title: Heat shock protein 90-dependent (geldanamycin-inhibited) movement of the glucocorticoid receptor through the cytoplasm to the nucleus requires intact cytoskeleton.
abstract: We use here a chimera of the green fluorescent protein (GFP) and the glucocorticoid receptor (GR) to test the notion that the protein chaperone heat shock protein-90 (hsp90) is required for steroid-dependent translocation of the receptor through the cytoplasm along cytoskeletal tracks. The GFP-GR fusion protein undergoes steroid-mediated translocation from the cytoplasm to the nucleus, where it is transcriptionally active. Treatment of 3T3 cells containing steroid-bound GFP-GR with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, inhibits dexamethasone-dependent translocation from the cytoplasm to the nucleus. The t1/2 for translocation in the absence of geldanamycin is approximately 5 min, and the t1/2 in the presence of geldanamycin is approximately 45 min. In cells treated for 1 h with the cytoskeletal disrupting agents colcemid, cytochalasin D, and beta,beta'-iminodipropionitrile to completely disrupt the microtubule, microfilament, and intermediate filament networks, respectively, the GFP-GR still translocates rapidly to the nucleus in a strictly dexamethasone-dependent manner but translocation is no longer affected by geldanamycin. After withdrawal of the cytoskeletal disrupting agents for 3 h, normal cytoskeletal architecture is restored, and geldanamycin inhibition of 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 conditions 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 protein that is not complexed with hsp90, undergoes GH-dependent translocation to the nucleus in a manner that is not dependent upon hsp90 chaperone activity.
authors.affiliation: Department of Pharmacology, The University of Michigan Medical School, Ann Arbor 48109, USA.
authors.forename: M D
authors.initials: MD
authors.lastname: Galigniana
authors.forename: J L
authors.initials: JL
authors.lastname: Scruggs
authors.forename: J
authors.initials: J
authors.lastname: Herrington
authors.forename: M J
authors.initials: MJ
authors.lastname: Welsh
authors.forename: C
authors.initials: C
authors.lastname: Carter-Su
authors.forename: P R
authors.initials: PR
authors.lastname: Housley
authors.forename: W B
authors.initials: WB
authors.lastname: Pratt
mesh_terms: D016475:3T3 Cells; D000818:Animals; D016227:Benzoquinones; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D018397:Equidae; D049452:Green Fluorescent Proteins; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D047029:Lactams, Macrocyclic; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008894:Milk Proteins; D011809:Quinones / Q000494:pharmacology*; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050799:STAT5 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D016227:Benzoquinones; D004268:DNA-Binding Proteins; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D008164:Luminescent Proteins; D008894:Milk Proteins; D011809:Quinones; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D050799:STAT5 Transcription Factor; C494095:STAT5B protein, human; C494096:Stat5b protein, mouse; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; D003907:Dexamethasone; C001277:geldanamycin