Glycogen synthase kinase 3 has roles in a great range of biological processes that include not only its regulation of metabolism and insulin action through phosphorylation of glycogen synthase but also functions in wnt signaling (through β-catenin), inflammation (through phosphorylation of NF-κB), cell cycle control (through cyclin D1), and translation (through eukaryotic initiation factor 2B). Galliher-Beckley et al. extend its known actions even further by identifying a new target site of GSK-3 phosphorylation in the human glucocorticoid receptor (GR). The glucocorticoid receptor is phosphorylated at several sites and undergoes other covalent modifications as well that may help tune it to appropriate activities in various tissues and cell types. The authors detected dexamethasone (a GR agonist)–enhanced phosphorylation of affinity-tagged GR expressed in osteosarcoma cells at Ser404 by mass spectrometry and used antibodies that specifically recognized GR phosphorylated at this site to show that Ser404 was phosphorylated on the endogenous protein in cultured bone, liver, cervix, and lung cells. Motif scanning showed that Ser404 lies within a consensus site for phosphorylation by GSK-3. GSK-3β phosphorylated GR in vitro, and mouse embryo fibroblasts lacking GSK-3β were not phosphorylated on the residue corresponding to Ser404 in the human enzyme. Expression in the osteosarcoma cells of GR mutants in which the phosphorylation site was removed or a charged residue was inserted to mimic phosphorylation indicated that phosphorylation enhanced export of the GR from the nucleus, increased GR susceptibility to degradation, and altered the spectrum of genes whose transcription was altered by GR. Analysis with Ingenuity Pathways software of the genes regulated indicated that the unphosphorylatable receptor influenced transcription of genes participating in lipid metabolism, small molecule biochemistry, and cancer that were not regulated by the wild-type receptor. Studies of transcription reporter genes showed that phosphorylation of GR decreased GR function at genes regulated directly through a glucocorticoid response element or transrepressed by the GR. Compared to interactions with wild-type GR, association of the phosphorylated GR with the cofactor CBP/300 was weakened, whereas association with the p65(RelA) subunit of the transcription factor NF-κB was enhanced. Cell death of osteoblasts in culture in response to GR stimulation was enhanced in cells expressing the nonphosphorylatable mutant. Given that altered GSK-3 activity is associated with widespread diseases such as diabetes, Alzheimer’s disease, and cancer and the broad use of glucocorticoids to treat inflammation, the influences of GSK-3 on GR signaling described may have implications for human health.
A. J. Galliher-Beckley, J. G. Williams, J. B. Collins, J. A. Cidlowski, Glycogen synthase kinase 3β-mediated serine phosphorylation of the human glucocorticoid receptor redirects gene expression profiles. Mol. Cell. Biol. 28, 7309–7322 (2008). [Abstract] [Full Text]