Cyclic AMP stimulates the protein tyrosine kinase activity of Acinetobacter calcoaceticus

The protein tyrosine kinase activity of Acinetobacter calcoaceticus was analyzed in vitro through the specific phosphorylation of an endogenous protein which is modified exclusively at tyrosine residues. A strong stimulation of this activity by cyclic AMP was observed. This finding represents the first example of a protein tyrosine kinase, in prokaryotes as well as in eukaryotes, whose functioning is cyclic nucleotide-dependent.     

Localization in the synaptic junction of the cyclic AMP stimulated intrinsic protein kinase activity of synaptosomal plasma membranes.

Synaptosomal plasma membrane fragments contain a tightly bound protein kinase which can catalyse the phosphorylation of endogenous protein the reaction bein stimulated by cyclic AMP. A fraction enriched in synaptic junctions, which can be isolated from Triton X-100-treated synaptosomal plasma membranes, is also enriched in the cyclic AMP stimulated intrinsic protein kinase. The location of the enzyme in the synaptic junction suggests that cyclic AMP-stimulated phosphorylation may have some role in synaptic transmission.     

Cyclic AMP stimulated protein kinase activity within the secretory vesicle fraction of rat islets.

The isolated rat islet secretory vesicle fraction (24,000xg) was incubated with gamma [³²P ATP] and the TCA-insoluble material was pelleted. The pellet was acid hydrolyzed, lyophilized, redissolved, then subjected to two-dimensional chromatographic separation. Two labeled compounds were identified, i.e., phosphoserine and inorganic phosphorus. With the addition of cyclic AMP (3.5 × 10^?6M), there was a 235% increase in phosphoserine radioactivity (P<.01) within endogenous protein subunits. When histones were added to the incubation media, the addition of cyclic AMP resulted in a threefold increase in phosphoserine radioactivity in the TCA-insoluble material (P<.01). A comparison was made of cyclic AMP-stimulated protein kinase activity in the homogenate and various islet subcellular fractions. Cyclic AMP-stimulated protein kinase activity is associated with the 24,000xg (secretory vesicle) fraction.     

Cyclic adenosine monophosphate-dependent and -independent protein kinase activity of renal brush border membranes.

Kinase(s) in brush border membranes, isolated from rabbit renal proximal tubules, phosphorylated proteins intrinsic to the membrane and exogenous proteins. cAMP stimulated phosphorylation of histone; phosphorylation of protamine was cAMP independent. cAMP-dependent increases in phosphorylation of endogenous membrane protein were small, but highly reproducible. Most of the ³²P incorporated into membranes represented phosphorylation of serine residues, with phosphorylthreonine comprising a minor component. cAMP did not alter the electrophoretic pattern of ³²P-labeled membrane polypeptides. The small cAMP-dependent phosphorylation of brush border membrane proteins was not due to membrane phosphodiesterase or adenylate cyclase activities. Considerable cAMP was found “endogenously” bound to the membranes as prepared. However, this did not result in preactivation of the kinase since activity was not inhibited by a heat-stable protein inhibitor of cAMP-dependent protein kinases. With intrinsic membrane protein as phosphate acceptor, the relationship between rate of phosphorylation and ATP concentration appeared to follow Michaelis-Menton kinetics. With histone the relationship was complex. cAMP did not affect the apparent K_m for histone. One-half maximal stimulation of the rate of histone phosphorylation was obtained with 7 × 10^?8m cAMP. The K_a values for dibutyryl cAMP, cIMP, and cGMP were one to two orders of magnitude greater. Treatment of brush border membranes with detergent greatly increased the dependency of histone phosphorylation on cAMP. Phosphorylations of intrinsic membrane protein and histone were nonlinear with time, due in part to the lability of the protein kinase, the hydrolysis of ATP, and minimally to the presence of phosphoprotein phosphatase in the border membrane. The membrane phosphoprotein phosphatase was unaffected by cyclic nucleotides. Protein kinase activity was also found in cytosolic and crude particulate fractions of the renal cortex. Activity was enriched in the brush border membrane relative to that in the crude membrane preparation. The kinase activities in the different loci were distinct both in relative activities toward different substrates and in responsiveness to cAMP.     

Cyclic AMP induced inhibition of pyruvate kinase flux in the intact liver cell.

The rate of pyruvate kinase flux in the intact cell is estimated by a new procedure, involving trapping of ¹⁴C from NaH¹⁴CO₃ in a large pyruvate + lactate pool, and calculation of the specific activity of phosphoenol pyruvate. With high concentrations of pyruvate as substrate for isolated rat liver cells, cyclic AMP (0.1 mM) depresses pyruvate kinase flux by about 45%, in addition to inhibiting both glucose and lactate formation. The inhibition of pyruvate kinase may cause an inhibition of hydrogen translocation from the mitochondria to the cytosol.     

Cyclic AMP metabolism by swine adipocyte microsomal and plasma membranes

Extracellular cyclic AMP is source of extracellular adenosine in brain and kidney. Whether this occurs in adipose tissue is unknown. The present study evaluated the capacity of swine adipocyte plasma membranes to metabolize cyclic AMP to AMP and adenosine, via phosphodiesterase (PDE) and 5'-nucleotidase (5'-NT), respectively. Plasma membranes (PM) and microsomal membranes (MM) were isolated from over-the-shoulder subcutaneous adipose tissue of 3 month-old male miniature swine. The purity of the membrane fractions was determined and PDE and 5'-NT activities in PM and MM fractions were corrected for cross-contamination. The maximal activity of MM-PDE was 7-fold greater than that of PM-PDE. MM-PDE was 100% inhibited by 5 microM cilostamide, while PM-PDE was unaffected by this PDE3B inhibitor. Inhibitors of PDE1, PDE2, PDE4 and PDE5 also failed to inhibit PM-PDE. However, 1 mM DPSPX inhibited PM-PDE activity by 72%. When PM were incubated with 0.8 microM cyclic AMP for 20 min, AMP accumulation was four times that of adenosine. These data demonstrate that cyclic AMP can be converted to AMP and adenosine by the PM-bound enzymes 5'-NT and PDE, and suggest that the PM-PDE responsible for extracellular cyclic AMP metabolism to AMP is distinct from the intracellular MM-PDE.     

Characterization of the protein kinase activity in beet root plasma membranes

Two protein kinase activities were found in plasma membrane-enriched preparations from red beet ( Beta vulgarix L.). The kinases in these preparations produced the phosphorylation of several membrane polypeptides. These kinases also phosphorylated histone III-S and casein. The activities of two different kinases could be distinguished: one was half-maximally stimulated by 1 μ M free Ca²⁺ phosphorylated histone III-S better than casein, showed half-maximal activity at an ATP concentration of 0.071 m M . had an optimum pH of 7, and was poorly inhibited by GTP, CTP or UTP. Another, much lower, kinase activity that phosphorylated casein was also observed; it was Ca²⁺ independent, showed half-maximal activity at ATP concentrations of 0.017 and 0.287 m M , exhibited a broad pH optimum about pH 7 and was inhibited by GTP, CTP, UTP or GDP to a greater extent than the calcium-stimulated activity. When plasma membrane proteins were solubilized with lysophosphatidyicholine and treated with [γ-³²P]ATP at several dilutions, a 125-kDa polypeptide was autophosphorylated in the absence of Ca²⁺, while 77-, 71- and 65-kDa polypeptides were autophosphorylated in its presence. Autophosphorylation in gels after electrophoresis showed a Ca²⁺-stimulated phosphoprotein band at 64 kDa.     

Cyclic AMP-dependent protein kinase activity in Trypanosoma cruzi.

A cyclic AMP-dependent protein kinase activity from epimastigote forms of Trypanosoma cruzi was characterized. Cytosolic extracts were chromatographed on DEAE-cellulose columns, giving two peaks of kinase activity, which were eluted at 0.15 M- and 0.32 M-NaCl respectively. The second activity peak was stimulated by nanomolar concentrations of cyclic AMP. In addition, a cyclic AMP-binding protein co-eluted with the second kinase activity peak. Cyclic AMP-dependent protein kinase activity was further purified by gel filtration, affinity chromatography on histone-agarose and cyclic AMP-agarose, as well as by chromatography on CM-Sephadex. The enzyme ('holoenzyme') could be partially dissociated into two different components: 'catalytic' and 'regulatory'. The 'regulatory' component had specific binding for cyclic AMP, and it inhibited phosphotransferase activity of the homologous 'catalytic component' or of the 'catalytic subunit' from bovine heart. Cyclic AMP reversed these inhibitions. A 'holoenzyme preparation' was phosphorylated in the absence of exogenous phosphate acceptor and analysed by polyacrylamide-gel electrophoresis. A 56 kDa band was phosphorylated. The same preparation was analysed by Western blotting, by using polyclonal antibodies to the regulatory subunits of protein kinases type I or II. Both antibodies reacted with the 56 kDa band.     

Tyrosine protein kinase activity in renal brush-border membranes.

Tyrosine protein kinase (TPK) activity was detected in rat renal brush-border membranes (BBM) using poly(Glu80Na,Tyr20) as a substrate. Maximal TPK activity required prior detergent dispersion of the membranes with 0.05% Triton X-100 and the presence of vanadate, a potent inhibitor of phosphotyrosine protein phosphatases, in the phosphorylation medium. Optimal conditions for measurement of TPK activity were 10 mM of MgCl2 and MnCl2, at 30 degrees C and pH 7.0. TPK activity was inhibited by genistein, with a IC50 value of 15 microM, while no inhibition was observed in the presence of 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H7), an inhibitor of serine-threonine kinases. TPK activity was enriched 4-fold in the BBM fraction relative to cortex homogenate. It was co-enriched with BBM enzyme markers, but not with those of the basolateral membrane (BLM). The endogenous substrates of TPK in brush-border and basolateral membranes were determined by Western blot analysis using an antiphosphotyrosine monoclonal antibody (PY20). Various phosphotyrosine-containing proteins were found in the BBM (31, 34, 46, 50, 53, 72, 90, 118 and 170 kDa) and in the BLM (37, 48, 50, 53, 72, 90, 130 and 170 kDa). Addition of exogenous insulin receptor to BBM and BLM increased the phosphorylation of most of the substrates. Solubilization of the TPK activity from BBM with 0.5% CHAPS and subsequent gel filtration on Superdex 75 yielded two peaks of tyrosine protein kinase activity with apparent molecular masses of 49 and 66 kDa. These results provide evidence for a non-receptor TPK activity associated with the renal tubular luminal membrane.     

Cyclic AMP binding proteins and cyclic AMP-dependent protein kinase from Blastocladiella emersonii.

The stoichiometry of cyclic AMP binding protein to cyclic AMP in sporulating cells of Blastocladiella emersonii and the resistance of protein-bound cyclic AMP to enzyme-catalyzed hydrolysis suggest that the distribution of cyclic AMP between free and protein-bound pools is an important factor in cyclic AMP metabolism. Most but not all of the cyclic AMP binding protein in sporulating cells is associated with a cyclic AMP-dependent protein kinase.     

Bilirubin inhibition of protein kinase: its prevention by cyclic AMP.

The effect of bilirubin on protein kinase activity has been studied. Bilirubin inhibits the binding of cyclic AMP to protein kinase. It also inhibits the phosphorylation of histone. The inhibitory action of bilirubin is competitive, and increased amounts of cyclic AMP relieved the inhibition.     

A GTP-specific protein kinase in plasma membranes of mouse fibroblasts.

Human erythrocytes synthesize 5-deoxy-D-xylulose-1-phosphate from added acetaldehyde and endogenous dihydroxyacetone phosphate. Acetaldehyde is not only a substrate for the reaction but also undergoes oxidation, causing reversal of glycolysis and thus accumulation of the product.     

Cyclic AMP-dependent protein kinase stimulates the formation of polyphosphoinositides in the plasma membranes of different blood cells

Plasma membrane preparations from lymphocytes, platelets and red cells were phosphorylated in the presence of [gamma-32 P]ATP. The dissociated catalytic subunit of cyclic AMP-dependent protein kinase increased the 32P-labelling of proteins and polyphosphoinositides in lymphocyte, platelet and in some red cell membranes. In the majority of red cell membrane preparations the 32P-labelling of proteins and polyphosphoinositides seemed to be stimulated by the catalytic subunit of the endogenous protein kinase, since the phosphorylation was not increased by the addition of the catalytic subunit but it was decreased by the heat-stable inhibitor protein of the protein kinase. Different sets of 32P-labelled proteins were shown by SDS-gel electrophoresis in the membranes of the 3 cell types. A 24000-Mr protein was the only one which was phosphorylated by the catalytic subunit in each membrane.     

HeLa cell plasma membranes. Changes in membrane protein composition during the cell cycle.

HeLa cells grown in suspension culture were synchronized by amethopterin block and thymidine reversal. In some cases an additional Colcemid block was used to obtain mitotic cells. From the various phases of the cell cycle, cells were harvested and the plasma membranes isolated. The membrane proteins were solubilized in sodium dodecyl sulphate and separated by gel electrophoresis in the presence of sodium dodecyl sarcosinate. About 35 protein bands, five of which were stained with periodic acid-Schiff reagent, appeared. Most of the bands were identical in all membrane preparations, but a few minor bands seemed to be associated with limited periods of the cell cycle. In particular, the cells in mitosis apparently contained plasma membrane proteins which did not occur in other phases. Amino acid analyses of the plasma membranes revealed no significant cell cycle-dependent changes in the amino acid composition.     

Cyclic AMP stimulated phosphorylation of liver pyruvate kinase in hepatocytes.

The addition of glucagon (10^?6 M) to an incubation mixture containing ³²Pi and hepatocytes isolated from livers of rats fed $\text{ad libitum}$ results in both a 3-fold increased incorporation of ³²P into L-type pyruvate kinase and a decreased catalytic activity. The ³²P incorporated into pyruvate kinase was covalently bound to the enzyme as evidenced by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In addition, exogenous cyclic AMP (10^?3 M) stimulated the phosphorylation and the suppression of catalytic activity to a similar extent. On the other hand, insulin (10^?7 M) had essentially no effect on the incorporation of ³²P into pyruvate kinase or on its catalytic activity under the conditions used in this study. These results suggest that phosphorylation of pyruvate kinase $\text{invivo}$ is stimulated by glucagon via cyclic AMP and cyclic AMP-dependent protein kinase and that the activity of the enzyme is, at least in part, regulated by a phosphorylation-dephosphorylation mechanism.     

Cyclic AMP phosphodiesterase activity in three Morris hepatomas.

Rat liver cAMP phosphodiesterase has been fractionated into four peaks of activity with isoelectrofocusing column chromatography. The major two liver peaks (high Km enzymes) decreased with increasing growth rate while the minor two liver peaks (low Km enzymes) increased in one fast growing Morris hepatoma. There was also less total phosphodiesterase activity in the fast growing hepatoma.     

Loss of lymphocyte cyclic AMP dependent protein kinase activity in malignant melanoma.

Cyclic AMP dependent protein kinase activity was depressed in whole thymus and spleen as well as isolated splenic lymphocytes from B16 melanoma bearing C57B1/6J mice as compared to control animals. A similar loss of enzyme activity was observed in human peripheral blood lymphocytes from melanoma bearing patients as compared to normal subjects. An unaltered level of activity in the heart of tumor bearing mice suggested some specificity for the lymphoid system. This depressed enzyme activity was the result of a diminished Vmax for cAMP stimulated calf histone phosphorylation. The tumor bearing state in the mouse was also accompanied by a depletion of small lymphocytes from both thymus and spleen and it is hypothesized that the losses of lymphocytes and cAMP dependent protein kinase activity are related.