Effects of okadaic acid, a protein phosphatase inhibitor, on glucocorticoid receptor-mediated enhancement.
The effects of okadaic acid (OA), a protein phosphatase inhibitor, on transcriptional enhancement activity of rat glucocorticoid receptor (GR) were examined in transiently transfected cells. In the absence of hormone, GRs expressed in CV-1 and COS-1 fibroblasts were capable of enhancing transcription from cotransfected chloramphenicol acetyltransferase reporter plasmids in response to OA treatment. Synergistic enhancement resulted from combined hormone and OA treatment. The effects of OA on GR-mediated enhancement required the presence of linked glucocorticoid response elements and were observed with reporter plasmids that contained different promoters and glucocorticoid response elements. Since OA did not affect nuclear translocation of the receptor, enhancement mediated by unliganded GR was most likely accounted for by the accumulation of some unliganded GRs within nuclei of transfected CV-1 and COS-1 cells. Deletion of individual GR transactivation domains and point mutations within DNA- and hormone-binding domains severely reduced the response of receptors to OA, although some mutant receptors retained the capacity to elicit a synergistic response when exposed to OA and hormone. The effects of OA on transcriptional enhancement did not appear to correlate with major changes in GR phosphorylation, as visualized by two-dimensional tryptic mapping of in vivo 32P-labeled GRs. Thus, phosphorylation of various components of the GR signal transduction pathway, and not necessarily the receptor itself, may influence its transcriptional enhancement activity.
Somers
J P
JP
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260.
DeFranco
D B
DB
D000818:Animals; D002460:Cell Line; D004742:Enhancer Elements, Genetic / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D004988:Ethers, Cyclic / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed / Q000235:genetics / Q000502:physiology; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000502:physiology; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology; D015533:Transcriptional Activation / Q000235:genetics / Q000502:physiology
D004988:Ethers, Cyclic; D011965:Receptors, Glucocorticoid; D014157:Transcription Factors; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases