scholarly journals ADP-ribosylation of Rho proteins by Clostridium botulinum exoenzyme C3 is influenced by phosphorylation of Rho-associated factors

1994 ◽  
Vol 300 (1) ◽  
pp. 133-139 ◽  
Author(s):  
G Fritz ◽  
K Aktories
Keyword(s):  
Alkaline Phosphatase ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Protein Kinase A ◽  
Clostridium Botulinum ◽  
Rho Proteins ◽  
Cho Cell ◽  
Adp Ribosylation ◽  
Cell Extracts ◽  
Kinase A

Specific [32P]ADP-ribosylation by Clostridium botulinum exoenzyme C3 was used to study the involvement of phosphorylation in the regulation of the low-molecular-mass GTP-binding protein Rho. Dephosphorylation of CHO cell extracts by alkaline phosphatase treatment resulted in a 80-90% reduction in the C3-catalysed [32P]ADP-ribosylation of Rho proteins in both cytosolic and membrane fractions. Similar results were obtained after dephosphorylation with protein phosphatase type-1 from bovine retina, whereas type-2B and type-2C phosphatases had no effect on the level of subsequent [32P]ADP-ribosylation of Rho by C3. Incubation of CHO cell lysate under phosphorylation conditions increased the subsequent C3-mediated [32P]ADP-ribosylation of Rho proteins. The protein kinase inhibitors H7 and H9 had no effect on [32P]ADP-ribosylation at concentrations which are specific for inhibition of protein kinase A or C. Recombinant glutathione S-transferase-RhoA fusion protein (GST-RhoA) was phosphorylated by protein kinase A; however, the phosphorylation had no stimulatory effect on the ADP-ribosylation of GST-RhoA by C3. An approx. 48 kDa phosphoprotein was identified which bound specifically to recombinant GST-RhoA fusion protein. By gel-permeation chromatography, Rho-containing complexes of approx. 50 kDa and 130-170 kDa were detected. The ADP-ribosylation of Rho in the 130-170 kDa complex was reduced by alkaline phosphatase pretreatment. The data suggest that Rho activity is influenced by phosphorylation of Rho-associated regulatory factors. Phosphorylation/dephosphorylation of these Rho-regulating factors appears to alter the ability of Rho to serve as a substrate for C3-induced [32P]ADP-ribosylation.

scholarly journals Effect of Protein Kinase A Activity on the Association of ADP-ribosylation Factor 1 to Golgi Membranes

2000 ◽  
Vol 275 (25) ◽  
pp. 19050-19059 ◽  
Author(s):  
Maria Esther Martı́n ◽  
Josefina Hidalgo ◽  
Jose Luis Rosa ◽  
Pascal Crottet ◽  
Angel Velasco
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Adp Ribosylation ◽  
Golgi Membranes ◽  
Adp Ribosylation Factor ◽  
Kinase A

cAMP increases liver Na+-taurocholate cotransport by translocating transporter to plasma membranes

1997 ◽  
Vol 273 (4) ◽  
pp. G842-G848 ◽  
Author(s):  
Sunil Mukhopadhayay ◽  
M. Ananthanarayanan ◽  
Bruno Stieger ◽  
Peter J. Meier ◽  
Frederick J. Suchy ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Protein Kinase A ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Rat Hepatocytes ◽  
Plasma Membrane Vesicles ◽  
Short Term ◽  
Kinase A

Adenosine 3′,5′-cyclic monophosphate (cAMP), acting via protein kinase A, increases transport maximum of Na+-taurocholate cotransport within 15 min in hepatocytes (S. Grüne, L. R. Engelking, and M. S. Anwer. J. Biol. Chem. 268: 17734–17741, 1993); the mechanism of this short-term stimulation was investigated. Cycloheximide inhibited neither basal nor cAMP-induced increases in taurocholate uptake in rat hepatocytes, indicating that cAMP does not stimulate transporter synthesis. Studies in plasma membrane vesicles showed that taurocholate uptake was not stimulated by the catalytic subunit of protein kinase A but was higher when hepatocytes were pretreated with cAMP. Immunoblot studies with anti-fusion protein antibodies to the cloned Na+-taurocholate cotransport polypeptide (Ntcp) showed that pretreatment of hepatocytes with cAMP increased Ntcp content in plasma membranes but not in homogenates. Ntcp was detected in microsomes, endosomes, and Golgi fractions, and cAMP pretreatment resulted in a decrease only in endosomal Ntcp content. It is proposed that cAMP increases transport maximum of Na+-taurocholate cotransport, at least in part, by translocating Ntcp from endosomes to plasma membranes.

scholarly journals The cross-regulation of Gi-protein by cholera toxin involves a phosphorylation by protein kinase A

1995 ◽  
Vol 306 (3) ◽  
pp. 765-769 ◽  
Author(s):  
R Levistre ◽  
M Berguerand ◽  
G Bereziat ◽  
J Masliah
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Cholera Toxin ◽  
Inhibitory Effect ◽  
Negative Control ◽  
Adp Ribosylation ◽  
Gi Proteins ◽  
Kinase A

Pretreatment of alveolar macrophages with cholera toxin inhibits the release of arachidonic acid induced by the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine. The results presented here show that cholera toxin might exert its inhibitory effect through the phosphorylation of Gi alpha by protein kinase A (PKA). (1) Gi-proteins from cells pretreated with cholera toxin showed parallel increases in their sensitivity to ADP-ribosylation by toxins in vitro and in Gi alpha phosphorylation. By contrast, the Gi alpha concentration was unchanged. (2) Cholera toxin pretreatment also decreased the functional activity of Gi, as assessed by the inhibition (80%) of agonist-induced binding of guanosine-5′-[gamma-thio]triphosphate (GTP[gamma S]). (3) These effects of cholera toxin were blocked by a specific PKA inhibitor, N-(2-[methyl-amino]ethyl)-3-isoquinolinesulphonamide dihydrochloride (H8) and mimicked by a cyclic AMP (cAMP) analogue and a phosphatase inhibitor. (4) Gi alpha was also phosphorylated in vitro by the catalytic subunit of PKA. In contrast with other cell systems, the stimulation of protein kinase C seems to have no effect on the sensitivity of Gi to ADP-ribosylation or on its phosphorylation. Therefore, the phosphorylation of Gi-proteins by PKA seems to be the actual target of the negative control of arachidonic acid release via the cAMP-mediated pathway.

AtPIP5K1, an Arabidopsis thaliana phosphatidylinositol phosphate kinase, synthesizes PtdIns(3,4)P2 and PtdIns(4,5)P2 in vitro and is inhibited by phosphorylation

2001 ◽  
Vol 359 (3) ◽  
pp. 583-589 ◽  
Author(s):  
Tomas WESTERGREN ◽  
Stephen K. DOVE ◽  
Marianne SOMMARIN ◽  
Christophe PICAL
Keyword(s):  
Arabidopsis Thaliana ◽  
Alkaline Phosphatase ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Substrate Specificities ◽  
Phosphatidylinositol Phosphate ◽  
Dual Substrate Specificity ◽  
Kinase A ◽  
Dual Substrate

PtdIns phosphate kinases (PIPkins), which generate PtdInsP2 isomers, have been classified into three subfamilies that differ in their substrate specificities. We demonstrate here that the previously identified AtPIP5K1 gene from Arabidopsis thaliana encodes a PIPkin with dual substrate specificity in vitro, capable of phosphorylating PtdIns3P and PtdIns4P to PtdIns(3,4)P2 and PtdIns(4,5)P2 respectively. We also show that recombinant AtPIP5K1 is phosphorylated by protein kinase A and a soluble protein kinase from A. thaliana. Phosphorylation of AtPIP5K1 by protein kinase A is accompanied by a 40% inhibition of its catalytic activity. Full activity is recovered by treating phosphorylated AtPIP5K1 with alkaline phosphatase.

scholarly journals Candida albicans Lacking the Gene Encoding the Regulatory Subunit of Protein Kinase A Displays a Defect in Hyphal Formation and an Altered Localization of the Catalytic Subunit

2004 ◽  
Vol 3 (1) ◽  
pp. 190-199 ◽  
Author(s):  
Alejandro Cassola ◽  
Marc Parrot ◽  
Susana Silberstein ◽  
Beatrice B. Magee ◽  
Susana Passeron ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Protein Kinase A ◽  
Double Mutant ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Gfp Fusion Protein ◽  
Kinase A

ABSTRACT The fungal pathogen Candida albicans switches from a yeast-like to a filamentous mode of growth in response to a variety of environmental conditions. We examined the morphogenetic behavior of C. albicans yeast cells lacking the BCY1 gene, which encodes the regulatory subunit of protein kinase A. We cloned the BCY1 gene and generated a bcy1 tpk2 double mutant strain because a homozygous bcy1 mutant in a wild-type genetic background could not be obtained. In the bcy1 tpk2 mutant, protein kinase A activity (due to the presence of the TPK1 gene) was cyclic AMP independent, indicating that the cells harbored an unregulated phosphotransferase activity. This mutant has constitutive protein kinase A activity and displayed a defective germinative phenotype in N-acetylglucosamine and in serum-containing medium. The subcellular localization of a Tpk1-green fluorescent protein (GFP) fusion protein was examined in wild-type, tpk2 null, and bcy1 tpk2 double mutant strains. The fusion protein was observed to be predominantly nuclear in wild-type and tpk2 strains. This was not the case in the bcy1 tpk2 double mutant, where it appeared dispersed throughout the cell. Coimmunoprecipitation of Bcy1p with the Tpk1-GFP fusion protein demonstrated the interaction of these proteins inside the cell. These results suggest that one of the roles of Bcy1p is to tether the protein kinase A catalytic subunit to the nucleus.

Insulin sensitizes beta-agonist and forskolin-stimulated lipolysis to inhibition by 2',5'-dideoxyadenosine

1996 ◽  
Vol 270 (2) ◽  
pp. C562-C569 ◽  
Author(s):  
Y. Gokmen-Polar ◽  
E. C. Coronel ◽  
S. W. Bahouth ◽  
J. N. Fain
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Beta Agonist ◽  
Camp Accumulation ◽  
Fat Cell ◽  
Beta Agonists ◽  
Cell Extracts ◽  
Dependent Protein ◽  
Cellular Compartments ◽  
Kinase A

In isolated rat adipocytes incubated in the absence of insulin, 2',5'-dideoxyadenosine blocked the increase in total adenosine 3',5'-cyclic monophosphate (cAMP) accumulation due to beta 1- or beta 3-catecholamine agonists and forskolin without affecting their stimulation of lipolysis. The inhibition of cAMP accumulation by 2',5'-dideoxyadenosine was not reflected in the total cytosolic cAMP-dependent protein kinase A activity, suggesting that the inhibition of cAMP occurred in cellular compartments distinct from those involved in the regulation of bulk protein kinase A activity. However, there was a good correlation between effects of lipolytic agents on cytosolic protein kinase A activity in fat cell extracts and lipolysis. Furthermore, it was possible to see an inhibition of the increase due to beta-agonists in cAMP accumulation, protein kinase A activity, and lipolysis by 2',5'-dideoxyadenosine in the presence of insulin. These data suggest that the readily measurable accumulation of cAMP seen with catecholamines in the absence of insulin is in a compartment separate from that involved in protein kinase A activation.

scholarly journals Phosphorylation-Dependent SERS Readout for Activity Assay of Protein Kinase A in Cell Extracts

Nanomaterials ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 575
Author(s):  
Renyong Liu ◽  
Chenggen Xie ◽  
Yehan Yan ◽  
Lin Hu ◽  
Suhua Wang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Biological Samples ◽  
Cell Function ◽  
Activity Assay ◽  
Cell Extracts ◽  
Disease Diagnostics ◽  
Wide Range ◽  
Surface Enhanced Raman ◽  
Kinase A

Protein kinases are key regulators of cell function, the abnormal activity of which may induce several human diseases, including cancers. Therefore, it is of great significance to develop a sensitive and reliable method for assaying protein kinase activities in real biological samples. Here, we report the phosphorylation-dependent surface-enhanced Raman scattering (SERS) readout of spermine-functionalized silver nanoparticles (AgNPs) for protein kinase A (PKA) activity assay in cell extracts. In this assay, the presence of PKA would phosphorylate and alter the net charge states of Raman dye-labeled substrate peptides, and the resulting anionic products could absorb onto the AgNPs with cationic surface charge through electrostatic attraction. Meanwhile, the Raman signals of dyes labeled on peptides were strongly enhanced by the aggregated AgNPs with interparticle hot spots formed in assay buffer. The SERS readout was directly proportional to the PKA activity in a wide range of 0.0001–0.5 U·μL−1 with a detection limit as low as 0.00003 U·μL−1. Moreover, the proposed SERS-based assay for the PKA activity was successfully applied to monitoring the activity and inhibition of PKA in real biological samples, particularly in cell extracts, which would be beneficial for kinase-related disease diagnostics and inhibitor screening.

scholarly journals Protein Kinase A-dependent Transactivation by the E2A-Pbx1 Fusion Protein

1995 ◽  
Vol 270 (43) ◽  
pp. 25340-25343 ◽  
Author(s):  
Atsushi Ogo ◽  
Michael R. Waterman ◽  
Mark P. Kamps ◽  
Norio Kagawa
Keyword(s):  
Protein Kinase ◽  
Fusion Protein ◽  
Protein Kinase A ◽  
Kinase A
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