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 (Thr(171)), serine 224 (Ser(224)), serine 232 (Ser(232)), and serine 246 (Ser(246)). We have previously demonstrated that Ser224 and Ser(232) are phosphorylated by cyclin-dependent kinases, while Ser(246) is phosphorylated by the c-Jun N-terminal kinase. We report here that the remaining GR phosphorylation site, Thr(171), 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 Thr(171). Correspondingly, over expression of a constitutively active form of the GSK-3 inhibitor, protein kinase B/Akt, increases GR transcriptional enhancement. Overexpression of GSK-3 bad no effect on GR-mediated transcriptional repression of AP1-dependent gene expression. Importantly, transcriptional activation by the human GR (hGR), which contains an alanine (Ala(150)) at the position equivalent to Thr(171) 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.