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N L Weigel

Publications and source records attributed to N L Weigel.

At least 55 records · Page 3Linked to original sources
PubMed1994

1,25-dihydroxyvitamin D3 modulates phosphorylation of serine 205 in the human vitamin D receptor: site-directed mutagenesis of this residue promotes alternative phosphorylation.

The vitamin D receptor (VDR) from a variety of animal species is a hormone-modulated substrate for phosphorylation in vivo. In this report, we utilize an expression vector to produce recombinant human VDR (hVDR) in 1,25-dihydroxyvitamin D3-treated COS-1 cells. Immunoprecipitation of the phosphorylated hVDR followed by gel purification and phosphoamino acid analysis revealed modification exclusively on one or more serine residues, consistent with previous studies of the VDR in other species. To identify the region of phosphorylation, immunoprecipitated and gel-purified hVDR from COS-1 cells was first mixed with purified hVDR isolated to homogeneity from Saccharomyces cerevisiae and then digested with trypsin or V8 protease, and the peptides were resolved on HPLC. The single phosphate-containing peptides were recovered and subjected to amino acid sequence analysis, revealing the modification to reside in a region extending from residue 171 to residue 206 common to both the tryptic- and the V8 protease-derived peptides. Sequential cleavage of similar VDR mixtures using trypsin and then CNBr, alpha-chymotrypsin, or thermolysin demonstrated an amino-terminal boundary of the phosphorylated peptide at 202. Selective manual Edman degradation of phosphorylated peptides beginning at 171, 195, and 200 revealed phosphate release only at serine 205. This peptide contained an average of 8-fold less radioactive phosphate in the absence of prior treatment of the culture cells with 1,25(OH)2D3. Site-directed modification of VDR serine 205 to alanine, aspartate, or glutamate each led to fully functional proteins when assessed in a transactivation assay using several VDRE-linked natural promoters. Unexpectedly, evaluation of the serine 205 to alanine hVDR mutant revealed that this protein continued to be phosphorylated in a hormone-dependent manner on an alternative site. These studies show directly that hVDR serine residue 205, a consensus site for casein kinase II, is modified in vivo in response to hormone.

Amino Acid Sequence↗
PubMed1994

Phosphorylation of Ser530 facilitates hormone-dependent transcriptional activation of the chicken progesterone receptor.

Phosphorylation regulates the activities of many proteins, including transcription factors. However, the evidence for the significance of phosphorylation in steroid hormone receptor action is mainly indirect. In this study, one of the hormone-induced phosphorylation sites of chicken progesterone receptor, Ser530, was mutated to alanine, a nonphosphorylatable amino acid, and the transcriptional activity of the mutant receptor was compared with that of wild type in a transient cotransfection assay. The results showed that this mutation resulted in reduced transcriptional activity of chicken progesterone receptor at low hormone concentrations but did not affect the maximal activity of the receptor at saturating levels of hormone, suggesting that the phosphorylation at Ser530 influences the response of the receptor to its ligand. The decreased sensitivity of the mutant receptor is not due to a decrease in hormone-binding affinity, leading to our hypothesis that Ser530 phosphorylation stabilizes the receptor in its active state, perhaps by preventing its reassociation with heat shock proteins or by maintaining a conformation suitable for interaction with other transcription factors.

Animals↗
PubMed1994

Multiple signaling pathways activate the chicken progesterone receptor.

We have previously reported that treatment of CV1 cells, transiently transfected with DNA encoding the chicken progesterone receptor (cPR) and a reporter plasmid PREtkCAT, with either 8-Br-cAMP or okadaic acid resulted in ligand-independent transcriptional activation of the cPR. The surprising finding that cPR can be activated in the absence of hormone has been followed by numerous studies from other laboratories examining the effects of modulators of kinases and phosphatases on the activity of other steroid receptors. These studies have yielded mixed results: ligand-independent activation has been observed in some cases, but not in others. In order to determine whether the ligand-independent activation of cPR was restricted to a specific reporter and cell type and to better characterize this phenomenon, the studies in this report were undertaken. Using both the original reporter, PREtkCAT, and a simpler reporter, GRE2E1bCAT, we found that ligand-independent activation of the cPR can be induced in both CV1 and HeLa cells. The magnitude of the response and the response of the reporter alone differed in the two cell types. Further analysis of the activation of cPR by inhibitors of protein phosphatases showed that inhibition of phosphatase 1 rather than phosphatase 2A was necessary for activation of cPR. Finally, treatment with vanadate, an inhibitor of phosphotyrosine phosphatases, or epidermal growth factor resulted in activation of cPR. These studies suggest that signals transduced through multiple signaling pathways can activate cPR.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Chicken progesterone receptor expressed in Saccharomyces cerevisiae is correctly phosphorylated at all four Ser-Pro phosphorylation sites.

This study describes the phosphorylation of chicken progesterone receptor (cPR) produced in yeast, Saccharomyces cerevisiae, and examines the dependence of specific phosphorylations on hormone and DNA binding. The chicken progesterone receptor is expressed in vivo as two forms, cPRB and a smaller form, cPRA. Characterization of the phosphorylation sites in the cPRB form expressed in yeast shows that progesterone receptor is phosphorylated on the three serines (Ser211, Ser260, and Ser530) reported previously in chicken oviduct. An additional site which was phosphorylated in response to hormone was also detected and was subsequently identified as Ser367. Although cPRB and cPRA are phosphorylated identically in chicken oviduct, cPRA expressed in yeast is phosphorylated on Ser211, Ser260, and Ser367, but phosphorylation of Ser530 is almost undetectable. In contrast, cPRB expressed in yeast is phosphorylated on all four sites. No phosphorylations were found in or near the region required for hormone binding, indicating that phosphorylation is not required for hormone binding. In order to determine whether any of the phosphorylations were DNA-dependent, phosphorylation was also studied using cPRA containing a partial deletion of the DNA binding domain. Two of the sites, Ser211 and Ser367, showed reduced phosphorylation in this mutant, suggesting a possible requirement for DNA binding activity for the phosphorylation of these sites. To our knowledge, this is one of the first demonstrations that a eucaryotic protein expressed in yeast is correctly phosphorylated.

Amino Acid Sequence↗
PubMed1993

The progesterone antagonist RU486 acquires agonist activity upon stimulation of cAMP signaling pathways.

The protein kinase A stimulator cAMP can potentiate the ability of progestins to induce the transactivation function of the human progesterone receptor (hPR). We questioned in the present study whether cAMP could functionally cooperate with the progestin antagonist RU486. In T47D human breast cancer cells, RU486 behaves as a pure antagonist with respect to induction of the progesterone-responsive mouse mammary tumor virus chloramphenicol acetyltransferase (MMTV-CAT) reporter gene. It fails to stimulate MMTV-CAT expression and completely inhibits induction by the synthetic progestin R5020. However, when RU486 is combined with 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), MMTV-CAT is induced to levels approaching that stimulated by R5020 alone. Also, RU486 in the presence of 8-Br-cAMP is only partially effective in antagonizing R5020 action. The agonist activity exhibited under these conditions appears to be due to RU486 acting through hPR as evidenced by the fact that 8-Br-cAMP alone has no effect on MMTV-CAT, whereas induction by the combination of 8-Br-cAMP and RU486 is dose responsive to RU486 in a saturable manner and can be inhibited by the type I antiprogestin (prevents hPR-DNA binding) ZK98299, which does not exhibit positive functional cooperation with cAMP. Acquisition of agonist activity in the presence of 8-Br-cAMP also extends to the type II antiprogestin (permits hPR-DNA binding) ZK112993. Since RU486 is also a type II antagonist, these results suggest that detection of functional synergism between cAMP and antiprogestins may require binding of the hPR-antagonist complex to DNA. We propose that cross-talk between second messenger and steroid receptor signal transduction pathways may be one mechanism for resistance to steroid antagonists that frequently develops in breast cancer.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Modulators of cellular protein phosphorylation alter the trans-activation function of human progesterone receptor and the biological activity of progesterone antagonists.

Addition of progesterone to breast cancer cells in vivo increases phosphorylation of human progesterone receptor (PR), suggesting that phosphorylation has a regulatory role in producing the activated form of receptor. Kinetic analysis indicates that hormone-dependent phosphorylation is sequential and that early stages of phosphorylation(s) are closely associated with enhancement of PR-DNA binding while later stages are associated with a trans-activation function. Various agents that stimulate cellular protein phosphorylation (8-Br cAMP, okadaic acid, TPA) functionally synergize with progesterone to enhance progesterone-dependent PR trans-activation in intact cells. These results suggest that protein phosphorylation does have a role in modulating the trans-activation function of PR in vivo. They also demonstrate cross-talk between second messenger signal transduction pathways and nuclear steroid receptors. Whether the phosphorylated target that provides the link between these two signal transduction pathways is PR itself or another protein involved in PR-mediated gene transcription is not known. Positive cooperative interactions were also observed between cAMP signaling pathways and the progesterone antagonist RU486, that resulted in RU486 exerting substantial agonist activities. This ability of cross-talk between second messenger and steroid receptor signal transduction pathways to override the antagonistic effects of RU486 suggests a novel mechanism to explain the problem of resistance to clinically important steroid antagonists.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Identification of a hormone-dependent phosphorylation site adjacent to the DNA-binding domain of the chicken progesterone receptor.

We have previously identified three phosphorylation sites in chicken progesterone receptor (PR) isolated from the cytosol of chicken oviducts. Several lines of evidence suggested that there might be additional phosphorylations that occur only in the nuclear compartment. We have reexamined the phosphorylation of the PR using whole cell extracts and have identified a second hormone-dependent phosphorylation site (Ser367) which is located on the amino-terminal side of the DNA-binding domain. This site also contains a Ser-Pro consensus sequence, as do the three previously reported sites. There are four Ser-Pro sequences in chicken PR, and all four are phosphorylated. Two of the sites showed substantial phosphorylation in the absence of hormone, with some enhancement in response to hormone, whereas two of the sites showed very little phosphorylation in the absence of hormone. This suggests either that despite the Ser-Pro consensus sequences, the sites are phosphorylated by different enzymes, or that binding hormone changes the conformation of the protein sufficiently to expose the two hormone-dependent phosphorylation sites.

Amino Acid Sequence↗
PubMed1992

Hormone and antihormone induce distinct conformational changes which are central to steroid receptor activation.

Antihormones are potent antagonists of hormone action in vivo, but the mechanism underlying this antagonism is not understood. Several steroid hormones transform (activate) their receptors from a cytosolic, non-DNA binding 8 S sedimentation form to a nuclear, DNA binding 4 S form. Transformation is accompanied by the loss of associated heat shock proteins. We have previously demonstrated that an additional hormone-dependent step, separate from heat shock protein removal, is required for activation of the human progesterone receptor. We have devised an assay in which the human progesterone receptor translated in vitro binds to its specific response element in a hormone-dependent manner. As assessed by limited proteolytic digestion, hormone treatment of the nascent receptor induces a dramatic conformational change within the protein. The conformational change occurs in the absence of DNA and renders the entire ligand binding domain resistant to digestion by proteases. A number of antiprogestins, including RU486, induce an equally dramatic, but distinct, structural alteration of the ligand binding domain. The distinction centers upon the final 30 to 40 amino acids at the carboxyl terminus. The conformational change can be induced by ligand prior to dissociation of the 8 S complex and is not induced by heat shock protein removal in the absence of hormone. Remarkably, virtually identical hormone-induced conformational changes were detected following proteolytic analysis of in vitro translated retinoic acid receptors. Our data indicate that the sole necessary event in the activation of steroid receptors is conformational modification by the ligand. Furthermore, we conclude that transcriptional inactivation of steroid receptors by antihormones involves the induction of an inappropriate structural conformation at the extreme carboxyl terminus of the ligand binding domain.

Base Sequence↗
PubMed1992

A novel, highly regulated, rapidly inducible system for the expression of chicken progesterone receptor, cPRA, in Saccharomyces cerevisiae.

A rapidly inducible and tightly regulated system for the expression of protein in yeast is based on a chimeric promoter constructed of two copies of a vitellogenin-estrogen-response element (ERE) which are inserted upstream from the promoter of the yeast gene encoding iso-1-cytochrome c. The chimeric promoter was inserted in a yeast expression plasmid upstream from the coding sequence of ubiquitin fused in frame to a cDNA encoding the full-length chicken progesterone receptor A (cPRA). The resultant plasmid (YEpA2) was co-transformed in Saccharomyces cerevisiae with a plasmid which encodes the human estrogen receptor. Estradiol (E2)-induced transactivation of the chimeric promoter results in transcription of the cPRA gene from YEpA2, and synthesis of cPRA. The fusion protein, ubiquitin-cPRA, is rapidly cleaved in vivo to produce cPRA. Analysis of samples by Western immunoblot shows that cPRA is almost undetectable in the absence of E2, and that treatment with 50 nM E2 results in a 500-1000-fold induction of cPRA (0.06-0.3% of the total protein) after 1 h. The plasmid-expressed soluble receptor is stable and demonstrates the correct affinity for its ligand. We have prepared yeast extracts using enzymatic digestion of the cell wall with oxalyticase followed by hypotonic shock. This has resulted in a dramatic increase in the % of receptor which binds hormone compared to previous studies which used mechanical disruption techniques. The cPRA is biologically active since it activates transcription of a co-transformed reporter gene containing its response element.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Chicken progesterone receptor is phosphorylated by a DNA-dependent protein kinase during in vitro transcription assays.

We have reported previously that chicken progesterone receptor (PR) is phosphorylated in vivo in response to progesterone administration. Three phosphorylation sites have been reported, two of which show increased phosphorylation in response to hormone and one which is phosphorylated only in response to hormone administration. We found previously that PR lacking the hormone-dependent phosphorylation is active in an in vitro transcription assay. Since the source of general transcription factors is a HeLa nuclear extract which contains many kinases, we have analyzed the receptor for phosphorylation during the in vitro transcription assay. We report here that the receptor is rapidly and efficiently phosphorylated on new sites, causing a change in receptor mobility on sodium dodecyl sulfate-gels. This phosphorylation is strictly dependent upon the presence of double stranded DNA. A DNA-activated protein kinase with similar properties has been isolated previously from HeLa cell nuclei. We find that phosphorylation of PR with this purified enzyme mimics the phosphorylation observed in the transcription assay. These data suggest that a previously undetected additional series of DNA-dependent phosphorylations may be required for activation of the PR.

Animals↗
PubMed1992

Ligands induce conformational changes in the carboxyl-terminus of progesterone receptors which are detected by a site-directed antipeptide monoclonal antibody.

We have prepared a monoclonal antibody, C-262, to a synthetic peptide that contains the carboxy-terminal 14 amino acids from progesterone receptors (PR). This sequence is 100% conserved in all species of PRs that have been cloned to date, suggesting that this antibody will recognize all mammalian and avian PR. The C-262 antibody recognizes both native and denatured forms of the receptor. However, it does not recognize PR when they are bound to the hormone agonists progesterone or R5020. Surprisingly the antibody does recognize PR when they are bound to the steroid antagonist RU486. This suggests that progestin agonists induce a conformational change in the receptor that occludes the C-262 epitope in the carboxyl-terminus, whereas unliganded receptors and receptors bound with RU486 assume distinct conformations that leaves the C-terminal tail accessible to the C-262 antibody.

Amino Acid Sequence↗
PubMed1992

Effects of hormone and cellular modulators of protein phosphorylation on transcriptional activity, DNA binding, and phosphorylation of human progesterone receptors.

Human progesterone receptors (PR) in T47D breast cancer cells are synthesized as two different sized proteins, PR-A [94 kilodaltons (kDa)] and PR-B (120 kDa). Progestin addition to cells (in vivo) causes a 2-fold increase in total phosphorylation of PR and an increase in the apparent mol wt of both PR-A and PR-B on sodium dodecyl sulfate (SDS)-gels. Time-course experiments showed that increased PR phosphorylation that results from hormone addition is a multistep process and involves a rapid increase into total 32P labeling that takes place before the more slowly occurring phosphorylation(s) responsible for the change in electrophoretic mobility of PR on SDS-gels. As an approach to test whether phosphorylation is involved in regulating PR activity, we have examined the effects of cellular modulators of protein phosphorylation on PR-mediated target gene transcription in vivo using a T47D cloned cell line containing a stably transfected mouse mammary tumor virus-chloramphenicol acetyltransferase construct. Treatment with 8-bromo-cAMP (activator of cAMP-dependent protein kinases) or okadaic acid (protein phosphatase-1 and -2A inhibitor) did not stimulate target gene expression in the absence of progestin. When added together with progestin, either compound augmented PR-mediated target gene transcription by 3- to 4-fold. The cyclic nucleotide-dependent protein kinase inhibitor H8 completely blocked target gene responsiveness to hormone. Neither 8-bromo-cAMP, okadaic acid, nor H8 altered the hormone- or DNA-binding activities of PR, as measured in vitro or affected cellular concentrations of PR. These agents, therefore, appeared to selectively modulate PR transcriptional activity. Moreover, none of these compounds altered expression from a control reporter gene, pSV2CAT, indicating that these agents affect PR-mediated processes directly and are not acting through a general effect on transcription. Effects on PR phosphorylation were assessed by measuring 32P labeling of PR in vivo. None of these treatments had a substantial effect on the extent of total 32P labeling of immune isolated PR or on the phosphorylation(s) responsible for PR up-shifts on SDS-gels. This suggests that these agents modulate PR transcriptional activity either through phosphorylation of another protein intimately involved in PR-mediated transcription or through modification of a key site(s) not measurable as a change in total PR phosphorylation or electrophoretic mobility on SDS gels.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Synergism between steroid response and promoter elements during cell-free transcription.

We have analyzed quantitatively the influence of distal promoter elements on steroid-responsive gene expression in vitro. Functional synergism between enhancer and distal promoter elements was examined using two model promoters, one containing a natural promoter (mouse mammary tumor virus long terminal repeat) and one constructed artificially. Human glucocorticoid receptor (GR) expressed in baculovirus induces transcription from a mouse mammary tumor virus long terminal repeat-containing DNA template. Transcription is diminished by oligonucleotides containing a nuclear factor 1 (NF-1)-binding site or a glucocorticoid/progesterone response element. Quantitative analysis indicates that NF-1 and GR act synergistically during transcriptional activation. In contrast, efficient activation by GR or purified chick progesterone receptor of a glucocorticoid/progesterone response element-linked ovalbumin promoter does not require interaction with the chicken ovalbumin upstream promoter (COUP) element in the distal promoter. Lack of synergism is not related to enhancer strength, since the glucocorticoid/progesterone response elements can be moved further from the promoter or reduced to a single copy response element without increasing the dependence upon COUP. Strong synergism is restored following substitution of an NF-1 distal promoter element for the COUP element in this construct. Our results suggest that synergism between steroid response and distal promoter elements is dependent upon the identity of the promoter element rather than upon the inherent strength of the enhancer element.

Animals↗
PubMed1991

High level expression of biologically active estrogen receptor in Saccharomyces cerevisiae.

Biochemical over-expression of the human estrogen receptor was achieved using a Saccharomyces cerevisiae expression system. The receptor was produced as a novel ubiquitin fusion protein. This fusion protein is short lived in the cell and is processed to produce unfused receptor shortly after folding. Conventional high copy expression plasmids produced receptor to about 0.04% of the total soluble protein. By incorporating a defective leu2 allele into these vectors, an additional 5-fold increase in receptor production was obtained. The recombinant receptor was undergraded, soluble and biologically active. Conventional methods of disrupting cells using glass beads had a detrimental effect on the ability of the receptor to bind hormone. Enzymatic digestion of the cell wall followed by hypotonic shock liberates the receptor that quantitatively binds estrogen.

Alleles↗
PubMed1990

Regulation of progesterone receptor-mediated transcription by phosphorylation.

The progesterone receptor (PR) in the chicken oviduct is a phosphoprotein that regulates gene transcription in the presence of progesterone. Treatment with progesterone in vivo stimulates phosphorylation of the progesterone receptor. With transient transfection assays, the present work has tested whether phosphorylation participates in the regulation of PR-mediated transcription. Treatment with 8-bromo-cyclic adenosine monophosphate (8-Br cAMP), a stimulator of cAMP-dependent protein kinase [protein kinase A (PKA)], mimicked progesterone-dependent, receptor-mediated transcription in the absence of progesterone. Inhibition of PKA blocked hormone action. Treatment with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, stimulated transcription in a manner similar to that of progesterone. These observations suggest that phosphorylation of the PR or other proteins in the transcription complex can modulate PR-mediated transcription in vivo.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Hormonal regulation and identification of chicken progesterone receptor phosphorylation sites.

The present studies examine the effects of in vivo and in situ progesterone treatment in the regulation of site-specific phosphorylation of the chicken oviduct progesterone receptor (PR). By gas-phase protein sequencing we have identified three hormonally regulated phosphorylation sites: Ser-211, Ser-260, and Ser-530. We determined phosphorylation stoichiometries by analyzing the amounts of phosphorylated and dephosphorylated serine at each site. Stoichiometries of sites 211 and 260 were about 20% under basal conditions and increased 1.5-2-fold by in situ progesterone treatment. Site 530 was virtually absent under basal conditions and induced to greater than 33% by in situ progesterone treatment. We tested several protein kinases for phosphorylation of the PR in vitro on these sites or peptides containing these sites. We found that the catalytic subunit of cAMP-dependent protein kinase mimicked the in vivo, hormone-induced altered mobility of PRs in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the in vivo and in vitro alterations were reversed by alkaline phosphatase. Finally, we showed that cAMP-dependent protein kinase phosphorylated Ser-528.

Amino Acid Sequence↗
PubMed1990

The steroid-dependent regulatory element in the ovalbumin gene does not function as a typical steroid response element.

Transcription of the chicken ovalbumin gene is induced both in vivo and in vitro by four classes of steroid hormones. Recent experiments identified a steroid-dependent regulatory element (SDRE) in the 5'-flanking region of the ovalbumin gene between -900 and -521. To characterize the regulatory properties of the SDRE more precisely, additional mutations were created in this region, and fusion genes prepared by linking the ovalbumin 5'-flanking region and promoter to the chloramphenicol acetyltransferase structural gene. When the ovalbumin-chloramphenicol acetyltransferase fusion genes were transfected into steroid-responsive primary oviduct cells, mutants lacking sequences between -900 and -732 were no longer responsive to estrogen, corticosterone, progesterone, or dihydrotestosterone. The SDRE did not confer steroid-dependent expression on the heterologous thymidine kinase promoter by itself but did in conjunction with the negative regulatory element identified between -350 and -100. This suggests that the two elements act as a single functional entity and that the SDRE is not behaving as a typical steroid response element. Gel shift analyses revealed that two SDRE.protein complexes were formed when nuclear protein extracts were derived from estrogen-treated chicken oviduct but that only one complex was formed with extracts from estrogen-withdrawn oviduct or from other tissues. Neither an estrogen response element oligomer nor a glucocorticoid/progesterone response element oligomer competed for either of the DNA.protein complexes. Partially purified progesterone receptor also did not bind to the SDRE. These data indicate that induction of the ovalbumin gene by steroid hormones requires complex interactions involving both the SDRE and the negative regulatory element.

Animals↗