期刊
BMC BIOCHEMISTRY
卷 10, 期 -, 页码 -出版社
BMC
DOI: 10.1186/1471-2091-10-36
关键词
-
资金
- NIH/NIDDK [RO1DK06832]
- Department of Defense Breast Cancer Research Program [DAMD17-02-1-0531]
- Career Development Award [(DAMD17-02-1-0530]
- NIH/NCI [K01CA129078]
- UNCF/Merck
Background: Estrogen receptor alpha (ER alpha) phosphorylation is important for estrogen-dependent transcription of ER-dependent genes, ligand-independent receptor activation and endocrine therapy response in breast cancer. However ER alpha phosphorylation at the previously identified sites does not fully account for these receptor functions. To determine if additional ER alpha phosphorylation sites exist, COS-1 cells expressing human ER alpha were labeled with [P-32]H3PO4 in vivo and ER alpha 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 ER alpha 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 ER alpha 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 ER alpha at S282 and S559 using motif analysis, in vitro kinase assays, and incubation of cells with CK2 kinase inhibitor. Conclusion: These novel ER alpha phosphorylation sites represent new means for modulation of ER alpha activity. S559 represents the first phosphorylation site identified in the extreme C-terminus ( F domain) of a steroid receptor.
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