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)

The participation of the actin cytoskeleton in the control of PRL secretion by dopamine (DA) is not yet fully understood. Recently, we demonstrated that DA induces cortical actin assembly and stabilization in anterior pituitary PRL-secreting cells (lactotropes) that can be linked to DA-induced inhibition of PRL secretion. Here we show that DA prevents cell flattening and the formation of cytoplasmic actin cables in cultured rat lactotropes. The effects of DA were reversible, mediated by D2 receptors, exclusive to lactotropes, and independent of other anterior pituitary cells present in the cultures. Because cAMP and Ca2+ mediate DA-induced inhibition of PRL secretion and synthesis, we investigated whether morphological responses to DA were dependent on these second messengers. Either inhibition of protein kinase A activity with the specific inhibitor KT5720 or blockade of Ca2+ channels with nifedipine inhibited cell flattening and induced cytoplasmic actin filament breakdown. Nifedipine was as effective as DA, but KT5720 was less effective than DA. Increased intracellular cAMP levels provoked cell flattening, which was blocked by nifedipine and KT5720, but not by DA. The results suggest that Ca2+-dependent pathways control cell shape in most lactotropes; however, in a subpopulation of lactotropes, cAMP-dependent pathways may also contribute to DA morphological responses. Next, we studied the participation of the Rho family of guanosine triphosphatases, which is known to regulate the dynamics of actin filaments. Inactivation of Rho by C3 exoenzyme induced cytoplasmic actin cable disassembly and lactotrope rounding up. No additive effects were observed among Rho-, cAMP-, and Ca2+-dependent pathways. However, C3-induced morphological responses were blocked by increased cAMP levels, suggesting that Rho-dependent steps are upstream cAMP-dependent steps. DA-induced actin cytoskeleton reorganization in lactotropes may involve modifications in the expression and localization of actin-binding proteins. DA increased expression of the actin anchoring proteins talin and alpha-actinin, but not of vinculin. DA enhanced association of talin to cell membranes. Increased talin-membrane interaction may be implicated in DA-induced maintenance of a round phenotype in lactotrope cells.
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PMID:Intracellular mechanisms involved in dopamine-induced actin cytoskeleton organization and maintenance of a round phenotype in cultured rat lactotrope cells. 1043 2

Human endometrial stromal (ES) cells in culture express PRL, a marker of decidualization, in response to sustained activation of protein kinase A (PKA). Cotreatment with the progestin medroxyprogesterone acetate (MPA) enhanced decidual PRL gene activation in the presence of elevated intracellular cAMP levels. This synergy became apparent, at protein and promoter level, after a lag period of 2 days and increased in a time-dependent manner thereafter. Pretreatment with cAMP advanced the time at which synergy between cAMP and MPA was apparent, suggesting that PKA activation sensitized ES cells to the effects of progestins. Analysis of the progesterone receptor (PR) indicated that PR-A was the predominant form in differentiating ES cells, but its abundance decreased markedly during the course of the decidualization response. The decline in PR levels was of functional relevance, as expression of PR-B or PR-A, by transient transfection, dramatically inhibited the activity of a decidual PRL promoter-reporter construct in response to cAMP. Furthermore, the expression of endogenous PRL protein in response to cAMP or cAMP plus MPA was substantially decreased by constitutive expression of green fluorescence protein-tagged PR, which was localized in the nucleus even in the absence of added ligand. Ligand-independent PR inhibition of the decidual PRL promoter was receptor specific, independent of known PR phosphorylation sites, and required minimally a functional DNA-binding domain. Transient expression of steroid receptor coactivator-1e (SRC-1e), but not SRC-1a, allowed synergy between cAMP and MPA without the requirement of sensitization by pretreatment with cAMP. This raised the possibility that SRC-1e was a component of cAMP-dependent sensitization of ES cells, but there was no evidence of altered messenger RNA expression of either SRC-1 isoform during decidualization. In conclusion, cellular PR levels determine the onset of the decidualization response. Initiation of this process requires elevated intracellular cAMP levels that sensitize ES cells to the actions of progestins through down-regulation of cellular PR levels and possibly via modulation of function of an intermediate factor(s) such as SRC-1e.
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PMID:Progesterone receptor regulates decidual prolactin expression in differentiating human endometrial stromal cells. 1049 41

Our previous studies have identified a role for annexin 1 (also called lipocortin 1) in the regulatory actions of glucocorticoids (GCs) on the release of PRL from the rat anterior pituitary gland. In the present study we used antisense and immunoneutralization strategies to extend this work. Exposure of rat anterior pituitary tissue to corticosterone (1 nM) or dexamethasone (100 nM) in vitro induced 1) de novo annexin 1 synthesis and 2) translocation of the protein from intracellular to pericellular sites. Both responses were prevented by the inclusion in the medium of an annexin 1 antisense oligodeoxynucleotide (ODN; 50 nM), but not by the corresponding sense and scrambled ODN sequences. Unlike the GCs, 17beta-estradiol, testosterone, and aldosterone (1 nM) had no effect on either the synthesis or the cellular disposition of annexin 1; moreover, none of the steroids or ODNs tested influenced the expression of annexin 5, a protein closely related to annexin 1. The increases in PRL release induced in vitro by drugs that signal via cAMP/protein kinase A [vasoactive intestinal polypeptide (10 nM), forskolin (100 microM), 8-bromo-cAMP (0.1 microM)] or phospholipase C (TRH, 10 nM) were attenuated by preincubation of the pituitary tissue with either corticosterone (1 nM) or dexamethasone (100 nM). The inhibitory actions of the steroids on the secretory responses to vasoactive intestinal polypeptide, forskolin, and 8-bromo-cAMP were specifically quenched by inclusion in the medium of the annexin 1 antisense ODN (50 nM) or a neutralizing antiannexin 1 monoclonal antibody (antiannexin 1 mAb, diluted 1:15,000). By contrast, the ability of the GCs to suppress the TRH-induced increase in PRL release was unaffected by both the annexin 1 antisense ODN and the antiannexin 1 mAb. In vivo, interleukin-1beta (10 ng, intracerebroventricularly) produced a significant increase in the serum PRL concentration (P < 0.01), which was prevented by pretreatment of the rats with corticosterone (100 microg/100 g BW, sc). The inhibitory actions of the steroid were specifically abrogated by peripheral administration of an antiannexin 1 antiserum (200 microl, sc); by contrast, when the antiserum was given centrally (3 microl, intracerebroventricularly), it was without effect. These results support our premise that annexin contributes to the regulatory actions of GCs on PRL secretion and suggest that it acts at point distal to the formation of cAMP.
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PMID:Annexin 1 (lipocortin 1) mediates the glucocorticoid inhibition of cyclic adenosine 3',5'-monophosphate-stimulated prolactin secretion. 1083 Mar 10

GH and PRL stimulate proliferation and insulin production of pancreatic beta-cells. Whereas GH- and PRL-regulated transcription of the insulin gene in insulinoma cells has been shown to depend on STAT5 (signal transducer and activator of transcription 5), the signaling pathways involved in GH/PRL-induced beta-cell replication are unknown. The roles of various signaling pathways in human GH (hGH)-induced DNA synthesis were studied by analysis of the effect of specific inhibitors in both the insulin-producing cell line, INS-1, and in primary beta-cells. The mitogen-activated protein kinase kinase (MEK)-inhibitor, PD98059, as well as the mitogen-activated protein kinase p38 (MAPKp38) inhibitor, SB203580, partially inhibited hGH- induced proliferation in INS-1 cells but had no significant effect in primary beta-cells. Staurosporine, a protein kinase C (PKC) and protein kinase A (PKA) inhibitor, blocked both basal and hGH-induced proliferation in INS-1 cells, but had no inhibitory effect in primary beta-cells. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited hGH-induced proliferation neither in INS-1 cells nor in primary beta-cells, whereas the tyrosine kinase inhibitor, genistein, completely inhibited hGH- induced proliferation in both primary beta-cells and INS-1 cells. To analyze the possible role of STAT5 in hGH-induced proliferation, a dominant negative STAT5 mutant, STAT5Delta749, was expressed in INS-1 cells under the control of a doxycycline- inducible promoter by stable transfection. Two clones were found to exhibit dose-dependent, doxycycline-inducible expression of STAT5Delta749 and suppression of hGH-stimulated transcriptional activation of a STAT5-regulated PRL receptor (PRLR) promoter-reporter construct. Furthermore, induction of STAT5Delta749 expression completely inhibited hGH-induced DNA synthesis. Analysis of endogenous gene expression revealed a doxycycline-dependent inhibition of hGH-stimulated PRLR and cyclin D2 mRNA levels. Our results suggest that GH/PRL-induced beta-cell proliferation is dependent on the Janus Kinase2 (JAK2)/STAT5 signaling pathway but not the MAPK, PI3K, and PKC signaling pathways. Furthermore, the cell cycle regulator cyclin D2 may be a crucial target gene for STAT5 in this process.
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PMID:Growth hormone- and prolactin-induced proliferation of insulinoma cells, INS-1, depends on activation of STAT5 (signal transducer and activator of transcription 5). 1114 45

Although decidualization of endometrial stromal cells (ESC) is crucial for blastocyst implantation and maintenance of pregnancy, its complex mechanism still remains largely unknown. It has long been believed that hCG can directly induce in vitro decidualization of ESC via cAMP signaling. Recently, however, it has been reported that the LH/CG receptor is not present in human endometrium, and the direct effect of hCG on decidualization has become controversial. To reevaluate the exact effect of hCG on decidualization, human ESC were isolated and cultured with hCG and/or ovarian steroids. ESC treated with 17beta-estradiol plus progesterone (E(2)/P) transformed morphologically and produced significant PRL, whereas ESC treated with hCG alone showed no significant increase in PRL in culture medium and exhibited no morphological changes. Moreover, hCG did not promote E(2)/P-induced PRL production or intracellular cAMP accumulation, and protein kinase A inhibitor failed to block E(2)/P-induced PRL production. These results suggest that hCG does not directly affect in vitro decidualization of human ESC and that the process of E(2)/P-induced in vitro decidualization might consist of several pathways, including the intracellular cAMP signaling cascade.
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PMID:The role of human chorionic gonadotropin on decidualization of endometrial stromal cells in vitro. 1150 45

The epidermal growth factor receptor (EGFR) and its ligands EGF and transforming growth factor-alpha (TGF alpha) are expressed in the anterior pituitary, and overexpression of TGF alpha in the lactotrope cells of the pituitary gland in transgenic mice results in lactotrope hyperplasia and adenomata, suggesting a role for EGFR signaling in pituitary cell proliferation. To address the role of EGFR signaling in pituitary development in vivo, we blocked EGFR signaling in transgenic mice using the dominant negative properties of a mutant EGFR lacking an intracellular protein kinase domain (EGFR-tr). We directed EGFR-tr expression to GH- and PRL- producing cells using GH and PRL promoters, and a tetracycline-inducible gene expression system, to allow temporal control of gene expression. EGFR-tr overexpression in GH-producing cells during embryogenesis resulted in dwarf mice with pituitary hypoplasia. Both somatotrope and lactotrope development were blocked. However, when EGFR-tr overexpression was delayed to the postnatal period either by directing its expression with the PRL promoter or by delaying the onset of induction with tetracycline in the GH cells, no specific phenotype was observed. Lactotrope hyperplasia during pregnancy also occurred normally in the PRL-EGFR-tr mice. These data suggest that EGFR signaling is required for the differentiation and/or maintenance of somatomammotropes early in pituitary organogenesis but not later in life. (Molecular Endocrinology 15: 600-613, 2001)
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PMID:Stage-sensitive blockade of pituitary somatomammotrope development by targeted expression of a dominant negative epidermal growth factor receptor in transgenic mice. 1126 11

We examined whether mitogen-activated protein (MAP) kinase was activated by stimulation of the cAMP pathway and whether MAP kinase activation was involved in synthesis of PRL and GH in GH(3) cells. Treatment of the cells with a cAMP analog, 8-(4-chlorophenylthio)cAMP (CPT-cAMP), activated MAP kinase and increased PRL at both the protein and messenger RNA levels. The protein and messenger RNA of GH were decreased by the treatment. We constructed the luciferase reporter genes after the promoters of PRL and GH and found the activation of both promoters by the CPT-cAMP treatment. We confirmed that overexpression of the catalytic subunit of cAMP-dependent protein kinase had essentially the same effects on MAP kinase activation and synthesis of PRL and GH as the CPT-cAMP treatment. Furthermore, treatment of the cells with pituitary adenylate cyclase-activating polypeptide 27 activated MAP kinase. The activation of PRL promoter by CPT-cAMP and pituitary adenylate cyclase-activating polypeptide 27 was abolished by pretreatment with PD098059 and H89. Although the increase in PRL and GH secretion by CPT-cAMP was inhibited by H89, PD098059 had no effect on secretion. These results suggest that cAMP-induced MAP kinase activation is essential for PRL gene expression, but not for secretion of PRL and GH.
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PMID:Involvement of mitogen-activated protein kinase in cyclic adenosine 3',5'-monophosphate-induced hormone gene expression in rat pituitary GH(3) cells. 1141

Fibroblast growth factors play a critical role in cell growth, development, and differentiation and are also implicated in the formation and progression of tumors in a variety of tissues including pituitary. We have previously shown that fibroblast growth factor activation of the rat PRL promoter in GH4T2 pituitary tumor cells is mediated via MAP kinase in a Ras/Raf-1-independent manner. Herein we show using biochemical, molecular, and pharmacological approaches that PKCdelta is a critical component of the fibroblast growth factor signaling pathway. PKC inhibitors, or down-regulation of PKC, rendered the rat PRL promoter refractory to subsequent stimulation by fibroblast growth factors, implying a role for PKC in fibroblast growth factor signal transduction. FGFs caused specific translocation of PKCdelta from cytosolic to membrane fractions, consistent with enzyme activation. In contrast, other PKCs expressed in GH4T2 cells (alpha, betaI, betaII, and epsilon) did not translocate in response to fibroblast growth factors. The PKCdelta subtype-selective inhibitor, rottlerin, or expression of a dominant negative PKCdelta adenoviral construct also blocked fibroblast growth factor induction of rat PRL promoter activity, confirming a role for the novel PKCdelta isoform. PKC inhibitors selective for the conventional alpha and beta isoforms or dominant negative PKCalpha adenoviral expression constructs had no effect. Induction of the endogenous PRL gene was also blocked by adenoviral dominant negative PKCdelta expression but not by an analogous dominant negative PKCalpha construct. Finally, rottlerin significantly attenuated FGF-induced MAP kinase phosphorylation. Together, these results indicate that MAP kinase-dependent fibroblast growth factor stimulation of the rat PRL promoter in pituitary cells is mediated by PKCdelta.
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PMID:Fibroblast growth factor activation of the rat PRL promoter is mediated by PKCdelta. 1151

Signal transducer and activator of transcription 5b (STAT5b), the major liver-expressed STAT5 form, is phosphorylated on both tyrosine and serine in GH-stimulated cells. Although tyrosine phosphorylation is known to be critical for the dimerization, nuclear translocation, and activation of STAT5b DNA-binding and transcriptional activities, the effect of STAT5b serine phosphorylation is uncertain. Presently, we identify Ser730 as the site of STAT5b serine phosphorylation in GH-stimulated liver cells. We additionally show that the serine kinase inhibitor H7 partially blocks the GH-stimulated formation of (Ser,Tyr)-diphosphorylated STAT5b without inhibiting STAT5b nuclear translocation. Evaluation of the functional consequences of STAT5b serine phosphorylation by mutational analysis revealed an approximately 50% decrease in GH-stimulated luciferase reporter gene activity regulated by an isolated STAT5-binding site when STAT5b Ser730 was mutated to alanine and under conditions where STAT5 DNA-binding activity was not diminished. No decrease in GH-stimulated reporter activity was seen with the corresponding STAT5a-Ser725Ala mutant; however, a decrease in reporter activity occurred when the second established STAT5a serine phosphorylation site, serine 779, was additionally mutated to alanine. Unexpectedly, STAT5a-Ser725,779Ala and STAT5b-Ser730Ala displayed approximately 2-fold higher GH- or PRL-stimulated transcriptional activity compared with wild-type STAT5b when assayed using an intact beta-casein promoter-luciferase reporter. Finally, STAT5b-stimulated gene transcription was abolished in cells treated with H7, but in a manner unrelated to the inhibitory effects of H7 on STAT5b Ser730 phosphorylation. These findings suggest that the effects of STAT5b and STAT5a serine phosphorylation on STAT-stimulated gene transcription can be modulated by promoter context. Moreover, in the case of STAT5a, phosphorylation of serine 779, but not serine 725, may serve to regulate target gene transcriptional activity.
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PMID:Serine phosphorylation of GH-activated signal transducer and activator of transcription 5a (STAT5a) and STAT5b: impact on STAT5 transcriptional activity. 1173 17

Trophoblast differentiation is a key event in human placental development. During extravillous trophoblast (EVT) differentiation, stem cells from the anchoring villi detach from their basement membrane and proliferate to form aggregates called trophoblast cell columns (TCCs). They subsequently invade the decidua and differentiate into interstitial and endovascular trophoblasts. The influence of the decidua on EVT differentiation is controversial. We therefore compared the pattern of trophoblast differentiation marker expression in viable intrauterine and tubal pregnancies, as decidual cell markers (prolactin [PRL] and insulin-like growth factor binding Protein-1 [IGFBP1]) were only expressed in endometrial implantation sites. Extravillous trophoblast differentiation in anchoring villi from uterine and ectopic pregnancies exhibited a comparable phenotypical switch: alpha6 integrin subunit, E-cadherin, EGF receptor, Ki 67 and connexin 40 were localized in the proximal part of the TCC, while alpha5beta1 and alpha1 integrins, c-erb B2, hPL and HLA-G were expressed by invasive cytotrophoblasts. The cyclin-dependent kinase inhibitors p16 and p57 were mainly detected in invasive cytotrophoblasts some distance from the columns. However, the TCC was markedly longer in tubal pregnancy than in intrauterine pregnancy. These findings suggest that the decidua is not necessary to trigger EVT invasion, but that it is likely to limit the extent of the TCC and to accelerate the onset of EVT migration.
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PMID:Evidence of a limited contribution of feto-maternal interactions to trophoblast differentiation along the invasive pathway. 1288 91


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