Angelo C. Notides

Phosphorylation of the human estrogen receptor by mitogen-activated protein kinase and casein kinase II: Consequence on DNA binding

We determined the amino acid and radiolabel sequences of tryptic [32P]phosphopeptides of the purified human estrogen receptor (hER) from MCF-7 cells and Sf9 cells. Serine 118 was identified as a site that was phosphorylated independently of estradiol-binding in MCF-7 cells. Proline is on the carboxy terminus of serine 118, which suggests that the serine-proline may be a consensus phosphorylation site motif for either the mitogen-activated protein (MAP) kinase or p34cdc2 kinase. MAP kinase selectively phosphorylated the recombinant hER in vitro on serine 118 independent of estradiol-binding, whereas p34cdc2 did not phosphorylate the hER. We demonstrated previously that serine 167 of the hER was phosphorylated in an estradiol-dependent manner. We therefore compared the consequence of hER phosphorylation at serine 118 by MAP kinase and phosphorylation at serine 167 by casein kinase II on the receptors affinity for specific DNA binding. The binding of the hER to an estrogen response element was not altered by phosphorylation with MAP kinase at serine 118 but was significantly increased when phosphorylated at serine 167 by casein kinase II. These data suggest that phosphorylation of the hER by MAP kinase(s) pathways may influence receptor action by a mechanism other than the estradiol-dependent phosphorylation of hER by casein kinase II.

Thin-layer chromatographic separation of pineal gland derivatives of serotonin-14C.

Abstract A two-dimensional TLC method has been developed which separates N -acetylserotonin, hydroxytryptophol, hydroxyindoleacetic acid, methoxyindoleacetic acid, methoxytryptophol, and melatonin.

In vivo and in vitro phosphorylation of the human estrogen receptor

We report here that the human estrogen receptor (hER) overexpressed in Sf9 insect cells is phosphorylated similarly to hER from the human MCF-7 mammary carcinoma cell line. The recombinant and native hER labeled to steady-state with [32P]phosphate were purified to homogeneity using specific DNA-affinity chromatography followed by SDS-gel electrophoresis. Resolution of the hER tryptic digests by reverse phase-high performance liquid chromatography revealed that five [32P]phosphopeptides from the hER expressed in the Sf9 cells had retention times identical to five of the seven [32P]phosphopeptides from the hER in MCF-7 cells. Uniquely, a dephosphorylation of a single 32P-labeled peptide occurred in response to estradiol treatment of MCF-7 cells. In vitro protein kinase assays with the purified recombinant hER revealed that the DNA-dependent protein kinase (DNA-PK) phosphorylated the receptor and induced a decrease in the receptors mobility as demonstrated by SDS-gel electrophoresis. In contrast, protein kinases A and C did not phosphorylate the purified recombinant hER. These results suggest that in the process of becoming transcriptionally active the estrogen receptor undergoes a dephosphorylation after estrogen-binding and subsequent phosphorylations, in part by the DNA-PK.

A molecular and kinetic analysis of estrogen receptor transformation.

The rate of the 4 to 5 S estrogen-binding protein (EBP) in vitro transformation was measured by sucrose gradient centrifugation analysis. The temperature-activated 4 to 5 S EBP transformation is found to be highly reproducible without loss of [3H]estradiol-binding activity in a buffer containing an excess of [3H]estradiol, 40 mM Tris, 1 mM dithiothreitol, and 1 M urea at pH 7.4. The presence of [3H]estradiol is necessary for the 4 to 5 EBP transformation. A kinetic analysis of the 4 to 5 EBP transformation shows that it is a bimolecular reaction, the dimerization of the 4 S EBP with a second (similar or dissimilar) monomer or subunit. In buffers containing 0.4 M KCl the apparent second order rate constant is 2.3 plus or minus 0-2 times 10-7 M minus 1 min minus 1 at 28 degrees. The reaction is independent of the initial receptor concentration, suggesting that the 4 S EBP is dissociated into monomeric units in buffers of high ionic strength. In buffers without KCl or with 0.1 M KCl the apparent second order rate constant of receptor transformation increases with decreasing receptor concentration. This suggests that the 4 S EBP is associated weakly with another macromolecule (or macromolecules) in buffers of low ionic strength. The rate of 4 to 5 S EBP transformation shows a 200-fold increase between 0 and 35 degrees. The Arrhenius energy of activation is 21.3 kcal mol minus 1 in buffer without KCl and 19.1 kcal mol minus 1 in buffer with 0.4 M KCl. Following the temperature-activated dimerization, the avidity of binding between the 4 S EBP and its complementary subunit is increased, 0.4 M KCl can no longer cause dissociation, and the 5 S EBP dimer appears. This kinetic analysis indicates that the avidity of binding between the subunits of the estrogen receptor is modulated by estradiol, temperature, and ionic strength. We propose that these interactions of the estrogen receptors subunits reflect conformational changes involved in receptor activation.

Estrogen-binding proteins of the human uterus☆

Abstract 1. 1. Sucrose gradient centrifugation analysis and agarose gel chromatography of the human uterine cytosols, equilibrated with [3H]estradiol, have demonstrated the presence of two specific estrogen-binding proteins. The endometrial cytosol contained estrogen-binding proteins which sediment in sucrose gradients at 8 S, with a secondary estrogen-binding protein sedimenting at 3 S, while the myometrial cytosol contained almost exclusively a 3-S (3.1 ± 0.1 S) estrogen-binding protein. A non-specific [3H]-estradiol-binding protein with a sedimentation coefficient of 4.6 S was shown to be serum albumin. 2. 2. The addition of diisopropylfluorophosphate (DFP) to the homogenization buffer resulted in the appearance of the 8-S and no 3-S estrogen-binding protein in the myometrial cytosol, suggesting that the 3-S species may be obtained from the 8-S estrogen-binding protein by limited proteolysis, but without loss of the estradiol-binding capacity. 3. 3. The myometrial 3-S estrogen-binding protein has a molecular Stokes radius of 26.7 (+ 0.4) A, with a frictional ratio ( ƒ ƒ 0 ) of 1.20–1.25, and a molecular weight of 35 000–38 000 as approximated by agarose gel chromatography and sucrose gradient analysis. 4. 4. The apparent dissociation constant of the myometrial estrogen-binding protein was 1·10−9 M and the binding capacity was 67 (± 10)·10−15 mole of [3H]-estradiol bound per mg protein, with large variation among patients, 25·10−15 140·10−15 mole of estradiol bound per protein. Test compounds competed with the [3H]estradiol for binding by the myometrial estrogen-binding protein in the following sequence: 17β-estradiol > estrone > ethynylestradiol ≥ diethylstilbestrol > 17α-estradiol > estriol > CI-628 > U11, 100A >cis-clomiphene > 5-androsten-3β,17β-diol > 4-androsten-3β,17β-diol. Dihydrotestosterone, testosterone, androstenedione, progesterone or cortisol were not effective competitors of [3H]estradiol for the myometrial estrogen-binding protein.

A molecular analysis of the human estrogen receptor.

Abstract The human myometrial estrogen receptor, isolated in buffer containing diisopropylftuorophosphate (DFP) to inhibit proteolytic activity, was partially purified by ammonium sulfate fractionation. Sucrose gradient analysis in dilute Tris buffer, pH 7.5, without KCl indicates that the cytoplasmic estrogen receptor sediments as an 8 S, 5 S and 4 S estrogen-binding protein (EBP). Isolation of the estrogen receptor without DFP yields predominantly a proteolytic fragment of the receptor which sediments as a 3 S EBP in gradients having low salt or high salt concentrations, and has a molecular weight of 30,000 to 40,000. The nuclear form of the estrogen receptor has a sedimentation coefficient of 3.8 ± 0.04 S in sucrose gradients containing high salt concentrations, a molecular Stokes radius of 38.5 ± 0.9 A , and a molecular weight of 60,000 to 70,000, values similar to the cytoplasmic form of the receptor. The cytoplasmic estrogen receptor shows a temperature-enhanced activation as indicated by its increased affinity for isolated uterine nuclei. In contrast to the activated estrogen receptor from the rat or calf uterus, the human uterus does not produce a 5 S EBP (a dimer of the 4 S EBP with another macromolecule) in high salt concentration at 0–4°C. In sucrose gradients containing 0.15 M KCl, centrifugation at 0, 6, 12, 20, and 25°C indicates that the human estrogen receptor undergoes a temperature dependent increases in its sedimentation coefficient from 4 S to 5 S and a transformation similar to that of the rat or calf estrogen receptor, but it is more readily dissociated by high salt or low temperature; the preferred form of the human estrogen receptor under more physiological conditions is the 5 S EBP dimer.

Computer modeling of estradiol interactions with the estrogen receptor

Two computer models for the binding of estradiol to estrogen receptors were constructed, based solely upon the thermodynamic constraints of the most likely equilibria involved and known equilibrium constants. Previous data had suggested that the positive cooperativity of the system was dependent upon a monomer-dimer equilibrium (Notides et al., Proc. natn. Acad. Sci., U.S.A. 78 (1981) 4926-4930). Using computer modeling, we confirmed that the thermodynamic constraints of a monomer-dimer equilibrium system result in convex Scatchard plots in agreement with experimental data, including the progression to linearity at low receptor concentrations. This technique yielded estimates of the equilibrium constant for dimerization (approx. 10(10) to 10(14) M-1). The dose-response characteristics of the monomer-dimer equilibrium system revealed steep dose-response curves that were sensitive to the receptor concentration. In contrast, the dose-response curves that did not undergo a monomer-dimer equilibrium system and had a single step equilibrium process were more gradual.

The role of phosphorylation in human estrogen receptor function.

We have studied the role of phosphorylation of the human estrogen receptor (hER) at serine 118, which has been previously identified 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 significance 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 affinity 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 findings indicate that phosphorylation of serine 118 of the hER plays a role in regulating its transcriptional activity.

A MOLECULAR AND KINETIC ANALYSIS OF ESTROGEN RECEPTOR TRANSFORMATION

Recent studies of the uterine estrogen receptor indicate it is a multi-subunit protein. Estradiol and temperature induce a conformational change in the 4 S estrogen-binding protein (EBP) or monomer (mol. wt. 7–8 × 104) as indicated by the high energy of activation (19–21 kcal mol–1) accompanying the formation of the 4 S EBP dimer, 5 S EBP (mol. wt. 13–14 × 104). The formation of the 5 S EBP suggests that new intramolecular bonding forces have been established, presumably hydrophobic interactions, which were not available in the inactive 4 S EBP monomer. Kinetic analysis of the 4 S to 5 S EBP transformation indicates a second-order reaction, the dimerization of the 4 S EBP with a second (similar or dissimilar) subunit. The 5 S EBP produced by the in vitro warming of the cytosol-[3H]-estradiol and the 5 S EBP extracted from isolated nuclei have similar molecular weights, sedimentation coefficients, molecular radii and rates of formation. These results suggest that an estradiol and temperature induced conformational change in the 4 S EBP leads to a macromolecular association process and receptor activation.

Kinetic analysis of the interaction of human estrogen receptor with an estrogen response element

The kinetics of the interaction between recombinant human estrogen receptor and chicken vitellogenin gene II estrogen response element (ERE) were determined by ERE-Sepharose chromatography. The association constant of the interaction between the ERE and the human estrogen receptor was dependent on receptor concentration, estradiol binding and temperature. The highest association constant (80-100 x 10(6)M-1) was measured for the estradiol-bound receptor prepared at 25 degrees C and at concentrations higher than 7 nM. At high receptor concentrations (>7 nM) the binding mechanism of estradiol to the receptor was positive cooperative, indicating receptor homodimerization. At lower concentrations the binding mechanism was partially cooperative and the association constant of the liganded receptor was significantly lower. The binding mechanism at 4 degrees C was cooperative as well, and the association constants were similarly dependent upon receptor concentration, but were 50% lower than the receptor prepared at 25 degrees C. The association constant of the unliganded receptor was 4- to 5-fold lower than that of the liganded receptor at 25 degrees C. These data suggest that in addition to estradiol-induced conformational changes in the receptor, the receptor dimers are subjected to temperature-dependent changes, which further increase their affinity for an ERE.