scholarly journals Phosphatidylinositol 3-Kinase/Protein Kinase Cζ-Induced Phosphorylation of Sp1 and p107 Repressor Release Have a Critical Role in Histone Deacetylase Inhibitor-Mediated Depression of Transcription of the Luteinizing Hormone Receptor Gene

2006 ◽  
Vol 26 (18) ◽  
pp. 6748-6761 ◽  
Author(s):  
Ying Zhang ◽  
Mingjuan Liao ◽  
Maria L. Dufau
Keyword(s):  
Protein Kinase ◽  
Luteinizing Hormone ◽  
Histone Deacetylase ◽  
Hormone Receptor ◽  
Gene Activation ◽  
Gene Promoter ◽  
Luteinizing Hormone Receptor ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

ABSTRACT We have demonstrated that silencing of luteinizing hormone receptor (LHR) gene transcription is mediated via a proximal Sp1 site at its promoter. Trichostatin A (TSA) induced histone acetylation and gene activation in JAR cells that prevailed in the absence of changes in Sp1/Sp3 expression, their binding activity, disassociation of the histone deacetylase/mSin3A complex from the Sp1 site, or demethylation of the promoter. This indicated a different mechanism involved in TSA-induced derepression. The present studies have revealed that phosphatidylinositol 3-kinase/protein kinase Cζ (PI3K/PKCζ)-mediated Sp1 phosphorylation accounts for Sp1 site-dependent LHR gene activation. TSA caused marked phosphorylation of Sp1 at serine 641 in JAR and MCF-7 cells. Blockade of PI3K or PKCζ activity by specific inhibitors, kinase-deficient mutants, or small interfering RNA abolished the effect of TSA on the LHR gene and Sp1 phosphorylation. PKCζ was shown to associate with Sp1, and this association was enhanced by TSA. Sp1 phosphorylation at serine 641 was required for the release of the pRb homologue p107 from the LHR gene promoter, while p107 acted as a repressor of the LHR gene. Inhibition of PKCζ activity blocked the dissociation of p107 from the LHR gene promoter and markedly reduced Sp1 phosphorylation and transcription. These results have demonstrated that phosphorylation of Sp1 by PI3K/PKCζ is critical for TSA-activated LHR gene expression. These studies have revealed a novel mechanism of TSA action through derecruitment of a repressor from the LHR gene promoter in a PI3K/PKCζ-induced Sp1 phosphorylation-dependent manner.

343 PHOSPHATIDYLINOSITOL 3-KINASE IS INVOLVED IN THE MAINTENANCE OF METAPHASE II ARREST IN PORCINE OOCYTES MATURED IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 328
Author(s):  
N. Kashiwazaki ◽  
M. Shimada ◽  
J. Ito
Keyword(s):  
Protein Kinase ◽  
Time Dependent ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Mapk Activity ◽  
Pronuclear Formation ◽  
Pkb Phosphorylation ◽  
Phosphatidylinositol 3

It has been reported that phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB) pathway plays a crucial role in the meiotic resumption and progression to the metaphase II (MII) stage of oocytes. However, the role of this pathway in meiotic arrest at the MII stage (cytostatic activity) is not well understood. In this study, the effect of a PI3K inhibitor, LY294002, on the mitogen-activated protein kinase (MAPK) and p34cdc2 kinase activities of matured porcine oocytes was examined. Immature oocytes were collected from ovaries and cultured in modified NCSU37 up to 48 hr. After culture, cumulus cells were removed and oocytes were cultured up to 24 h in medium supplemented with 25 or 50 μM LY294002. Groups of 10 or 20 oocytes were collected at each culture period for in vitro kinase assay of p34cdc2 kinase and MAPK, respectively. Groups of 40 oocytes were also used for detection of PKB phosphorylation by Western blotting. After maturation culture, both the p34cdc2 kinase and MAPK activities in the oocytes were gradually decreased in a time-dependent manner. Although 25 μM LY294002 did not affect either the p34cdc2 kinase or MAPK activities, 50 μM LY294002 suppressed the PKB phosphorylation and slightly decreased MAPK activity, but not the p34cdc2 kinase activity. Next, the effect of 10 μM Ca2+ ionophore which was reported as inducing a transient decrease of p342+ kinase but not MAPK activities, was examined in LY294002-treated oocytes. Pronuclear formation of the oocytes was also evaluated by the aceto-orcein staining. By additional treatment with LY294002 after Ca2+ ionophore, both the MAPK and p34cdc2 kinase activities were decreased in a time-dependent manner, concomitantly with improvement of pronuclear formation. Therefore, we concluded that PI3K is possibly involved in the maintenance of MAPK activity in matured porcine oocytes. The work was supported in part by Grant-in-Aid for Scientific Research from JSPS (KAKENHI) (21789253) to J.I. This work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan through a Grant-in-Aid for Matching Fund Subsidy for Private Universities to J.I. and N.K.

scholarly journals Phosphatidylinositol 3-Kinase-mediated Endocytosis of Renal Na+,K+-ATPase α Subunit in Response to Dopamine

1998 ◽  
Vol 9 (5) ◽  
pp. 1209-1220 ◽  
Author(s):  
Alexander V. Chibalin ◽  
Juleen R. Zierath ◽  
Adrian I. Katz ◽  
Per-Olof Berggren ◽  
Alejandro M. Bertorello
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Α Subunit ◽  
Kinase C ◽  
Dose Dependent ◽  
Α Subunits ◽  
K Activity ◽  
Phosphatidylinositol 3

Dopamine (DA) inhibition of Na+,K+-ATPase in proximal tubule cells is associated with increased endocytosis of its α and β subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na+,K+-ATPase α subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na+,K+-ATPase α subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D1 receptor subtype and the sequential activation of phospholipase A2, arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na+,K+-ATPase α subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.

scholarly journals Characterization of constitutive activation and inactivation mutants in conserved regions of the eel luteinizing hormone receptor

2019 ◽  
Author(s):  
Munkhzaya Byambaragchaa ◽  
Dong-An Kim ◽  
Dae-Jung Kim ◽  
Sun-Mee Hong ◽  
Myung-Hwa Kang ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Luteinizing Hormone ◽  
Hormone Receptor ◽  
Fold Increase ◽  
Luteinizing Hormone Receptor ◽  
Maximal Response ◽  
Dependent Manner ◽  
Time Resolved ◽  
Inactivating Mutations

Abstract Background: We analyzed signal transduction of three constitutively activating mutants (designated M410T, L469R, and D590Y) and two inactivating mutants (D383N and Y546F) of the eel luteinizing hormone receptor (LHR), known to be naturally occurring in humans as LHR. The objective of the present study is to assess the functional effects of these mutations in signal transduction. Methods: Site-directed mutant receptors were transiently expressed in CHO-K1 cells and cAMP accumulation, stimulated by recombinant eelLH (rec-eelLH), was measured by homogeneous time-resolved fluorescence (HTRF) assays. Results: The cAMP response in cells expressing the wild-type eel LHR (eLHR-WT) increased in a dose-dependent manner with rec-eelLH ligand stimulation. Cells expressing the activating eelLHR mutants, M410T, L469R, and D590Y, exhibited a 4.0-, 19.1-, and 7.8-fold increase in the basal cAMP response, respectively. However, their maximal responses to agonist were approximately 73, 53, and 92%, respectively, of the maximal response of the LHR-WT. The L469R mutant exhibited a particularly marked increase in cAMP concentration in the absence of ligand. The inactivating mutations did not completely impair signal transduction. The maximal responses of the inactivating mutants, D383N and Y546F, were 32% and 24% of the LHR-WT, respectively. Conclusion: We report here the first characterization of activating and inactivating mutations in eelLHR. These results provide important data on the signal transduction of constitutively active and inactive eelLHR mutants. Keywords: eel LHR, constitutively activating/inactivating mutation

scholarly journals Coordinated Changes in DNA Methylation and Histone Modifications Regulate Silencing/Derepression of Luteinizing Hormone Receptor Gene Transcription

2005 ◽  
Vol 25 (18) ◽  
pp. 7929-7939 ◽  
Author(s):  
Ying Zhang ◽  
Naheed Fatima ◽  
Maria L. Dufau
Keyword(s):  
Dna Methylation ◽  
Luteinizing Hormone ◽  
Histone Modifications ◽  
Hormone Receptor ◽  
Promoter Region ◽  
Gene Promoter ◽  
Luteinizing Hormone Receptor ◽  
Activated State ◽  
Jar Cells ◽  
Mcf 7

ABSTRACT We have previously demonstrated that transcription of the luteinizing hormone receptor (LHR) gene is subject to repression by histone deacetylation at its promoter region, where a histone deacetylase (HDAC)/mSin3A complex is anchored at a proximal Sp1 site. The present studies have shown that epigenetic silencing and activation of the LHR gene is achieved through coordinated regulation at both the histone and DNA levels. The HDAC inhibitor trichostatin A (TSA) evoked robust but significantly lower activation of the LHR gene in JAR than in MCF-7 cells. This effect was localized to the 176-bp promoter region, which is highly methylated in JAR and lightly methylated in MCF-7 cells. Consequently, TSA and the DNA demethylating reagent 5-azacytidine (5-AzaC) caused marked synergistic activation of the LHR gene in JAR but not in MCF-7 cells. Multiple site-specific lysine acetylation of H3/H4 is associated with such LHR gene activation. Methylation or acetylation of H3 at K9 is present at the silenced and derepressed LHR promoter, respectively. While DNA methylation levels did not affect the histone code of the LHR gene promoter, demethylation of the promoter CpG sites was necessary for maximal stimulation of this gene. Mechanistically, the combined actions of TSA and 5-AzaC, but not either 5-AzaC or TSA alone, resulted in complete demethylation of the LHR gene promoter in JAR cells. Release of the repressive HDAC/mSin3A complex from the LHR gene promoter in both cell types required both TSA-induced changes of histone modifications and, concurrently, a demethylated promoter. Also, Dnmt1 was largely dissociated from the LHR gene promoter in the presence of TSA or TSA plus 5-AzaC, and binding of MBD2 in JAR cells was diminished upon conversion of the promoter to a demethylated state. Such changes induced a more permissive chromatin where recruitment of polymerase II and TFIIB to the promoter was significantly increased. The activated state of the LHR gene induced by TSA and 5-AzaC in JAR and MCF-7 cells was observed basally in LHR-expressing PLC cells, in which the promoter is unmethylated and associated with hyperacetylated histones. Consequently, PLC cells are unresponsive to drug treatment. These findings have elucidated a regulatory mechanism whereby concurrent dissociation of repressors and association of activators and basal transcriptional components, resulting from coordinated histone hyperacetylation and DNA demethylation, lead to derepression of the LHR gene expression.

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