Site-Specific Phosphorylation Induces Functionally Active Conformation in the Intrinsically Disordered N-Terminal Activation Function (AF1) Domain of the Glucocorticoid Receptor

Abstract

Intrinsically disordered (ID) regions are disproportionately higher in cell signaling proteins and are predicted to have much larger frequency of phosphorylation sites than ordered regions, suggesting an important role in their regulatory capacity. In this study, we show that AF1, an ID activation domain of the glucocorticoid receptor (GR), adopts a functionally folded conformation due to its site-specific phosphorylation by p38 mitogen-activated protein kinase, which is involved in apoptotic and gene-inductive events initiated by the GR. Further, we show that site-specific phosphorylation-induced secondary and tertiary structure formation specifically facilitates AF1's interaction with critical coregulatory proteins and subsequently its transcriptional activity. These data demonstrate a mechanism through which ID activation domain of the steroid receptors and other similar transcription factors may adopt a functionally active conformation under physiological conditions.

Ligand-Independent Phosphorylation of the Glucocorticoid Receptor Integrates Cellular Stress Pathways with Nuclear Receptor Signaling

“…Our results also demonstrate a decreased association of p38 MAPK with the GR in the presence of hormone, suggesting that GR nuclear translocation decreases p38 MAPK-GR complex formation, or ligand binding results in a conformational change preventing p38 MAPK from binding the GR. The ability of p38 MAPK to phosphorylate the GR on May 10, 2018 by guest http://mcb.asm.org/ in a ligand-dependent manner at Ser211 was reported previously; however, recent studies challenge this notion (17,22,32). To determine if p38 MAPK activity is necessary for the phosphorylation of serines 134 and 211 of the GR, we pretreated U2-OS cells expressing WT-GR with the p38 MAPK inhibitor SB203580 before stimulation with Dex and/or irradiation with UVC.…”

“…The phosphorylation of the GR also affects the stability of the protein by altering nuclear/cytoplasmic shuttling and targeting the receptor for ubiquitin-mediated proteasomal degradation. Thus, the ligand-dependent phosphorylation of the GR significantly impacts the cellular response to steroids (4,7,(15)(16)(17)26). In response to glucocorticoids, cyclin-dependent kinases (CDKs), mitogen activated protein kinases (MAPKs), and glycogen synthase kinase 3(GSK-3) gain access to the human GR (hGR) and phosphorylate it at several sites, with serines 203, 211, 226, and 404 being the most thoroughly characterized at the molecular level (15).…”

Glucocorticoid receptor control of transcription: precision and plasticity via allostery

“…Phosphorylation of sites Ser203, Ser211 and Ser226, which are located within AF1, are predicted to affect exposure of protein surfaces critical for cofactor interactions 77,78 . Phosphorylation of Ser211 results in increased recruitment of GR to GORs and subsequent regulation of GR target genes 79 .…”

Regulation of the Structurally Dynamic N-terminal Domain of Progesterone Receptor by Protein-induced Folding

“…Fluorescence Emission (FE) Spectroscopy-Fluorescence emission spectra of purified recombinant PR NTD in solution were recorded in the absence or presence of varying concentrations of TMAO using a Spex FluoroMax spectrometer at excitation wavelengths of 278 or 295 nm as described (32). Measurements were taken in 1-cm rectangular cuvettes thermostated at 22°C, and all data were corrected for the contribution of the buffer.…”