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)

We have shown previously that exposure of rat uterine cells in primary culture to estradiol (E2), insulin-like growth factor-I (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 ER-mediated 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 10(-9) M E2, CT (1 micrograms/ml) + IBMX (10(-4) M), 8-Br-cAMP (10(-4) M), or IGF-I (20 ng/ml) resulted in an 8- to 10-fold induction of CAT activity. CAT activity stimulated by all agents was nearly completely suppressed by coincubation with the antiestrogen ICI 164,384 (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 E2, 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 E2, CT + IBMX, 8-Br-cAMP, or IGF-I. Of interest, the antiestrogen ICI also elicited a similar increase in overall ER phosphorylation. The PK inhibitors H8 and PKI suppressed the increase in overall ER phosphorylation stimulated by these agents by 50-75%. The results of our study indicate that E2, IGF-I, and agents which raise intracellular cAMP are able to stimulate ER-mediated trans-activation and ER phosphorylation. The fact that antiestrogen (ICI) evokes a similar increase in ER phosphorylation without a similar increase in transcription activation indicates that an increase in overall ER phosphorylation does not necessarily result in increased transcriptional activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Stimulation of estrogen receptor-mediated transcription and alteration in the phosphorylation state of the rat uterine estrogen receptor by estrogen, cyclic adenosine monophosphate, and insulin-like growth factor-I. 768 95

The human progesterone receptor (PR), a member of the steroid/thyroid receptor superfamily of ligand-activated transcription factors, is expressed in most tissues as two forms that exhibit differential transcriptional activation potentials, full-length PR-B and NH2-terminally truncated PR-A. In human breast cancer cells (T47D) both forms of PR are constitutively phosphorylated but phosphorylation is increased in response to hormone treatment, suggesting that this modification has a role in regulating the activation state of the receptor. To more directly define the functional role of phosphorylation in the action of A and B receptors requires knowledge of the phosphorylated amino acid residues and the protein kinase(s) involved. Toward this end we have developed a strategy that combines isolation of PR phosphotryptic peptides by reverse phase high performance liquid chromatography, secondary analytical protease digestion, manual Edman degradation, and release of 32P that resulted in identification of two major phosphorylation sites, Ser81 and Ser162. Both sites are located in the amino-terminal region unique to PR-B, and one of these sites (Ser81) is encompassed in a casein kinase II (CKII) consensus sequence. Although human PR contains 11 potential CKII consensus sequences, CKII in vitro phosphorylated purified PR-B only at Ser81 suggesting that this may be an authentic site for CKII in vivo.
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PMID:Identification of phosphorylation sites unique to the B form of human progesterone receptor. In vitro phosphorylation by casein kinase II. 798 41

Signal amplification is fundamental to the normal operation of the preovulatory LH surge and is achieved through processes such as GnRH self-priming and augmentation of stimulated LH secretion by progesterone. We have proposed a model for GnRH self-priming that requires cross-communication between a GnRH receptor-activated protein kinase A pathway and the progesterone receptor (PR) to achieve amplification of the GnRH signal. We found that a pulse of GnRH administered to gonadotrope-enriched pituitary cells cultured in medium containing charcoal-treated serum plus estradiol (E2) potentiated the LH secretory response to subsequent GnRH pulses, and this potentiation could be blocked by a PR antagonist, RU486, in the absence of progesterone. Similarly, exposure of gonadotrope-enriched cultures to forskolin augmented the response to a pulse of GnRH, and the augmentation due to cAMP elevation could be reduced by RU486 in the absence of progesterone. To directly test whether stimulation with either GnRH or a cAMP analog results in transactivation of the endogenous PR, we used rat anterior pituitary cells cultured in the presence of E2 and transfected with reporter plasmids containing progesterone-responsive elements (PRE) and either a E1b or a thymidine kinase (tk) promoter linked to the chloramphenicol acetyltransferase (CAT) gene. For pituitary cells transfected with the PRE-E1b-CAT plasmid, exposure to either progesterone, GnRH, or 8-bromo-cAMP (8BrcAMP) for 6 h resulted in an induction of CAT activity which could be suppressed by coincubation with RU486. RU486 by itself had no effect on CAT activity. Similar results were obtained when a plasmid containing a different promoter (PRE-tk-CAT) was used. For cells transfected with a construct lacking a PRE (pSV2CAT), 8BrcAMP was without effect on CAT expression. When cells were made PR-deficient by omission of E2 from the incubation medium and transfected with PRE-E1b-CAT, neither progesterone, GnRH, nor 8BrcAMP was able to induce CAT activity. In summary we found that either GnRH or 8BrcAMP is able to stimulate transcription of reporter genes linked to two different PRE-containing promoters in anterior pituitary cells that contain endogenous PR; this occurred in the absence of progesterone and was suppressed by a PR antagonist. A simple interpretation of these data is that a GnRH-triggered signaling cascade can result in progesterone-independent transactivation of the PR. We propose that, in the normal operation of the preovulatory LH surge, the pathways for GnRH self-priming and progesterone augmentation converge at the PR and that the pathways serve as physiological redundancies to ensure the LH surge.
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PMID:Activation of the progesterone receptor by the gonadotropin-releasing hormone self-priming signaling pathway. 798 48

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.
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PMID:The progesterone antagonist RU486 acquires agonist activity upon stimulation of cAMP signaling pathways. 838 50

Estrogens regulate the growth, differentiation, and functioning of diverse target tissues, both within and outside of the reproductive system. Most of the actions of estrogens appear to be exerted via the estrogen receptor (ER) of target cells, an intracellular receptor that is a member of a large superfamily of proteins that function as ligand-activated transcription factors, regulating the synthesis of specific RNAs and proteins. To understand how the ER discriminates between estrogen ligands, which activate the ER, and antiestrogen ligands, which fail to effectively activate the ER, we have generated and analyzed human estrogen receptors with mutations in the ER hormone binding domain. These studies provide evidence for the promoter-specific and cell-specific actions of the estrogen-occupied and antiestrogen-occupied and antiestrogen-occupied ER, highlight a regional dissociation of the hormone binding and transcription activation functions in domain E of the receptor, and indicate that some of the contact sites of estrogens and antiestrogens in the ER are likely different. In addition, multiple interactions among different cellular signaling pathways are involved in the regulation of gene expression and cell proliferation by the ER. In several cell types, protein kinase activators and some growth factors enhance the transcriptional activity of the ER. Cyclic AMP also alters the agonist/antagonist balance of some antiestrogens. Estrogens and, to a lesser extent, antiestrogens, as well as protein kinase activators and growth factors increase phosphorylation of the ER and possibly other proteins involved in the ER-specific response pathway, suggesting that changes in cellular phosphorylation state will be important in determining the biological activity of the ER and the effectiveness of antiestrogens as estrogen antagonists. The ER also has important interrelationships with the progesterone receptor (PR) system in modulation of biological responses. Liganded PR-A and PR-B can each suppress estradiol-stimulated ER activity, with the magnitude of repression dependent on the PR isoform, progestin ligand, promoter, and cell type. These findings underscore the mounting evidence for the importance of interactions between members of the steroid hormone receptor family.
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PMID:Estrogen receptors: bioactivities and interactions with cell signaling pathways. 878 78

Data obtained in our laboratory about the membrane receptor for sex steroid-binding protein (SBP-R) in human breast cancer are reported. SBP-R was detected in MCF-7 cells (estrogen receptor positive, ER+), while MDA-MB 231 cells (ER-) did not bind SBP. MCF-7 cells treated with SBP and E2 showed a marked increase of intracellular cAMP, and a significant reduction of both E2 induced cell proliferation and E2-mediated increase of progesterone receptor (PGR). The inhibition of E2 effects in MCF-7 cells was shown to be highly specific for SBP and mediated by protein kinase A, the target of cAMP. Membrane SBP-R was also evaluated in primary breast cancers. SBP-R was detectable only on ER+/PR+ samples and SBP-R+ samples presented a lower proliferation rate than negative samples. Our data, thus suggest that SBP-R and ER could be functionally related and also that SBP could modulate estrogen action at target cell site.
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PMID:Sex steroid-binding protein and its membrane receptor in estrogen-dependent breast cancer: biological and pathophysiological impact. 896 84

Progestin antagonists inhibit the proliferation of progesterone receptor-positive cells, including breast cancer cells, by G1 phase-specific actions, but the molecular targets involved are not defined. Reduced phosphorylation of pRB, a substrate for G1 cyclin-dependent kinases (CDKs) in vivo, was apparent after 9 h treatment of T-47D breast cancer cells with the antiprogestins RU 486 or ORG 31710, accompanying changes in S phase fraction. Although the abundance of cyclin D1, Cdk4, and Cdk6 did not decrease cyclin D1-associated kinase activity was reduced by approximately 50% at 9-18 h. Similarly, cyclin E-associated kinase activity decreased by approximately 60% at 12-24 h in the absence of significant changes in the abundance of cyclin E and Cdk2. The CDK inhibitor p21 increased in mRNA and protein abundance and was present at increased levels in cyclin D1 and cyclin E complexes at times when their kinase activity was decreased. Increased p21 protein abundance was observed in another antiprogestin-sensitive cell line, BT 474, but not in two breast cancer cell lines insensitive to antiprogestins. These data suggest increased p21 abundance and concurrent inhibition of CDK activity as a mechanism for antiprogestin induction of growth arrest. Antiprogestin effects on proliferation were markedly reduced after ectopic expression of cyclin D1, indicating that inhibition of cyclin D1 function is a critical element in antiprogestin inhibition of proliferation. However, these data also implicate regulation of cyclin E function in antiprogestin regulation of cell cycle progression.
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PMID:Antiprogestin inhibition of cell cycle progression in T-47D breast cancer cells is accompanied by induction of the cyclin-dependent kinase inhibitor p21. 899 88

In species that ovulate spontaneously, two key events mediate the stimulation of preovulatory gonadotropin surges: 1) neurosecretion of a preovulatory LHRH surge and 2) an acute increase in responsiveness of the pituitary gland to the LHRH neurosecretory trigger. These processes, in turn, depend upon both the positive feedback actions of preovulatory estrogen secretions and specific neural signals for initiation of the surge. In female rats, the neural signals for the surge are principally derived from the 24-h neural clock, thereby limiting the timing of surges to the afternoon of proestrus. It remains unclear, however, how neural signals converge with endocrine signals (estrogen) in specific brain cells and how their cellular integration leads to appropriate secretion of gonadotropin surges. Previous work has suggested that estrogen may exert its facilitatory actions by opening a neural "gate," thereby allowing transmission of the daily neural signal to surge-initiating neuronal groups. How may estrogen act to render a neural pathway patent? A conventional view holds that steroid hormones can exert permissive effects on signaling efficacy by modulating neurotransmitter receptor expression, intracellular second messenger production, and protein kinase activity. However, recent evidence has suggested that estrogen may also have the capacity to permit cross-talk between neurotransmitter signaling pathways and parallel transcriptional regulatory pathways. The progesterone receptor is an estrogen-inducible transcription factor that has been shown to be transactivated--even in the absence of its cognate ligand--after stimulation of neurotransmitter receptors coupled to adenylate cyclase stimulation. Thus, the convergence of neural and endocrine signals for the stimulation of gonadotropin surges could occur at the level of the progesterone receptor: estrogen may stimulate expression of progesterone receptors, which in turn may be initially transactivated by synaptic signals. Activated progesterone receptors may thereafter regulate transcription of target genes that control transmitter synthesis and release in neural circuitries governing LHRH gene expression and/or pulsatile LHRH release. An analogous mechanism may operate in pituitary gonadotrophs, in which ligand-independent transactivation of progesterone receptors mediates integration of neurosecretory and estrogen positive feedback signals, leading to increased pituitary responsiveness to LHRH. It is proposed that the "seeding" of specific neuronal groups and pituitary gonadotrophs with progesterone receptors, and perhaps other inducible transcription factors, comprises an important basis of estrogen's permissive role in the stimulation of gonadotropin surges. The validity of this integrative model remains to be confirmed, as does its possible importance in generating gonadotropin surges in other species.
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PMID:New concepts of the neuroendocrine regulation of gonadotropin surges in rats. 911 24

Activators of protein kinase A have been shown to affect the transactivation potential of progestins and antiprogestins. To analyze the mechanisms and factors involved, we have created HeLa and CV1 cell clones stably expressing isoform B of progesterone receptor. In the HeLa cell background, the progesterone antagonist RU486 significantly induces progesterone-regulatable reporter genes, and this agonistic effect is synergistically enhanced by elevating cAMP or through overexpression of protein kinase A catalytic subunit. In contrast, in CV1 cells containing functional progesterone receptors no agonist activity of RU486 could be detected, suggesting the involvement of cell specifically expressed factors. In a PR(B)-positive HeLa cell clone containing stably integrated copies of a thymidine kinase-luciferase reporter gene with two progesterone response elements, we observed a complete loss of RU486 antagonist potential upon cotreatment with cAMP for 25 h while partial antagonist potential was maintained in a 5-h experiment. This result shows that, particularly in the presence of protein kinase A activators, the duration of hormone treatment is a crucial parameter in the evaluation of antagonist properties of antiprogestins. A detailed analysis of the kinetics of the hormone effects on transcription revealed that the onset of cAMP/RU486 synergism is delayed relative to the responses induced by RU486 or R5020 alone. Moreover, partial inhibition of protein synthesis by cycloheximide completely abolished cAMP/RU486 synergism while R5020 and RU486 responses were not inhibited. Together, these data indicate that cAMP/RU486 synergism is a delayed primary response requiring the intermediate induction of an essential factor.
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PMID:Synergistic enhancement of PRB-mediated RU486 and R5020 agonist activities through cyclic adenosine 3',5'-monophosphate represents a delayed primary response. 948 68

The molecular chaperone Hsp90 plays an essential role in the folding and function of important cellular proteins including steroid hormone receptors, protein kinases and proteins controlling the cell cycle and apoptosis. A 15 A deep pocket region in the N-terminal domain of Hsp90 serves as an ATP/ADP-binding site and has also been shown to bind geldanamycin, the only specific inhibitor of Hsp90 function described to date. We now show that radicicol, a macrocyclic antifungal structurally unrelated to geldanamycin, also specifically binds to Hsp90. Moreover, radicicol competes with geldanamycin for binding to the N-terminal domain of the chaperone, expressed either by in vitro translation or as a purified protein, suggesting that radicicol shares the geldanamycin binding site. Radicicol, as does geldanamycin, also inhibits the binding of the accessory protein p23 to Hsp90, and interferes with assembly of the mature progesterone receptor complex. Radicicol does not deplete cells of Hsp90, but rather increases synthesis as well as the steady-state level of this protein, similar to a stress response. Finally, radicicol depletes SKBR3 cells of p185erbB2, Raf-1 and mutant p53, similar to geldanamycin. Radicicol thus represents a structurally unique antibiotic, and the first non-benzoquinone ansamycin, capable of binding to Hsp90 and interfering with its function.
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PMID:Antibiotic radicicol binds to the N-terminal domain of Hsp90 and shares important biologic activities with geldanamycin. 967 45


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