Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this study we analyzed the covalent binding to proteins of 17 beta-estradiol (E2), retinoic acid (RA), and progesterone in MCF-7 and MCF-7/AdrR cells. MCF-7 cells have receptors for E2 and progesterone. MCF-7/AdrR cells do not have these receptors. After a 1-day incubation period with either [3H]E2, [3H]progesterone, or [3H]RA the levels of covalently bound radioactivity was between 1.4- to 2-fold greater in MCF-7 cells than in MCF-7/AdrR cells. We analyzed the labeled proteins with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. About 40 proteins were labeled by E2 in MCF-7 cells and about 10 of these proteins were the only proteins labeled by E2 in MCF-7/AdrR cells. We saw that the same 8 proteins were labeled by RA in both cell lines. Progesterone labeled 2 proteins with M(r) values of 37,000 and 20,000 in MCF-7 cells. These 2 proteins had mobilities that were the same as proteins that were labeled by either E2 or RA in both MCF-7 and MCF-7/AdrR cells. Besides these 2 proteins, we saw proteins of M(r) 51,000 (p51) and 55,000 that were covalently labeled by E2 in MCF-7 cells and by RA in both MCF-7 and MCF-7/AdrR cells. The p51 had the same mobility on 2D-PAGE as an 8-azido-[32P]cAMP-labeled protein. This protein is probably RII alpha, the type II cAMP-binding regulatory subunit of type II cAMP-dependent protein kinase. These results suggest that the estrogen receptor, while not obligatory, might still modulate the covalent linkage of E2 to protein. In addition, our results raise the possibility that some effects of some ligands of the thyroid/steroid hormone receptor family may involve the covalent linking of these hormones to proteins, including RII alpha.
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PMID:The covalent labeling of proteins by 17 beta-estradiol, retinoic acid, and progesterone in the human breast cancer cell lines MCF-7 and MCF-7/AdrR. 132 24

Primary uterine cell cultures were used to study multifactor regulation of progesterone receptor (PR) and the signal transduction pathways which may serve to mediate that regulation. Increases in intracellular cAMP, brought about by treatment with cholera toxin plus isobutyl methyl xanthine or by addition of 8-bromo-cAMP, result in 6- to 7-fold increases in the intracellular content of PR as monitored by [3H]R5020 binding and by Western immunoblot using anti-PR antibodies. In these primary cultures of uterine cells isolated from 19-day-old immature rats, 8-bromo-cAMP evokes significant increases in PR by 8 h with maximal increases by 24 h. This time course and magnitude of PR stimulation are similar to those evoked by maximally effective concentrations of estradiol (3 x 10(-9) M) or IGF-I (20 ng/ml). Dose-response studies reveal that 10(-6) to 10(-4) M concentrations of 8-bromo-cAMP (8-Br-cAMP) elicit a maximal response. In contrast, 8-bromo-cGMP over a wide concentration range was unable to elevate cellular PR levels. Under these culture conditions, cell proliferation was not altered by treatment with any of these agents. Although estrogen, cAMP, and insulin-like growth factor I (IGF-I) may act via different pathways to increase PR, the effects evoked by maximally effective concentrations of these agents are not additive implying involvement of a common component. The increases in PR evoked by estradiol, cAMP, or IGF-I are markedly suppressed by treatment with antiestrogen (ICI 164,384) or the cyclic nucleotide-dependent protein kinase inhibitor H8 or the protein kinase A inhibitor PKI, indicating the involvement of the estrogen receptor and phosphorylation pathways in PR regulation by these three agents. The present studies identify cAMP, as well as estrogen and IGF-I, as important regulators of the level of PR in uterine cells and suggest that multiple factors, including those affecting intracellular cAMP levels, might influence responsiveness to progestins via regulation of the intracellular PR content.
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PMID:Progesterone receptor regulation in uterine cells: stimulation by estrogen, cyclic adenosine 3',5'-monophosphate, and insulin-like growth factor I and suppression by antiestrogens and protein kinase inhibitors. 170 63

All proteins of this world are constructed in compliance with the same rule. Accordingly, two totally unrelated proteins, on the average, share 30 identical tripeptides, two tetrapeptides, and one pentapeptide per 500 residues. With this in mind, the 221-residue-long influenza virus hemagglutinin II (IVHA-II), as a representative of alien antigens, was compared with three diverse proteins representing the host: 533-residue-long chicken c-src protein kinase (c-src product of the cellular oncogene of Rous sarcoma virus), 595-residue-long human estrogen receptor, and 585-residue-long human serum albumin. Forty-three tripeptides, two tetrapeptides, and one pentapeptide of IVHA-II were also found in one or the other of the three host proteins. Six regions of IVHA-II (9-22 residues long) in which oligopeptides were clustered that were identical to their host oligopeptides were defined as "host-homologous" regions, and the remaining regions were called "nonself" or "pathogen-specific" regions. Because the total number of host proteins is vastly more than three, host-homologous regions were no doubt underestimated, while only one or two regions of IVHA-II must remain as truly pathogen-specific. Nevertheless, oligopeptide analysis of two known T-cell response-eliciting peptide fragments and one known inert peptide fragment of a virus and a malarial protozoan readily revealed the latter to be a host-homologous region. Of the two known T-cell response-eliciting peptide fragments, one was more nonself than the other. Not surprisingly, the more nonself fragment elicited helper T-cell response from individuals of diverse major histocompatibility complex haplotypes, whereas the less nonself fragment elicited cytotoxic T-cell response only from HLA-A2 human individuals.
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PMID:Many peptide fragments of alien antigens are homologous with host proteins, thus canalizing T-cell responses. 170 30

We have examined the ability of estradiol (E2) to regulate the expression of three mRNAs [for pS2, progesterone receptor (PR), and estrogen receptor (ER)], known to be under E2 regulation in the parental E2 growth-responsive MCF-7 cells, in an E2 growth-independent MCF-7 K3), previously isolated from the parental estrogen-dependent MCF-7 K1 human breast cancer cells after long term growth in vitro in the absence of estrogen, acquired estrogen-independent growth in vitro as well as the ability to form tumors in nude mice in vivo without estrogen. We find that the content of pS2 mRNA and the transcription rate of the pS2 gene, while being markedly increased by E2 in MCF-7 K1 cells, are no longer stimulated by E2 in this subline, although protein kinase activators tremendously increase (greater than 10-fold) pS2 mRNA in both K1 and K3 cells. In fact, basal pS2 mRNA levels are elevated 2.8 +/- 0.4-fold in MCF-7 K3 cells, and E2 evokes a concentration-dependent suppression of the pS2 mRNA level. In contrast, PR mRNA in the K3 subline, as in the parental K1 cells, is still up-regulated by E2, and ER mRAN content and the ER mRNA transcription rate are still down-regulated by E2 and show normal E2 dose-response relationships, implying that the ER in this subline is functional. These results demonstrate that the progression to estrogen-independent growth in K3 cells is accompanied by a change in the regulation of some estrogen-induced genes by estrogen. While PR and ER retain normal patterns of regulation by E2, the pS2 gene in the estrogen growth-independent K3 subline is differentially affected and is no longer stimulated by E2. Our data suggest that this altered regulation of the pS2 gene is probably not caused by a defect of the ER or ER regulation in this subline.
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PMID:Differential regulation of gene expression by estrogen in estrogen growth-independent and -dependent MCF-7 human breast cancer cell sublines. 172 71

In this report we show that the mRNA level for the estrogen receptor (ER) is regulated by 8-bromo cyclic AMP (8-Br-cAMP) and human chorionic gonadotropin in a mouse tumor Leydig cell line (MA-10 cells). When the MA-10 cells were cultured in the presence of the cAMP analogue for varying time periods, a transient increase in the level of ER mRNA was observed. Short time incubation (0-2 h) with 8-Br-cAMP enhanced the expression of ER mRNA (2-fold), whereas longer times of incubation (6 h) had the opposite effect (the level of ER mRNA was reduced by 60-70%). The inhibitory effect of 8-Br-cAMP on ER mRNA was not counteracted by aminoglutethimide, an inhibitor of steroidogenic enzymes, indicating that this effect is not mediated via steroids (progesterone). Treatment of 8-Br-cAMP for 6 h caused a concentration-dependent inhibition of ER mRNA with a half-maximal effect of approximately 150 microM. Increasing concentrations of human chorionic gonadotropin for 6 h was also associated with a biphasic effect on the ER mRNA level. Low concentrations (0.20-0.40 ng/ml) increased ER mRNA in the MA-10 cells whereas the highest concentration (20 ng/ml) caused a suppression of this mRNA. In contrast to the biphasic effects observed for the ER mRNA, the level of the regulatory subunit type II beta of the cAMP-dependent protein kinase (protein kinase A) was enhanced in a concentration-dependent manner by human chorionic gonadotropin. Furthermore, 8-Br-cAMP stimulated the mRNA for regulatory subunit type II beta (10- to 20-fold) by all concentrations examined (50-1000 microM). The observations reported here indicate that the expression of ER mRNA is regulated both by endogenously formed and exogenously added cAMP and that there may exist regulatory loops between the steroid and the cAMP/protein kinase A systems.
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PMID:Biphasic regulation of the messenger ribonucleic acid coding for the estrogen receptor by cyclic adenosine 3':5'-monophosphate in tumor Leydig cells. 215 26

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 (Ry, Kd = 0.8 nM) by a process requiring the gamma-phosphoryl moiety of ATP. The enzymatic activity (Fy) 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 Fy kinase characteristically phosphorylates casein, histones, and glycogen synthase. Comparison of the kinase with casein kinase II, which also phosphorylates casein and glycogen synthase, indicates that Fy represents a distinct protein kinase since its activity is not stimulated by spermine or inhibited by heparin. Fy-mediated conversion of Rnb to Ry is blocked by the phosphatase inhibitors vanadate, fluoride, and pyrophosphate. The substrate specificity of the Fy phosphatase activity is distinct from that of the two well-characterized protein phosphatases 1 and 2A. Moreover, the requirement for Fy phosphatase activity in converting Rnb to Ry could not be mimicked by its substitution with purified protein phosphatases 1 or 2A. The unique substrate specificity of the oviduct protein phosphatase and protein kinase, which are apparently necessary to confer estradiol binding characteristics to the receptor, implies that these enzymes play a key role in the control of the estrogen receptor in its function as a transcription factor.
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PMID:Receptor interconversion model of hormone action. 2. Requirement of both kinase and phosphatase activities for conferring estrogen binding activity to the estrogen receptor. 216 Dec 54

We investigated the effects of the antiestrogen tamoxifen on MCF-7 cell protein kinase C either by using the in vitro histone kinase assay or by studying the phosphorylation of its endogenous Mr 28,000 protein substrate in intact cells. In the in vitro assay, tamoxifen inhibited the enzyme competitively with respect to phospholipid, whereas estradiol and morpholinobenzyl phenoxy ethanamine, a specific ligand for antiestrogen binding sites, were considerably less efficient. In contrast, tamoxifen did not affect phosphorylation of the Mr 28,000 protein induced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate in intact MCF-7 cells. Estradiol and morpholinobenzyl phenoxy ethanamine also had no effect. At high concentration (100 microM), tamoxifen itself stimulated specific phosphorylation of this Mr 28,000 protein. Estradiol and morpholinobenzyl phenoxy ethanamine neither mimicked nor interfered with this effect. Our data suggest that the effect of tamoxifen on protein kinase C activity depends on the phospholipid environment of the enzyme, and opposite effects may be observed in intact cells to those seen in disrupted cells. The action of tamoxifen on endogenous protein phosphorylation was thought to be due to direct interaction with the phospholipid binding domain of the enzyme rather than by interaction with the estrogen receptor or the antiestrogen binding site. Nevertheless, our results do not rule out a possible activation by tamoxifen of specific protein kinase(s) and phosphatase(s). In any case, the antiproliferative activity of tamoxifen on MCF-7 cells cannot be attributed to its effects on protein kinase C.
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PMID:Opposite effects of tamoxifen on in vitro protein kinase C activity and endogenous protein phosphorylation in intact MCF-7 cells. 239 53

High performance liquid chromatography (HPLC) was employed as a means of analyzing estrogen receptor (ER)-antibody recognition. This technique takes advantage of the fact that the majority of gamma-globulin-antigen complexes do not interact with the anion-exchange resins selected. A variety of monoclonal (MAb) and polyclonal antibodies (PAb) raised against ER and ER-associated proteins were assessed for their chromatographic behaviour after association with charged ER, based upon properties of size, shape, and surface charge. ER exhibits polymorphism, several isoforms being present in target cells. The monoclonal antibody H222Sp gamma demonstrated discrete specificity for the 150 mM ER isoform (normally eluting at 150 mM phosphate) from the high-performance ion-exchange chromatography column which was eluted unretained when complexed with antibody. However, the monoclonal reagent D547Sp gamma interacted directly with anion-exchange columns (SynChropak AX-1000 and Altex DEAE-5PW), complicating a clear evaluation of ER-MAb association. Only 50-60% of the 150 mM ER isoform was eluted at a lower salt concentration. Few conclusions could be drawn with respect to MAb interaction with the 50-60 mM ER isoform (normally eluting at 50-60 mM phosphate) since the antibody-receptor complex was also eluted at the same phosphate concentration. In addition, polyclonal and monoclonal antibodies to the ER and ER-associated proteins were assessed by HPLC. At present, heat shock proteins and protein kinase activity have been shown by other techniques to be associated with the ER. Size-exclusion resins, such as TSK 3000 SW, were employed in a fast method of determining ER isoform-antibody recognition. Thus, HPLC may be used to analyze soluble antibody-antigen interactions rapidly, with high recovery of biological activity.
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PMID:Assessment of estrogen receptor-monoclonal antibody interaction by high-performance liquid chromatography. 244 25

These studies were undertaken to determine whether nonsteroidal antiestrogens would inhibit the calcium/lipid-dependent protein kinase (protein kinase C) activity in hormonally-responsive human reproductive tissues. Cytosol was prepared from human corpus luteum and term placenta. Protein kinase C activity was examined with various antiestrogens, estrogens, and catecholestrogens. The nonsteroidal antiestrogens tamoxifen, clomiphene and Z-4-hydroxytamoxifen inhibited protein kinase C in cytosol from human corpora lutea and placentae in a concentration-dependent manner. The IC50 values were 35-45 microM for tamoxifen, 58-66 microM for clomiphene, and 88 microM for hydroxytamoxifen. Protein kinase C purified 600-fold from human placenta was also inhibited by tamoxifen. The estrogens, estradiol and diethylstilbestrol (DES), and the catecholestrogens, 2-hydroxyestradiol and 4-hydroxyestradiol, had no effect on protein kinase C activity, nor were they able to prevent the inhibition of protein kinase C by the antiestrogens. Inhibition of the enzyme by the antiestrogens was competitive with phosphatidylserine and 1,2-diolein. In addition, tamoxifen inhibited enzyme activity stimulated by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). The data suggest that the action of these antiestrogens on protein kinase C was a direct inhibition of the enzyme. Furthermore, the site of interaction showed markedly different structural specificity from that of the estrogen receptor.
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PMID:Nonsteroidal antiestrogen inhibition of protein kinase C in human corpus luteum and placenta. 281 41

Quantitative polyacrylamide gel electrophoresis analysis of Ca2+, phospholipid-dependent protein kinase (PKC) of human mammary tumor cell lines (MCF-7, ZR-75, T-47-D, MDA-MB-231, BT-20, and HBL-100) revealed that 80% of the total cellular PKC resided in the cytosol. The tumor cells with no detectable levels of estrogen receptors (MDA-MB-231, HBL-100, and BT-20 cells) exhibited significantly larger (P less than 0.001) cytosolic PKC activities than those cells that contained estrogen receptors (MCF-7, T-47-D, and ZR-75 cells). In addition, in estrogen receptor-negative cell lines, relatively high levels of specific low-affinity (apparent Kd = 700 pM) epidermal growth factor (EGF) binding activities were found as compared with estrogen receptor-positive cells with significantly (P less than 0.001) lower levels of specific high-affinity (apparent Kd = 90 pM) EGT binding. A significant positive correlation (P less than 0.01) was observed between the number of EGF receptor (Rs = 0.50) and/or the EGF receptor dissociation constants (Rs = 0.78) with the cytosolic PKC activity levels. These data indicate that, in human breast cancer cells, a positive relationship may exist between PKC activity, estrogen, and EGF receptors.
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PMID:Epidermal growth factor binding and protein kinase C activities in human breast cancer cell lines: possible quantitative relationship. 300 98


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