Phosphorylation of steroid receptor coactivator-1. Identification of the phosphorylation sites and phosphorylation through the mitogen-activated protein kinase pathway.
Steroid receptor coactivator-1 (SRC-1) is a member of a coactivator family that enhance the activation of the steroid/nuclear receptor superfamily of ligand-stimulated transcription factors. To study the regulation of SRC-1 by signaling pathways in the cell, the major phosphorylation sites of SRC-1 were identified in COS-1 cells using a combination of in vivo labeling with [(32)P]H(3)PO(4), modified manual Edman degradation, phosphoamino acid analysis, endoproteinase digestion, and mutagenesis of the SRC-1 phosphorylation sites. Seven phosphorylation sites were identified in SRC-1: serine 372, serine 395, serine 517, serine 569, serine 1033, threonine 1179, and serine 1185. All the sites contained consensus sequences for the serine/threonine-proline-directed family of protein kinases, and two sites (serine 395 and threonine 1179) contained a perfect consensus sequence for the mitogen-activated protein kinase family (Erk-1 and Erk-2). Furthermore, Erk-2 phosphorylated threonine 1179 and serine 1185 (and to a lesser extent, serine 395) in vitro, suggesting the importance of this pathway for SRC-1 regulation. Treatment of cells expressing SRC-1 with epidermal growth factor enhanced the ligand-dependent, progesterone receptor-mediated activation of a target reporter gene. These results identify phosphorylation as a regulatory modification of SRC-1 and provide a basis upon which to identify signaling pathways that regulate SRC-1 function and, consequently, modify steroid/nuclear receptor action.
Rowan
B G
BG
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Weigel
N L
NL
O'Malley
B W
BW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D015180:Electrophoresis, Gel, Two-Dimensional; D004815:Epidermal Growth Factor / Q000494:pharmacology; D017930:Genes, Reporter; D051548:Histone Acetyltransferases; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D056920:Nuclear Receptor Coactivator 1; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D011374:Progesterone / Q000494:pharmacology; D011980:Receptors, Progesterone / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D010748:Phosphopeptides; D011980:Receptors, Progesterone; D014157:Transcription Factors; D011374:Progesterone; D004815:Epidermal Growth Factor; D051548:Histone Acetyltransferases; D056920:Nuclear Receptor Coactivator 1; D020928:Mitogen-Activated Protein Kinases