scholarly journals Differential effects of prostaglandin F2alpha on in vitro luteinized bovine granulosa cells

Reproduction ◽  
2001 ◽  
pp. 245-253 ◽  
Author(s):  
SJ Tsai ◽  
MC Wiltbank
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Granulosa Cells ◽  
Ovarian Function ◽  
Prostaglandin F2alpha ◽  
Induced Expression ◽  
Receptor Mrna ◽  
Kinase A

Prostaglandins have been implicated in various aspects of ovarian function including ovulation and luteolysis. In this study, the expression and regulation of inducible prostagland in G/H synthase (PGHS-2) and PGF(2alpha) receptors were investigated in bovine granulosa cells at various stages of differentiation. Firstly, the induction of PGF(2alpha) receptor mRNA and PGHS-2 mRNA in preovulatory granulosa cells was evaluated. Granulosa cells were collected from preovulatory follicles and cultured for 1, 4, 7 or 10 days. Cells were treated with hCG (10 iu) or with increasing doses of forskolin (0-10 micromol l(-1)) for 24 h. Forskolin increased steady-state concentrations of mRNA for PGHS-2 (> 20-fold) and PGF(2alpha) receptor (> 1000-fold) in a dose-dependent fashion. Use of selective protein kinase A inhibitor (H89) reduced both hCG- and forskolin-induced expression of PGF(2alpha) receptor mRNA and PGHS-2 mRNA. The hypothesis that luteinized granulosa cells would acquire PGF(2alpha) responsiveness similar to responses to PGF(2alpha) observed in vivo was also evaluated. Treatment with PGF(2alpha) (100 nmol l(-1)) reduced forskolin-induced expression of PGF(2alpha) receptor mRNA on days 4, 7 and 10, but not on day 1 of culture (n = 3). Treatment with PGF(2alpha) did not change forskolin-induced expression of PGHS-2 mRNA on or before day 4 of culture. In contrast, PGF(2alpha) significantly increased PGHS-2 mRNA expression in granulosa cells primed with forskolin for 7 or 10 days. In conclusion, expression of PGHS-2 and PGF(2alpha) receptor mRNA is protein kinase A-dependent in preovulatory bovine granulosa cells. Granulosa cells become PGF(2alpha)-responsive soon after expression of PGF(2alpha) receptor, whereas further differentiation is required before PGF(2alpha) induces PGHS-2 mRNA upregulation. These results demonstrate that at least two key transitions are required in PGF(2alpha)-induced luteal regression in the mid-cycle corpus luteum.

scholarly journals Membrane-Bound Protein Kinase A Inhibits Smooth Muscle Cell Proliferation In Vitro and In Vivo by Amplifying cAMP–Protein Kinase A Signals

2001 ◽  
Vol 88 (3) ◽  
pp. 319-324 ◽  
Author(s):  
Ciro Indolfi ◽  
Eugenio Stabile ◽  
Carmela Coppola ◽  
Adriana Gallo ◽  
Cinzia Perrino ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Smooth Muscle ◽  
Protein Kinase ◽  
Smooth Muscle Cell ◽  
Protein Kinase A ◽  
Membrane Bound ◽  
Proliferation In Vitro ◽  
Kinase A

scholarly journals Full-length cardiac Na+/Ca2+ exchanger 1 protein is not phosphorylated by protein kinase A

2011 ◽  
Vol 300 (5) ◽  
pp. C989-C997 ◽  
Author(s):  
Pimthanya Wanichawan ◽  
William E. Louch ◽  
Kristin H. Hortemo ◽  
Bjørg Austbø ◽  
Per Kristian Lunde ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Mutational Analysis ◽  
Phosphorylation Site ◽  
Full Length ◽  
Kinase A

The cardiac Na+/Ca2+ exchanger 1 (NCX1) is an important regulator of intracellular Ca2+ homeostasis and cardiac function. Several studies have indicated that NCX1 is phosphorylated by the cAMP-dependent protein kinase A (PKA) in vitro, which increases its activity. However, this finding is controversial and no phosphorylation site has so far been identified. Using bioinformatic analysis and peptide arrays, we screened NCX1 for putative PKA phosphorylation sites. Although several NCX1 synthetic peptides were phosphorylated by PKA in vitro, only one PKA site (threonine 731) was identified after mutational analysis. To further examine whether NCX1 protein could be PKA phosphorylated, wild-type and alanine-substituted NCX1-green fluorescent protein (GFP)-fusion proteins expressed in human embryonic kidney (HEK)293 cells were generated. No phosphorylation of full-length or calpain- or caspase-3 digested NCX1-GFP was observed with purified PKA-C and [γ-32P]ATP. Immunoblotting experiments with anti-PKA substrate and phosphothreonine-specific antibodies were further performed to investigate phosphorylation of endogenous NCX1. Phospho-NCX1 levels were also not increased after forskolin or isoproterenol treatment in vivo, in isolated neonatal cardiomyocytes, or in total heart homogenate. These data indicate that the novel in vitro PKA phosphorylation site is inaccessible in full-length as well as in calpain- or caspase-3 digested NCX1 protein, suggesting that NCX1 is not a direct target for PKA phosphorylation.

Protein kinase A mediates novel serine-584 phosphorylation of HDAC4

2019 ◽  
Vol 97 (5) ◽  
pp. 526-535 ◽  
Author(s):  
Shanmukha K. Doddi ◽  
Githavani Kummari ◽  
Jagannadham M.V. ◽  
Arunasree M. Kalle
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Cell Line ◽  
Target Genes ◽  
K562 Cells ◽  
Pcr Analysis ◽  
Wild Type ◽  
Kinase A

Given the well-established diversified signaling pathways for histone deacetylase 4 (HDAC4) and the regulation of HDAC4 by several post-translational modifications (PTMs), including phosphorylation, sumoylation, and ubiquitination, an unbiased and detailed analysis of HDAC4 PTMs is needed. In this study, we used matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF) to describe phosphorylation at serine 584 (Ser584) along with already-known dual phosphorylation at serines 265 and 266 (Ser265/266), that together regulate HDAC4 activity. Overexpression of site-specific HDAC4 mutants (S584A, S265/266A) in HEK 293T cells, followed by HDAC activity assays, revealed the mutants to be less active than the wild-type protein. In vitro kinase assays have established that Ser584 and Ser265/266 are phosphorylated by protein kinase A (PKA). Luciferase assays driven by the myocyte enhancer factor 2 (MEF2) promoter and real-time PCR analysis of the MEF2 target genes show that the S584A and S265/266A mutants are less repressive than the wild-type. Furthermore, treatment with PKA activators such as 8-Bromo-cAMP and forskolin, and silencing either by shRNA or its inhibitor H-89 in a mouse myoblast cell line (C2C12) and in a non-muscle human cell line (K562), confirmed in vivo phosphorylation of HDAC4 in C2C12 but not in K562 cells, indicating the specific functional significance of HDAC4 phosphorylation in muscle cells. Thus, we identified PKA-induced Ser584 phosphorylation of HDAC4 as a yet unknown regulatory mechanism of the HDAC4–MEF2 axis.

A2 adenosine receptors regulate CFTR through PKA and PLA2

2002 ◽  
Vol 282 (1) ◽  
pp. L12-L25 ◽  
Author(s):  
B. R. Cobb ◽  
F. Ruiz ◽  
C. M. King ◽  
J. Fortenberry ◽  
H. Greer ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Receptor Activation ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Δf508 Cftr ◽  
Kinase A

We investigated adenosine (Ado) activation of the cystic fibrosis transmembrane conductance regulator (CFTR) in vitro and in vivo. A2B Ado receptors were identified in Calu-3, IB-3-1, COS-7, and primary human airway cells. Ado elevated cAMP in Calu-3, IB-3-1, and COS-7 cells and activated protein kinase A-dependent halide efflux in Calu-3 cells. Ado promoted arachidonic acid release from Calu-3 cells, and phospholipase A2(PLA2) inhibition blocked Ado-activated halide efflux in Calu-3 and COS-7 cells expressing CFTR. Forskolin- and β2-adrenergic receptor-stimulated efflux were not affected by the same treatment. Cytoplasmic PLA2(cPLA2) was identified in Calu-3, IB-3-1, and COS-7 cells, but cPLA2 inhibition did not affect Ado-stimulated cAMP concentrations. In cftr(+) and cftr(−/−) mice, Ado stimulated nasal Cl− secretion that was CFTR dependent and sensitive to A2 receptor and PLA2 blockade. In COS-7 cells transiently expressing ΔF508 CFTR, Ado activated halide efflux. Ado also activated G551D CFTR-dependent halide efflux when combined with arachidonic acid and phosphodiesterase inhibition. In conclusion, PLA2 and protein kinase A both contribute to A2 receptor activation of CFTR, and components of this signaling pathway can augment wild-type and mutant CFTR activity.

scholarly journals Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate, and specificity of the phosphorylation site sequence in the whole protein

2006 ◽  
Vol 396 (1) ◽  
pp. 117-126 ◽  
Author(s):  
Paula Portela ◽  
Silvia Moreno ◽  
Silvia Rossi
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Pyruvate Kinase ◽  
Phosphorylation Site ◽  
Bovine Heart ◽  
Specificity Constant ◽  
Fructose 1,6 Bisphosphate ◽  
Kinase A

Pyk1 (pyruvate kinase 1) from Saccharomyces cerevisiae was characterized as a substrate for PKA (protein kinase A) from bovine heart and yeast. By designing Pyk1 synthetic peptides containing potential PKA sequence targets (Ser22, Thr94 and Thr478) we determined that the peptide S22 was a substrate for PKA in vitro, with a Ksp* (specificity constant) 10-fold and 3-fold higher than Kemptide for bovine heart and yeast PKA respectively. In vitro phosphorylation of the Pyk1 S22A mutant protein was decreased by as much as 90% when compared with wild-type Pyk1 and the Pyk1 T94A mutant. The Ksp* values for Pyk1 and Pyk1 T94A were the same, indicating that both proteins are phosphorylated at the same site by PKA. Two-dimensional PAGE of Pyk1 and Pyk1 S22A indicates that in vivo the S22A mutation prevented the formation of one of the Pyk1 isoforms. We conclude that in yeast the major PKA phosphorylation site of Pyk1 is Ser22.Phosphorylation of Ser22 leads to a Pyk1 enzyme that is more active in the absence of FBP (fructose 1,6-bisphosphate). The specificity of yeast and mammalian PKA towards the S22 peptide and towards whole Pyk1 protein was measured and compared. The Ksp* for the S22 peptide is higher than that for Pyk1, indicating that the peptide modelled on Pyk1 is a much better substrate than Pyk1, regardless of which tissue was used as the source of PKA. However, the Km of Pyk1 protein is lower than that of the better substrate, the S22 peptide, indicating that ground-state substrate binding is not the major determinant of substrate specificity for PKA.

scholarly journals Forskolin, 8-Br-3′,5′-Cyclic Adenosine 5′-Monophosphate, and Catalytic Protein Kinase A Expression in the Nucleus Increase Radioiodide Uptake and Sodium/Iodide Symporter Protein Levels in RET/PTC1-Expressing Cells

2004 ◽  
Vol 89 (12) ◽  
pp. 6168-6172 ◽  
Author(s):  
Anjli Venkateswaran ◽  
Derek K. Marsee ◽  
Steven H. Green ◽  
Sissy M. Jhiang
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Sodium Iodide ◽  
Sodium Iodide Symporter ◽  
Thyroid Cells ◽  
Protein Levels ◽  
And Function ◽  
Kinase A

Abstract RET/PTC1, a thyroid-specific oncogene, has been reported to down-regulate sodium/iodide symporter (NIS) expression and function in vitro and in vivo. Recently, RET/PTC1 has been shown to interfere with TSH signaling at multiple levels in thyroid cells. The objective of this study was to investigate whether RET/PTC1-mediated NIS reduction can be rescued by activating cAMP-protein kinase A (PKA) pathways. We showed that both forskolin and 8-Br-cAMP increase radioiodide uptake and NIS protein in RET/PTC1-expressing cells to the same extent as the parental PC Cl 3 cells. We found that RET/PTC1 decreases nuclear localization of catalytic PKA, and forskolin treatment was able to counteract this RET/PTC1 effect. Furthermore, transient expression of catalytic PKA in the nucleus increased radioiodide uptake and NIS protein in RET/PTC1-expressing cells. Taken together, these studies suggest that RET/PTC1 down-regulates NIS expression by interrupting TSH/cAMP signaling, and this RET/PTC1 effect can be reversed by activating cAMP-PKA pathways.

scholarly journals Impaired platelet activation and cAMP homeostasis in MRP4-deficient mice

Blood ◽  
2015 ◽  
Vol 126 (15) ◽  
pp. 1823-1830 ◽  
Author(s):  
Benoit Decouture ◽  
Elise Dreano ◽  
Tiphaine Belleville-Rolland ◽  
Orjeta Kuci ◽  
Blandine Dizier ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Key Points ◽  
Platelet Signaling ◽  
Deficient Mice ◽  
Kinase A

Key PointsIn vivo and in vitro thrombus formation is altered in MRP4-deficient mice. MRP4 modulates the cAMP–protein kinase A platelet signaling pathway.

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