Inhibition of glucocorticoid receptor nucleocytoplasmic shuttling by okadaic acid requires intact cytoskeleton.
It has been shown previously that glucocorticoid receptors (GRs) that have undergone hormone-dependent translocation to the nucleus and have subsequently exited the nucleus upon hormone withdrawal are unable to recycle into the nucleus if cells are treated during hormone withdrawal with okadaic acid, a cell-permeable inhibitor of certain serine/threonine protein phosphatases. Using a green fluorescent protein (GFP) GR chimera (GFP-GR), we report here that okadaic acid inhibition of steroid-dependent receptor recycling to the nucleus is abrogated in cells treated for 1 h with colcemid to eliminate microtubule networks prior to steroid addition. After withdrawal of colcemid, normal cytoskeletal architecture is restored and okadaic acid inhibition of steroid-dependent GFP-GR nuclear recycling is restored. When okadaic acid is present during hormone withdrawal, GR that is recycled to the cytoplasm becomes complexed with hsp90 and binds steroid, but it does not undergo the normal agonist-dependent dissociation from hsp90 upon retreatment with steroid. However, when the cytoskeleton is disrupted by colcemid, the GR in okadaic acid-treated cells recycles from the cytoplasm to the nucleus in an agonist-dependent manner without dissociating from hsp90. This suggests that under physiological conditions where the cytoskeleton is intact, a dephosphorylation event is required for loss of high affinity binding to hsp90 that is required for receptor translocation through the cytoplasm to the nucleus along cytoskeletal tracts.
Galigniana
M D
MD
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Housley
P R
PR
DeFranco
D B
DB
Pratt
W B
WB
D016475:3T3 Cells; D000818:Animals; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D003345:Corticosterone / Q000494:pharmacology; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism*; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D003703:Demecolcine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D049452:Green Fluorescent Proteins; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D019319:Okadaic Acid / Q000008:administration & dosage / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D008164:Luminescent Proteins; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019319:Okadaic Acid; C067024:okadaic acid-sensitive phosphatase; D010749:Phosphoprotein Phosphatases; D003345:Corticosterone; D003703:Demecolcine