Calmodulin-stimulated phosphorylation of 17 beta-estradiol receptor on tyrosine (original) (raw)
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Properties of a purified estradiol-dependent calf uterus tyrosine kinase
Biochemistry, 1993
A uterus tyrosine kinase has been purified to a single 67-kDa protein when analyzed by SDS-PAGE. Under nondenaturing conditions the molecular weight of the enzyme ranges from 1 14 to 136 kDa, depending on the procedure employed. The kinase binds calmodulin in a Caz+-dependent manner and the ATP analog [ (fluorosulfonyl) benzoyl] adenosine. The purified enzyme phosphorylates the phosphatasetreated uterus estradiol receptor on tyrosine and activates its hormone binding. The kinase phosphorylates actin, calmodulin, and histone H2B. Whatever the substrate, the enzymic activity is dependent on purified estradiol-receptor complex and is activated by Ca2+-calmodulin. The kinase activates and phosphorylates the human estradiol receptor (HEO) within the hormone binding domain (HBD) Mol Endocrinol. 3, 1061-10691 as well as four of the five mutants of the H E 0 obtained by substituting each of the five tyrosine residues present in the HBD of the receptor with phenylalanine by site-directed mutagenesis. The mutant substituted at tyrosine 537 is the only one that is neither phosphorylated nor activated by the kinase. This proves a causal relationship between the phosphorylation of estradiol receptor on tyrosine 537 and its hormone binding activity. A synthetic peptide corresponding to 11 out of 13 amino acids surrounding tyrosine at position 537 of the human estrogen receptor can be phosphorylated by the kinase. This and other findings indicate that this kinase, unlike other tyrosine kinases, phosphorylates tyrosyl residues with acidic amino acids close to the carboxyl side.
Phosphorylation of the estradiol receptor in MCF-7 huma breast cancer cells in culture
Molecular and Cellular Endocrinology, 1990
Double labelling and Western blot techniques were used to demonstrate phosphorylation of estradiol receptor. Cells in monolayer culture were incubated with [32P]orthophosphate for 18 h followed by covalent whole cell labelling of the estradiol receptor with tritiated tamoxifen aziridine (f3H]TA). Labelled receptor was pr~pitated with the mono~lonal antibodies H222 or JS 34/32, coupled to protein A-Sepharose, and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or transferred to nitrocellulose paper. Receptor protein was detected on the Western blot with the monoclonal antibody H222 and rabbit anti-rat peroxidase conjugate. Phosphorylated receptor was visualized by autoradiography. Tritium and 32P activities were monitored in the gels.
Phosphorylation of the estradiol receptor in MCF-7 human breast cancer cells in culture
Molecular and Cellular Endocrinology, 1990
Double labelling and Western blot techniques were used to demonstrate phosphorylation of estradiol receptor. Cells in monolayer culture were incubated with [32P]orthophosphate for 18 h followed by covalent whole cell labelling of the estradiol receptor with tritiated tamoxifen aziridine (f3H]TA). Labelled receptor was pr~pitated with the mono~lonal antibodies H222 or JS 34/32, coupled to protein A-Sepharose, and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or transferred to nitrocellulose paper. Receptor protein was detected on the Western blot with the monoclonal antibody H222 and rabbit anti-rat peroxidase conjugate. Phosphorylated receptor was visualized by autoradiography. Tritium and 32P activities were monitored in the gels.
Biochemistry, 1990
Three interconvertible forms of the estrogen receptor have been identified in the oviduct of estrogen-stimulated chicks. The non-estradiol binding form (Rnb) can be converted to the lower affinity binding form (R,, Kd = 0.8 nM) by a process requiring the y-phosphoryl moiety of ATP. The enzymatic activity (F,) essential for this "receptor potentiation" has been isolated from oviduct cytosol using ammonium sulfate fractionation, DEAE chromatography, and HPLC size-exclusion chromatography. The potentiation appears to require both kinase and phosphatase activities. The F, kinase characteristically phosphorylates casein, histones, and glycogen synthase. Comparison of the kinase with casein kinase 11, which also phosphorylates casein and glycogen synthase, indicates that F, represents a distinct protein kinase since its activity is not stimulated by spermine or inhibited by heparin. F,-mediated conversion of Rnb to R, is blocked by the phosphatase inhibitors vanadate, fluoride, and pyrophosphate. The substrate specificity of the F phosphatase activity is distinct from that of the two well-characterized protein phosphatases 1 and 2A. Ihoreover, the requirement for F, phosphatase activity in converting Rnb to R, could not be mimicked by its substitution with purified protein phosphatases 1 or 2A. The unique substrate specificity of the oviduct 'Supported by National Institutes of Health Grant HD17727.
Molecular Endocrinology, 1993
We have shown previously that exposure of rat uterine cells in primary culture to estradiol (E2), insulin-like growth factor-l (IGF-I), or agents which alter intracellular CAMP levels, such as cholera toxin plus isobutylmethylxanthine (CT + IBMX) and 8-Br-CAMP, results in the up-regulation of cellular levels of the progesterone receptor, an effect believed to be mediated through the activation of estrogen receptor (ER) and phosphorylation pathways. We have therefore undertaken studies using transient transfection of these uterine cell cultures with a simple estrogen-responsive reporter gene in order to determine the ability of these agents to stimulate ERmediated gene transcription directly. We also compared the ability of these same agents to alter the phosphorylation state of the endogenous uterine ER protein. Plasmid DNA containing two tandem estrogen responsive elements and a TATA box linked to the chloramphenicol acetyl transferase (CAT) gene was introduced into immature rat uterine cells grown in primary culture. Treatment of transfected cells with lo-' M EP, CT (1 Fg/ml) + IBMX (lo-" M), 8-Br-CAMP (10m4 M), or IGF-I (20 rig/ml) resulted in an 8-to lofold induction of CAT activity. CAT activity stimulated by all agents was nearly completely suppressed by coincubation with the antiestrogen ICI 184,384 (ICI) or the protein kinase (PK) inhibitor H8. CAT activity induced by 8-Br-CAMP was more readily suppressed by ICI than that induced by EP, indicating that ER in cells exposed to 8-Br-CAMP is either unoccupied or minimally occupied by ligand. The level of ER phosphorylation in uterine cells was increased 3-to 5-fold upon exposure to EP, CT
Biology of …, 2008
The aim of the present study was to determine the mechanisms involved in estrogen actions in cultured rat Sertoli cells. RT-PCR detected transcripts for the estrogen receptors ESR1 and ESR2 in cultured immature Sertoli cells and in the testis of 15-, 28-, and 120-day-old rats. The expression of ESR1 and ESR2 was confirmed in Sertoli cells by immunofluorescence and Western blot. Immunohistochemistry with cryosections of testes from immature and adult rats revealed that ESR1 is present in Sertoli, Leydig, and some peritubular myoid cells, and ESR2 is present in multiple cell types, including germ cells. Treatment of Sertoli cells with 17beta-estradiol (E 2 ) induced a translocation of ESR1 and ESR2 to the plasma membrane and a concomitant phosphorylation of MAPK3/1. Both effects reached a maximum after 10 min and were blocked by PP2, an inhibitor of the SRC family of protein tyrosine kinases, and by the antiestrogen ICI 182,780 (ICI). MAPK3/1 phosphorylation was also decreased in the presence of AG 1478, an inhibitor of the epidermal growth factor receptor (EGFR) kinase, and in the presence of MAP2K1/2 inhibitor UO126. Treatment with E 2 for 24 h increased the incorporation of [methyl-3 H]thymidine, which was blocked by ICI. These results indicate that E 2 activates an SRC-mediated translocation of estrogen receptors to the plasma membrane, which results in the activation of EGFR and the mitogenactivated protein kinase signaling pathway. In addition, activation of ESR1 and/or ESR2 by E 2 is involved in proliferation of immature Sertoli cells. The estrogen actions in Sertoli cells might be a key step mediating cellular events important for spermatogenesis and fertility. estradiol, estradiol receptor, kinases, mechanisms of hormone action, Sertoli cells
The role of phosphorylation in human estrogen receptor function
The Journal of Steroid Biochemistry and Molecular Biology, 1998
We have studied the role of phosphorylation of the human estrogen receptor (hER) at serine 118, which has been previously identi®ed as a site important for transactivation. We have tested this transactivation in yeast and cell-free transcription assays, and have shown that mutation of serine 118 to alanine results in a 30±40% decrease in hER-dependent transcription. Furthermore, we investigated the functional signi®cance of phosphorylation at this site by hormone binding and DNA binding. The mutation of serine 118 to alanine in the hER caused no decrease in its af®nity for either estradiol or an ERE. The mutant receptor had an altered phosphorylation pattern when expressed in COS-1 and Sf9 cells, but not in HeLa cells. Our ®ndings indicate that phosphorylation of serine 118 of the hER plays a role in regulating its transcriptional activity.