scholarly journals cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets.

1994 ◽  
Vol 269 (20) ◽  
pp. 14509-14517 ◽  
Author(s):  
E. Butt ◽  
K. Abel ◽  
M. Krieger ◽  
D. Palm ◽  
V. Hoppe ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Focal Adhesion ◽  
Human Platelets ◽  
Phosphorylation Sites ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Kinase Phosphorylation ◽  
Camp And Cgmp

In vitro phosphorylation of ciliary dyneins by protein kinases from Paramecium

1993 ◽  
Vol 106 (4) ◽  
pp. 1369-1376 ◽  
Author(s):  
C.E. Walczak ◽  
D.L. Nelson
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Sucrose Gradient ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Ciliary Protein ◽  
Dependent Protein ◽  
Camp And Cgmp

Paramecium dyneins were tested as substrates for phosphorylation by cAMP-dependent protein kinase, cGMP-dependent protein kinase, and two Ca(2+)-dependent protein kinases that were partially purified from Paramecium extracts. Only cAMP-dependent protein kinase caused significant phosphorylation. The major phosphorylated species was a 29 kDa protein that was present in both 22 S and 12 S dyneins; its phosphate-accepting activity peaked with 22 S dynein. In vitro phosphorylation was maximal at five minutes, then decreased. This decrease in phosphorylation was inhibited by the addition of vanadate or NaF. The 29 kDa protein was not phosphorylated by a heterologous cAMP-dependent protein kinase, the bovine catalytic subunit. Phosphorylation of dynein did not change its ATPase activity. In sucrose gradient fractions from the last step of dynein purification, phosphorylation by an endogenous kinase occurred. This phosphorylation could not be attributed to the small amounts of cAMP- and cGMP-dependent protein kinases known to be present, nor was it Ca(2+)-dependent. This previously uncharacterized ciliary protein kinase used casein as an in vitro substrate.

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 Engineering a bioluminescent indicator for cyclic AMP-dependent protein kinase

1991 ◽  
Vol 279 (3) ◽  
pp. 727-732 ◽  
Author(s):  
G B Sala-Newby ◽  
A K Campbell
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Specific Activity ◽  
Phosphorylation Site ◽  
Live Cells ◽  
Phosphorylation Sites ◽  
Dependent Protein Kinase ◽  
Ph Optimum ◽  
Dependent Protein

cDNA coding for the luciferase in the firefly Photinus pyralis was amplified in vitro to generate cyclic AMP-dependent protein kinase phosphorylation sites. The DNA was transcribed and translated to generate light-emitting protein. A valine at position 217 was mutated to arginine to generate a site RRFS and the heptapeptide kemptide, the phosphorylation site of the porcine pyruvate kinase, was added at the N- or C-terminus of the luciferase. The proteins carrying phosphorylation sites were characterized for their specific activity, pI, effect of pH on the colour of the light emitted and effect of the catalytic subunit of protein kinase A in the presence of ATP. Only one of the recombinant proteins (RRFS) was significantly different from wild-type luciferase. The RRFS mutant had a lower specific activity, lower pH optimum, emitted greener light at low pH and when phosphorylated it decreased its activity by up to 80%. This latter effect was reversed by phosphatase. This recombinant protein is a good candidate to measure for the first time cyclic AMP-dependent phosphorylation in live cells.

Regulation of calcium mobilization and entry in human platelets by endothelium-derived factors

1994 ◽  
Vol 267 (1) ◽  
pp. C236-C244 ◽  
Author(s):  
J. Geiger ◽  
C. Nolte ◽  
U. Walter
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Human Platelets ◽  
Dependent Protein Kinase ◽  
Rapid Phase ◽  
No Donor ◽  
Cyclic Monophosphate ◽  
Dependent Protein ◽  
Camp And Cgmp ◽  
Stimulation Of

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.

Cyclic GMP-dependent protein kinase is required for thrombospondin and tenascin mediated focal adhesion disassembly

1996 ◽  
Vol 109 (10) ◽  
pp. 2499-2508 ◽  
Author(s):  
J.E. Murphy-Ullrich ◽  
M.A. Pallero ◽  
N. Boerth ◽  
J.A. Greenwood ◽  
T.M. Lincoln ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Focal Adhesion ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Focal Adhesions ◽  
Matrix Proteins ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

Focal adhesions are specialized regions of cell membranes that are foci for the transmission of signals between the outside and the inside of the cell. Intracellular signaling events are important in the organization and stability of these structures. In previous work, we showed that the counter-adhesive extracellular matrix proteins, thrombospondin, tenascin, and SPARC, induce the disassembly of focal adhesion plaques and we identified the active regions of these proteins. In order to determine the mechanisms whereby the anti-adhesive matrix proteins modulate cytoskeletal organization and focal adhesion integrity, we examined the role of protein kinases in mediating the loss of focal adhesions by these proteins. Data from these studies show that cGMP-dependent protein kinase is necessary to mediate focal adhesion disassembly triggered by either thrombospondin or tenascin, but not by SPARC. In experiments using various protein kinase inhibitors, we observed that selective inhibitors of cyclic GMP-dependent protein kinase, KT5823 and Rp-8-Br-cGMPS, blocked the effects of both the active sequence of thrombospondin 1 (hep I) and the alternatively-spliced segment (TNfnA-D) of tenascin-C on focal adhesion disassembly. Moreover, early passage rat aortic smooth muscle cells which have high levels of cGMP-dependent protein kinase were sensitive to hep I treatment, in contrast to passaged cGMP-dependent protein kinase deficient cells which were refractory to hep I or TNfnA-D treatment, but were sensitive to SPARC. Transfection of passaged smooth muscle cells with the catalytic domain of PKG I alpha restored responsiveness to hep I and TNfnA-D. While these studies show that cGMP-dependent protein kinase activity is necessary for thrombospondin and tenascin-mediated focal adhesion disassembly, kinase activity alone is not sufficient to induce disassembly as transfection of the catalytic domain of the kinase in the absence of additional stimuli does not result in loss of focal adhesions.

Physiological concentration of atrial natriuretic peptide induces endothelial regeneration in vitro

2003 ◽  
Vol 284 (4) ◽  
pp. H1388-H1397 ◽  
Author(s):  
Hyun Kook ◽  
Hiroshi Itoh ◽  
Bong Seok Choi ◽  
Naoki Sawada ◽  
Kentaro Doi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Protein Kinase ◽  
Dependent Protein Kinase ◽  
Physiological Concentration ◽  
Cell Numbers ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Regeneration In Vitro

Both nitric oxide (NO) and natriuretic peptides produce apoptosis of vascular smooth muscle cells. However, there is evidence that NO induces endothelial cell proliferation, which suggests that there is a difference in the response of endothelial cells to natriuretic peptides. The purpose of this study was to investigate the effect of atrial natriuretic peptide (ANP) on human endothelial cell survival. ANP within the physiological concentration (10−11mol/l) induced a 52% increase in the number of human coronary arterial endothelial cells and a 63% increase in human umbilical vein endothelial cells at a low concentration of serum. The increase in cell numbers was blocked by pretreatment with RP8-CPT-cGMP (RP8), a cGMP-dependent protein kinase inhibitor, with wortmannin, an Akt/PKB inhibitor, and with PD-98059, an ERK1/2 inhibitor. In a Transwell migration test, ANP also increased the cell migration, and RP8, wortmannin, and PD-98059 blocked this increase. A wound healing assay was performed to examine the effects of ANP on regeneration in vitro. ANP increased both cell numbers and migration, but the effects were blocked by the above three kinase inhibitors. ANP increased the expression of phospho-Akt and of phospho-ERK1/2 within 1.5 h. These results suggest that ANP can potentiate endothelial regeneration by cGMP-dependent protein kinase stimulation and subsequent Akt and ERK1/2 activations.

scholarly journals Phosphorylation of SOX9 by Cyclic AMP-Dependent Protein Kinase A Enhances SOX9's Ability To Transactivate aCol2a1 Chondrocyte-Specific Enhancer

2000 ◽  
Vol 20 (11) ◽  
pp. 4149-4158 ◽  
Author(s):  
Wendong Huang ◽  
Xin Zhou ◽  
Véronique Lefebvre ◽  
Benoit de Crombrugghe
Keyword(s):  
Protein Kinase ◽  
Dna Binding ◽  
Cyclic Amp ◽  
Protein Kinase A ◽  
Chondrocyte Differentiation ◽  
Phosphorylation Sites ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Kinase A

ABSTRACT Sox9 is a high-mobility-group domain-containing transcription factor required for chondrocyte differentiation and cartilage formation. We used a yeast two-hybrid method based on Son of Sevenless (SOS) recruitment to screen a chondrocyte cDNA library and found that the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA-Cα) interacted specifically with SOX9. Next we found that two consensus PKA phosphorylation sites within SOX9 could be phosphorylated by PKA in vitro and that SOX9 could be phosphorylated by PKA-Cα in vivo. In COS-7 cells cotransfected with PKA-Cα and SOX9 expression plasmids, PKA enhanced the phosphorylation of wild-type SOX9 but did not affect phosphorylation of a SOX9 protein in which the two PKA phosphorylation sites (S64 and S211) were mutated. Using a phosphospecific antibody that specifically recognized SOX9 phosphorylated at serine 211, one of the two PKA phosphorylation sites, we demonstrated that addition of cAMP to chondrocytes strongly increased the phosphorylation of endogenous Sox9. In addition, immunohistochemistry of mouse embryo hind legs showed that Sox9 phosphorylated at serine 211 was principally localized in the prehypertrophic zone of the growth plate, corresponding to the major site of expression of the parathyroid hormone-related peptide (PTHrP) receptor. Since cAMP has previously been shown to effectively increase the mRNA levels of Col2a1 and other specific markers of chondrocyte differentiation in culture, we then asked whether PKA phosphorylation could modulate the activity of SOX9. Addition of 8-bromo-cAMP to chondrocytes in culture increased the activity of a transiently transfected SOX9-dependent 48-bp Col2a1chondrocyte-specific enhancer; similarly, cotransfection of PKA-Cα increased the activity of this enhancer. Mutations of the two PKA phosphorylation consensus sites of SOX9 markedly decreased the PKA-Cα activation of this enhancer by SOX9. PKA phosphorylation and the mutations in the consensus PKA phosphorylation sites of SOX9 did not alter its nuclear localization. In vitro phosphorylation of SOX9 by PKA resulted in more efficient DNA binding. We conclude that SOX9 is a target of cAMP signaling and that phosphorylation of SOX9 by PKA enhances its transcriptional and DNA-binding activity. Because PTHrP signaling is mediated by cAMP, our results support the hypothesis that Sox9 is a target of PTHrP signaling in the growth plate and that the increased activity of Sox9 might mediate the effect of PTHrP in maintaining the cells as nonhypertrophic chondrocytes.

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