In vitro phosphorylated GR LBD LC-MSMS

Glucocorticoid receptor (GR) is a ligand dependent transcription factor that plays a central role in inflammation. Part of a complex cellular network, GR activity is further modulated via protein–protein interactions . The regulation of GR by 14-3-3 proteins has been previously reported, though with differing sites of phosphorylation identified and variable consequences assigned. Hence, we sought to examine this protein–protein interaction and, using phosphorylated GR peptides, biophysical studies and X-ray crystallography, we identified key residues within the ligand binding domain of GR, T524 and S617, whose phosphorylation results in recognition by 14-3-3. Investigation of the kinases responsible for phosphorylation of these sites assigned a key role for MINK1 and cell-based approaches confirmed the importance of these two GR phosphosites and MINK1 in GR–14-3-3 binding. Together our results provide a molecular-level insight into 14-3-3 regulation of GR and highlight both MINK1 and the GR–14-3-3 axis as potential targets for future therapeutic intervention.