The role of DNA-binding and phosphorylation on nucleocytoplasmic trafficking of the glucocorticoid receptor

To characterize the nuclear transfer defect in the mouse S49 lymphoma, I have used the Arg484-His and Tyr770-Asn single amino acid substituted glucocorticoid receptor (GR) mutants to examine the effects of four factors on nucleocytoplasmic (NC) transport (NCT): defective hormone binding, defective DNA binding, the GR antagonist RU486, and modulators of protein kinase and protein phosphatase activities. A quantitative indirect immunofluorescence assay was developed and used during G;Kinetic measurements of loss of nuclear receptors to the cytoplasm after hormone withdrawal, using combined modulators such as forskolin/(TPA) or forskolin/okadaic acid (OA) demonstrated a cancellation effect on apparent nuclear retention of WT receptor. However, the Arg484-His GR mutant in both cases demonstrated either the effects of TPA alone or the forskolin alone respectively. These preliminary data provide evidence of different phosphorylation events playing an important role in DNA binding and subsequent transcriptional activity of GR.