Identification of four novel phosphorylation sites in estrogen receptor alpha: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2.

BACKGROUND: Estrogen receptor alpha (ERalpha) phosphorylation is important for estrogen-dependent transcription of ER-dependent genes, ligand-independent receptor activation and endocrine therapy response in breast cancer. However ERalpha phosphorylation at the previously identified sites does not fully account for these receptor functions. To determine if additional ERalpha phosphorylation sites exist, COS-1 cells expressing human ERalpha were labeled with [32P]H3PO4 in vivo and ERalpha tryptic phosphopeptides were isolated to identify phosphorylation sites. RESULTS: Previously uncharacterized phosphorylation sites at serines 46/47, 282, 294, and 559 were identified by manual Edman degradation and phosphoamino acid analysis and confirmed by mutagenesis and phospho-specific antibodies. Antibodies detected phosphorylation of endogenous ERalpha in MCF-7, MCF-7-LCC2, and Ishikawa cancer cell lines by immunoblot. Mutation of Ser-282 and Ser-559 to alanine (S282A, S559A) resulted in ligand independent activation of ERalpha as determined by both ERE-driven reporter gene assays and endogenous pS2 gene expression in transiently transfected HeLa cells. Mutation of Ser-46/47 or Ser-294 to alanine markedly reduced estradiol dependent reporter activation. Additionally protein kinase CK2 was identified as a kinase that phosphorylated ERalpha at S282 and S559 using motif analysis, in vitro kinase assays, and incubation of cells with CK2 kinase inhibitor. CONCLUSION: These novel ERalpha phosphorylation sites represent new means for modulation of ERalpha activity. S559 represents the first phosphorylation site identified in the extreme C-terminus (F domain) of a steroid receptor.

Mechanistic differences in the activation of estrogen receptor-alpha (ER alpha)- and ER beta-dependent gene expression by cAMP signaling pathway(s).

Although increases in intracellular cAMP can stimulate estrogen receptor-alpha (ER alpha) activity in the absence of exogenous hormone, no studies have addressed whether ER beta can be similarly regulated. In transient transfections, forskolin plus 3-isobutyl-1-methylxanthine (IBMX), which increases intracellular cAMP, stimulated the transcriptional activities of both ER alpha and ER beta. This effect was blocked by the protein kinase A inhibitor H89 (N-(2-(p-bromocinnamylamino)-ethyl)-5-isoquinolinesulfonamide) and was dependent on an estrogen response element. A 12-O-tetradecanoylphorbol-13-acetate response element (TRE) located 5' to the estrogen response element was necessary for cAMP-dependent activation of gene expression by ER beta but not ER alpha, indicating that the former subtype requires a functional interaction with TRE-interacting factor(s) to stimulate transcription. Both p160 and CREB-binding protein coactivators stimulated cAMP-induced ER alpha and ER beta transcriptional activity. However, mutation of the two cAMP-inducible SRC-1 phosphorylation sites important for cAMP activation of chicken progesterone receptor or all seven known SRC-1 phosphorylation sites did not specifically impair cAMP activation of ER alpha. The E/F domains of ER alpha are sufficient for activation by forskolin/IBMX, and this is accompanied by an increase in receptor phosphorylation. In contrast, cAMP signaling reduces the phosphorylation of the corresponding region of ER beta, and this correlates with the lack of forskolin/IBMX stimulated transcriptional activity. Our data suggest that cAMP activation of ER alpha transcriptional activity is associated with receptor instead of SRC-1 phosphorylation. Moreover, differences in the cofactor requirements, domains of ER alpha and ER beta sufficient for forskolin/IBMX activation, and the effect of cAMP on receptor phosphorylation indicate that this signaling pathway utilizes distinct mechanisms to stimulate ER alpha and ER beta transcriptional activity.