pp90rsk1 regulates estrogen receptor-mediated transcription through phosphorylation of Ser-167.
The estrogen receptor alpha (ER), a member of the steroid receptor superfamily, contains an N-terminal hormone-independent transcriptional activation function (AF-1) and a C-terminal hormone-dependent transcriptional activation function (AF-2). Here, we used in-gel kinase assays to determine that pp90rsk1 activated by either epidermal growth factor (EGF) or phorbol myristate acetate specifically phosphorylates Ser-167 within AF-1. In vitro kinase assays demonstrated that pp90rsk1 phosphorylates the N terminus of the wild-type ER but not of a mutant ER in which Ser-167 was replaced by Ala. In vivo, EGF stimulated phosphorylation of Ser-167 as well as Ser-118. Ectopic expression of active pp90rsk1 increased the level of phosphorylation of Ser-167 compared to that of either a mutant pp90rsk1, which is catalytically inactive in the N-terminal kinase domain, or to that of vector control. The ER formed a stable complex with the mutant pp90rsk1 in vivo. Transfection of the mutant pp90rsk1 depressed ER-dependent transcription of both a wild-type ER and a mutant ER that had a defective AF-2 domain (ER TAF-1). Furthermore, replacing either Ser-118 or Ser-167 with Ala in ER TAF-1 showed similar decreases in transcription levels. A double mutant in which both Ser-118 and Ser-167 were replaced with Ala demonstrated a further decrease in transcription compared to either of the single mutations. Taken together, our results strongly suggest that pp90rsk1 phosphorylates Ser-167 of the human ER in vivo and that Ser-167 aids in regulating the transcriptional activity of AF-1 in the ER.
Joel
P B
PB
Center for Cell Signaling and Department of Pharmacology, University of Virginia, Charlottesville 22908, USA.
Smith
J
J
Sturgill
T W
TW
Fisher
T L
TL
Blenis
J
J
Lannigan
D A
DA
D000818:Animals; D019556:COS Cells; D002384:Catalysis; D002868:Chromosomal Proteins, Non-Histone; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005668:Furylfuramide / Q000378:metabolism; D056488:Histone Chaperones; D006801:Humans; D016350:Leucine Zippers; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000378:metabolism*; D017471:Receptors, Interferon / Q000378:metabolism; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D012694:Serine / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D056488:Histone Chaperones; C086185:IFNGR2 protein, human; D011960:Receptors, Estrogen; D017471:Receptors, Interferon; C513473:SET protein, human; D014157:Transcription Factors; D005668:Furylfuramide; D012694:Serine; D004815:Epidermal Growth Factor; D011494:Protein Kinases; C069329:RPS6KA1 protein, human; D038744:Ribosomal Protein S6 Kinases, 90-kDa