scholarly journals Nuclear Progestin Receptor Phosphorylation by Cdk9 Is Required for the Expression of Mmp15, a Protease Indispensable for Ovulation in Medaka

Cells ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 215 ◽  
Author(s):  
Katsueki Ogiwara ◽  
Takayuki Takahashi
Keyword(s):  
Critical Role ◽  
Cdk Inhibitor ◽  
Vertebrate Species ◽  
Dependent Protein Kinase ◽  
Progestin Receptor ◽  
Follicle Rupture ◽  
Dependent Protein ◽  
Ecm Degradation ◽  
Cdk9 Inhibitor

Ovulation denotes the discharge of fertilizable oocytes from ovarian follicles. Follicle rupture during ovulation requires extracellular matrix (ECM) degradation at the apex of the follicle. In the teleost medaka, an excellent model for vertebrate ovulation studies, LH-inducible matrix metalloproteinase 15 (Mmp15) plays a critical role during rupture. In this study, we found that follicle ovulation was inhibited not only by roscovitine, the cyclin-dependent protein kinase (CDK) inhibitor, but also by CDK9-inhibitor II, a specific CDK9 inhibitor. Inhibition of follicle ovulation by the inhibitors was accompanied by the suppression of Mmp15 expression in the follicle. In follicles treated with the inhibitors, the formation of the phosphorylated nuclear progestin receptor (Pgr) was inhibited. Roscovitine treatment caused a reduction in the binding of Pgr to the promoter region of mmp15. The expression of Cdk9 and cyclin I (Ccni), and their association in the follicle was demonstrated, suggesting that Cdk9 and Ccni may be involved in the phosphorylation of Pgr in vivo. LH-induced follicular expression of ccni/Ccni was also shown. This study is the first to report the involvement of CDK in ECM degradation during ovulation in a vertebrate species.

scholarly journals LINC-PINT impedes DNA repair and enhances radiotherapeutic response by targeting DNA-PKcs in nasopharyngeal cancer

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
You-hong Wang ◽  
Zhen Guo ◽  
Liang An ◽  
Yong Zhou ◽  
Heng Xu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nasopharyngeal Cancer ◽  
Noncoding Rnas ◽  
Dependent Protein Kinase ◽  
Damage Repair ◽  
Dependent Protein ◽  
Cancer Tissues

AbstractRadioresistance continues to be the leading cause of recurrence and metastasis in nasopharyngeal cancer. Long noncoding RNAs are emerging as regulators of DNA damage and radioresistance. LINC-PINT was originally identified as a tumor suppressor in various cancers. In this study, LINC-PINT was significantly downregulated in nasopharyngeal cancer tissues than in rhinitis tissues, and low LINC-PINT expressions showed poorer prognosis in patients who received radiotherapy. We further identified a functional role of LINC-PINT in inhibiting the malignant phenotypes and sensitizing cancer cells to irradiation in vitro and in vivo. Mechanistically, LINC-PINT was responsive to DNA damage, inhibiting DNA damage repair through ATM/ATR-Chk1/Chk2 signaling pathways. Moreover, LINC-PINT increased radiosensitivity by interacting with DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and negatively regulated the expression and recruitment of DNA-PKcs. Therefore, these findings collectively support the possibility that LINC-PINT serves as an attractive target to overcome radioresistance in NPC.

scholarly journals Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hai-Jun Gao ◽  
Xu-Dong Sun ◽  
Yan-Ping Luo ◽  
Hua-Sheng Pang ◽  
Xing-Ming Ma ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mrna Expression ◽  
Echinococcus Granulosus ◽  
Calcium Content ◽  
Mrna Levels ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein

Abstract Background Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. Methods The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. Results In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 μg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. Conclusions Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

scholarly journals Chronic Elevation of Calmodulin in the Ventricles of Transgenic Mice Increases the Autonomous Activity of Calmodulin-Dependent Protein Kinase II, Which Regulates Atrial Natriuretic Factor Gene Expression

2000 ◽  
Vol 14 (8) ◽  
pp. 1125-1136 ◽  
Author(s):  
Josep M. Colomer ◽  
Anthony R. Means
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Transgenic Mice ◽  
Atrial Natriuretic Factor ◽  
Ventricular Myocytes ◽  
Dependent Protein Kinase ◽  
Natriuretic Factor ◽  
Dependent Protein ◽  
Autonomous Activity

Abstract Although isoforms of Ca2+/calmodulin-dependent protein kinase II (CaMKII) have been implicated in the regulation of gene expression in cultured cells, this issue has yet to be addressed in vivo. We report that the overexpression of calmodulin in ventricular myocytes of transgenic mice results in an increase in the Ca2+/calmodulin-independent activity of endogenous CaMKII. The calmodulin transgene is regulated by a 500-bp fragment of the atrial natriuretic factor (ANF) gene promoter which, based on cell transfection studies, is itself known to be regulated by CaMKII. The increased autonomous activity of CaMKII maintains the activity of the transgene and establishes a positive feedforward loop, which also extends the temporal expression of the endogenous ANF promoter in ventricular myocytes. Both the increased activity of CaMKII and transcriptional activation of ANF are highly selective responses to the chronic overexpression of calmodulin. These results indicate that CaMKII can regulate gene expression in vivo and suggest that this enzyme may represent the Ca2+-dependent target responsible for reactivation of the ANF gene during ventricular hypertrophy.

Mutation of amino acids in pp60c-src that are phosphorylated by protein kinases C and A

1989 ◽  
Vol 9 (6) ◽  
pp. 2453-2463
Author(s):  
P Yaciuk ◽  
J K Choi ◽  
D Shalloway
Keyword(s):  
Protein Kinase ◽  
3T3 Cells ◽  
Triple Mutant ◽  
Dependent Protein Kinase ◽  
Mutant Proteins ◽  
Tetradecanoyl Phorbol Acetate ◽  
Dependent Protein ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The product of the c-src proto-oncogene, pp60c-src, is phosphorylated at Ser-17 by cyclic AMP-dependent protein kinase A and at Ser-12 by calcium-phospholipid-dependent protein kinase C (when stimulated by 12-O-tetradecanoyl phorbol acetate). We tested the effects of Ser----Ala and Ser----Glu mutations at these sites in pp60c-src and in pp60c-src(F527) (a mutant whose transforming activities are enhanced by Tyr-527----Phe mutation) by transfecting single-, double-, and triple-mutant src expression plasmids into NIH 3T3 cells. Tryptic phosphopeptide analyses of the mutant proteins confirmed prior biochemical identifications of the phosphorylation sites and showed that neither separate nor coordinate mutations at Ser-12 and Ser-17 affected Tyr-416, Tyr-527, or Ser-48 phosphorylation or prevented mitosis-specific phosphorylations of either pp60c-src or pp60c-src(F527). Ser-12 mutation did not affect phosphorylation of the Ser-17-containing peptide, but mutation of Ser-17 significantly increased phosphorylation at Ser-12. Specific kinase activities (both with and without in vivo 12-O-tetradecanoyl phorbol acetate treatment) and the abilities of pp60c-src and pp60c-src(F527) to induce foci, transformed morphologies, and anchorage-independent growth were unaffected by any of the serine mutations. Thus, pp60c-src transforming activity in NIH 3T3 cells is relatively insensitive to phosphorylation at these sites, but there is a suggestion that Ser-17 phosphorylation may have a subtle regulatory effect.

ADR1c mutations enhance the ability of ADR1 to activate transcription by a mechanism that is independent of effects on cyclic AMP-dependent protein kinase phosphorylation of Ser-230

1992 ◽  
Vol 12 (4) ◽  
pp. 1507-1514
Author(s):  
C L Denis ◽  
S C Fontaine ◽  
D Chase ◽  
B E Kemp ◽  
L T Bemis
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Growth Conditions ◽  
Dependent Protein Kinase ◽  
Inactive Form ◽  
Dependent Protein ◽  
Kinase Phosphorylation

Four ADR1c mutations that occur close to Ser-230 of the Saccharomyces cerevisiae transcriptional activator ADR1 and which greatly enhance the ability of ADR1 to activate ADH2 expression under glucose-repressed conditions have been shown to reduce or eliminate cyclic AMP-dependent protein kinase (cAPK) phosphorylation of Ser-230 in vitro. In addition, unregulated cAPK expression in vivo blocks ADH2 depression in an ADR1-dependent fashion in which ADR1c mutations display decreased sensitivity to unregulated cAPK activity. Taken together, these data have suggested that ADR1c mutations enhance ADR1 activity by blocking cAPK phosphorylation and inactivation of Ser-230. We have isolated and characterized an additional 17 ADR1c mutations, defining 10 different amino acid changes, that were located in the region defined by amino acids 227 through 239 of ADR1. Three observations, however, indicate that the ADR1c phenotype is not simply equivalent to a lack of cAPK phosphorylation. First, only some of these newly isolated ADR1c mutations affected the ability of yeast cAPK to phosphorylate corresponding synthetic peptides modeled on the 222 to 234 region of ADR1 in vitro. Second, we observed that strains lacking cAPK activity did not display enhanced ADH2 expression under glucose growth conditions. Third, when Ser-230 was mutated to a nonphosphorylatable residue, lack of cAPK activity led to a substantial increase in ADH2 expression under glucose-repressed conditions. Thus, while cAPK controls ADH2 expression and ADR1 is required for this control, cAPK acts by a mechanism that is independent of effects on ADR1 Ser-230. It was also observed that deletion of the ADR1c region resulted in an ADR1c phenotype. The ADR1c region is, therefore, involved in maintaining ADR1 in an inactive form. ADR1c mutations may block the binding of a repressor to ADR1 or alter the structure of ADR1 so that transcriptional activation regions become unmasked.

scholarly journals Cyclic AMP-dependent protein kinase inhibits the activity of myogenic helix-loop-helix proteins.

1992 ◽  
Vol 12 (10) ◽  
pp. 4478-4485 ◽  
Author(s):  
L Li ◽  
R Heller-Harrison ◽  
M Czech ◽  
E N Olson
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Signal Transduction Pathway ◽  
Specific Gene ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Differentiation of skeletal muscle cells is inhibited by the cyclic AMP (cAMP) signal transduction pathway. Here we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) can substitute for cAMP and suppress muscle-specific transcription by silencing the activity of the MyoD family of regulatory factors, which includes MyoD, myogenin, myf5, and MRF4. Repression by the PKA catalytic (C) subunit is directed at the consensus sequence CANNTG, the target for DNA binding and transcriptional activation by these myogenic regulators. Phosphopeptide mapping of myogenin in vitro and in vivo revealed two PKA phosphorylation sites, both within the basic region. However, repression of myogenin function by PKA does not require direct phosphorylation of these sites but instead involves an indirect mechanism with one or more intermediate steps. Regulation of the transcriptional activity of the MyoD family by modulation of the cAMP signaling pathway may account for the inhibitory effects of certain peptide growth factors on muscle-specific gene expression and may also determine the responsiveness of different cell types to myogenic conversion by these myogenic regulators.

scholarly journals Domain Swapping Used To Investigate the Mechanism of Protein Kinase B Regulation by 3-Phosphoinositide-Dependent Protein Kinase 1 and Ser473 Kinase

1999 ◽  
Vol 19 (7) ◽  
pp. 5061-5072 ◽  
Author(s):  
Mirjana Andjelković ◽  
Sauveur-Michel Maira ◽  
Peter Cron ◽  
Peter J. Parker ◽  
Brian A. Hemmings
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Protein Kinase B ◽  
Second Messengers ◽  
Activation Mechanism ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Phosphoinositide 3 Kinase ◽  
Regulatory Sites

ABSTRACT Protein kinase B (PKB or Akt), a downstream effector of phosphoinositide 3-kinase (PI 3-kinase), has been implicated in insulin signaling and cell survival. PKB is regulated by phosphorylation on Thr308 by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and on Ser473 by an unidentified kinase. We have used chimeric molecules of PKB to define different steps in the activation mechanism. A chimera which allows inducible membrane translocation by lipid second messengers that activate in vivo protein kinase C and not PKB was created. Following membrane attachment, the PKB fusion protein was rapidly activated and phosphorylated at the two key regulatory sites, Ser473 and Thr308, in the absence of further cell stimulation. This finding indicated that both PDK1 and the Ser473 kinase may be localized at the membrane of unstimulated cells, which was confirmed for PDK1 by immunofluorescence studies. Significantly, PI 3-kinase inhibitors prevent the phosphorylation of both regulatory sites of the membrane-targeted PKB chimera. Furthermore, we show that PKB activated at the membrane was rapidly dephosphorylated following inhibition of PI 3-kinase, with Ser473 being a better substrate for protein phosphatase. Overall, the results demonstrate that PKB is stringently regulated by signaling pathways that control both phosphorylation/activation and dephosphorylation/inactivation of this pivotal protein kinase.

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