Role of protein kinases in estrogen receptor function
Table of Contents
Ligand-independent activation of estrogen receptor
Estrogen receptor-alpha is a member of the superfamily of class I nuclear receptors. After activation by its ligand estradiol it acts as a transcription factor and binds to specific target sequences in the promoter of estrogen-dependent genes. However, there is strong evidence that the estrogen receptor is not only activated by estrogens. There is an interaction or cross-talk of growth factor - activated signalling pathways such as the insulin-like growth factor and the epidermal growth factor activated pathways with estrogen receptor function. This interaction is achieved by phosphorylation of serine residues located in the N-terminal domain of the estrogen receptor, which possesses an activation function. Protein kinases that phosphorylate the different serine residues, are effectors of growth factor activated signalling pathways such as the mitogen-activated protein kinase (MAPK), glycogen synthase kinase-3 (GSK-3), protein kinase A (PKA) and protein kinase B (Akt/PKB). Phosphorylation results in weak activation of the estrogen receptor in the absence of estradiol and in modulation of estrogen receptor activity in the presence of estradiol. Phosphorylation at Ser118 is required for full activation of the ER.
Role of GSK-3beta in the activation of estrogen receptor
Glycogen synthase kinase-3 (GSK-3) exists as two isoforms, GSK-3alpha and GSK-3beta, the beta-isoform being more important. GSK-3 plays a key role in the the regulation of the activity of transcription factors including steroid receptors. We could show that GSK-3 is related to ligand-dependent and ligand-independent estrogen receptor activation.
1. Glycogen synthase kinase-3 is a key regulator of estrogen receptor stability and function
Treatment of breast cancer cells with estradiol results in rapid activation of the phosphatidylinositol 3-kinase/Akt/protein kinase B pathway as well as in rapid phosphorylation of the estrogen receptor at Ser118. This raised the hypothesis that Akt/PKB activation is related to estrogen receptor activation. However, Akt/PKB does not phosphorylate the estrogen receptor at Ser118. Moreover, overexpression of a constitutively active form of Akt/PKB resulted in inhibition of estrogen receptor transactivation. This suggested the involvement of a negative regulator downstream of Akt/PKB. We have identified estrogen receptor as a substrate for GSK-3, which is a downstream effector of Akt/PKB. GSK-3 is a terminal serine/threonine kinase that plays a central role as a negatively regulated docking kinase in a large number of signalling pathways that control metabolism, proliferation and differentiation. GSK-3 phosphorylates estrogen receptor at two different motifs, at the Ser102,104,106 motif and at Ser118, both located in the AF-1 domain of the receptor. The Ser104,106 residues are found phosphorylated in the cytoplasm in unstimulated cells. After treatment of the cells with estradiol, phosphorylation at the Ser104,106 domain disappears rapidly, and the estrogen receptor translocates into the nucleus where Ser118 is phosphorylated. GSK-3 is required for estradiol-induced estrogen receptor phosphorylation at Ser118 in the nucleus as well as for full transcriptional activation of the receptor upon estradiol-stimulation.
GSK-3 silencing results in the significant reduction of estrogen receptor levels and transcriptional activity. The reduction of estrogen receptor levels is due to increased ubiquitination and proteasomal degradation of receptor. We conclude that GSK-3 protects estrogen receptor from degradation and plays a crucial role in estrogen receptor stabilization and turnover.
These findings describe not only a novel role for GSK-3beta in the regulation of estrogen receptor stability and function, but also uncover an entirely novel mechanism for the regulation of estrogen receptor-mediated estrogen signalling controlled by multiple actions of GSK-3.
2. GSK-3 mediates interaction between protein kinase C-signalling and estrogen receptor activity
Protein kinase C (PKC) is a protein kinase system whose stimulation activates estrogen receptor in a ligand-independent manner. Treatment of estrogen receptor positive cells with the phorbol ester TPA results in rapid nuclear translocation and phosphorylation of the receptor. We identified PKC-delta as the PKC isoform that triggers estrogen receptor activation, however, PKC-delta did not phosphorylate the estrogen receptor at Ser118 in in vitro-kinase assays. We could show that GSK-3beta is a substrate for PKC-delta. Phosphorylation of GSK-3beta after TPA-treatment coincided with nuclear translocation and Ser118 phosphorylation of the estrogen receptor. We suggest that GSK-3beta plays a similar role in PKCdelta - mediated ligand-independent estrogen receptor activation as was described above for estrogen-dependent activation of the receptor.
