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)

Transferrin-specific cDNA clones were isolated from a rat liver cDNA library prepared from transferrin-enriched mRNA. Hybrid selection and sequence analysis confirmed that the selected clone contained the carboxy-terminal coding region of the transferrin mRNA. Northern blot analysis was used to demonstrate the presence of transferrin mRNA in liver and Sertoli cells. Transferrin mRNA levels were measured in total RNA isolated from cultured rat Sertoli cells after treatment with FSH, insulin, retinol, and testosterone. The results showed a 2- to 4-fold increase in the level of transferrin mRNA, which peaked on the fourth day of culture after initiation of treatment, with FSH, insulin, retinol, and testosterone. This induction is gene specific, since no change in the mRNA levels for either the catalytic or regulatory subunits of cAMP-dependent protein kinase was observed. The effects of hormones, vitamin A (retinol), and Bu2 cAMP on transferrin mRNA and transferrin secretion (measured by RIA) in cultured Sertoli cells were compared. In general, a direct relationship between the amount of transferrin mRNA present in the cells and the amount of transferrin secreted into the culture medium was observed. These results demonstrate the important role that vitamin A, testosterone, and peptide hormones play in modulating transferrin gene expression in Sertoli cells.
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PMID:Transferrin messenger ribonucleic acid: molecular cloning and hormonal regulation in rat Sertoli cells. 302 31

Several tumor promoters exert their effects by activating a Ca2+-phospholipid-dependent protein kinase (protein kinase C). To study the role of this protein kinase in the regulation of Sertoli cell function, we have evaluated the effect of phorbol esters, mezerein, and teleocidin on the response of the Sertoli cell to FSH. Cells were treated for different time intervals with the tumor promoters, and cell response was measured by stimulating the cell with FSH. 12-O-Tetradecanoylphorbol 13-acetate (TPA) had no significant effect on basal cAMP production but markedly inhibited the cAMP response to FSH. Significant inhibition of cAMP accumulation was observed after 15 min treatment with 100 nM TPA, and maximal inhibition developed within 1 h. The decrease in cAMP accumulation was dependent on the dose of phorbol ester used, with an estimated ED50 of 10-20 nM TPA. In a manner similar to TPA, mezerein and teleocidin also inhibited the cAMP response of the Sertoli cell, while the phorbol ester 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), inactive as a tumor promoter and unable to stimulate protein kinase C activity, was devoid of effect. The promoters that inhibited cAMP response also inhibited the FSH-stimulated androgen aromatization. The dose of TPA producing half-maximal inhibition of estrogen accumulation was again 10-20 nM TPA, mezerein, and teleocidin inhibited estrogen accumulation whether FSH, forskolin or cholera toxin was used to stimulate the Sertoli cell. In contrast, only FSH-dependent cAMP accumulation was inhibited by the tumor promoters, while forskolin and cholera toxin stimulations were not affected. These data suggest that tumor promoters which activate protein kinase C act at two sites of the Sertoli cell response. They alter receptor-mediated signal transduction across the membrane and affect steroidogenesis at a site distal to cAMP accumulation.
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PMID:Inhibition by phorbol esters and other tumor promoters of the response of the Sertoli cell to FSH: evidence for dual site of action. 303 Aug 55

The response of cultured Sertoli cells to short term FSH stimulation was studied to elucidate early events involved in the hormone response of this cell type. The phosphorylation of proteins by [32P]orthophosphate-labeled cells was examined using two-dimensional polyacrylamide gel electrophoresis and autoradiography. FSH stimulation resulted in a variety of changes in the phosphoprotein labeling pattern. Within 5 min, unique phosphoproteins appeared in autoradiograms of treated cells. Increased labeling of vimentin was also noted. After 25 min of FSH stimulation, increases and decreases in apparent labeling intensity were evident in additional phosphoproteins that were constitutively labeled in control cultures. Changes in protein patterns in stained gels were also noted after acute hormone treatment. These observations demonstrate that Sertoli cells respond to hormonal stimulation in a detectable manner within 5 min. Cytoskeletal involvement in initial phases of hormone response is indicated. Mechanisms such as increased protein kinase activity, changes in protein kinase substrate affinity, increased protein turnover, or phosphatase activation may all contribute to the early events involved in Sertoli cell hormone response.
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PMID:Two-dimensional gel analysis of Sertoli cell protein phosphorylation: effect of short term exposure to follicle-stimulating hormone. 308 Mar 8

The activity of the calcium-, phospholipid-dependent protein kinase (PKc) was partially characterized in Sertoli cell cultures prepared from 20-day-old rats. The calcium dependency, the requirements for phosphatidylserine and diolein, as well as the Km for ATP and for the tumor promoter TPA, were determined in total cell extracts. The specific activity of PKc was almost 3-fold higher in the soluble than in the particulate fraction of Sertoli cells. Treatment of cultured Sertoli cells with retinol inhibited, within 1 h of treatment, both the soluble and the particulate fraction-associated PKc activity, with an IC50 of 0.1 microM. Partial inhibition of PKc activity was obtained treating Sertoli cell cultures with FSH, while testosterone was ineffective. However, both FSH and testosterone potentiated the inhibitory effect of retinol. Less differentiated Sertoli cells, obtained from 8-day-old rats, displayed higher PKc activity and a pattern of subcellular distribution of the enzyme opposite to that of Sertoli cells obtained from 20-day-old rats. These data suggest that the actual PKc activity of rat Sertoli cells be negatively regulated by retinol and, spontaneously, during the progression of Sertoli cell differentiation.
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PMID:Calcium-, phospholipid-dependent protein kinase activity of cultured rat Sertoli cells and its modifications by vitamin A. 310 Mar 59

A method for culturing cells from human pituitaries obtained at autopsy for studies of the secretion of gonadotropins and other hormones is described. The EC50 of GnRH-stimulated LH secretion from human pituitary cells was 1.25 nmol/L, similar to that found with rat pituitary cells (2.45 nmol/L). The GnRH agonist (D-Ala6, N alpha MeLeu7, Pro9-NEt)GnRH was 10-fold more active in stimulating LH release from both human and rat pituitary cells (EC50, 0.21 and 0.35 nmol/L, respectively). The GnRH antagonists (Ac-D-Nal(2)1,D-alpha-Me-4-ClPhe2,D-3-Pal3,D-Arg6,D-Ala10 )GnRH and (D-pGlu1,D-Phe2,D-Trp3,6)GnRH both inhibited 3 nmol/L GnRH-stimulated LH release from human pituitary cells (IC50, 2.40 and 78.6 nmol/L, respectively) with potencies similar to those observed in the rat (IC50, 0.68 and 33.0 nmol/L, respectively). Stimulation of the human pituitary cells with 100 nmol/L GnRH for 44 min resulted in biphasic release of LH and FSH. The initial phase occurred during the first 4 min of stimulation, and the second protracted plateau phase continued for at least the ensuing 40 min. The kinetics of LH and FSH release were identical, with no evidence of differential release at any stage. GnRH-stimulated LH and FSH secretion was Ca2+ dependent. The presence of 3 mmol/L EGTA prevented the gonadotropin response to GnRH, and the Ca2+ ionophore A23187 stimulated release of both LH and FSH. The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate also stimulated gonadotropin secretion, indicating that activation of protein kinase-C is able to elicit LH release. GnRH desensitized the cells to subsequent stimulation with GnRH. After initial 2-h incubation with 0.1-100 nmol/L GnRH, LH release during a second 2-h incubation with the same doses was reduced by 50-100%. The reduction in FSH ranged from 63-92%. Depletion of gonadotropin pools may contribute to desensitization, since total LH and FSH cell content decreased 48% and 49%, respectively, after 100 nmol/L GnRH stimulation for 4 h. We conclude that functionally active cells can be cultured from human pituitaries obtained postmortem and that the mechanism of GnRH action and the relative potencies of GnRH analogs closely parallel those in the rat.
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PMID:Culture of functionally active human pituitary cells: investigation of gonadotropin regulation. 313 82

FSH and GnRH both stimulate rat granulosa cells to produce tissue-type plasminogen activator (tPA). We have studied the molecular mechanisms involved in the action of these hormones by measuring tPA mRNA levels in primary cultures of rat granulosa cells. When granulosa cells were cultured in the presence of FSH or GnRH the level of tPA mRNA was increased 20- and 12-fold, respectively. The induction of tPA mRNA by FSH and GnRH was additive and the kinetics of induction differed. The effect of FSH could be mimicked by bromo-cAMP or forskolin, and was drastically enhanced by cotreatment with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine. These findings are consistent with the notion that FSH mediates its effect through the protein kinase A pathway. GnRH is believed to augment phospholipid turnover in granulosa cells, leading to the activation of the protein kinase C pathway. Like GnRH, the protein kinase C activator phorbol myristate acetate also induced tPA mRNA in granulosa cells. In the presence of the protein synthesis inhibitor, cycloheximide, FSH-stimulated tPA message levels were enhanced by 30-fold, revealing superinduction of tPA mRNA levels by this pathway. In contrast the induction of tPA mRNA by GnRH was inhibited by cycloheximide indicating that the synthesis of an intermediate protein is required for the GnRH effect. Our data suggest that FSH and GnRH increase the tPA mRNA levels by two distinct pathways in cultured granulosa cells, providing a model system for studying the hormonal regulation of tPA gene expression.
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PMID:Hormonal regulation of tissue-type plasminogen activator messenger ribonucleic acid levels in rat granulosa cells: mechanisms of induction by follicle-stimulating hormone and gonadotropin releasing hormone. 313 93

The heat-stable protein inhibitor of cAMP-dependent protein kinase is specifically regulated by hormones in cultures of rat Sertoli cells maintained under completely defined conditions. Hormones that are known to elevate Sertoli cell cAMP concentrations, namely FSH and isoproterenol, produce a 4- to 5-fold increase in the specific activity of protein kinase inhibitor, whereas testosterone and LH have no effect. The stimulatory effects of FSH or isoproterenol on protein kinase inhibitor are completely mimicked by dibutyryl cAMP. The ability of FSH to stimulate protein kinase inhibitor is dependent upon the age of the animals used as a source for the Sertoli cells. FSH stimulates protein kinase inhibitor activity in cells from 9- and 16-day-old animals, but not in cells from 32-day-old animals. On the other hand, isoproterenol or dibutyryl cAMP will stimulate protein kinase inhibitor in cells from both young and old animals. FSH can stimulate protein kinase inhibitor activity in older cells only in the added presence of the phosphodiesterase inhibitor, 1-methyl-3-isobutyl xanthine. Using specific antibodies to protein kinase inhibitor, we have shown that this protein is regulated by hormones via preferential stimulation of de novo synthesis of the inhibitor. (Endocrinology 108: 427, 1981)
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PMID:Regulation of protein kinase inhibitor by follicle-stimulating hormone in Sertoli cells in vitro. 625 52

The Sertoli cell is the primary target for FSH action in the mammalian testis. These cells contain the majority of testicular plasma membrane receptors for this hormone. Receptor occupancy is directly correlated with a stimulation of adenylyl cyclase and a decrease in the activity of a cytoplasmic Ca++-sensitive cAMP phosphodiesterase. Regulation of these two enzymes allows increased intracellular accumulation of cAMP, activation of cAMP-dependent protein kinase and phosphorylation of a variety of protein substrates. All of these events occur within the first 30 min following exposure of isolated Sertoli cells to FSH. RNA and protein synthesis are also enhanced by FSH. Previous studies have suggested that this gonadotropin may augment the overall cellular synthesis of proteins. Our results reveal that protein kinase inhibitor (PKI) is selectively elevated by FSH both in vivo and in vitro. PKI thus becomes the initial intracellular protein whose synthesis is under FSH control. In addition to effects on protein synthesis, FSH also positively modulates the secretion of several specific proteins. One of the proteins in this latter category is androgen binding protein (ABP). Again, regulation can be observed both in vivo and in vitro. Elevated synthesis of PKI occurs prior to demonstrable secretion of ABP. Both of these events occur subsequent to the effects of FSH on cAMP metabolism. Indeed cAMP (or any of several nonhydrolyzable derivatives) can substitute for FSH in vitro. The temporal sequence of events subsequent to hormone binding and cAMP production are identical, but occur more rapidly. Together these data support the hypothesis that most of the biochemical steps leading to the synthesis and secretion of proteins by FSH are regulated by elevated levels of cAMP.
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PMID:Factors affecting Sertoli cell function in the testis. 626 10

The responsiveness of granulosa cells to the gonadotropins and cAMP increases as ovarian follicles mature. To determine if this change in response might be related to either the content or cAMP-dependent phosphorylation of specific proteins, we labeled proteins in 30,000 X g supernatant fractions (cytosol) with [gamma-32P] ATP in the presence or absence of cAMP. Using two-dimensional gel electrophoresis, we observed that granulosa cells of preantral follicles exhibited low amounts of cAMP-dependent phosphorylation of two proteins with apparent molecular weights of 54,000-56,000 and 43,000. Using [32P]8-N3cAMP and photoaffinity labeling procedures, the Mr = 54,000-56,000 protein was identified as RII, the regulatory subunit of type II protein kinase. Polychromatic silver staining, as well as the photoaffinity labeling, revealed that RII exists in three forms, each of which was also labeled by [gamma-32P] ATP. Based on the relative isoelectric points and specific silver staining of highly purified actin and phosphorylated actin, the Mr = 43,000 protein has been provisionally identified as actin. Five proteins (Mr = 37,500, 27,500, 22,500, 19,000, and 15,000), in addition to RII and actin, were phosphorylated in cytosol of granulosa cells from preovulatory follicles. By adding increasing concentrations of exogenous catalytic subunit to the cytosols, we demonstrated that the content, as well as the phosphorylation of these proteins, was increased selectively in granulosa cells of antral follicles. By using hypophysectomized rats, we demonstrated that these five proteins are induced by follitropin (FSH). Because they were not present in cytosols of thecal cells or corpora lutea, they appear to be specific markers for granulosa cells. The content and phosphorylation of RII was also dramatically increased in cytosols of granulosa cells from antral follicles, whereas that of actin remained unchanged. These observations indicate that granulosa cell differentiation involves regulation by FSH of specific proteins which are substrates for cAMP-dependent protein kinase. Thus, FSH and cAMP appear to regulate the intracellular content and phosphorylation of a cAMP response system in granulosa cells. The extent to which RII and the five specific phosphoproteins themselves regulate granulosa cell responsiveness remains to be determined.
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PMID:Changes in content and cAMP-dependent phosphorylation of specific proteins in granulosa cells of preantral and preovulatory ovarian follicles and in corpora lutea. 630 Jan 22

The mechanisms by which estradiol enhances the actions of FSH (and cAMP), including the induction of LH receptors in rat ovarian granulosa cells, remain unclear. These studies were conducted to determine the extent to which changes in the activity, content, or intracellular distribution of the catalytic subunit of cAMP-dependent protein kinase might be altered in granulosa cells as a consequence of estradiol, FSH, and hCG administration in vivo. Dose-dependent stimulation of protein kinase activity (measured by histone phosphorylation in the presence of [gamma 32P]ATP and cAMP) demonstrated that the EC50 for cAMP was consistently 20 X 10(-8) M in cytosols prepared from granulosa cells of hypophysectomized rats before and after treatment with estradiol alone or estradiol and FSH. However, estradiol alone caused a 1.5 to 2.0-fold increase in the total amount of enzyme activity. When the cytosol content of the catalytic subunit (C) was quantitated directly, using immunoblotting procedures, the amount of C was 40 pmol/mg protein in all tissues, regardless of hormone treatments in vivo. When the content of RII, the regulatory subunit of type II cAMP-dependent protein kinase, was measured by similar immunoblotting procedures, a 10-fold increase was observed in granulosa cells exposed to both estradiol and FSH compared to that in cells exposed to estradiol alone. Greater than 80% of the intracellular content of both C and RII was present in the cytosol fraction (30,000 X g supernatant) rather than in the particulate nuclear fraction (30,000 X g pellet) of granulosa cells. This distribution of subunits was not altered by rapidly elevating intracellular concentrations of cAMP in vivo with 10 IU hCG, iv. We conclude that the catalytic subunit of protein kinase is a constitutive component of granulosa cells and that the sensitivity of the enzyme for cAMP is not affected by hormones or by a 10-fold increase in RII. Thus, the ability of estradiol to enhance FSH and cAMP action in granulosa cells appears to come primarily from the induction of specific substrates for the enzyme and a small increase in the catalytic activity but not from a change in the content of the catalytic subunit.
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PMID:Adenosine 3',5'-monophosphate-dependent protein kinase and granulosa cell responsiveness to gonadotropins. 632 38


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