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)

Catecholamines stimulate proopiomelanocortin (POMC) gene expression in corticotrope cells, but the molecular mechanisms of these effects are not known. While beta-adrenergic receptors stimulate the protein kinase A (PKA) system, the POMC promoter does not have classical cAMP-response elements (CREs). Therefore, we investigated the induction of the c-fos protooncogen, previously shown to increase POMC transcription in AtT20 cells. In this corticotrope-derived cell line, we show that activation of beta-receptors with isoprenaline (Iso) induces a transient rise in c-fos mRNA levels. Gel mobility shift assays with a labeled AP1 consensus sequence (TGACTCA) showed induction of specific binding activity after Iso treatment. Cotransfection experiments with dominant inhibitory PKA mutants and reporter genes containing c-fos promoter sequences showed that c-fos induction by Iso is entirely dependent on a functional PKA activity. Furthermore, we show that beta-receptor induction of c-fos in corticotrophs is mediated by at least two distinct cAMP-responsive sequences. cAMP regulatory element binding (CREB)-dependent induction is observed on the CRE located at -60 bp on the c-fos promoter. A region located in the vicinity of the dyad symetry element (-290) is also found to mediate tissue-specific cAMP induction. Transcriptional activation by this site, although sensitive to PKA antagonism, is not blocked by CREB mutants.
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PMID:Beta-adrenergic stimulation of cFOS via protein kinase A is mediated by cAMP regulatory element binding protein (CREB)-dependent and tissue-specific CREB-independent mechanisms in corticotrope cells. 133 Oct 87

Gonadotropins (follicle-stimulating hormone (FSH), luteinizing hormone, and human chorionic gonadotropin) and beta-adrenergic agonists have been shown to stimulate expression of the proopiomelanocortin (POMC) gene in ovarian granulosa cells. The current studies investigate the intracellular mechanisms by which gonadotropins regulate gene expression. Primary cultures of rat granulosa cells were transfected with the plasmid POMC-CAT-150, which expresses the chloramphenicol acetyltransferase (CAT) reporter gene under the regulation of the rat POMC 5'-flanking region. CAT activity was stimulated by treatment of the cells with either 20 ng/ml FSH or 1 microM isoproterenol. To assess the role of protein kinase A (ATP:protein phosphotransferase; EC 2.7.1.37) in the gonadotropin and adrenergic response, an expression vector, MtR-AB, encoding a mutant RI regulatory subunit was cotransfected with POMC-CAT-150. The mutant protein kinase A regulatory subunit encoded by MtR-AB lacks functional cAMP-binding sites but effectively binds and specifically inhibits the catalytic activity of protein kinase A. The results of this analysis demonstrated that gonadotropin and adrenergic agonist stimulation of the POMC-CAT reporter construct in primary cultures of rat granulosa cells were abolished by cotransfection with MtR-AB; whereas a control SV40-promoter construct was unaffected by either gonadotropin treatment or cotransfection with MtR-AB. Basal expression directed by the POMC promoter was also decreased by cotransfection with the MtR-AB, implying that basal expression from the POMC promoter may also depend on protein kinase A. Deletion analysis of the POMC sequence indicated regions (-40 to -33 and +4 to +63) important for basal and FSH-stimulated expression. These studies suggest that both gonadotropin and adrenergic stimulation of the POMC promoter are mediated by protein kinase A and that regions proximal to the promoter are essential for gonadotropin-regulated expression from the promoter.
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PMID:Specific inhibition of protein kinase A in granulosa cells abolishes gonadotropin regulation of the proopiomelanocortin promoter. 190 60

Forskolin, an activator of adenylate cyclase, stimulates adrenocorticotropin (ACTH) release and increases proopiomelanocortin mRNA levels in anterior pituitary cells by enhancing cyclic AMP (cAMP)-dependent protein kinase activity. The phorbol ester phorbol 12-myristate 13-acetate (PMA) evokes these same responses from anterior pituitary cells by activating protein kinase C. Both protein kinases most likely induce their cellular effects by catalyzing the phosphorylation of specific proteins. To elucidate the mechanisms by which cAMP-dependent protein kinase and protein kinase C promote ACTH secretion and synthesis, the phosphoproteins regulated by forskolin and PMA were identified in the cell line AtT-20, which consists of a homogeneous population of corticotrophs. Phosphoproteins were analyzed in different subcellular fractions by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Forskolin increased phosphate incorporation into two proteins in the cytoplasmic fraction of 24 kilodaltons (kd) (pI 6.8) and 40 kd (pI 5.8), two proteins in the plasma membrane fraction of 32 kd (pI 8.3) and 60 kd (pI 8), and one protein in the nuclear fraction of 20 kd (pI 8.7). Insertion of the inhibitor of cAMP-dependent protein kinase into the AtT-20 cells, using a liposome technique, blocked the rise in phosphate incorporation induced by forskolin. PMA also stimulated phosphate incorporation into proteins in AtT-20 cells. PMA increased the phosphorylation of three cytoplasmic proteins of 25 kd (pI 7.6), 40 kd (pI 5.8), and 40 kd (pI 8.1) as well as two membrane proteins of 32 kd (pI 8.3) and 60 kd (pI 8) and one nuclear protein of 20 kd (pI 6.3).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protein phosphorylation induced by phorbol esters and cyclic AMP in anterior pituitary cells: possible role in adrenocorticotropin release and synthesis. 253 66

Corticotropin-releasing factor (CRF) is the most potent and effective natural stimulant of corticotropin (ACTH) secretion. In a tumor cell line of the mouse anterior pituitary (AtT-20/D16-16) consisting of a homogeneous population of corticotrophs, CRF is known to increase adenylate cyclase and cAMP-dependent protein kinase activities as well as to release ACTH. To determine whether activation of cAMP-dependent protein kinase is essential for CRF to evoke the secretion of ACTH, an inhibitor (PKI) of this kinase was inserted into AtT-20 cells. This was accomplished by first encapsulating PKI into liposomes and then covalently coupling them to protein A for binding to antibodies directed against an AtT-20 cell surface antigen, N-CAM (neural cell adhesion molecule). The binding of the liposomes to the anti-N-CAM antibodies led to the internalization of the PKI into the tumor cells. The PKI treatment greatly attenuated CRF-stimulated ACTH release as well as the secretory response to beta-adrenergic agonists. However, ACTH release in response to caerulein, an agonist of cholecystokinin 8 receptors, was not altered by the PKI treatment. CRF treatment also increased the levels of mRNA for proopiomelanocortin (POMC), the precursor for ACTH in AtT-20 cells. Application of liposomes containing PKI to AtT-20 cells blocked the ability of CRF and 8-bromo-cAMP, but not phorbol ester, to increase POMC mRNA levels. The results revealed an essential role for cAMP in mediating the effect of CRF on ACTH release and POMC gene expression.
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PMID:Corticotropin-releasing factor-induced adrenocorticotropin hormone release and synthesis is blocked by incorporation of the inhibitor of cyclic AMP-dependent protein kinase into anterior pituitary tumor cells by liposomes. 299 99

The biological effects of pituitary adenylate cyclase-activating peptide (PACAP) 27 and 38 on peptide secretion and gene regulation were studied in the mouse corticotrope-derived cell line AtT20. Treatment of these cells with PACAP 27/38 led to a dose-dependent increase in cAMP content and ACTH accumulation in the medium with an apparent ED50 value close to 10(-9) M. The genomic effects of PACAP were first investigated by using a reporter gene containing a cAMP responsive element (CRE: TGACGTCA) PACAP 27/38 stimulate transcription from this construction and the effect is further increased when cells are cotreated with the phosphodiesterase inhibitor rolipram. Furthermore, we show by measuring nuclear heterologous proopiomelanocortin (POMC) RNA levels or by using a reporter gene containing the POMC promoter region, that PACAP stimulates POMC transcription. This transcriptional stimulation is mediated by the cAMP-dependent protein kinase (PKA) since genetic inactivation of PKA by a dominant inhibitory mutant of this enzyme completely abolished the effect of PACAP on POMC transcription. Finally, we show that the transcriptional stimulation of POMC by PACAP is repressed by the glucocorticoid receptor agonist dexamethasone. Taken together, these data suggest that PACAP is a hypophysiotropic hormone that exert similar if not identical functions as corticotropin-releasing hormone (CRH) on corticotrope cells.
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PMID:Pituitary adenyl cyclase-activating peptide: a hypophysiotropic factor that stimulates proopiomelanocortin gene transcription, and proopiomelanocortin-derived peptide secretion in corticotropic cells. 784 39

A pheochromocytoma from a 59-year-old woman was found to be immunoreactive to adrenocorticotropin (ACTH), chromogranin, neurofilament-200, neuron-specific enolase and S-100 protein. Northern blot analysis showed that both proopiomelanocortin (POMC) and corticotropin-releasing hormone (CRH) genes were expressed in the pheochromocytoma but not in the surrounding adrenal cortex. In primary culture, the POMC and CRH mRNAs were increased by dexamethasone (500 micrograms/l for 3 days) up to 10- and 15-fold of the control, respectively. The secretion of ACTH also was stimulated eightfold with the same treatment. The stimulatory effect of dexamethasone on POMC gene expression was inhibited 70% by nerve growth factor (NGF, 200 micrograms/l), 30% by 12-O-tetradecanoyl phorbol 13-acetate (TPA, 160 nmol/l) (a protein kinase-C activator) and 30% by (Bu)2cAMP (1 mmol/l). On the other hand, NGF alone increased the CRH mRNA accumulation up to 10-fold, and further enhanced the stimulatory effect of dexamethasone on the CRH mRNA twofold, and TPA inhibited (30%) the dexamethasone-induced CRH mRNA accumulation. Furthermore, the conditioned medium of the pheochromocytoma cells increased secretion of corticosterone fourfold in the primary culture of rat fetal adrenal cells. Our results indicate abnormal expression and regulation of POMC and CRH genes in this pheochromocytoma.
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PMID:Pheochromocytoma expressing adrenocorticotropin and corticotropin-releasing hormone; regulation by glucocorticoids and nerve growth factor. 792 Dec 4

The most potent, physiologic activator of proopiomelanocortin (POMC) gene transcription is corticotropin releasing hormone (CRH) and increased intracellular cAMP is critical for this effect. The 5'-flanking region of the murine POMC gene has several potential binding sites for regulatory proteins. To characterize the region between nucleotides -141 and -106, which includes a TRE-like site and an adjacent AP-2 consensus sequence, and to study its role in signal-transcription coupling, gel mobility shift assays and transient expression of CAT chimeras were performed. In transient transfections of AtT-20 cells with pCATp-141/-106, CRH treatment led to significant increases in CAT expression compared with CRH treatment of cells transfected with the enhancerless vector. However, no response to direct activation of cAMP dependent protein kinase or protein kinase C was detected. Despite the high homology of the sequence -137/-131 to the consensus AP-1 binding site (TRE), the nuclear factor(s) in AtT-20 cells binding to this region appears to be different than authentic AP-1 since neither a competitor oligonucleotide having the authentic TRE sequence nor antibodies against Jun or Fos affected the gel shift pattern of a probe having the -137/-131 sequence. We conclude that the -141 to -106 region of the murine POMC gene contains a functional CRH responsive element and that second messenger systems that transduce the CRH signal to this element do not exert their actions solely through activation of PKA or PKC.
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PMID:Characterization of a corticotropin releasing hormone responsive region in the murine proopiomelanocortin gene. 814

The effect of the antidiarrheal drug loperamide, a mu-opiate agonist, on ACTH secretion and biosynthesis, cAMP generation and phosphoinositide turnover was studied in rat anterior pituitary cell cultures. The cAMP-dependent protein kinase A pathway was stimulated with both corticotropin-releasing hormone (CRH; 2-5 nM) and the membrane-permeable Bu(2)cAMP (0.5-2.5 mM). The protein kinase C pathway was stimulated with 1 microM arginine vasopressin (AVP) and 1-10 nM phorbol 12-myristate 13-acetate (PMA). After 3.5 h, loperamide (10 microM) had no effect on basal ACTH levels but significantly suppressed CRH-induced ACTH release, in a dose-dependent manner, to 60 +/- 4% of control (100%) (p < 0.0001). After 24 h, basal proopiomelanocortin mRNA was significantly decreased to 50% of control by loperamide (p < 0.05). The suppressive effect of loperamide on CRH-induced ACTH secretion was not reversible by naloxone (0.1-1,000 microM). Morphine (0.01-10 microM) had no effect on basal and CRH-induced ACTH secretion. Loperamide did not influence basal and CRH-induced adenylate cyclase activity in anterior pituitary cell membrane preparations, but it significantly blunted Bu(2)cAMP-induced ACTH secretion in cell culture from 100 +/- 4 to 77 +/- 4% (p < 0.05). In Ca(2+)-depleted medium (Ca2+ < 0.1 mM), loperamide had no suppressive effect on CRH-induced ACTH secretion. AVP-induced ACTH secretion was significantly suppressed by loperamide from 100 +/- 5 to 74 +/- 3% (p < 0.0001), while basal and AVP-induced inositol 1-phosphate generation and PMA-induced ACTH secretion were not affected by loperamide.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Loperamide inhibits corticotrophic cell function by a naloxone-insensitive mechanism in the rat in vitro. 823 60

Besides acting as an important cofactor in the biosynthesis of catecholamine, ascorbic acid (AA) also modulates the activity of peptidylglycine-alpha-amidating monooxygenase for the post-translational modification of neuropeptides such as alpha-MSH and TRH. We report here a novel action of AA in modulating the secretion of immunoreactive beta-endorphin (ir-beta EP) and mRNA expression of proopiomelanocortin (POMC) following the activation of cAMP-dependent protein kinase A pathway in rat hypothalamic neurons. Primary cultures of hypothalamic neurons from neonatal rats as previously described were employed in the present studies. Six days after plating, cultures were replenished with serum-free media and incubated with vehicle or various doses of AA in the presence or absence of forskolin, 3-isobutyl-1-methylxanthine (IBMX), N6,2'-O-dibutyryladenosine 3'5'-(cyclic)monophosphate [(Bu)2cAMP]. Whereas the basal ir-beta EP release was 22.0 +/- 0.4 pg/well (mean +/- S.E.; n = 3), 10 microM of forskolin treatment increased ir-beta EP release approximately 4.2-fold. Co-incubation with AA enhanced forskolin induced ir-beta EP release and that this enhancing effect of AA was both time related and dose-dependent, with an ED50 of approximately 10 microM and an Emax of 100 microM. At the concentration of 10 microM, AA augmented ir-beta EP release approximately 6.1-fold that of cultures treated with forskolin alone. A similar potentiating effect of AA was also seen in cultures co-treated with IBMX or with (Bu)2cAMP. These enhancing effects of AA were similarly found in the abundance of total cAMP and of POMC mRNA of cultures which received identical treatments. However, it is important to point out that AA alone did not modulate ir-beta EP release or the abundance of POMC mRNA or total cAMP levels of the hypothalamic cultures when protein kinase A pathway was not activated. We thus conclude that AA augments cAMP-dependent protein kinase A pathway-induced production and release of beta EP from rat hypothalamic neurons in culture. Furthermore, this biological effect of AA is, at least in part, mediated through enhancing the responsiveness of the adenylyl cyclase-cAMP system.
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PMID:Ascorbic acid augments the adenylyl cyclase-cAMP system mediated POMC mRNA expression and beta-endorphin secretion from hypothalamic neurons in culture. 882 63

We have previously shown that 10-12 kDa N-terminal fragments of rat proopiomelanocortin (POMC) and human POMC1-76 stimulate mitosis and/or differentiation in lactotrophs of early postnatal rat pituitary. A truncated form, POMC1-26, mimics the differentiation-inducing but not the mitogenic action of the former peptides. To further characterize these two biological responses, the present study compared changes in the intracellular free calcium concentration ([Ca2+]i) in response to POMC1-76 and POMC1-26 in isolated pituitary cells from 14-day-old female rats. Calcium (Ca2+) responses were also used as a guide to determine whether the responsive cells belong to the lactosomatotroph lineage. Application of POMC1-76 or POMC1-26 induced a maintained oscillating [Ca2+]i increase in a small population of cells. Increasing doses of the peptides did not affect the magnitude and the frequency of [Ca2+]i oscillations but clearly augmented the number of responding cells. Approximately 2% of the cells responded at 0.1 nM POMC1-76 or 5 nM POMC1-26, and 11-13% of the cells responded at 10 nM and 500 nM of the respective peptides. About one-third of the cells responsive to these peptides also showed a [Ca2+]i increase in response to growth hormone-releasing peptide-6 (GHRP-6) while, in a small number of responsive cells, [Ca2+]i was depressed by dopamine, suggesting that the former cells are somatotrophs and the latter lactotrophs. This interpretation was confirmed by immunocytochemical identification of prolactin and growth hormone (GH) in the cells. Of the cells showing Ca2+ response to POMC1-76, approximately one-third contained GH and another third prolactin. The remainder contained neither GH nor prolactin. Comparable results were obtained with POMC1-26. The rise of [Ca2+]i induced by the N-terminal POMC peptides persisted after depletion of intracellular Ca2+ stores by thapsigargin. Removal of Ca2+ from the extracellular medium or addition of cadmium completely abolished both the POMC1-76- and POMC1-26-induced [Ca2+]i increase. Nifedipine inhibited the Ca2+ response to both peptides, although only in 55% of the responsive cells. Depletion of some isoforms of protein kinase C by preincubation with the phorbol ester PMA for 24 h did not modify the Ca2+ responses. In contrast, blockade of the protein kinase A pathway with Rp-cAMPs partially inhibited the POMC1-76- or POMC1-26-induced [Ca2+]i increase. The present data show that, in immature pituitary cells, POMC1-76 induces an increase in [Ca2+]i through extracellular Ca2+ influx, possibly mediated in part by protein kinase A activation. The active domain of POMC1-76 seems to comprise its N-terminal moiety. The data support the hypothesis that POMC1-76 exerts a specific function in the development of different members of the lactosomatotroph lineage and that the peptide mobilizes different subsets of cells within this lineage, by a mechanism determined by its concentration.
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PMID:Stimulation of Ca2+ entry in lactotrophs and somatotrophs from immature rat pituitary by N-terminal fragments of proopiomelanocortin. 957 10


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