Phosphorylation and inhibition of rat glucocorticoid receptor transcriptional activation by glycogen synthase kinase-3 (GSK-3). Species-specific differences between human and rat glucocorticoid receptor signaling as revealed through GSK-3 phosphorylation.
Transcriptional activation by the glucocorticoid receptor (GR) is regulated by both glucocorticoid binding and phosphorylation. The rat GR N-terminal transcriptional regulatory domain contains four major phosphorylation sites: threonine 171 (Thr171), serine 224 (Ser224), serine 232 (Ser232), and serine 246 (Ser246). We have previously demonstrated that Ser224 and Ser232 are phosphorylated by cyclin-dependent kinases, while Ser246 is phosphorylated by the c-Jun N-terminal kinase. We report here that the remaining GR phosphorylation site, Thr171, is a target for glycogen synthase kinase-3 (GSK-3) in vitro and in cultured mammalian cells. Increasing GSK-3 activity through its overexpression in cultured cells inhibits GR transcriptional enhancement, an effect dependent upon Thr171. Correspondingly, overexpression of a constitutively active form of the GSK-3 inhibitor, protein kinase B/Akt, increases GR transcriptional enhancement. Overexpression of GSK-3 had no effect on GR-mediated transcriptional repression of AP1-dependent gene expression. Importantly, transcriptional activation by the human GR (hGR), which contains an alanine (Ala150) at the position equivalent to Thr171 in rat GR, is not affected by GSK-3 overexpression. Introduction of a threonine residue at this position (A150T) establishes GSK-3-mediated inhibition of hGR transcriptional activation. These findings demonstrate species-specific differences in GR signaling, as revealed through GSK-3 phosphorylation, which suggests that GR function in rodents may not fully recapitulate receptor action in humans and that hGR is capable of adopting the GSK-3 signaling pathway through a somatic mutation.
Rogatsky
I
I
Department of Microbiology and the Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, New York 10016, USA.
Waase
C L
CL
Garabedian
M J
MJ
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D010769:Phosphothreonine / Q000032:analysis; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics*; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured
D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D010769:Phosphothreonine; D038341:Glycogen Synthase Kinases; C494918:AKT1 protein, human; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3