Modulation of Glucocorticoid Receptor Function via Phosphorylation

M. J. Garabedian

Departments of Microbiology and Urology, New York University School of Medicine, New York, U.S.A.

The glucocorticoid receptor (GR) is phosphorylated at multiple serine residues in a hormone-dependent manner. It has been suggested that GR phosphorylation modulates turnover, subcellular trafficking, or transcriptional regulatory functions of the receptor, yet the contribution of individual GR phosphorylation sites to the modulation of GR activity remains enigmatic. To gain further insight into the function of GR phosphorylation in vivo, we have produced antibodies that specifically recognize phosphorylation sites within human GR at serines 203, 211 and 226 [1]. We found that GR transcriptional activity correlates with the amount of phosphorylation at S211, which then can serve as an important surrogate marker for activated GR in vivo (Fig. [1]). Our GR phosphorylation state-specific antibodies have allowed for the first time a detailed analysis of the localization of the GR phospho-isoforms in vivo. We found that differentially phosphorylated receptor species are located in unique subcellular compartments (Fig. [2]). For example, the GR phospho-S211 form is found in the nucleus after hormone treatment, consistent with its agonist-dependent phosphorylation. Thus, the GR phospho-S211 form corresponds to the nuclear transcriptionally active subpopulation of the receptor. In contrast, in hormone-treated cells the GR phospho-S203 is predominantly cytoplasmic and remains largely confined to the perinuclear region of the cell, with only a small fraction of this receptor isoform transiently entering the nucleus. Thus, the GR phospho-S203 form may participate in receptor function(s) at the cytoplasmic/nuclear border, such as nuclear import or export.

Fig. 1 Effects of agonists and antagonists on GR phosphorylation and transcriptional activation.

Fig. 2 The subcellular distribution of endogenous GR phospho-S203 and phospho-S211 GR in A459 cells.

We and others have proposed that GR phosphorylation modulates interaction with accessory proteins involved in transcriptional regulation, protein stability or subcellular trafficking, thereby affecting receptor function. Utilizing a yeast two-hybrid approach that allows transcriptional activators to be used as bait, we have previously identified the tumor susceptibility gene 101 (Tsg101) and the Vitamin D receptor-interacting protein 150 (DRIP150) as proteins that specifically interact with the GR AF-1 [2]. Interestingly, these protein:protein interactions appear sensitive to GR phosphorylation in a reciprocal manner: DRIP150 associates more efficiently with phospho-GR, whereas Tsg101 interacts more favorably with the non-phosphorylated receptor. Tsg101 shows significant sequence similarity to E2 ubiquitin ligases but is unable to catalyze ubiquitin transfer as it lacks the active site cysteine that forms the transient thioester bond with the C-terminus of ubiquitin. Interestingly, overexpression of Tsg101 stabilizes the non-phosphorylated form of GR in the absence of hormone, whereas hormone-dependent down-regulation of GR remains largely intact. Thus, Tsg101 is recruited to the GR AF-1 hypo-phosphorylated form in the absence of ligand, and protects GR from degradation. This may represent general mechanism for maintaining the steady state levels of steroid receptors in vivo in the absence of ligand. In contrast, upon ligand-binding when GR phosphorylation is high, DRIP150 is recruited to GR, which facilitates transcriptional enhancement. Indeed, we have shown differential occupancy of GR phospho-isoforms at endogenous GR target genes in vivo by chromatin immunoprecipitation (ChIP) using GR phosphorylation site-specific antiserum (Fig. [3]).

Fig. 3 Occupancy of GREs by GR and phospho-GR at the endogenous tyrosine amino transferase gene by ChIP. Analysis of GR phosphorylation from rat hepatoma HTC cells with GR phospho-S211 antibody. Soluble chromatin was prepared from HTC cells either untreated either untreated (0) or treated with 100 nM Dex for the times indicated and analyzed with an antibody to total GR [GR N499: Total GR], GR phospho-S203 (203-P), GR phospho-S211 (S211-P), or GR phospho-226 (S226-P). Precipitated DNAs were amplified using pairs of primers that cover the two functional GREs that mediate dexamethasone induction of the TAT gene in liver cells.

Thus, GR phosphorylation appears to be a versatile mechanism to modulate protein:protein interactions, which in turn stabilize the hypo-phosphorylated form of the receptor in the absence of ligand as well as facilitate transcriptional activation by the hyper-phosphorylated GR via cofactor recruitment upon ligand binding.

References

• 1 Wang Z, Frederick J, Garabedian M J. Deciphering the phosphorylation ”code” of the glucocorticoid receptor in vivo.  J Biol Chem. 2002;  277 26 573-26 580
• 2 Hittelman A B, Burakov D, Iñiguez-Lluhí J A, Freedman L P, Garabedian M J. Differential regulation of glucocorticoid receptor transcriptional activation via AF-1-associated proteins.  EMBO J. 1999;  18 5380-5388