Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation

Abstract

Post-translational modifications such as phosphorylation are known to play an important role in the gene regulation by the transcription factors including the nuclear hormone receptor superfamily of which the glucocorticoid receptor (GR) is a member. Protein phosphorylation often switches cellular activity from one state to another. Like many other transcription factors, the GR is a phosphoprotein, and phosphorylation plays an important role in the regulation of GR activity. Cell signaling pathways that regulate phosphorylation of the GR and its associated proteins are important determinants of GR function under various physiological conditions. While the role of many phosphorylation sites in the GR is still not fully understood, the role of others is clearer. Several aspects of transcription factor function, including DNA binding affinity, interaction of transactivation domains with the transcription initiation complex, and shuttling between the cytoplasmic compartments, have all been linked to site-specific phosphorylation. All major phosphorylation sites in the human GR are located in the N-terminal domain including the major transactivation domain, AF1. Available literature clearly indicates that many of these potential phosphorylation sites are substrates for multiple kinases, suggesting the potential for a very complex regulatory network. Phosphorylated GR interacts favorably with critical coregulatory proteins and subsequently enhances transcriptional activity. In addition, the activities and specificities of coregulators may be subject to similar regulation by phosphorylation. Regulation of the GR activity due to phosphorylation appears to be site-specific and dependent upon specific cell signaling cascade. Taken together, site-specific phosphorylation and related kinase pathways play an important role in the action of the GR, and more precise mechanistic information will lead to fuller understanding of the complex nature of gene regulation by the GR- and related transcription factors. This review provides currently available information regarding the role of GR phosphorylation in its action, and highlights the possible underlying mechanisms of action.

Keywords: coactivators; gene regulation; glucocorticoid receptor; phosphorylation; transactivation activity.

Figure 1

Classical action of the glucocorticoid signaling mediated by the GR. Unliganded receptor is located in the cytosol associated with several heat shock and other chaperone proteins including HSP90, HSP70, CyP-40, P23, and FKBPs (shown by different shapes and shades in the cytosol). Ligand binding leads to conformational alterations in the GR, and by doing so GR dissociates from these associated proteins, and ligand bound GR is free to translocate to the nucleus. This process appears to be phosphorylation-dependent. Once in the nucleus, GR dimerizes and binds to site-specific DNA binding sequences and interacts with several other coregulatory proteins including coactivators and proteins from the basal transcription machinery including SRCs, CBP/p300, DRIP/TRAP, TBP, GRIP1, and several others (shown by different shapes and shades) in the nucleus, and subsequently leading to transcriptional regulation.

Figure 2

A topological diagram of the human GR (amino acids 1–777) showing its modular structure and known phosphorylation sites in it (based on ref. 38). Numbers on the bottom indicate amino acid positions of different functional domains. 1–420, NTD; 77–262, AF1; 421–481; DBD; and remaining C-terminal part, the LBD are shown. P denotes known phosphorylation sites in the NTD of the human GR. Shown from left to right: S113, S141, S203, S211, S226, and S308. Corresponding amino acids in the rat GR are S134, S162, S224, S232, S246, and S329, whereas in the mouse GR these correspond to S122, S150, S212, S220, S234, and S315. In the rat and mouse GR, there is one Threonine residue (T171 in rat and T159 in mouse) that is known to be phosphorylable, and is not conserved in the human GR. In the human GR except for S308, all other residues are located within the AF1 domain, and S203, S211, and S226 are reported to have to have some functional roles in the action of the GR. Phosphorylation of these sites are kinase- and cell-dependent.

Figure 3

A hypothetical model of the role of site-specific phosphorylation on the structure and functions of the AF1 domain. The GR AF1 domain exists in an intrinsically disordered (ID) conformation ie, as an ensemble of conformers that collectively appear to lack any significant secondary/tertiary structure. Phosphorylation of one or more sites within the AF1 alters its conformation in such a way that AF1 adopts a partially folded (PD) conformation that facilitates AF1’s interaction with one or more of the coregulatory proteins. This protein:protein interaction allows structurally modified forms of AF1 to suit for its varied interactions with other critical coregulatory proteins, and possibly additional modulations in receptor structure essential for gene regulation by the GR. This AF1:coregulators assembly provides optimal ordered conformation in the AF1 for subsequent transactivation activity. Alternatively, one or more of the coregulator proteins directly interact with one or more unphosphorylated AF1 conformers that may trigger phosphorylation of AF1. The resultant assembly of proteins may depend upon the cellular environment, specific promoters used, and the kinase pathway involved. All the available phosphorylation sites in the GR could be involved simultaneously or there may be a coordinated synergy between each site. In the full length GR, AF1:coregulators assembly may also be influenced by other factors such as DNA binding to its DBD, ligand binding and the cross talk between AF1 and AF2. In addition to the level of AF1 phosphorylation, phosphorylation of different coregulators may also influence the outcome, and certain coregulators may be included or excluded from the assembly depending upon the interacting surfaces of the AF1 and/or coregulator proteins.