scholarly journals The amounts of rat liver cyclic AMP-dependent protein kinase I and II are differentially regulated by diet

1988 ◽  
Vol 256 (2) ◽  
pp. 447-452 ◽  
Author(s):  
R Ekanger ◽  
O K Vintermyr ◽  
S O Døskeland
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Restriction ◽  
Type I ◽  
Short Term ◽  
Rat Hepatocyte ◽  
Dependent Protein Kinase ◽  
Key Enzyme ◽  
Dependent Protein ◽  
Fasted Rats

1. The fluctuations in rat hepatocyte volume and protein content in response to dietary perturbations (starvation, protein restriction, refeeding) were accompanied by corresponding fluctuations in the amount of the regulatory (R) and catalytic (C) subunits of cyclic AMP-dependent protein kinase. Thus the intracellular concentration of this key enzyme was adjusted to be near constant. 2. The adjustment of cellular R was accomplished almost exclusively by regulating cytosolic RI (R subunit of type I kinase). The preferential down-regulation of cytosolic RI in response to starvation/protein restriction indicates that particulate RI and cytosolic as well as particulate RII are more resistant to breakdown during general catabolism in the hepatocyte. 3. The diet-induced fluctuations of kinase subunits were uniformly distributed in all populations of parenchymatous hepatocytes, regardless of their size and density. It is thus possible to isolate hepatocytes with uniformly altered RI/RII ratio from livers of rats with different feeding regimens. 4. The binding of endogenous cyclic AMP to RI and RII was similar in livers with high RI/RII ratio (fed rats) and low RI/RII ratio (fasted rats) as well as in hepatocytes isolated from fasted rats. Under the conditions of the experiment (short-term stimulation by glucagon), therefore, neither the dietary state nor the RI/RII ratio seemed to affect the apparent affinity of the isoreceptors for cyclic AMP. However, RI appeared to show a slightly higher co-operativity of intracellular cyclic AMP binding than did RII in all states.

scholarly journals Photoaffinity labelling of central-nervous-system myelin. Evidence for an endogenous type I cyclic AMP-dependent kinase phosphorylating the larger subunit of 2′,3′-cyclic nucleotide 3′-phosphodiesterase

1984 ◽  
Vol 221 (2) ◽  
pp. 361-368 ◽  
Author(s):  
J M Bradbury ◽  
R J Thompson
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Cyclic Nucleotide ◽  
Type I ◽  
Phosphodiesterase Activity ◽  
Dependent Protein Kinase ◽  
Photoaffinity Labelling ◽  
Dependent Protein

Endogenous cyclic AMP-stimulated phosphorylation of a 49700-Mr Wolfgram protein component in rabbit central nervous system was investigated by using photoaffinity labelling and 2′,3′-cyclic nucleotide 3′-phosphodiesterase activity staining after electroblotting on to nitrocellulose paper. Photoaffinity labelling with 8′-azidoadenosine 3′,5′-cyclic monophosphate showed a cyclic AMP-binding protein that appeared to be intrinsic to the myelin membrane and appeared to represent the R-subunit of a type I cyclic AMP-dependent protein kinase. This photoaffinity-labelled protein was of larger apparent Mr than the protein showing cyclic AMP-stimulated phosphorylation. Blotting of one-dimensional sodium dodecyl sulphate/polyacrylamide-gel electrophoretograms followed by staining for 2′,3′-cyclic nucleotide 3′-phosphodiesterase activity showed two activity bands corresponding to the two components of the Wolfgram protein doublet. Cyclic AMP-stimulated protein phosphorylation corresponded to the upper component of this doublet. Electroblotting of two-dimensional non-equilibrium pH-gradient electrophoretograms also showed co-migration of cyclic AMP-stimulated protein phosphorylation with enzyme activity. It is proposed that central-nervous-system myelin contains an endogenous type I cyclic-AMP dependent protein kinase that phosphorylates the larger subunit of 2′,3′-cyclic nucleotide 3′-phosphodiesterase.

scholarly journals Subcellular Localization and Biological Actions of Activated RSK1 Are Determined by Its Interactions with Subunits of Cyclic AMP-Dependent Protein Kinase

2006 ◽  
Vol 26 (12) ◽  
pp. 4586-4600 ◽  
Author(s):  
Deepti Chaturvedi ◽  
Helen M. Poppleton ◽  
Teresa Stringfield ◽  
Ann Barbier ◽  
Tarun B. Patel
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Nuclear Accumulation ◽  
Regulatory Subunit ◽  
Type I ◽  
Dependent Protein Kinase ◽  
Anchoring Proteins ◽  
Dependent Protein ◽  
Ribosomal S6 Kinase ◽  
Biological Actions

ABSTRACT Cyclic AMP (cAMP)-dependent protein kinase (PKA) and ribosomal S6 kinase 1 (RSK1) share several cellular proteins as substrates. However, to date no other similarities between the two kinases or interactions between them have been reported. Here, we describe novel interactions between subunits of PKA and RSK1 that are dependent upon the activation state of RSK1 and determine its subcellular distribution and biological actions. Inactive RSK1 interacts with the type I regulatory subunit (RI) of PKA. Conversely, active RSK1 interacts with the catalytic subunit of PKA (PKAc). Binding of RSK1 to RI decreases the interactions between RI and PKAc, while the binding of active RSK1 to PKAc increases interactions between PKAc and RI and decreases the ability of cAMP to stimulate PKA. The RSK1/PKA subunit interactions ensure the colocalization of RSK1 with A-kinase PKA anchoring proteins (AKAPs). Disruption of the interactions between PKA and AKAPs decreases the nuclear accumulation of active RSK1 and, thus, increases its cytosolic content. This subcellular redistribution of active RSK1 is manifested by increased phosphorylation of its cytosolic substrates tuberous sclerosis complex 2 and BAD by epidermal growth factor along with decreased cellular apoptosis.

scholarly journals Isolation of two myosin light-chain kinases from bovine carotid artery and their regulation by phosphorylation mediated by cyclic AMP-dependent protein kinase

1982 ◽  
Vol 203 (3) ◽  
pp. 583-592 ◽  
Author(s):  
Ramesh C. Bhalla ◽  
Ram V. Sharma ◽  
Ramesh C. Gupta
Keyword(s):  
Protein Kinase ◽  
Carotid Artery ◽  
Cyclic Amp ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Activity ◽  
Type I ◽  
Type Ii ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Myosin light-chain kinase was purifed from bovine carotid artery. Approx. 90% of myosin kinase was extracted in the supernatant fraction with buffer containing EDTA during myofibril preparation. The soluble fraction yielded two distinct peaks on DEAE-Sephacel chromatography. Peak I was eluted at a conductance of 11–12mmho and was completely dependent on Ca2+–calmodulin for its activity. Peak II was eluted at a conductance of 13–14mmho and showed approx. 15% Ca2+-independent activity. The myosin kinases I and II were further purified by affinity chromatography by using calmodulin coupled to Sepharose 4B, which resulted in 960-and 650-fold purification of type I and type II kinases respectively. Myosin kinase II activity was completely Ca2+-dependent after affinity chromatography on the calmodulin–Sepharose column. Myosin kinases I and II were phosphorylated by cyclic AMP-dependent protein kinase. In the presence of bound calmodulin 0.5–0.7mol of phosphate was incorporated/mol of myosin kinases I and II. On the other hand, in the absence of bound calmodulin 1–1.4mol of phosphate was incorporated/mol of kinases I and II. Phosphorylation in the absence of calmodulin significantly decreased the myosin kinase activity of both enzymes, and the decrease in myosin kinase activity was due to a 3–5-fold increase in the amount of calmodulin required for half-maximal stimulation of both type I and type II kinases. The regulation of myosin kinase activity by cyclic AMP-dependent phosphorylation would suggest that β-adrenergic-mediated relaxation of vascular smooth muscle may be partly due to the direct interaction of cyclic AMP at the site of contractile proteins.

scholarly journals Inhibition of foetal pulmonary choline-phosphate cytidylyltransferase under conditions favouring protein phosphorylation

1985 ◽  
Vol 232 (3) ◽  
pp. 833-840 ◽  
Author(s):  
K Radika ◽  
F Possmayer
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Lung Surfactant ◽  
Microsomal Enzyme ◽  
Dependent Protein Kinase ◽  
Surfactant Production ◽  
Key Enzyme ◽  
Dependent Protein ◽  
Choline Phosphate ◽  
Atp Analogues

Choline-phosphate cytidylyltransferase (EC 2.7.7.15) activity from 25- and 29-day-foetal rabbit lungs was inhibited in both the cytosolic and the microsomal fractions by preincubation with MgATP. The inhibition of the cytosolic enzyme was greater when measured with added phosphatidylglycerol (PG) than without (78-89% versus 50-55%), whereas the inhibition of the microsomal enzyme did not exhibit this distinction (66-72% versus 60-70%). When preincubated with the buffer alone, the cytosolic enzyme was activated to a greater extent by added PG than was the microsomal enzyme (13-14-fold versus 2-3-fold). However, after preincubation with MgATP, the cytosolic enzyme was activated to a smaller extent by added PG (3-6-fold). The inhibition of the enzyme by MgATP required a preincubation and was absent when ADP or AMP was substituted for ATP. Moreover, ATP analogues such as adenosine 5′-[beta, gamma-methylene]triphosphate and adenosine 5′-[γ-thio]triphosphate also failed to inhibit the enzyme when substituted for ATP in the preincubation. The inhibition by MgATP was not affected by including cyclic AMP in the preincubation, but Ca2+ ions alone or plus diacylglycerol in the preincubation increased the inhibition slightly. The inhibition was abolished by including an inhibitor of cyclic-AMP-dependent protein kinase in the preincubation. These observations, taken collectively, point to the inhibition of foetal pulmonary cytidylyltransferase through the phosphorylation of a protein and suggest that this key enzyme in lung surfactant production may be regulated through this mechanism.

scholarly journals Bacterial expression of Chinese hamster regulatory type-I and catalytic subunits of cyclic AMP-dependent protein kinase and mutational analysis of the type-I regulatory subunit

1994 ◽  
Vol 297 (1) ◽  
pp. 79-85 ◽  
Author(s):  
M E Gosse ◽  
R Fleischmann ◽  
M Marshall ◽  
N Wang ◽  
S Garges ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Mutational Analysis ◽  
Novel Mutation ◽  
Chinese Hamster ◽  
Alpha Subunit ◽  
Regulatory Subunit ◽  
Type I ◽  
Dependent Protein Kinase ◽  
Dependent Protein

The type-I regulatory subunit (RI) of the cyclic AMP-dependent protein kinase (PKA) from Chinese hamster ovary (CHO) cells has been cloned and expressed in a strain of BL21(DE3) Escherichia coli lacking adenylate cyclase [BL21(DE3)/delta cya]. RI expressed in this bacterial system free of cyclic AMP is soluble and can reconstitute functional PKA. Recombinant CHO C alpha is predominantly insoluble with some active soluble protein. C beta is entirely insoluble and inactive. Soluble recombinant RI and soluble recombinant C alpha can associate in vitro and be activated by cyclic AMP. Six site-directed mutations of RI were generated to study the interaction of cyclic AMP with RI and RI-C alpha subunit interactions. Four cyclic AMP-binding-site point mutants were generated [W261R (tryptophan to arginine at position 261), a novel mutation in site A; V376G, a novel mutation in site B; G200E (site A), and Y370F (site B), previously described in bovine RI were introduced into the CHO RI for comparison purposes]. Mutants W261R, Y370F, and G200E demonstrated decreased 8-N3-[3H]cyclic AMP binding as well as 5-fold reduced affinity for [3H]cyclic AMP, with threefold increased EC50 values for cyclic AMP activation of kinase activity from reconstituted mutant holoenzymes. The mutation at V376G did not alter cyclic AMP binding or activation by cyclic AMP of mutant holoenzyme. A truncation mutant, G200Stop, which lacks both cyclic AMP-binding sites, did not bind cyclic AMP but can inhibit C alpha subunit activity. A novel mutation outside the cyclic AMP-binding regions of RI (V89A) weakened the interaction with C alpha indicated by a 7-fold lower EC50 for mutant holoenzyme activation by cyclic AMP.

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