ReviewEstrogen receptor alpha phosphorylation and its functional impact in human breast cancer
Introduction
Estrogen receptor alpha (ERα) is a member of the nuclear receptor superfamily of transcription factors whose activity is primarily regulated by binding of estrogen/estradiol (E2). E2 plays an indispensable role in growth, development, reproduction and maintenance of numerous physiological systems in mammals. Sustained exposure to E2 is a well-known stimulatory signal for breast cancer growth (Colditz, 1998, Hankinson et al, 2004). ERα is responsible for many of the effects of E2 on normal mammary and breast cancer tissue via its ligand induced transcriptional program (genomic actions) (McDonnell and Norris, 2002), as well as elicitation of rapid, cytoplasmic signaling cascades (non-genomic actions) (Levin, 2009).
ERα is a modular protein consisting of a number of functional domains including an N-terminal domain, two transcriptional activation functions (AF-1, AF-2), a centrally located DNA binding domain, hinge region, and a C-terminal ligand binding domain (Fig. 1). The C-terminal domain of ERα is highly structured upon ligand binding and this domain is highly conserved across many species. The N-terminal domain is less structured and poorly conserved across species, and is activated by both ligand-dependent and ligand-independent mechanisms. Though ERα function is strongly activated by ligand binding, ERα function is also regulated by posttranslational modifications (PTMs), most significantly by phosphorylation. ERα phosphorylation occurs at multiple sites, most located in the N-terminal domain, is regulated by ligand binding and by ligand-independent mechanisms such as peptide growth factor signaling (Lannigan, 2003, Murphy et al, 2011, Ward, Weigel, 2009). ERα phosphorylation sites contribute to regulation of multiple functional activities including hormone sensitivity, nuclear localization, DNA binding, protein/chromatin interactions, protein stability and gene transcription (Al-Dhaheri, Rowan, 2007, Duplessis et al, 2011, Huderson et al, 2012, Joel et al, 1995, Shah, Rowan, 2005, Williams et al, 2009).
In the absence of ligand, ERα like other steroid hormone receptors exists as a monomer bound to chaperone protein heat shock protein 90 (Hsp90). In the ‘classical genomic’ action of ERα, upon ligand binding ERα dissociates from heat shock proteins and binds either directly to estrogen response element (ERE) sequences in genes, or is tethered to promoters via interaction with other transcription factors [e.g. activation protein 1 (AP1); specificity protein 1 (SP1)], and recruits coregulators and the transcription machinery to induce transcription of ERα regulated genes. Dependent upon cell and promoter context, ERα regulates transcription through its two activation domains, AF-1 and AF-2. AF-2 is contained within the LBD and becomes active upon E2 binding. AF-1 however, is activated by phosphorylation of ERα at several sites, most notably serine 118 (S118) and serine 167 (S167), sites that are regulated by multiple signaling pathways.
This review will discuss evidence from the author’s laboratory on the role of ERα phosphorylation sites, particularly S118 and S167, on ERα function in breast cancer, including effects on chromatin interaction, gene expression, the impact on protein complexes at target promoters, breast cancer growth and endocrine therapy response.
Section snippets
ERα phosphorylation sites
There are fourteen serine residues in the NTD (10, 46, 47, 84, 91, 102, 104, 106, 118, 137, 154, 167, 173, and 178), of which serines 102, 104, 106, 118, and 167 have been identified as phosphorylation sites by numerous laboratories [for review, see Le Romancer et al. (2011)]. A number of these phosphorylation sites have been functionally characterized including serines 102 (S102), 104 (S104), 106 (S106), 118 (S118), and 167 (S167) in the AF-1 domain, serine 236 (S236) in the DNA binding
Phosphorylation of Ser118 exerts gene selective effects on promoter complex assembly and transcription
Gene-specific assembly of ERα and coregulator complexes at promoters may be dictated by unique DNA sequences acting as allosteric ligands that change receptor conformation thereby recruiting a distinct set of coregulators (Hall et al, 2002, Lefstin, Yamamoto, 1998, Tora et al, 1989). However, it is equally likely that conformational changes in ERα induced by phosphorylation could also dictate coregulator assembly. p160 steroid receptor coactivator (SRC) family members/nuclear receptor
Loss of ERα phosphorylation at S118 or S167 markedly impacts breast cancer growth, cellular morphology, and ERα signaling
To date, stable disruption of ERα phosphorylation sites in vivo has not been reported. Given the importance of S118 and S167 as surrogate markers for favorable outcome for tamoxifen adjuvant therapy in breast cancer, and as markers for a functional ERα signaling pathway, it was of interest to assess the impact of stable disruption of S118 and S167 phosphorylation on breast cancer growth, morphology and E2-regulated gene expression. Remarkably, MCF-7 breast cancer cells in which endogenous ERα
Src kinase promotes ERα interaction with promoters through phosphorylation at S167
A molecular link between Src activation and ERα function was demonstrated by Src-mediated changes in ERα phosphorylation at S118 (Feng et al., 2001). S167 phosphorylation is remarkable for the profound effect loss of S167 phosphorylation has on in vitro DNA binding (Arnold et al, 1995, Castano et al, 1997). Our laboratory was the first to report the connection between activated Src and ERα S167 phosphorylation. Src kinase activated PI3K/AKT and AKT that in turn, directly phosphorylated S167
Dim light exposure at night (dLEN) impacts the circadian clock and ERα phosphorylation
The circadian clock in peripheral tissues coordinates intrinsic timing in most organisms including humans, and controls various physiological, biochemical and behavioral processes that include sleep/wake cycles, body temperature, blood pressure, hormone production, digestive juice secretion, and the immune system [reviewed in Fu and Lee (2003)]. The master clock in the brain controls and coordinates the rhythmicity in various tissues according to the light input it receives. In the present 24/7
Summary
ERα phosphorylation serves as a cellular sensor of environmental cues that will modulate the gene expression program of ERα signaling in breast cancer. Even in the presence of saturating levels of E2, specific ERα phosphorylation sites regulate a distinct temporal recruitment of ERα to promoters, as well as direct recruitment of distinct coregulator complexes that are unique for each ERα regulated gene. We have directed our efforts toward study of single ERα phosphorylation sites to establish
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