scholarly journals Knockdown of PRKAR2B Results in the Failure of Oocyte Maturation

2018 ◽  
Vol 45 (5) ◽  
pp. 2009-2020 ◽  
Author(s):  
Hyemin Yoon ◽  
Hoon Jang ◽  
Eun-Young Kim ◽  
Sohyeon Moon ◽  
Sangho Lee ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Time Lapse ◽  
Pentose Phosphate ◽  
Regulatory Subunit ◽  
Spindle Formation ◽  
Qrt Pcr ◽  
Dependent Protein

Background/Aims: Cyclic adenosine monophosphate (cAMP)-dependent type 2 regulatory subunit beta (Prkar2b) is a regulatory isoform of cAMP-dependent protein kinase (PKA), which is the primary target for cAMP actions. In oocytes, PKA and the pentose phosphate pathway (PPP) have important roles during the germinal vesicle (GV) stage arrest of development. Although the roles of the PKA signal pathway have been studied in the development of oocyte, there has been no report on the function of PRKAR2B, a key regulator of PKA. Methods: Using reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR (qRT-PCR), immunohistochemistry, and immunofluorescence, we determined the relative expression of Prkar2b in various tissues, including ovarian follicles, during oocyte maturation. Prkar2b-interfering RNA (RNAi) microinjection was conducted to confirm the effect of Prkar2b knockdown, and immunofluorescence, qRT-PCR, and time-lapse video microscopy were used to analyze Prkar2b-deficient oocytes. Results: Prkar2b is strongly expressed in the ovarian tissues, particularly in the growing follicle. During oocyte maturation, the highest expression of Prkar2b was during metaphase I (MI), with a significant decrease at metaphase II (MII). RNAi-mediated Prkar2b suppression resulted in MI-stage arrest during oocyte development, and these oocytes exhibited abnormal spindle formation and chromosome aggregation. Expression of other members of the PKA family (except for Prkaca) were decreased, and the majority of the PPP factors were also reduced in Prkar2b-deficient oocytes. Conclusion: These results suggest that Prkar2b is closely involved in the maturation of oocytes by controlling spindle formation and PPP-mediated metabolism.

Development of a Scintillation Proximity Assay for Calcineurin Phosphatase Activity

1997 ◽  
Vol 2 (1) ◽  
pp. 19-23 ◽  
Author(s):  
Elaine Sullivan ◽  
Paul Hemsley ◽  
Anne Pickard
Keyword(s):  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Regulatory Subunit ◽  
Dependent Protein Kinase ◽  
Scintillation Proximity Assay ◽  
Large Numbers ◽  
Dependent Protein ◽  
Biotinylated Peptide ◽  
Calcineurin Phosphatase ◽  
Calcineurin Activity

We have developed a scintillation proximity assay (SPA) that allows the Ca2+/calmodulin (CaM)-dependent Serine/threoine (Ser/Thr) phosphoprotein phosphatase 2B (calcineurin) activity to be analyzed. A [33P] labeled and biotinylated peptide containing a partial sequence of the regulatory subunit (Rn) of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase was synthesized and used as a synthetic substrate for calcineurin. Following incubation of the peptide with calcineurin, which removes the [33P] label, streptavidin-coated SPA beads were added to capture the biotinylated peptide (the level of the signal detected is inversely proportional to that of the calcineurin activity). Sensitivity is increased in this system by settling or centrifuging the streptavidin-coated SPA beads after binding has occurred. This method allows calcineurin phosphatase assays to be carried out in a 96-well format that is amenable to screening large numbers of compounds.

scholarly journals Inhibition of mos-induced oocyte maturation by protein kinase A.

1993 ◽  
Vol 120 (5) ◽  
pp. 1197-1202 ◽  
Author(s):  
I Daar ◽  
N Yew ◽  
G F Vande Woude
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Regulatory Subunit ◽  
Germinal Vesicle Breakdown ◽  
Downstream Target ◽  
Dependent Protein ◽  
Pka Regulatory Subunit ◽  
The Relationship

The relationship between the mos protooncogene protein and cAMP-dependent protein kinase (PKA) during the maturation of Xenopus oocytes was investigated. Microinjection of the PKA catalytic subunit (PKAc) into Xenopus oocytes inhibited oocyte maturation induced by the mos product but did not markedly affect the autophosphorylation activity of injected mos protein. By contrast, PKAc did not inhibit maturation promoting factor (MPF) activation or germinal vesicle breakdown (GVBD) that was initiated by injecting crude MPF preparations. In addition, inhibiting endogenous PKA activity by microinjecting the PKA regulatory subunit (PKAr) induced oocyte maturation that was dependent upon the presence of the endogenous mos product. Moreover, PKAr potentiated mos protein-induced MPF activation in the absence of progesterone and protein synthesis. These data are consistent with the hypothesis that progesterone-induced release from G2/M is regulated via PKAc and that PKAc negatively regulates a downstream target that is positively regulated by mos.

scholarly journals Identification of Novel Substrates for cGMP Dependent Protein Kinase (PKG) through Kinase Activity Profiling to Understand Its Putative Role in Inherited Retinal Degeneration

2021 ◽  
Vol 22 (3) ◽  
pp. 1180
Author(s):  
Akanksha Roy ◽  
John Groten ◽  
Valeria Marigo ◽  
Tushar Tomar ◽  
Riet Hilhorst
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cell System ◽  
Guanosine Monophosphate ◽  
Retinal Cell ◽  
Regulatory Subunit ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Inherited retinal degenerative diseases (IRDs), which ultimately lead to photoreceptor cell death, are characterized by high genetic heterogeneity. Many IRD-associated genetic defects affect 3′,5′-cyclic guanosine monophosphate (cGMP) levels. cGMP-dependent protein kinases (PKGI and PKGII) have emerged as novel targets, and their inhibition has shown functional protection in IRDs. The development of such novel neuroprotective compounds warrants a better understanding of the pathways downstream of PKGs that lead to photoreceptor degeneration. Here, we used human recombinant PKGs in combination with PKG activity modulators (cGMP, 3′,5′-cyclic adenosine monophosphate (cAMP), PKG activator, and PKG inhibitors) on a multiplex peptide microarray to identify substrates for PKGI and PKGII. In addition, we applied this technology in combination with PKG modulators to monitor kinase activity in a complex cell system, i.e. the retinal cell line 661W, which is used as a model system for IRDs. The high-throughput method allowed quick identification of bona fide substrates for PKGI and PKGII. The response to PKG modulators helped us to identify, in addition to ten known substrates, about 50 novel substrates for PKGI and/or PKGII which are either specific for one enzyme or common to both. Interestingly, both PKGs are able to phosphorylate the regulatory subunit of PKA, whereas only PKGII can phosphorylate the catalytic subunit of PKA. In 661W cells, the results suggest that PKG activators cause minor activation of PKG, but a prominent increase in the activity of cAMP-dependent protein kinase (PKA). However, the literature suggests an important role for PKG in IRDs. This conflicting information could be reconciled by cross-talk between PKG and PKA in the retinal cells. This must be explored further to elucidate the role of PKGs in IRDs.

scholarly journals RASD1 Knockdown Results in Failure of Oocyte Maturation

2016 ◽  
Vol 40 (6) ◽  
pp. 1289-1302 ◽  
Author(s):  
Youngeun Lee ◽  
Kyeoung-Hwa Kim ◽  
Hyemin Yoon ◽  
Ok-Hee Lee ◽  
Eunyoung Kim ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Oocyte Maturation ◽  
Iron Homeostasis ◽  
Hormone Secretion ◽  
Growth Hormone Secretion ◽  
Germinal Vesicle ◽  
Time Lapse ◽  
Small Gtpases ◽  
Spindle Formation ◽  
Chromosome Alignment

Background: Ras dexamethasone-induced protein (RASD1) is a member of Ras superfamily of small GTPases. RASD1 regulates various signaling pathways involved in iron homeostasis, growth hormone secretion, and circadian rhythm. However, RASD1 function in oocyte remains unknown. Methods: Using immunohistochemistry, immunofluorescence, and quantitative real-time RT-PCR, RASD1 expression in mouse ovary and RASD1 role in oocyte maturation-related gene expression, spindle formation, and chromosome alignment were analyzed. RNAi microinjection and time-lapse video microscopy were used to examine the effect of Rasd1 knockdown on oocyte maturation. Results: RASD1 was highly detected in oocytes transitioning from primordial to secondary follicles. Rasd1 was highly expressed in germinal vesicle (GV), during GV breakdown, and in metaphase I (MI) stage as oocytes mature, and its expression was significantly downregulated in MII stage. With knockdown of Rasd1, maturation in GV oocytes was arrested at MI stage, showing disrupted meiotic spindling and chromosomal misalignment. In addition, Obox4 and Arp2/3, engaged in MI-MII transition and cytokinesis, respectively, were misregulated in GV oocytes by Rasd1 knockdown. Conclusion: These findings suggest that RASD1 is a novel factor in MI-MII oocyte transition and may be involved in regulating the progression of cytokinesis and spindle formation, controlling related signaling pathways during oocyte maturation.

scholarly journals Dibutyryl cyclic adenosine monophosphate reduces expression of c-myc during HL-60 differentiation

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 412-416
Author(s):  
SS Jr McCachren ◽  
J Nichols ◽  
RE Kaufman ◽  
JE Niedel
Keyword(s):  
Leukemia Cell ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Mechanisms Of Action ◽  
Promyelocytic Leukemia ◽  
Leukemia Cell Line ◽  
Dependent Protein Kinase ◽  
Rna Transcripts ◽  
Dependent Protein ◽  
Promyelocytic Leukemia Cell Line

The human promyelocytic leukemia cell line HL-60 is induced to differentiate along a myelocytic pathway by dibutyryl cyclic adenosine monophosphate (dbcAMP). Other cAMP analogs are ineffective as inducing agents. The effect of these compounds on expression of c-myc was investigated using a DNA probe for c-myc to detect RNA transcripts. The dose response and time to commitment for reduction in c-myc expression with dbcAMP was similar to the findings for phenotypic changes. Bromo- cyclic AMP and butyrate alone caused no changes in c-myc expression in 24 hours, but demonstrated dramatic synergism together, suggesting that butyrate contributes in part to the effects of dbcAMP. Evidence for mechanisms of action of cAMP other than activation of the cAMP- dependent protein kinase is reviewed.

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