Dataset Information

In vitro phosphorylated GR LBD LC-MSMS

ABSTRACT: 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.

INSTRUMENT(S): AB SCIEX instrument model

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Claire Munier

LAB HEAD: Matthew W. D. Perry

PROVIDER: PXD022364 | Pride | 2021-09-09

REPOSITORIES: pride

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Publications

Glucocorticoid receptor Thr524 phosphorylation by MINK1 induces interactions with 14-3-3 protein regulators.

Munier Claire C CC De Maria Leonardo L Edman Karl K Gunnarsson Anders A Longo Marianna M MacKintosh Carol C Patel Saleha S Snijder Arjan A Wissler Lisa L Brunsveld Luc L Ottmann Christian C Perry Matthew W D MWD

The Journal of biological chemistry 20210101

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor that plays a central role in inflammation. The GR activity is also modulated via protein-protein interactions, including binding of 14-3-3 proteins induced by GR phosphorylation. However, the specific phosphorylation sites on the GR that trigger these interactions and their functional consequences are less clear. Hence, we sought to examine this system in more detail. We used phosphorylated GR peptides, biophysical studi ...[more]

PMID: 33744286

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Project description:In response to environmental stressors and a variety of inflammatory cytokines, p38 MAPKs become directly activated. Here we report the human glucocorticoid receptor (GR) Serine 134 as a novel target for p38 MAPK. Unlike most other phosphorylation events that occur on the GR, phosphorylation of Ser134 was found to be hormone-independent in several human and rat cell types. Instead we found phosphorylation of Ser134 was induced by a variety of stress-activating stimuli, including: glucose starvation, ultraviolet irradiation, osmotic shock, and oxidative stress. Pharmacological inhibitors and shRNA-mediated knockdown experiments correlate this phosphorylation with the activation of p38 MAPK. Compared to wild-type GR, cells expressing a mutant receptor incapable of phosphorylation at Ser134 (S134A GR) had a significantly altered hormone-dependent genome-wide transcriptional response to glucocorticoids. Moreover, we show that although WT GR regulated roughly half as many genes as S134A GR, WT receptor selectively activated significantly more genes associated with endocrine and inflammatory disease than the mutant receptor, suggesting that the phosphorylation status of Ser134 is critical for modulating GR function. Phosphorylation of Ser134 did not alter either nuclear translocation or the stability of the GR protein in the absence or presence of ligand. However, phosphorylation of Ser134 significantly increased the association of the GR with the zeta isoform the 14-3-3 class of signaling proteins, resulting in a blunted hormone-dependent transcriptional response of LAD1 and IGFBP1 but not GILZ. Together these data suggest that the phosphorylation of Ser134 acts as a molecular sensor on the GR, monitoring the level of cellular stress to allow for altered 14-3-3zeta cofactor association, ultimately modifying glucocorticoid signaling in a gene-dependent manner. Our results reveal one mechanism that may allow cellular stress to dictate the transcriptional response of cells to hormone. U2OS cells, a human osteosarcoma cell line, were transfected with either WT GR or S134A GR and put under antibiotic selection to produce a stable mixed population of cells expressing comparable levels of GR. 10^6 cells were treated with 100nM Dexamethasone (DEX) or vehicle control for 6 hours. Three biological and one hybridization replicate are included for each sample.

			Action	DRS
	290818GRMAP4K4.wiff.scan	Wiff
	290818GRMAP4K4.wiff2	Wiff
	290818GRMINK1.wiff.scan	Wiff
	290818GRMINK1.wiff2	Wiff
	290818GRMST1.wiff.scan	Wiff