Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells

1997 ◽  
Vol 272 (6) ◽  
pp. F816-F822 ◽  
Author(s):  
T. Katsura ◽  
C. E. Gustafson ◽  
D. A. Ausiello ◽  
D. Brown
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Phosphorylation Site ◽  
Transfected Cells ◽  
Aquaporin 2 ◽  
Intracellular Vesicles ◽  
Kinase A

Vasopressin-dependent translocation of aquaporin-2 (AQP2) between intracellular vesicles and the plasma membrane has been demonstrated in vivo and in vitro. Furthermore, the vasopressin-induced increase in apical membrane water permeability of renal principal cells is dependent on a rise in intracellular adenosine 3',5'-cyclic monophosphate and activation of protein kinase A (PKA). To determine whether trafficking of AQP2 is dependent on PKA phosphorylation, we first examined the effect of the PKA-inhibitor N-(2[[3-(4-bromophenyl)-2-propenyl]-amino]-ethyl)-5-isoquinolinesulfonam ide (H-89) on AQP2 translocation in transfected LLC-PK1 cells. Vasopressin-induced membrane insertion of AQP2 was completely inhibited by pretreatment of the cells for 60 min with H-89. This reagent also caused a dense accumulation of AQP2 in the Golgi region. Next, LLC-PK1 cells were stably transfected with AQP2 cDNA in which the PKA phosphorylation site, Ser256, was replaced with alanine (S256A). S256A-AQP2 was not phosphorylated in vitro by PKA, and S256A-AQP2 was mainly localized to intracellular vesicles in the basal condition, similar to wild-type AQP2. However, after stimulation with vasopressin or forskolin, the cellular distribution of S256A-AQP2 remained unchanged. In addition, the usual vasopressin-induced increase in endocytosis seen in AQP2-transfected cells was not observed in S256A-AQP2-transfected cells. These results demonstrate that the Ser256 PKA phosphorylation site is possibly involved in the vasopressin-induced trafficking of AQP2 from intracellular vesicles to the plasma membrane and in the subsequent stimulation of endocytosis.

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.

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.

Mucolipin 1 channel activity is regulated by protein kinase A-mediated phosphorylation

2008 ◽  
Vol 410 (2) ◽  
pp. 417-425 ◽  
Author(s):  
Silvia Vergarajauregui ◽  
Ross Oberdick ◽  
Kirill Kiselyov ◽  
Rosa Puertollano
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Transient Receptor Potential ◽  
Phosphorylation Site ◽  
Receptor Potential ◽  
Channel Activity ◽  
Lysosomal Storage ◽  
Kinase A

Mucolipins constitute a family of cation channels with homology with the transient receptor potential family. Mutations in MCOLN1 (mucolipin 1) have been linked to mucolipidosis type IV, a recessive lysosomal storage disease characterized by severe neurological and ophthalmologic abnormalities. At present, little is known about the mechanisms that regulate MCOLN1 activity. In the present paper, we addressed whether MCOLN1 activity is regulated by phosphorylation. We identified two PKA (protein kinase A) consensus motifs in the C-terminal tail of MCOLN1, containing Ser557 and Ser559. Ser557 was the principal phosphorylation site, as mutation of this residue to alanine caused a greater than 75% reduction in the total levels of phosphorylated MCOLN1 C-terminal tail. Activation of PKA with forskolin promoted MCOLN1 phosphorylation, both in vitro and in vivo. In contrast, addition of the PKA inhibitor H89 abolished MCOLN1 phosphorylation. We also found that PKA-mediated phosphorylation regulates MCOLN1 channel activity. Forskolin treatment decreased MCOLN1 channel activity, whereas treatment with H89 increased MCOLN1 channel activity. The stimulatory effect of H89 on MCOLN1 function was not observed when Ser557 and Ser559 were mutated to alanine residues, indicating that these two residues are essential for PKA-mediated negative regulation of MCOLN1. This paper presents the first example of regulation of a member of the mucolipin family by phosphorylation.

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

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 TSH Regulates Pendrin Membrane Abundance and Enhances Iodide Efflux in Thyroid Cells

Endocrinology ◽  
2012 ◽  
Vol 153 (1) ◽  
pp. 512-521 ◽  
Author(s):  
Liuska Pesce ◽  
Aigerim Bizhanova ◽  
Juan Carlos Caraballo ◽  
Whitney Westphal ◽  
Maria L. Butti ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Phosphorylation Site ◽  
Physiological Role ◽  
Thyroid Cells ◽  
Hormone Synthesis ◽  
Iodide Transport ◽  
Rat Thyroid ◽  
Kinase A

Thyroid hormones are essential for normal development and metabolism. Their synthesis requires transport of iodide into thyroid follicles. The mechanisms involving the apical efflux of iodide into the follicular lumen are poorly elucidated. The discovery of mutations in the SLC26A4 gene in patients with Pendred syndrome (congenital deafness, goiter, and defective iodide organification) suggested a possible role for the encoded protein, pendrin, as an apical iodide transporter. We determined whether TSH regulates pendrin abundance at the plasma membrane and whether this influences iodide efflux. Results of immunoblot and immunofluorescence experiments reveal that TSH and forskolin rapidly increase pendrin abundance at the plasma membrane through the protein kinase A pathway in PCCL-3 rat thyroid cells. The increase in pendrin membrane abundance correlates with a decrease in intracellular iodide as determined by measuring intracellular 125iodide and can be inhibited by specific blocking of pendrin. Elimination of the putative protein kinase A phosphorylation site T717A results in a diminished translocation to the membrane in response to forskolin. These results demonstrate that pendrin translocates to the membrane in response to TSH and suggest that it may have a physiological role in apical iodide transport and thyroid hormone synthesis.

Direct effect of protein kinase A on four putative phosphorylation sites of aquaporin 2 in vitro

2020 ◽  
Vol 525 (2) ◽  
pp. 505-511
Author(s):  
Zhiyi Chen ◽  
Jieqiu Zhuang ◽  
Qing Yang ◽  
Jianhuan Yang ◽  
Dexuan Wang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Direct Effect ◽  
Phosphorylation Sites ◽  
Aquaporin 2 ◽  
Kinase A

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.

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