The effects of thyroparathyroidectomy and 1,25 dihydroxyvitamin D3 on changes in the activities of some cytoplasmic and nuclear protein kinases during liver regeneration

Partial hepatectomy (HPX), which proliferatively activates the remaining liver cells, triggered two transient prereplicative surges in the total activities of cytoplasmic types I and II cyclic AMP-dependent protein kinase holoenzymes, and of nuclear catalytic subunits from cyclic AMP-dependent protein kinases. It also induced a transient prereplicative increase in the activities of a nuclear Ca2+-calmodulin-stimulable, protamine-phosphorylating protein kinase, and a nuclear poly(L-lysine)-phosphorylating, 105 kDa protein kinase. Thyroparathyroidectomy (TPTX) delayed and reduced the first surge and completely eliminated the second surge of both of the cytoplasmic cyclic AMP-dependent protein kinases, reduced the rises in the activity of nuclear catalytic subunits, and completely eliminated the surge of the Ca2+-calmodulin-stimulable protein kinase, but did not affect the surge of the nuclear 105 kDa protein kinase. The impairment of the responses of the two cyclic AMP-dependent protein kinases to HPX in TPTX rats was not accompanied by a rise in the level of heat-stable inhibitor of cyclic AMP-dependent protein kinase activity. One intraperitoneal injection of 1,25-dihydroxyvitamin D1 into TPTX rats immediately after HPX completely restored the post-HPX surges in the activity of type I cyclic AMP-dependent protein kinase, but the hormone, even in high doses, had little or not effect on the type II isoenzyme or the nuclear Ca2+-calmodulin-stimulable, protamine-phosphorylating enzyme.     

Developmentally regulated cyclic AMP-dependent protein kinases in Dictyostelium discoideum.

Two apparently distinct species of cyclic AMP-dependent protein kinase appear during the first 1-2 hr of development in Dictyostelium discoideum; no such activity can be detected in vegetative cell extracts. These two kinases are similar in properties to the type I and II cyclic AMP-dependent protein kinases found in a number of mammalian tissues. Their time of appearance supports the idea that one or both mediate the effects of cyclic AMP on gene expression early in Dictyostelium development.     

Protein kinase activities in rat pancreatic islets of Langerhans.

1. Protein kinase activities in homogenates of rat islets of Langerhans were studied. 2. On incubation of homogenates with [gamma-32P]ATP, incorporation of 32P into protein occurred: this phosphorylation was neither increased by cyclic AMP nor decreased by the cyclic AMP-dependent protein kinase inhibitor described by Ashby & Walsh [(1972) J. Biol. Chem. 247, 6637--6642]. 3. On incubation of homogenates with [gamma-32P]ATP and histone as exogenous substrate for phosphorylation, incorporation of 32P into protein was stimulated by cyclic AMP (approx. 2.5-fold) and was inhibited by the cyclic AMP-dependent protein kinase inhibitor. In contrast, when casein was used as exogenous substrate, incorporation of 32P into protein was not stimulated by cyclic AMP, nor was it inhibited by the cyclic AMP-dependent protein kinase inhibitor. 4. DEAE-cellulose ion-exchange chromatography resolved four peaks of protein kinase activity. One species was the free catalytic subunit of cyclic AMP-dependent protein kinase, two species corresponded to 'Type I' and 'Type II' cyclic AMP-dependent protein kinase holoenzymes [see Corbin, Keely & Park (1975) J. Biol. Chem. 250, 218--225], and the fourth species was a cyclic AMP-independent protein kinase. 5. Determination of physical and kinetic properties of the protein kinases showed that the properties of the cyclic AMP-dependent activities were similar to those described in other tissues and were clearly distinct from those of the cyclic AMP-independent protein kinase. 6. The cyclic AMP-independent protein kinase had an s20.w of 5.2S, phosphorylated a serine residue(s) in casein and was not inhibited by the cyclic AMP-dependent protein kinase inhibitor. 7. These studies demonstrate the existence in rat islets of Langerhans of multiple forms of cyclic AMP-dependent protein kinase and also the presence of a cyclic AMP-independent protein kinase distinct from the free catalytic subunit of cyclic AMP-dependent protein kinase. The presence of the cyclic AMP-independent protein kinase may account for the observed characteristics of 32P incorporation into endogenous protein in homogenates of rat islets.     

Differential responses of cyclic GMP-dependent and cyclic AMP-dependent protein kinases to synthetic peptide inhibitors.

The peptide Arg-Lys-Arg-Ala-Arg-Lys-Glu was synthesized and tested as an inhibitor of cyclic GMP-dependent protein kinase. This synthetic peptide is a non-phosphorylatable analogue of a substrate peptide corresponding to a phosphorylation site (serine-32) in histone H2B. The peptide was a competitive inhibitor of cyclic GMP-dependent protein kinase with respect to synthetic peptide substrates, with a Ki value of 86 microM. However, it did not inhibit phosphorylation of intact histones by cyclic GMP-dependent protein kinase under any conditions tested. Arg-Lys-Arg-Ala-Arg-Lys-Glu competitively inhibited the phosphorylation of either peptides or histones by the catalytic subunit of cyclic AMP-dependent protein kinase, with similar Ki values (550 microM) for both of these substrates. The peptide Leu-Arg-Arg-Ala-Ala-Leu-Gly, which was previously reported to be a selective inhibitor of both peptide and histone phosphorylation by cyclic AMP-dependent protein kinase, was a poor inhibitor of cyclic GMP-dependent protein kinase acting on peptide substrates (Ki = 800 microM), but did not inhibit phosphorylation of histones by cyclic GMP-dependent protein kinase. The selectivity of these synthetic peptide inhibitors toward either cyclic GMP-dependent or cyclic AMP-dependent protein kinases is probably based on differences in the determinants of substrate specificity recognized by these two enzymes. It is concluded that histones interact differently with cyclic GMP-dependent protein kinase from the way they do with the catalytic subunit of cyclic AMP-dependent protein kinase.     

Characterization of two adenosine 3':5'-phosphate-dependent protein kinase species from Chinese hamster ovary cells.

Chinese hamster ovary cells exhibit several characteristic morphological and physiological responses upon treatment with agents which increase the intracellular level of adenosine 3':5'-phosphate (cyclic AMP). To better understand the mechanism of these cyclic AMP-mediated responses, we separated two cyclic AMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) (protein kinase I and protein kinase II) from the cytosol of Chinese hamster ovary cells by DEAE-cellulose chromatography and studied their properties. Protein kinase I is eluted at a lower salt concentration than protein kinase II and is stimulable to 10 times its basal catalytic activity, while protein kinase II is stimulable only 2-fold. Both kinases are completely dissociated by cyclic AMP and inhibited by specific cyclic AMP-dependent protein kinase inhibitor. They have similar Km values for magnesium (approximately 1 mM), cyclic AMP (approximately 60 nM), and ATP (approximately 0.1 mM), and the dissociation constant (Kdis) for cyclic AMP (approximately 13 nM) is the same for both enzymes. However, they appear to have different substrate preferences and cyclic AMP-binding properties in that cyclic AMP bound to protein kinase II exchanges readily with free cyclic AMP, while that bound to protein kinase I is not exchangeable. The native enzymes have different sedimentation coefficients (6.4 S for protein kinase I and 4.8 S for protein kinase II), whereas those of the activated enzymes are the same (2.9--3.0 S). It appears that the two cyclic AMP-dependent protein kinases which differ from each other in their regulatory subunits may play different roles in the mediation of cyclic AMP action in Chinese hamster ovary cells.     

Selective regulation of the amount of catalytic subunit of cyclic AMP-dependent protein kinases during isoprenaline-induced growth of the rat parotid gland.

Stimulation of growth of the rat parotid gland by repeated injection of the beta-agonist isoprenaline led to a significant decrease in the activity of cyclic AMP-dependent protein kinases. Immunochemical quantification of the catalytic (C) and regulatory (RI and RII) subunits of the cyclic AMP-dependent protein kinases type I and type II revealed a loss of 65% of the immunochemically measurable amount of catalytic subunit C. The amount of the regulatory subunits, however, remained constant. The observed decrease in C-subunit was not due to a translocation of the molecule to cellular membranes or to an inhibiting effect of the heat-stable inhibitor of cyclic AMP-dependent protein kinases. A selective decrease in only the C-subunit was also observed after a brief exposure to isoprenaline leading to the stimulation of DNA synthesis. Under these conditions, the decrease was observed at the onset of DNA synthesis (17 h after injection), but not at the the time of an earlier small cyclic AMP peak (13 h after injection) or at the time of maximal DNA synthesis (24 h after injection). The results indicate that the amount of the catalytic subunit of cyclic AMP-dependent protein kinases can be regulated independently from that of the regulatory subunits. The time-limited occurrence of the specific change in the amount of the C-subunit suggests that such a regulation is of physiological significance and that it may participate in cyclic AMP-mediated events involved in the control of cellular proliferation.     

Localization of catalytic and regulatory subunits of cyclic AMP-dependent protein kinases in mitochondria from various rat tissues.

Observation and quantification of the catalytic subunit C of cyclic AMP-dependent protein kinases by immuno-gold electron microscopy suggested a high concentration of cyclic AMP-dependent protein kinases in mitochondria from liver, kidney, heart and skeletal muscle, pancreas, parotid gland and brain cells. The position of gold particles pointed to a localization in the inner membrane/matrix space. A similar distribution was obtained by immunolocalization of the cyclic AMP-dependent protein kinase regulatory subunits RI and RII in liver, pancreas and heart cells. The results indicated the presence of both the type I and the type II cyclic AMP-dependent protein kinases in mitochondria of hepatocytes, and the preferential occurrence of the type I protein kinase in mitochondria from exocrine pancreas and heart muscle. The immunocytochemical results were confirmed by immunochemical determination of cyclic AMP-dependent protein kinase subunits in fractionated tissues. Determinations by e.l.i.s.a. of the C-subunit in parotid gland cell fractions indicated about a 4-fold higher concentration of C-subunit in the mitochondria than in a crude 1200 g supernatant. Immunoblot analysis of subfractions from liver mitochondria supported the localization in situ of cyclic AMP-dependent protein kinases in the inner membrane/matrix space and suggested that the type I enzyme is anchored by its regulatory subunit to the inner membrane. In accordance with the immunoblot data, the specific activity of cyclic AMP-dependent protein kinase measured in the matrix fraction was about twice that measured in whole mitochondria. These findings indicate the importance of cyclic AMP-dependent protein kinases in the regulation of mitochondrial functions.     

Differential activation of type-I and type-II adenosine 3'':5''-cyclic monophosphate-dependent protein kinases in liver of glucagon-treated rats.

The protein-bound cyclic AMP and the activity of cytosolic protein kinases in the presence and absence of cyclic AMP were determined in rat liver up to 2h after injection of glucagon. On the basis of the different salt-sensitivities of the activated cyclic AMP-dependent proteinkinases I and II, an activation of protein kinase II restricted to the high cyclic AMP concentrations present in the first 30 min after hormone injection was found. Essentially the same result was obtained by chromatographic analysis on DEAE-cellulose of liver cytosol from untreated rats and from rats killed at 2 and 60 min after glucagon injection. Protein kinase II activation was only detected at 2 min after injection. In contrast, the cyclic AMP-dependent protein kinase I was found to be nearly totally activated at 2 min and to be still almost as active at 60 min after the hormone stimulus, whereas the amount of bound cyclic AMP and the activation of total cytosolic protein kinases had fallen to two-thirds of their maximal values during this time period. A third cyclic AMP-independent protein kinase, which co-chromatographed with protein kinase type II, could be clearly distinguished from the two cyclic AMP-dependent kinases by use of the heat-stable inhibitor from bovine muscle, which totally inhibited the cyclic AMP-dependent enzymes, but stimulated the cyclic AMP-independent protein kinase.     

Glycogen synthase kinases. Distribution in mammalian tissues of forms that are independent of cyclic AMP.

Extracts of rat tissues contain kinases which catalyze the conversion of glycogen synthease from the glucose 6-phosphate-independent (I) form to the glucose 6-phosphatate-dependent (D) form. These kinases were stimulated by adenosine 3':5' monophosphate (cyclic AMP). The glycogen synthase kinase activity ratio (activity in the absence of cyclic AMP divided by activity in the presence of cyclic AMP) varied from 0.28 to 0.97. The activity ratio for histone kinase in the same extracts ranged from 0.11 to 0.29. The levels of glycogen synthase kinase varied by a factor of 80 in the following rat tissues (given in order of decreasing enzyme activity): kidney, liver, stomach mucosa, lung, brain, heart, skeletal muscle, and adipose tissue. In the same tissues the levels of histone kinase varied by only a factor of 6 and did not correlate with the levels of glycogen synthase kinase. A modification of the method of Walsh et al. ((1971) J. Biol. Chem. 246, 1977-1985) was developed for purification of the heat-stable inhibitor of cyclic AMP-dependent protein kinases (inhibitor). The modified procedure resulted in good yields of highly purified inhibitor and was much simpler than the previously described procedure. This inhibitor completely inhibited cyclic AMP-dependent histone kinase activity of the extracts but much of the glycogen synthase kinase activity was not inhibited. The portion of glycogen synthase kinase that was insensitive to the inhibitor was: stomach mucosa, 95%; brain, 90%; liver, 82%; kidney, 81%; lung, 68%; adipose tissue, 65%; skeletal muscle, 63%; and heart, 54%. This histone kinase activity in the extracts and hte ratio of glycogen synthase kinase to histone kinase activity of purified catalytic subunit of the cyclic AMP-dependent protein kinase was used to calculate for each extract the glycogen synthase kinase activity contributed by the cyclic AMP-dependent protein kinase. Based on these calculations, the portion of the glycogen synthase kinase which was due to kinases independent of cyclic AMP was: kidney, 97%; liver, 91%; lung, 89%; brain, 87%, heart, 85%; stomach mucosa, 84%; adipose tissue, 38%; and skeletal muscle, 33%. A significant portion of the glycogen synthase kinase activity, but virtually none of the cyclic AMP-dependent histone kinase activity, of these extracts could be adsorbed to phosphocellulose columns. Liver extracts contained, in addition, a form of glycogen synthase kinase which was not adsorbed to phosphocellulose and which could be separated from the cyclic AMP-dependent protein kinase by additional chromatography. These studies demonstrate that kinases independent of cyclic AMP account for most of the glycogen synthase kinase activity of many tissues. The widespread distribution and high concentrations of these enzymes suggest that they are of physiological importance.     

Effects of Ca2+, calmodulin and cyclic AMP on the phosphorylation of endogenous proteins by homogenates of rt islets of langerhans

Activation of Ca2+ -calmodulin- and cyclic AMP-dependent protein kinases has been suggested to be involved in stimulus-secretion coupling in the pancreatic beta-cell. To study the properties of suc kinases and their endogenous protein substrates homogenates of rat islets of Langerhans were incubated with [gamma-32P]ATP. Phosphorylated proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and detected by autoradiography. The phosphorylation of certain proteins could be enhanced by Ca2+ plus calmodulin or by cyclic AMP. The major effect of Ca2+ and calmodulin was to stimulate the phosphorylation of a protein (P53) of molecular weight 53,100 +/- 500 (n = 15). Maximum phosphorylation of protein P53 occurred within 2 min with 2 micrometers free Ca2+ and 0.7 micrometers calmodulin. Incorporation of label into protein P53 was inhibited by trifluoperazine or W7 but not by cyclic AMP-dependent protein kinase inhibitor. Phosphorylation of a proteins of similar molecular weight could be enhanced to a lesser extent in the absence of Ca2+ but in the presence of cyclic AMP and 3-isobutylmethylxanthine: this phosphorylation was blocked by cyclic AMP-dependent protein kinase inhibitor. Cyclic AMP also stimulated incorporation of label into polypeptides of molecular weights 55,000 and 70-80,000. The results are consistent with the hypothesis that protein phosphorylation mechanisms may play a role in the regulation of insulin secretion.     

Inhibition of cerebral protein kinase activity and cyclic AMP-dependent ribosomal-protein phosphorylation in experimental hyperphenylalaninaemia

Studies were carried out to elucidate the mechanisms underlying the diminished phosphorylation of cerebral ribosomal protein in experimental hyperphenylalaninaemia [Roberts & Morelos (1980) Biochem. J.190, 405-419]. Administration of N(6),O(2)'-dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine, which increased phosphorylation of the S6 protein of cerebral 40S ribosomal subunits in control infant rats, did not counteract the decreased phosphorylation of this ribosomal protein resulting from intraperitoneal administration of a loading dose of l-phenylalanine. N(2),O(2)'-Dibutyryl cyclic GMP had no effect on cerebral ribosomal-protein phosphorylation in either control or hyperphenylalaninaemic animals. The phenylalanine-induced decrease in ribosomal-protein phosphorylation was associated with decreased protein kinase activity in cerebral cytosolic and microsomal preparations. However, the maximal protein kinase response to cyclic AMP added in vitro was unaltered by prior administration of phenylalanine in vivo. The heat-stable protein inhibitor of cyclic AMP-dependent protein kinases decreased the activity of these enzymes by about 90% and eliminated the phenylalanine-induced difference in protein kinase activity in the absence of added cyclic AMP. Intracisternal administration of doses of dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine which increased the cyclic AMP-dependent protein kinase activity ratio in control infant rats was without effect on this index in phenylalanine-treated animals. Dibutyryl cyclic GMP had no effect on the protein kinase activity ratio in either group of animals. These results suggest that inhibition of cerebral cyclic AMP-dependent protein kinases by abnormally high concentrations of phenylalanine may contribute to the decrease in cerebral ribosomal-protein phosphorylation in experimental hyperphenylalaninaemia.     

A specific substrate from rabbit cerebellum for guanosine-3':5'-monophosphate-dependent protein kinase. III. Amino acid sequences at the two phosphorylation sites

G-substrate is a protein present in cerebellum which is a major endogenous substrate for cyclic GMP-dependent protein kinase, and one of the few known proteins phosphorylated more effectively by cyclic GMP-dependent protein kinase than by cyclic AMP-dependent protein kinase. G-substrate has been shown to be phosphorylated on two threonine residues, and the amino acid sequences surrounding these sites, which correspond to about 30% of the primary structure, are: Leu-Asn-Val-Glu-Ser-Asp-Gln-Lys-Lys-Pro-Arg-Arg-Lys-Asp-Thr(P)-Pro-Ala-Leu-His- Ile-Pro-Pro-Phe-Ile-Ser-Gly-Val-Ile-Ser-Gln-Asn SITE 1 Leu-His-Asn-Thr-Asp-Leu-Glu-Gln-Gln-Lys-Pro-Arg-Arg-Lys-Asp-Thr(P)-Pro-Ala-Leu- His-Thr-Ser-Pro-Phe-Gln-Ser-Gly-Val-Arg SITE 2 The amino acid sequences surrounding the phosphorylated residues show 18 identities over a sequence of 26 residues, and suggest that G-substrate contains an internal gene duplication. Site-1 appears to be located 17 residues from the COOH terminus of the protein. Site 1 and site 2 are phosphorylated at similar rates by cyclic GMP-dependent protein kinase. In contrast, cyclic AMP-dependent protein kinase phosphorylates site 1 4-fold more rapidly than site 2. A decapeptide sequence surrounding the phosphothreonine residues in G-substrate shows 5 identities with that surrounding the phosphothreonine residue in protein phosphatase inhibitor 1. Inhibitor 1, a specific substrate for cyclic AMP-dependent protein kinase, also resembles G-substrate in its physical properties. The possible function of G-substrate and the molecular specificities of cyclic AMP-dependent protein kinase and cyclic GMP-dependent protein kinase are discussed in the light of these results.     

Activation of Tyrosine Hydroxylase in PC12 Cells by the Cyclic GMP and Cyclic AMP Second Messenger Systems

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is subject to regulation by a variety of agents. Previous workers have found that cyclic AMP-dependent protein kinase and calcium-stimulated protein kinases activate tyrosine hydroxylase. We wanted to determine whether cyclic GMP might also be involved in the regulation of tyrosine hydroxylase activity. We found that treatment of rat PC12 cells with sodium nitroprusside (an activator of guanylate cyclase), 8-bromocyclic GMP, forskolin (an activator of adenylate cyclase), and 8-bromocyclic AMP all produced an increase in tyrosine hydroxylase activity measured in vitro or an increased conversion of [14C]tyrosine to labeled catecholamine in situ. Sodium nitroprusside also increased the relative synthesis of cyclic GMP in these cells. In the presence of MgATP, both cyclic GMP and cyclic AMP increased tyrosine hydroxylase activity in PC12 cell extracts. The heat-stable cyclic AMP-dependent protein kinase inhibitor failed to attenuate the activation produced in the presence of cyclic GMP. It eliminated the activation produced in the presence of cyclic AMP. Sodium nitroprusside also increased tyrosine hydroxylase activity in vitro in rat corpus striatal synaptosomes and bovine adrenal chromaffin cells. In all cases, the cyclic AMP-dependent activation of tyrosine hydroxylase was greater than that of the cyclic GMP-dependent second messenger system. These results indicate that both cyclic GMP and cyclic AMP and their cognate protein kinases activate tyrosine hydroxylase activity in PC12 cells.     

Purification and characterization of two cyclic AMP-independent casein/glycogen synthase kinases from rat liver cytosol

Two cyclic AMP-independent protein kinases (ATP: protein phosphotransferase, EC 2.7.1.37) (casein kinase 1 and 2) have been purified from rat liver cytosol by a method involving chromatography on phosphocellulose and casein-Sepharose 4B. Both kinases were essentially free of endogeneous protein substrates and capable of phosphorylating casein, phosvitin and I-form glycogen synthase, but were inactive on histone IIA, protamine and phosphorylase b. They were neither stimulated by cyclic AMP, Ca2+ and calmodulin, nor inhibited by the cyclic AMP-dependent protein kinase inhibitor protein. The casein and glycogen synthase kinase activities of each enzyme decreased at the same rate when incubated at 50 degrees C. Casein kinase 1 and casein kinase 2 showed differences in molecular weight, sensitivity to KCl, Km for casein and phosvitin and Ka for Mg2+, whereas their Km values for ATP and I-form glycogen synthase were similar. The phosphorylation of glycogen synthase by these kinases correlated with a decrease in the +/- glucose 6-phosphate activity ratio (independence ratio). However, casein kinase 1 catalyzed the incorporation of about 3.6 mol of 32P/85000 dalton subunit, decreasing the independence ratio from 83 to about 15, whereas the phosphorylation achieved by casein kinase 2 was only about 1.9 mol of 32P/850000 dalton subunit, decreasing the independence ratio to about 23. The independence ratio decrease was prevented by the presence of casein but was unaffected by phosphorylase b. These data indicate that casein/glycogen synthase kinases 1 and 2 are different from cyclic AMP-dependent protein kinase and phosphorylase kinase.     

Phosphorylation of rabbit and human erythrocyte membranes by soluble adenosine 3':5'-monophosphate-dependent and -independent protein kinases.

Previous reports from this laboratory and others have established that both the rabbit and human erythrocyte membranes contain multiple protein kinase and phosphate acceptor activities. We now report that these membranes also contain phosphoryl acceptor sites for the soluble cyclic AMP-dependent and -independent protein kinases from rabbit erythrocytes. The rabbit erythrocyte membrane, which does not contain a cyclic AMP-dependent protein kinase, has at least four polypeptides (Bands 2.1, 2.3, 4.5, and 4.8) which are phosphorylated in the presence of the soluble cyclic AMP-dependent protein kinases I, IIa, and IIb isolated from rabbit erythrocyte lysates. The resulting phosphoprotein profile is very similar to that obtained for the cyclic AMP-mediated autophosphorylation of human erythrocyte membranes. The activities of the soluble cyclic AMP-dependent protein kinases toward the membranes have been studied at several pH values. Although the substrate specificity of the three kinases is similar, polypeptide 2.3 appears to be phosphorylated to a greater extent by kinase IIa than by I or IIb. This occurs at all pH values studied. Also apparent is that the pH profile for membrane phosphorylation is different from that of histone phosphorylation. The phosphorylation of membrane proteins can also be catalyzed by the soluble erythrocyte casein kinases. These enzymes are not regulated by cyclic nucleotides and can use either ATP or GTP as their phosphoryl donor. Polypeptides 2.1, 2.9, 4.1, 4.5, 4.8, and 5 of both human and rabbit erythrocyte membranes are phosphorylated in the presence of GTP and the casein kinases. This reaction is optimal at pH 7.5. Experiments were performed to determine whether the phosphorylation of the membranes by the soluble and membrane-bound kinases is additive or exclusive. Our results indicate that after maximal autophosphorylation of the erythrocyte membranes, phosphoryl acceptor sites are available to the soluble cyclic AMP-dependent and -independent protein kinases. Furthermore, after maximal phosphorylation of the membranes with one type of soluble kinase, further 32P incorporation can occur as a result of exposure to the other type of soluble kinase.     

Adenosine 3',5'-monophosphate-dependent protein kinase(s) in diploid and SV40 transformed human fibroblasts.

Cyclic AMP-dependent protein kinases (EC 2.7.1.37; ATP:protein phosphotransferase) in the human diploid fibroblast WI-38 and an SV40-transformant WI-38-VA13-2RA (VA13) have been compared on the basis of their concentrations in cells, isoenzyme composition and susceptibility to hormonal activation. In high population density cultures, total soluble cyclic AMP-dependent kinase activities measured with histone were essentially the same in WI-38 and VA13. Two soluble protein kinase forms separated by chromatography on DEAE-cellulose were present in both cell lines. The concentration of cyclic AMP required for half-maximal activation of both enzyme forms was 10-30 nM. Overall kinase stimulation was greater for the Peak I enzymes. Kinase activation induced in the presence of 0.5 M KCl was more rapid and complete for the Peak I enzymes. Under conditions which elevated the concentration of cyclic AMP in WI-38 and VA13 cells the activities of the soluble histone kinases were increased. Incubation of the cells with either of 5.7 micronM prostaglandin E1 or 1 micronM isopropylnorepinephrine induced complete activation of the cyclic AMP-dependent histone kinases within 5 min and maintained the effect for 20 min. When intracellular cyclic AMP levels were raised by prostaglandin E1, activation of glycogen phosphorylase (assayed-AMP) suggested that this enzyme cascade involving cyclic AMP-dependent protein kinase(s) was intact and responsive in both cell lines.