Cyclic Nucleotide-independent Protein Kinase Research Articles

Polyamine-like effects of cobalt(III) hexaammine on various cyclic nucleotide-independent protein phosphokinase reactions

The chemically inert trivalent ion cobalt(III) hexaammine, Co 3+(NH 3) 6, was found to exert polyamine-like effects in enhancing certain cyclic nucleotide-independent protein kinase reactions catalyzed by nuclear enzyme preparations from rat ventral prostate or liver. At 1 mM, Co 3+(NH 3) 6 stimulated chromatin- and also non-histone-protein-associated kinase activities with partially dephosphorylated phosvitin as substrate by 38% and 72% respectively, whereas chromatin-associated kinase-catalyzed phosphorylation of lysine-rich histones was not affected under the same conditions. 32P incorporation (from γ- 32P-ATP) into endogenous protein substrates of chromatin or non-histone protein fractions catalyzed by their erdogencus kinase activity was increased by 47% and 153%, respectively. These effects of Co 3+(NH 3) 6 were similar to those produced by 1mM spermine. Autoradiographic analysis of endogenous 32P-labelled nonhistone proteins revealed similar enhancements of the phosphorylation of several of the same proteins, induced by 1mM spermine or 1 mM Co 3+(NH 3) 6 or 2mM spermidine. The stimulatory actions of polyamines or Co 3+(NH 3) 6 were not mimicked by raising the ionic strength by addition of comparable concentrations of NaCl. The effects of 1 mM spermine and of 1 mM Co 3+(NH 3) 6 tested separately were not additive. Phosphorylation of lysine-rich histones by beef heart cyclic AMP-dependent protein kinase was not affected by polyamines or Co 3+(NH 3) 6 Various findings hint that the enhancement of cyclic nucleotide-independent kinase-catalyzed phosphorylation of certain protein substrates by spermidine, spermine and Co 3+(NH 3) 6 is primarily due to interaction of these cations with appropriate protein substrates affecting their conformational status. Further, these effects of polyamines may be a reflection of their cationic charge properties rather than being dependent on any particular conformations assumed by the polyamines.

Purification and characterization of a type II casein kinase from Drosophila melanogaster.

A cyclic nucleotide-independent protein kinase has been isolated from Drosophila melanogaster by chromatography on phosphocellulose and hydroxylapatite followed by gel filtration and glycerol gradient sedimentation. As determined by sodium dodecyl sulfate gel electrophoresis, the purified enzyme is greater than 95% homogeneous and is composed of two distinct subunits, alpha and beta, having Mr = 36,700 and 28,200, respectively. The native form of the enzyme is an alpha 2 beta 2 tetramer having a Stokes radius of 48 A, a sedimentation coefficient of 6.4 S, and Mr approximately 130,000. The purified kinase undergoes an autocatalytic reaction resulting in the specific phosphorylation of the beta subunit, exhibits a low apparent Km for both ATP and GTP as nucleoside triphosphate donor (17 and 66 microM, respectively), phosphorylates both casein and phosvitin but neither histones nor protamine, modifies both serine and threonine residues in casein, and is strongly inhibited by heparin (I50 = 21 ng/ml). These properties are remarkably similar to those of casein kinase II, an enzyme previously described in several mammalian and avian species. The strong similarities among the insect, avian, and mammalian enzymes suggest that casein kinase II has been highly conserved during evolution.

Regulation of polyamine-responsive protein kinase by certain highly specific polyamines and charged carbohydrates

Polyamine-responsive protein kinase, a cyclic nucleotide-independent protein kinase from the cytosol of Morris hepatoma 3924A, was stimulated 8–9 fold by several different polymers of polylysine, polyornithine and random copolymers of lysine-alanine; spermidine; spermine, and mixture of spermine and spermidine stimulated 2, 3, and 5 fold, respectively. The protein kinase was not stimulated by poly-carboxybenzyl-lysine, random copolymer of lysine-tyrosine, polyhistidine, polymethionine, polyglutamic acid, polyaspartic acid, dipeptide (Lys-Lys), lysine, ornithine, and putresine. The polyamine stimulation of the protein kinase was prevented by certain specific charged carbohydrated: heparin, chondroitin sulfates A, B, and C, dextran sulfate and hyaluronic acid. It was not prevented by noncharged carbohydrates: dextran, glycogen, starch, sucrose, etc; or by sulfate salts: ammonium sulfate, potassium sulfate, sodium thiosulfate, etc. The inhibition was reversed by increased polylysine. Heparin was non-competitive inhibitor of Mg 2+--ATP. It would appear that this enzyme is regulated by certain highly specific molecules with certain sizes and charges; plus charge is stimulatory, negative charge prevents the stimulation.

Oligomeric structure and catalytic activity of G type casein kinase. Isolation of the two subunits and renaturation experiments.

Casein kinase G purified from bovine tissue is an oligomeric cyclic nucleotide-independent protein kinase made of two different monomers, namely an alpha (Mr = 38 kilodaltons) and a self-phosphorylatable beta (Mr = 27 kilodaltons) subunit. Treatment of the native enzyme under denaturing conditions (0.5 M NaCl, 4 M LiCl, and 20 to 35% formamide) resulted in a progressive selective removal of the beta subunit following gel filtration and a correlated loss of activity of the corresponding renatured enzyme. Mild digestion with papain resulted in a proteolytic alteration limited to the beta monomer with a concomitant partial loss of the enzyme activity. Isolation of the alpha and beta casein kinase G subunits was achieved by preparative reversed polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Renaturation of the proteins following sodium dodecyl sulfate removal by acetone and/or Triton X-100 treatment allowed reconstitution of a functional casein kinase G. Whereas the isolated alpha subunit was found to exhibit a weak catalytic activity, addition of the beta subunit was required for recovery of a maximal casein kinase activity. The process was dose-dependent and reached a plateau for an alpha:beta subunit molar ratio of approximately 1 to 1. These data suggest that while the casein kinase G alpha subunit bears the catalytic site, stoichiometric combination with the beta subunit is required for optimal enzymatic activity. A possible role of the beta subunit as a regulatory component of casein kinase G activity in the intact cell remains to be examined.

RNA polymerase I in hepatoma 3924A: Mechanism of enhanced activity relative to liver

Two major cyclic nucleotide-independent protein kinases, NI and NII, have been identified in Morris hepatoma 3924A and rat liver. When expressed per unit DNA, the activities of protein kinase NI and NII were 1.3 and 12 times greater, respectively, in the hepatoma than in liver. Protein kinase NII, but not NI, was capable of phosphorylating and activating the DNA-dependent RNA polymerases I and II. Phosphorylation of RNA polymerase I was accompanied by an increase in average size of the RNA synthesized in vitro, whereas, phosphorylation of RNA polymerase II was concomitant with an elevation in the number of RNA chains initiated. RNA polymerase I polypeptides of M r 120,000, 65,000 and 25,000 were phosphorylated by protein kinase NII; RNA polymerase II polypeptides of M r 214,000, 140,000 and 21,000 were modified by this kinase. In contrast to the purified hepatoma enzyme, RNA polymerase I activity in nuclear lysates was not affected by addition of protein kinase NII. In vitro phosphorylation of the tumor lysate followed by immunoprecipitation of RNA polymerase I polypeptides indicated little or no phosphate transfer to the 65,000 M r polypeptide of the enzyme. These data suggested that the tumor enzyme, particularly the 65,000 M r polypeptide, was highly phosphorylated in vivo, but becomes dephosphorylated during purification. Unlike the tumor enzyme, RNA polymerase I in the liver lysate responded to protein kinase addition; phosphorylation of the liver polymerase I polypeptides of M r 120,000, 65,000 and 25,000 was observed. These observations indicate that the liver enzyme is not completely phosphorylated (activated) in vivo and that the relatively rapid rate of ribosomal RNA synthesis in the rapidly growing hepatoma may result, at least in part, from a polymerase I which is maximally phosphorylated.

Occurrence of NI and NII type protein kinases in the nuclei from various tissues of the rat

Cyclic nucleotide-independent protein kinases that preferentially phosphorylated casein and phosvitin as substrate were surveyed in various tissue nuclei of the rat. Enzymes were extracted from the isolated nuclei of liver, kidney, spleen, brain, heart, or testis tissue with a buffer solution containing 0.4 m NaCl, and analyzed by DEAE-Sephadex, phosphocellulose, and Bio-Gel A-1.5m column chromatographies. The chromatographic study together with characterization of the enzymes demonstrated that all the tissues contained in their cell nuclei commonly two protein kinases, the NI and NII types, and that these were exclusively found as main nuclear casein kinases. NII enzyme activity was stimulated by polyamines and strongly inhibited by heparin. By contrast, the NI enzymes were little influenced by these compounds. We interpret the present results as suggesting that NI and NII type protein kinases may be found in the cell nuclei from many tissues of rat, and have distinct functions in the cell nuclei.

Cyclic nucleotide-dependent protein kinases in airway smooth muscle.

Because of the potential importance of cyclic nucleotide-dependent protein kinases in the regulation of airway smooth muscle tone, we have examined some of the characteristics of these enzymes in the soluble fraction of canine trachealis homogenates. In the absence of added cAMP, the heat-stable cAMP-dependent protein kinase inhibitor (PKI) abolished only a half of the 32P incorporation into mixed histones. The remaining activity appeared to be contributed by a cyclic nucleotide-independent enzyme. Phosphotransferase activity was enhanced 5-fold by 5 microM cAMP but only 70% of the cAMP-stimulated activity could be inhibited by PKI. The sensitivity of the cyclic nucleotide-dependent, PKI-resistant enzyme to cAMP, cGMP, and Mg2+ indicated that it was cGMP-dependent protein kinase. Because of the large amount of cyclic nucleotide-independent activity, and the ability of cAMP to activate cGMP-dependent protein kinase, the traditional "-cAMP/+cAMP" ratio did not provide an accurate assessment of the in vivo activation state of cAMP-dependent protein kinase. However, a modified assay was developed which allowed the precise measurement of cAMP-dependent, cGMP-dependent, and cyclic nucleotide-independent protein kinase activities. Using this new method, the cAMP-dependent protein kinase activity ratio of 0.239 in untreated trachealis strips was increased to 0.355 and 0.386 by prior exposure of the intact tissue to the smooth muscle relaxants isoproterenol and prostaglandin E2, respectively. The results of this study are consistent with the proposed role of cAMP-dependent protein kinase in the regulation of smooth muscle contractile function.

Bovine adrenocortical self-phosphorylating protein kinase, SPK 380: Purification and characterization

Recently, we described the partial purification and characterization of a novel adrenocortical cyclic nucleotide-independent protein kinase, PK 380, that catalyzes the phosphorylation of an endogenous peptide (120,000 daltons) and a serine residue(s) of the α subunit (38,000 daltons) of the eucaryotic initiation factor eIF-2 ( Y. Kuroda, W. C. Merrick, and R. K. Sharma, 1982, Arch. Biochem. Biophys. 213, 271–275 ). In the present communication we describe the purification to apparent homogeneity and characterization of this protein kinase (SPK 380). As shown by sucrose density sedimentation, the native enzyme has a molecular weight of 356,000. The protein is composed of three identical subunits of M r 120,000. Polyacrylamide-gel isoelectric focusing electrophoresis revealed a single peak with p I 4.5. SPK 380 self-phosphorylated a histidine residue(s) of its 120,000-dalton peptide. This reaction utilized the terminal phosphate of ATP; GTP was inactive. Divalent cations (5 m m Mn 2+ or 10 m m Mg 2+) were essential for optimum activity. Thiol reagents ( N-ethylmaleimide, p-chloromercuriphenylsulfonic acid) inhibited the kinase, indicating a sulfhydryl-group requirement for enzyme activity.

The intrinsic substrate specificity of a cyclic nucleotide-independent protein (histone) kinase

Phosphorylation and dephosphorylation of proteins play a pivotal role in the regulation of intracellular processes (reviewed in [l-3]). Beside several well-characterized protein kinases a number of protein phosphorylating enzymes have been found; however, their natural substrates and regulatory properties are not known. A possible way to identify these enzymes is the determination of their intrinsic substrate specificity; i.e., the most important residues of the amino acid sequence that they recognize in their substrate. A cyclic nucleotide-independent histone kinase (designated as HK II) was described in 1451. We have also reported a type of cyclic nucleotide-independent histone kinase found in the cytoplasm and nucleus of human tonsillar lymphocytes [6,7] and in the extract of bovine thymus [8]. This enzyme phosphorylates the Ser-32 residue of calf thymus H2b histone and a synthetic peptide containing the amino acid sequence of H2b from Gly-26-Lys-34, but it does not act on Ser-36 which is phosphorylated preferentially by the catalytic subunit of the cyclic AMP-dependent protein kinase 19-l 11. Separation of the cyclic nucleotide-independent histone kinase from the catalytic subunit of the cyclic AMP-dependent protein kinase was carried out by DEAE-cellulose chromatography in the presence of cyclic AMP, as in 181. The histone kinase fraction obtained by this procedure from bovine thymus was dialyzed against 0.005 M potassium phosphate (pH 7.5) and it was applied onto a hydroxylapatite (Bio-Gel HTP) column. The column was washed by 5 vol. of each of 0.01 M, 0.07 M, 0.085 M, 0.1 M and 0.4 M potassium phosphate (pH 7.5). The histone kinase was eluted by the last volumes of 0.07 M and by the first volume of 0.085 M potassium phosphate. The specific activity of the enzyme preparation obtained was lo-12 nmol phosphate transferred mg protein -’ . min -‘, as measured in the presence of 1 mg histone Hi/ml and 2 x lo-’ M ATP. This preparation yielded a single (Mr 55 000) histone kinase peak on Sephadex G-100 gel-chromatography.

Kinetics of Phosphorylation of eIF-2 by the hemin-controlled repressor and casein kinase II. Inhibition by hemin and 2,3-diphosphoglyceric acid.

The kinetics of phosphorylation of eukaryotic initiation factor 2 (eIF-2) by two cyclic nucleotide-independent protein kinases from rabbit reticulocytes have been studied. The hemin-controlled repressor (HCR) and casein kinase II phosphorylate the alpha and beta subunits of eIF-2, respectively. The apparent Km for eIF-2 with HCR and casein kinase II was determined to be in the range of 0.5-1.0 microM. Using fully dephosphorylated eIF-2, it was possible to show that the phosphorylation state of the beta subunit did not affect the apparent Km of either HCR or casein kinase II for eIF-2. The effects of two inhibitory compounds, hemin and 2,3-diphosphoglyceric acid (2,3-DPG), were studied also. Hemin acted as a noncompetitive inhibitor of HCR with respect to eIF-2 with an apparent Ki of 3 microM, while inhibition of casein kinase II by 2,3-DPG was competitive with respect to eIF-2. The apparent inhibitory constant was determined to be 1 mM, a concentration within the physiological range of 2,3-DPG in red blood cells. From the kinetic values obtained with hemin and 2,3-DPG, it appears that these compounds could directly regulate the respective protein kinases in vivo.

Selective inhibition of a cyclic nucleotide independent protein kinase (G type casein kinase) by quercetin and related polyphenols

The effect of quercetin and a number of structurally related phenolic compounds upon the activity of three different purified protein kinases was examined. Whereas the catalytic subunit of a cyclic AMP-dependent protein kinase and an A type (using only ATP) cyclic nucleotide-independent casein kinase (CKA) were not affected, a G type (using GTP as well as ATP) casein kinase (CKG) was selectively inhibited by several bioflavonoid structures. Kinetic studies showed that quercetin behaved as a competitive inhibitor toward the nucleotidic substrate and exhibited a high affinity for the ATP ( K i = 0.75 μM) and GTP ( K i = 0.22 μM) site of the enzyme. Considering the CKG inhibitory potency of a series of flavonoid, cinnamic acid and coumarin derivatives, it is suggested that the biological activity lays upon a common structural feature involving a phenolic ring bearing a side chain with conjugated double bonds and an oxygenated function, as found in the coumaroyl residue. These observations suggest that quercetin and related compounds may lead to a shift in intracellular protein phosphorylations by selectively inhibiting a particular type of protein kinase activity (CKG). It remains to be established whether this process may contribute to the mechanism of action of flavonoids upon cellular metabolism, particularly in the case of malignant cells.

Avian sarcoma virus UR2 encodes a transforming protein which is associated with a unique protein kinase activity.

UR2 is a newly characterized avian sarcoma virus whose genome contains a unique sequence that is not related to the sequences of other avian sarcoma virus transforming genes thus far identified. This unique sequence, termed ros, is fused to part of the viral gag gene. The product of the fused gag-ros gene of UR2 is a protein of 68,000 daltons (P68) immunoprecipitable by antiserum against viral gag proteins. In vitro translation of viral RNA and in vivo pulse-chase experiments showed that P68 is not synthesized as a large precursor and that it is the only protein product encoded in the UR2 genome, suggesting that it is involved in cell transformation by UR2. In vivo, P68 was phosphorylated at both serine and tyrosine residues. Immunoprecipitates of P68 with anti-gag antisera had a cyclic nucleotide-independent protein kinase activity that phosphorylated P68, rabbit immunoglobulin G in the immune complex, and alpha-casein. The phosphorylation by P68 was specific to tyrosine of the substrate proteins. P68 was phosphorylated in vitro at only one tyrosine site, and the tryptic phosphopeptide of in vitro-labeled P68 was different from those of Fujinami sarcoma virus P140 and avian sarcoma virus Y73-P90. A comparison of the protein kinases encoded by UR2, Rous sarcoma virus, Fujinami sarcoma virus, and avian sarcoma virus Y73 revealed that UR2-P68 protein kinase is distinct from the protein kinases encoded by those viruses by several criteria. Our results suggest that several different protein kinases encoded by viral transforming genes have the same functional specificity and cause essentially the same cellular alterations.

Modulation of protein phosphorylation in human colon adenocarcinoma cell membrane preparations by epidermal growth factor in vitro.

Incubation of membranes prepared from the human colon adenocarcinoma cell line LoVo in vitro with [gamma-32P]ATP demonstrated numerous components whose phosphorylation was stimulated several fold by epidermal growth factor (EGF). One major component of Mn 170 K, which was either undetectable or very weakly phosphorylated in the absence of EGF, was primarily affected by exposure to EGF. The phosphorylation of the 170 K Mr membrane component required the presence of Mn2+; Mg2+ was ineffective. Although the phosphorylation of many LoVo membrane components was significantly modified by cAMP or dibutyryl-cAMP, none of these cyclic nucleotides appeared to be involved in the phosphorylation of the 170 K membrane component in the presence or absence of EGF. The phosphorylation system of the LoVo membranes efficiently utilized [gamma-32P]GTP in both the basal and EGF-stimulated reactions. All the membrane components phosphorylated in the absence or presence of EGF, except a band comigrating with the tracking dye front, were digested by trypsin. The possible glycoprotein nature of the 170 K dalton phosphoprotein was indicated by the fact that the 170 K dalton band comigrated with a periodic acid-Schiff base-stained band. These findings suggest that one of the biochemical steps in the mechanism of action of EGF in LoVo cells is enhanced phosphorylation of several membrane proteins, especially that of a glycoprotein of Mr 170 K, by a membrane-bound cyclic nucleotide independent protein kinase.

Calmodulin and acidic compounds alter basic protein phosphorylation by the protein kinase from human platelets

A cyclic nucleotide-independent protein kinase of human platelets, which phosphorylated histones, myelin basic protein and protamine and did not catalyze the phosphorylation of acidic proteins such as casein, phosvitin and myosin light chain, has been purified approx. 1,500-fold from the crude extract by steps of DEAE-cellulose, Sephadex G-200, hydroxylapatite and phosphoryl cellulose column chromatography. The substrate phosphorylation by this kinase was markedly enhanced by calmodulin even in the absence of Ca 2+, when mixed histone was used as a substrate. The interaction of the kinase with mixed histone resulted in an irreversible inactivation of the enzyme. Calmodulin prevented this inactivation, and this compound produced an apparent increase in histone phosphorylation by the kinase. It should be noted that acidic polypeptides such as troponin-C, phospholipids and nucleic acids have a similar ability. The addition of Ca 2+ reduced the effect of calmodulin more than the effects of other acidic compounds.

Specific phosphorylation of eukaryotic initiation factor 2α, elF-2α, by bovine adrenocortical cyclic nucleotide-independent protein kinase, PK 380

Recently, we characterized a novel cyclic nucleotide-independent protein kinase, PK 380, from bovine adrenal cortex ( Y. Kuroda and R. K. Sharma (1980) Biochem. Biophys. Res. Commun. 96, 601–610 ). PK 380 is independent of cyclic AMP, cyclic GMP, calcium, and calcium-calmodulin for its activity. It does not phosphorylate any of the commonly used exogenous substrates but phosphorylates an endogenous 120,000-dalton peptide. In the present study we demonstrate that PK 380 specifically phosphorylates the serine residue of eukaryotic initiation factor 2α, eIF-2α. PK 380 can be differentiated from two other protein kinases, hemin-controled repressor (HCR) or double-stranded RNA-activated inhibitor (dsRI), that are also known to phosphorylate eIF-2α. Unlike the activity of HCR, PK 380 activity is independent of hemin (5–10 μ m) and dependent on sulfhydryl groups. Poly(I) · poly(C), which is known to activate dsRI, does not affect the activity of PK 380. The possibility exists that the physiological substrate of PK 380 is eIF-2α. Thus, this novel protein kinase could play an important role in the translational control processes of adrenocortical cell.

Purification and characterization of the cytoplasmic casein kinase I from rat liver

Two cyclic nucleotide-independent protein kinases, which preferentially utilize casein and phosvitin as substrates, exist in rat liver. In contrast to cytosol the ‘light’ form of these enzymes was predominant in the ‘microsomal extract’. This form (30000–40000 daltons, casein kinase I) was separated from the ‘heavy’ form (130000 daltons, casein kinase II) by gel filtration. This enzyme was then purified by successive chromatography on carboxymethyl-Sephadex, phosvitin-Sepharose and hydroxyapatite. The activity of the purified enzyme was 2000–3000-fold the casein kinase activity of the cytosol. It had a s 20,w of approx. 3S as determined on sucrose density gradient. After iodination or incubation with [ γ −32 P ]ATP, it was analyzed on polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) and appeared to be composed of a single polypeptide (36000±1000 daltons) which self-phosphorylated. In contrast to casein kinase II, casein kinase I preferentially utilized ATP over GTP. The K m value for ATP was determined to be 14 μ M. The K m value for phosvitin was 0.17 mg/ml. Casein kinase I phosphorylated sites different from those of casein kinase II (as shown with ribosomes or SV40 T antigen). Casein kinase I was further characterized by studying its thermal stability. The half-life at 37°C was 6 min and 1 min 30s at 54°C. In the presence of two substrates (ATP and phosvitin), the half-life at 54°C increased from 1 min 30s to 4 min. Hemin strongly increased the rate of inactivation of the casein kinase I at 37°C in the absence or presence of the substrates. Nethylmaleimide also inactivated casein kinase I. Phosvitin, though not ATP, protected the enzyme. This observation may indicate that thiols are involved at the binding site of the enzyme for the protein substrate.

Purification and ATP substrate site of a cyclic nucleotide-independent protein kinase (NI) from porcine liver nuclei.

Purification and ATP substrate site of a cyclic nucleotide-independent protein kinase (NI) from porcine liver nuclei.

Cyclic Nucleotide-independent protein kinases from rabbit reticulocytes. Identification and characterization of a protein kinase activated by proteolysis.

Cyclic Nucleotide-independent protein kinases from rabbit reticulocytes. Identification and characterization of a protein kinase activated by proteolysis.

Regulation of cardiac cyclic GMP-dependent protein kinase

An assay method based on the ability of high concentrations of Mg 2+ to stimulate phosphorylation of histone in the presence of low concentrations of ATP was developed for the measurement of cyclic GMP-dependent protein kinase activity ratios (activity -cyclic GMP/activity + cyclic GMP). In tissues which contain only trace amounts of cyclic GMP-dependent protein kinase, the basal activity ratios were high due to interference from a cyclic nucleotide-independent protein kinase. In order to study the regulation of the cardica cyclic GMP-dependent protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0–4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0–4°C did not significantly alter the activity ratio of the enzyme under conditions of basal or elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMP-dependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2–3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated witth the acetylcholine-induced protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0–4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0–4°C did not significantly alter the activity ratio of the enzyme under conditions of basal elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMP-dependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2–3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated with the acetylcholine-induced increase in cyclic GMP and the cyclic GMP-dependent protein kinase activity ratio was a reduction in the force of contraction. In contrast, nitroprusside produced little or no increase in the cyclic GMP-dependent protein kinase activity ratio despite increasing the level of cyclic GMP 8–10-fold. Nitroprusside also had no effect on contractile force. In combination, nitroprusside and acetylcholine produced additive effects on cyclic GMP levels, but protein kinase activation and force of contraction were similar to those seen with acetylcholine alone. The results suggest that the cyclic GMP produced by acetylcholine in the rat heart is coupled to activation of the cyclic GMP-dependent protein kinase, while that produced by nitroprusside is not.

Quercetin inhibits tyrosine phosphorylation by the cyclic nucleotide-independent, transforming protein kinase, pp60src.

The bioflavonoid quercetin is a potent inhibitor of a cyclic nucleotide-independent, tumor virus-coded protein kinase which phosphorylates tyrosine residues and acts as a cellular transforming protein. Half-maximal inhibition of the protein kinase occurred at 3-4 microM quercetin whereas rutin was much less effective. The finding, that quercetin inhibits a cyclic nucleotide-independent protein kinase activity, may provide clues to the diverse pharmacological effects of the bioflavonoids.