[42] Peptide-based affinity labeling of adenosine cyclic monophosphate-dependent protein kinase

Stimulation of Ca2+ mobilization and entry by agonists such as ADP, thrombin, and thromboxane is an early step of platelet activation. Here, we compared the effects of adenosine 3',5'-cyclic monophosphate (cAMP)-elevating prostaglandins, guanosine 3',5'-cyclic monophosphate (cGMP)-elevating nitrovasodilators, membrane-permeant selective activators of cAMP- or cGMP-dependent protein kinases, and physiological endothelium-derived factors on the agonist-evoked Ca2+ mobilization and entry in human platelets. Prostaglandin E1, the prostacyclin analogue Iloprost, the nitric oxide (NO) donor 3-morpholinosydnonimine hydrochloride, and selective activators of cGMP- or cAMP-dependent protein kinase strongly inhibited the agonist-evoked Ca2+ mobilization from intracellular stores and associated late Ca2+ entry but had little effects on the rapid (1st) phase of ADP-evoked Ca2+ entry. During coincubation of platelets with endothelial cells, endothelium-derived factors that were released strongly inhibited platelet agonist-evoked Ca2+ mobilization and only moderately affected the rapid phase of ADP-evoked Ca2+ entry. These effects were partially prevented when endothelial cells were preincubated with cyclooxygenase and/or NO synthase inhibitors. Endothelial cells therefore produce sufficient quantities of labile platelet inhibitors whose effects on the platelet Ca2+ response resemble those observed with selective cAMP- and cGMP-dependent protein kinase activators.

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Affinity labeling of the ATP-binding site of type II calmodulin-dependent protein kinase by 5′-p-fluorosulfonylbenzoyl adenosine

Archives of Biochemistry and Biophysics ◽

10.1016/0003-9861(88)90052-5 ◽

1988 ◽

Vol 267 (2) ◽

pp. 467-473 ◽

Cited By ~ 16

Author(s):

Marita M. King ◽

David J. Shell ◽

Ann P. Kwiatkowski

Keyword(s):

Protein Kinase ◽

Binding Site ◽

Affinity Labeling ◽

Atp Binding ◽

Type Ii ◽

Dependent Protein Kinase ◽

Atp Binding Site ◽

Dependent Protein

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Effects of cAMP on serotonin evoked calcium transients in cultured rat airway smooth muscle cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.272.5.l865 ◽

1997 ◽

Vol 272 (5) ◽

pp. L865-L871 ◽

Cited By ~ 1

Author(s):

B. Tolloczko ◽

Y. L. Jia ◽

J. G. Martin

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

Airway Smooth Muscle ◽

Airway Smooth Muscle Cells ◽

Intracellular Camp ◽

Dependent Protein Kinase ◽

Cyclic Monophosphate ◽

Dependent Protein ◽

High Concentrations

Agents increasing intracellular adenosine 3',5'-cyclic monophosphate (cAMP) cause relaxation of airway smooth muscle. However, the mechanisms of their action are not fully understood. We investigated the role of cAMP in the modulation of intracellular Ca2+ concentration ([Ca2+]i) transients evoked by serotonin (5-HT) in cultured rat tracheal smooth muscle (TSM) cells. Forskolin (10(-7) M) caused a significant elevation of intracellular cAMP and a 60% relaxation of tracheal rings contracted with 5-HT but did not affect [Ca2+]i in TSM cells. Forskolin (10(-5) M) completely relaxed tracheal rings and significantly decreased [Ca2+]i during the sustained phase of the 5-HT response. Forskolin-induced relaxation was attenuated by the cAMP-dependent protein kinase A (PKA) inhibitor Rp diastereomer of cAMP (Rp-cAMPS; 10(-4) M) and by the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor [Rp isomer of 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate, 10(-4) M]. The effects of forskolin on [Ca2+]i were not altered by the PKA inhibitor but were abolished by the PKG inhibitor and thapsigargin. These results indicate that, in rat TSM, the relaxant effects of high concentrations of cAMP may be mediated, at least in part, by facilitating the sequestration of Ca2+ into intracellular stores by a mechanism involving PKG.

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cAMP-dependent protein kinase activation decreases cytokine release in bronchial epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00373.2013 ◽

2014 ◽

Vol 307 (8) ◽

pp. L643-L651 ◽

Cited By ~ 15

Author(s):

Todd A. Wyatt ◽

Jill A. Poole ◽

Tara M. Nordgren ◽

Jane M. DeVasure ◽

Art J. Heires ◽

...

Keyword(s):

Cell Migration ◽

Protein Kinase ◽

Epithelial Cells ◽

Wound Repair ◽

Bronchial Epithelial Cells ◽

Dependent Protein Kinase ◽

Bronchial Epithelial ◽

Cyclic Monophosphate ◽

Tnf Α ◽

Dependent Protein

Lung injury caused by inhalation of dust from swine-concentrated animal-feeding operations (CAFO) involves the release of inflammatory cytokine interleukin 8 (IL-8), which is mediated by protein kinase C-ε (PKC-ε) in airway epithelial cells. Once activated by CAFO dust, PKC-ε is responsible for slowing cilia beating and reducing cell migration for wound repair. Conversely, the cAMP-dependent protein kinase (PKA) stimulates contrasting effects, such as increased cilia beating and an acceleration of cell migration for wound repair. We hypothesized that a bidirectional mechanism involving PKA and PKC regulates epithelial airway inflammatory responses. To test this hypothesis, primary human bronchial epithelial cells and BEAS-2B cells were treated with hog dust extract (HDE) in the presence or absence of cAMP. PKC-ε activity was significantly reduced in cells that were pretreated for 1 h with 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) before exposure to HDE ( P < 0.05). HDE-induced IL-6, and IL-8 release was significantly lower in cells that were pretreated with 8-Br-cAMP ( P < 0.05). To exclude exchange protein activated by cAMP (EPAC) involvement, cells were pretreated with either 8-Br-cAMP or 8-(4-chlorophenylthio)-2'- O-methyladenosine-3',5'-cyclic monophosphate (8-CPT-2Me-cAMP) (EPAC agonist). 8-CPT-2Me-cAMP did not activate PKA and did not reduce HDE-stimulated IL-6 release. In contrast, 8-Br-cAMP decreased HDE-stimulated tumor necrosis factor (TNF)-α-converting enzyme (TACE; ADAM-17) activity and subsequent TNF-α release ( P < 0.001). 8-Br-cAMP also blocked HDE-stimulated IL-6 and keratinocyte-derived chemokine release in precision-cut mouse lung slices ( P < 0.05). These data show bidirectional regulation of PKC-ε via a PKA-mediated inhibition of TACE activity resulting in reduced PKC-ε-mediated release of IL-6 and IL-8.

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Characterization of the proteins of the intestinal Na(+)-K(+)-2Cl- cotransporter

AJP Cell Physiology ◽

10.1152/ajpcell.1994.267.2.c375 ◽

1994 ◽

Vol 267 (2) ◽

pp. C375-C384 ◽

Cited By ~ 21

Author(s):

W. Suvitayavat ◽

P. B. Dunham ◽

M. Haas ◽

M. C. Rao

Keyword(s):

Protein Kinase ◽

Subcellular Fractionation ◽

Inhibitory Effect ◽

Polyclonal Antiserum ◽

Calyculin A ◽

Dependent Protein Kinase ◽

Cyclic Monophosphate ◽

Microsomal Membranes ◽

Dependent Protein

Absorptive intestinal epithelia, such as that of the winter flounder, absorb salt via a bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport mechanism on the brush-border membrane (BBM). The present study demonstrates the first molecular characterization of the intestinal Na(+)-K(+)-2Cl- cotransporter and its unique regulation. The photoaffinity bumetanide analogue, 4-[3H]benzoyl-5-sulfamoyl-3- (3-thenyloxy)benzoic acid, specifically labeled three groups of proteins in flounder intestinal microsomal membranes (MM): a approximately 180-kDa peptide, prominently labeled, and diffuse bands at approximately 110-70 and 50 kDa, less intensely labeled. Subcellular fractionation revealed a single prominently labeled protein of approximately 170 kDa in BBM but not in basolateral membranes (BLM) and little or no labeling of proteins of approximately 110-70 or 50 kDa. Polyclonal antiserum raised against the Ehrlich ascites cell cotransporter identified a 180-kDa peptide in MM and a 175-kDa peptide (pI approximately 5.4) in BBM but none in BLM or in the cytosol of flounder intestine. As predicted from the regulation of cotransport in this tissue, phosphorylation of this protein is increased by guanosine 3',5'-cyclic monophosphate (cGMP)-dependent but not by adenosine 3',5'-cyclic monophosphate-dependent protein kinase. In addition, phosphorylation of the protein is not increased by protein kinase C or Ca2+/calmodulin-dependent protein kinase but is increased by the phosphatase inhibitor calyculin A. Finally, calyculin A preserves the inhibitory effect of cGMP on ion transport, even in the absence of the nucleotide, suggesting that phosphorylation-dephosphorylation mechanisms are crucial in cotransporter regulation. Thus the flounder intestinal cotransporter is a approximately 175-kDa BBM protein that can be regulated by phosphorylation.

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