scholarly journals Activity and regulation of calcium-, phospholipid-dependent protein kinase in differentiating chick myogenic cells.

1989 ◽  
Vol 108 (1) ◽  
pp. 153-158 ◽  
Author(s):  
S Adamo ◽  
C Caporale ◽  
C Nervi ◽  
R Ceci ◽  
M Molinaro
Keyword(s):  
Protein Kinase ◽  
Chick Embryo ◽  
Myogenic Differentiation ◽  
Tumor Promoter ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Myogenic Cells ◽  
Dramatic Decline ◽  
Dependent Protein ◽  
The Difference

The activity of calcium-, phospholipid-dependent protein kinase (PKc) was measured in (a) total extracts, (b) crude membrane, and (c) cytosolic fractions of chick embryo myogenic cells differentiating in culture. Total PKc activity slowly declines during the course of terminal myogenesis in contrast to the activity of cAMP-dependent protein kinase, which was also measured in the same cells. Myogenic cells at day 1 of culture possess high particulate and low soluble PKc activity. A dramatic decline of particulate PKc activity occurs during myogenic cell differentiation and is accompanied, through day 4, by a striking rise of the soluble activity. The difference in the subcellular distribution of PKc between replicating myoblasts and myotubes is confirmed by phosphorylation studies conducted in intact cells. These studies demonstrate that four polypeptides whose phosphorylation is stimulated by the tumor promoter 12-O-tetradecanoyl phorbol 13-acetate in myotubes, are spontaneously phosphorylated in control myoblasts. Phosphoinositide turnover under basal conditions in [3H]inositol-labeled cells is faster in myoblasts than in myotubes, a finding that may in part explain the different distribution of PKc observed during the course of myogenic differentiation.

scholarly journals The Commonly Used cGMP-dependent Protein Kinase Type I (cGKI) Inhibitor Rp-8-Br-PET-cGMPS Can Activate cGKIin Vitroand in Intact Cells

2008 ◽  
Vol 284 (1) ◽  
pp. 556-562 ◽  
Author(s):  
Nadejda Valtcheva ◽  
Peter Nestorov ◽  
Alexander Beck ◽  
Michael Russwurm ◽  
Matthias Hillenbrand ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Type I ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

scholarly journals Phosphorylation of β-Catenin by Cyclic AMP-Dependent Protein Kinase Stabilizes β-Catenin through Inhibition of Its Ubiquitination

2005 ◽  
Vol 25 (20) ◽  
pp. 9063-9072 ◽  
Author(s):  
Shin-ichiro Hino ◽  
Chie Tanji ◽  
Keiichi I. Nakayama ◽  
Akira Kikuchi
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Wnt Signaling ◽  
Protein Level ◽  
Glycogen Synthase ◽  
Wnt Signaling Pathway ◽  
Prostaglandin E ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Dependent Protein

ABSTRACT The mechanism of cross talk between the Wnt signaling and cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]) pathways was studied. Prostaglandin E1 (PGE1), isoproterenol, and dibutyryl cAMP (Bt2cAMP), all of which activate PKA, increased the cytoplasmic and nuclear β-catenin protein level, and these actions were suppressed by a PKA inhibitor and RNA interference for PKA. PGE1 and Bt2cAMP also increased T-cell factor (Tcf)-dependent transcription through β-catenin. Bt2cAMP suppressed degradation of β-catenin at the protein level. Although PKA did not affect the formation of a complex between glycogen synthase kinase 3β (GSK-3β), β-catenin, and Axin, phosphorylation of β-catenin by PKA inhibited ubiquitination of β-catenin in intact cells and in vitro. Ser675 was found to be a site for phosphorylation by PKA, and substitution of this serine residue with alanine in β-catenin attenuated inhibition of the ubiquitination of β-catenin by PKA, PKA-induced stabilization of β-catenin, and PKA-dependent activation of Tcf. These results indicate that PKA inhibits the ubiquitination of β-catenin by phosphorylating β-catenin, thereby causing β-catenin to accumulate and the Wnt signaling pathway to be activated.

scholarly journals Regulation of Microtubule Dynamics by Extracellular Signals: cAMP-dependent Protein Kinase Switches Off the Activity of Oncoprotein 18 in Intact Cells

1998 ◽  
Vol 140 (1) ◽  
pp. 131-141 ◽  
Author(s):  
Helena Melander Gradin ◽  
Niklas Larsson ◽  
Ulrica Marklund ◽  
Martin Gullberg
Keyword(s):  
Protein Kinase ◽  
Genetic System ◽  
Cell Surface Receptor ◽  
In Vitro Assays ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Camp Dependent Protein Kinase ◽  
Oncoprotein 18 ◽  
Dependent Protein

Oncoprotein 18 (Op18, also termed p19, 19K, metablastin, stathmin, and prosolin) is a recently identified regulator of microtubule (MT) dynamics. Op18 is a target for both cell cycle and cell surface receptor-coupled kinase systems, and phosphorylation of Op18 on specific combinations of sites has been shown to switch off its MT-destabilizing activity. Here we show that induced expression of the catalytic subunit of cAMP-dependent protein kinase (PKA) results in a dramatic increase in cellular MT polymer content concomitant with phosphorylation and partial degradation of Op18. That PKA may regulate the MT system by downregulation of Op18 activity was evaluated by a genetic system allowing conditional co-expression of PKA and a series of kinase target site–deficient mutants of Op18. The results show that phosphorylation of Op18 on two specific sites, Ser-16 and Ser-63, is necessary and sufficient for PKA to switch off Op18 activity in intact cells. The regulatory importance of dual phosphorylation on Ser-16 and Ser-63 of Op18 was reproduced by in vitro assays. These results suggest a simple model where PKA phosphorylation downregulates the MT-destabilizing activity of Op18, which in turn promotes increased tubulin polymerization. Hence, the present study shows that Op18 has the potential to regulate the MT system in response to external signals such as cAMP-linked agonists.

scholarly journals Hormone-induced protein phosphorylation. III. regulation of the phosphorylation of the secretagogue-responsive 29,000-dalton protein by both Ca2+ and cAMP in vitro.

1982 ◽  
Vol 95 (3) ◽  
pp. 918-923 ◽  
Author(s):  
S D Freedman ◽  
J D Jamieson
Keyword(s):  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Exocrine Pancreas ◽  
Second Messengers ◽  
Subcellular Fractions ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Dependent Protein

In the preceding papers, we demonstrated that the endogenous phosphorylation of a 29,000-dalton protein is stimulated in response to secretagogue application to intact cells from the rat exocrine pancreas and parotid and dephosphorylated upon termination of secretagogue action. One- and two-dimensional gel analysis of 32Pi-labeled pancreatic and parotid lobules as well as their respective subcellular fractions revealed that the same protein was covalently modified in both tissues and was localized to the ribosomal fraction. To identify the intracellular second messengers which may mediate or modulate the phosphorylation of the 29,000-dalton protein in intact cells, the effects of Ca2+, cAMP, and cGMP on the endogenous phosphorylation of this protein were assessed in subcellular fractions from the rat pancreas and parotid. Our results demonstrate that the phosphorylation of the 29,000-dalton polypeptide may be regulated by both Ca2+ and cAMP in the pancreas and in the parotid. No cGMP-dependent protein phosphorylation was found in either tissue. As in the in situ phosphorylation studies, the Ca2+- and cAMP-dependent phosphorylation of this same protein was localized to the ribosomal fraction. The cAMP-dependent protein kinase activity was found primarily in the postmicrosomal supernatant in contrast to the Ca2+-dependent protein kinase that appeared to be tightly associated with the substrate in addition to being present in the postmicrosomal supernatant. The data suggest that, in cells from the exocrine pancreas and parotid, secretagogues may regulate the phosphorylation of the 29,000-dalton protein through Ca2+ and/or cAMP.

scholarly journals Characterization of Sp-5,6-dichloro-1-β-d-ribofuranosylbenzimidazole- 3′,5′-monophosphorothioate (Sp-5,6-DCl-cBiMPS) as a potent and specific activator of cyclic-AMP-dependent protein kinase in cell extracts and intact cells

1991 ◽  
Vol 279 (2) ◽  
pp. 521-527 ◽  
Author(s):  
M Sandberg ◽  
E Butt ◽  
C Nolte ◽  
L Fischer ◽  
M Halbrügge ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Prostaglandin E1 ◽  
Human Platelets ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Cell Extracts ◽  
Platelet Membranes ◽  
Camp Analogue ◽  
Dependent Protein

A newly designed cyclic AMP (cAMP) analogue, Sp-5,6-dichloro-1-beta-D- ribofuranosylbenzimidazole-3′,5′-monophosphorothioate (Sp-5,6-DCl-cBiMPS), and 8-(p-chlorophenylthio)-cAMP (8-pCPT-cAMP) were compared with respect to their chemical and biological properties in order to assess their potential as activators of the cAMP-dependent protein kinases (cAMP-PK) in intact cells. Sp-5,6-DCl-cBiMPS was shown to be both a potent and specific activator of purified cAMP-PK and of cAMP-PK in platelet membranes, whereas 8-pCPT-cAMP proved to be a potent activator of cAMP-PK and cyclic-GMP-dependent protein kinase (cGMP-PK) both as purified enzymes and in platelet membranes. Sp-5,6-DCl-cBiMPS was not significantly hydrolysed by three types of cyclic nucleotide phosphodiesterases, whereas 8-pCPT-cAMP (and 8-bromo-cAMP) was hydrolysed to a significant extent by the Ca2+/calmodulin-dependent phosphodiesterase and by the cGMP-inhibited phosphodiesterase. The apparent lipophilicity, a measure of potential cell-membrane permeability, of Sp-5,6-DCl-cBiMPS was higher than that of 8-pCPT-cAMP. Extracellular application of Sp-5,6-DCl-cBiMPS to intact human platelets reproduced the pattern of protein phosphorylation induced by prostaglandin E1, a cAMP-increasing inhibitor of platelet activation. In intact platelets, Sp-5,6- DCl-cBiMPS was also more effective than 8-pCPT-cAMP in inducing quantitative phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein (VASP), a major substrate of cAMP-PK in platelets. As observed with prostaglandin E1, pretreatment of human platelets with Sp-5,6-DCl-cBiMPS prevented the aggregation induced by thrombin. The results suggest that Sp-5,6-DCl-cBiMPS is a very potent and specific activator of cAMP-PK in cell extracts and intact cells and, in this respect, is superior to any other cAMP analogue used for intact-cell studies. In contrast with 8-pCPT-cAMP, Sp-5,6-DCl-cBiMPS can be used to distinguish the signal-transduction pathways mediated by cAMP-PK and cGMP-PK.

scholarly journals Enzymic characteristics of an ecto-cyclic AMP-dependent protein kinase in rat epididymal spermatozoa

1981 ◽  
Vol 195 (1) ◽  
pp. 111-117 ◽  
Author(s):  
G C Majumder
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
External Surface ◽  
Cell Damage ◽  
Surface Protein ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Dependent Protein

Intact spermatozoa from rat cauda epididymides possess an ecto-(cyclic AMP-dependent protein kinase) activity that causes the transfer of the terminal phosphate group of ATP to the serine residues of all the histone fractions. The enzyme showed a high degree of substrate specificity for the phosphorylation of histones rather than protamine, casein and phosvitin. The cell-external-surface protein kinase requires Mg2+ for activity, and other bivalent cations such as Mn2+ and Co2+ can substitute partially for Mg2+, whereas Ca2+ and Zn2+ are potent inhibitors of the enzyme. The enzyme has markedly higher affinity for cyclic AMP than for other cyclic nucleotides for its activation, with an apparent Km value for cyclic AmP of 80 nM. Spermatozoal ecto-kinase activity is not due to contamination of broken cells or any possible cell damage during incubation and isolation of spermatozoa. There was no loss of kinase activity from the cells when washed with 2 mM-EDTA, and the histones phosphorylated by intact spermatozoa were located outside the cells. Protein kinase activity of intact cells was strongly inhibited (approx. 90%) by p-chloromercuribenzenesulphonic acid (10 microM), which is believed not to enter the cells. These data provide further support for the localization of a protein kinase on the external surface of spermatozoa.

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