title: Identification of four novel phosphorylation sites in estrogen receptor alpha: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 1Division of Basic Pharmaceutical Sciences, College of Pharmacy, Xavier University of Louisiana, New Orleans, LA, USA. cwillia0@tulane.edu
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Williams
authors.forename: Aninda
authors.initials: A
authors.lastname: Basu
authors.forename: Abeer
authors.initials: A
authors.lastname: El-Gharbawy
authors.forename: Latonya M
authors.initials: LM
authors.lastname: Carrier
authors.forename: Carolyn L
authors.initials: CL
authors.lastname: Smith
authors.forename: Brian G
authors.initials: BG
authors.lastname: Rowan
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000276:immunology / Q000378:metabolism; D019556:COS Cells; D047390:Casein Kinase II / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D047628:Estrogen Receptor alpha / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation
chemical_list: D045163:Antibodies, Phospho-Specific; D047628:Estrogen Receptor alpha; D047390:Casein Kinase II