Abstract
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.
Highlights
Coactivators are a class of proteins that interact with sequence-specific transcription factors to enhance their effect on gene transcription
Recent evidence has suggested that certain functions of coactivators and corepressors can be modulated by a number of different signal transduction pathways
To assess the effect of progesterone treatment on Steroid receptor coactivator-1 (SRC-1) phosphorylation, SRC-1, human progesterone receptor B and the GRE2E1bCAT reporter were coexpressed in COS-1 cells, and the cells were labeled in vivo with [32P]H3PO4 in the presence or absence of 10Ϫ8 M progesterone for 16 h
Summary
Materials—All cell culture reagents and LipofectAMINE were purchased from Life Technologies, Inc. Following centrifugation at 100,000 ϫ g for 30 min at 4 °C, 47.5 ml of phosphate-buffered saline containing 0.2% bovine serum albumin was added to the supernatant to reduce the urea concentration to 0.4 M and prepare the supernatant for SRC-1 immunopurification. SRC-1 phosphopeptides were spotted onto 20 ϫ 20-cm thin layer cellulose plates and separated in the first dimension by electrophoresis at 1000 V for 35 min in pH 1.9 buffer (2.2% v/v formic acid, 7.8% v/v acetic acid). Modified Manual Edman Degradation and Secondary Endoproteinase Digestion—Phosphopeptides purified by alkaline polyacrylamide gels or two-dimensional phosphopeptide analysis were subjected to modified manual Edman degradation as described previously to identify the phosphorylation sites [26]. The released SRC-1 phosphotryptic peptides were separated by C-18 reversed-phase HPLC or two-dimensional phosphopeptide maps and subjected to secondary protease digestion, phosphoamino acid analysis, and alkaline polyacrylamide gel electrophoresis.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
