence of geldanamycin is similar to 45 min. In cells treated for 1 h with the c ytoskeletal disrupting agents colcemid, cytochalasin D, and beta,beta' -iminodipropionitrile to completely disrupt the microtubule, microfila ment, and intermediate filament networks, respectively, the GFP-GR sti ll translocates rapidly to the nucleus in a strictly dexamethasone-dep endent manner but translocation is no longer affected by geldanamycin. After withdrawal of the cytoskeletal disrupting agents for 3 h, norma l cytoskeletal architecture is restored, and geldanamycin inhibition o f dexamethasone-dependent GFP-GR translocation is restored. We suggest that in cells without an intact cytoskeletal system, the GFP-GR moves through the cytoplasm by diffusion. However, under physiological cond itions in which the cytoskeleton is intact, diffusion is limited, and the GFP-GR utilizes a movement machinery that is dependent upon hsp90 chaperone activity. In contrast to the GR, GFP-STAT5B, a signaling pro tein that is not complexed with hsp90, undergoes OH-dependent transloc ation to the nucleus in a manner that is not dependent upon hsp90 chap erone activity.