Abstract
We have shown that the 32P-phosphorylation of the nuclear estrogen receptor from human MCF-7 cells or the calf uterus is estrogen-dependent. Within 2 min of estradiol treatment the phosphorylation of the estrogen receptor from MCF-7 cells doubled, and increased 4-fold within 20-40 min of estradiol treatment. Progesterone was ineffective in stimulating the phosphorylation of the estrogen receptor. Phosphoamino acid analysis indicated that the estrogen-stimulated phosphorylation of the human or calf estrogen receptor occurred only on serine residue(s). Phosphotryptic peptide analysis of the human estrogen receptor by two-dimensional peptide mapping or reverse-phase high pressure liquid chromatography revealed that only a single tryptic peptide (site) was phosphorylated. Treatment of the estrogen receptor with potato acid phosphatase resulted in the dephosphorylation of the 32P-labeled estrogen receptor and a decrease of the receptor's affinity for specific DNA sequences. These data suggest that transcriptional activation by the estrogen receptor involves an estrogen-dependent phosphorylation of the receptor resulting in its increased affinity for specific DNA sequences.
Highlights
From the Departmentof Biophysicsand Environmental Health Sciences Center, University of Rochester Schoolof Medicine and Dentistry, Rochester, New York 14642
Proges- response element-binding protein andglnALG haveindicated terone was ineffective in stimulating the phosphoryl- the importance of phosphorylation at specific amino acid ation of theestrogenreceptor.Phosphoaminoacid analysis indicated that the estrogen-stimulated phosphorylationofthehuman or calfestrogenreceptor occurred onlyon serineresidue(s).Phosphotryptic peptide analysis of the human estrogen receptor by twodimensionalpeptidemapping or reverse-phasehigh pressure liquid chromatography revealed that only a residues in theregulation of their transcriptionalactivity (Lee et al, 1990)
Aurrichio and co-workers (Migliaccio et al, The estrogen receptor is a transcriptionalregulatory protein 1989) have reported that theestrogen receptor is phosphorylthat is allosterically regulated by estrogen binding (Notides ated on tyrosine residues in response to estradiol treatment, et al, 1981; Sasson and Notides, 1988).The estrogen-receptor and that phosphorylation of the estrogen receptor is related complex interacts with specific nucleotide sequences termed to theability of the estrogen receptor to bind hormone
Summary
From the Departmentof Biophysicsand Environmental Health Sciences Center, University of Rochester Schoolof Medicine and Dentistry, Rochester, New York 14642. Receptor from MCF-7 cells doubled, and increased 4- Site-directed mutagenesis and gene transfer studiesof cAMP fold within 20-40 min of estradiol treatment. Many steroid hormone receptors have been shown to be phosphorylated proteins. Aurrichio and co-workers (Migliaccio et al., The estrogen receptor is a transcriptionalregulatory protein 1989) have reported that theestrogen receptor is phosphorylthat is allosterically regulated by estrogen binding (Notides ated on tyrosine residues in response to estradiol treatment, et al, 1981; Sasson and Notides, 1988).The estrogen-receptor and that phosphorylation of the estrogen receptor is related complex interacts with specific nucleotide sequences termed to theability of the estrogen receptor to bind hormone. Characteristics of phosphorylation of the estrogen receptor in It has been shown, using gene transfer experiments that the the MCF-7 breast adenocarcinoma cell line using a highly. The gel was swollen, homogenized, and extracted with 50 mM NH4C03and 10% P-mercaptoethanol for
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