Analysis of FK506, timcodar (VX‐853) and FKBP51 and FKBP52 chaperones in control of glucocorticoid receptor activity and phosphorylation

The immunosuppressive ligand FK 506 and the FK 506‐binding protein FKBP 52 are stimulatory to glucocorticoid receptor ( GR ) activity. Here, we explore the underlying mechanism by comparing GR activity and phosphorylation status in response to FK 506 and the novel nonimmunosuppressive ligand timcodar ( VX ‐853) and in the presence and absence of FKBP 52 and the closely related protein FKBP 51. Using mouse embryonic fibroblast cells ( MEF s) deficient knockout ( KO ) in FKBP 51 or FKBP 52, we show decreased GR activity at endogenous genes in 52 KO cells, but increased activity in 51 KO cells. In 52 KO cells, elevated phosphorylation occurred at inhibitory serine 212 and decreased phosphorylation at the stimulatory S220 residue. In contrast, 51 KO cells showed increased GR phosphorylation at the stimulatory residues S220 and S234. In wild‐type ( WT ) MEF cells, timcodar, like FK 506, potentiated dexamethasone‐induced GR transcriptional activity at two endogenous genes. Using 52 KO and 51 KO MEF cells, FK 506 potentiated GR activity in 51 KO cells but could not do so in 52 KO cells, suggesting FKBP 52 as the major target of FK 506 action. Like FK 506, timcodar potentiated GR in 51 KO cells, but it also increased GR activity in 52 KO cells. Knock‐down of FKBP 51 in the 52 KO cells showed that the latter effect of timcodar required FKBP 51. Thus, timcodar appears to have a dual specificity for FKBP 51 and FKBP 52. This work demonstrates phosphorylation as an important mechanism in FKBP control of GR and identifies the first nonimmunosuppressive macrolide capable of targeting GR action.