Independent modulation of hepatic protein kinase activities.

The effects of hormonal status on protein kinase activity was examined in homogenates of rat liver. Protein kinase activity was evaluated from incorporation of 32P from [gamma-32P]ATP into protamine or histone as receptor substrates. Protamine phosphorylation in the presence or absence of cyclic AMP exceeded histone phosphorylation by at least a factor or two. Hypophysectomy markedly increased protamine phosphorylation in the presence or absence of saturating amounts of cyclic AMP. In contrast, hypophysectomy only slightly increased cyclic AMP independent phosphorylation of histone. These results could not be amounted for by differences in ATPase or protein phosphase activities. Cortisone (2 mg/day x 3) decreased total protein kinase activity in livers of hypophysectomized rats when protamine was substrate, but had no effect on the total activity toward histone. Growth hormone (100 mug/day x 3) significantly increased histone, but not protamine phosphorylation in livers of hypophysectomized rats. Administration of 5 mug of triiodothyonine/day to hypophysectomized rats also markedly increased the phosphorylation of histone, but not protamine when saturating amounts of cyclic AMP were present. These results support the hypothesis that liver may contain more than one type of protein kinase activity and that the different protein kinase activities can be separately affected by hormones. Such control distal to cyclic AMP might allow selective modulation of cyclic AMP-dependent processes in cells which carry out more than one such process.     

Protein kinase activity associated with pancreatic zymogen granules.

Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues.     

Differences in the cyclic AMP-dependent phosphorylation of plasma membrane proteins of differentiated and undifferentiated L6 myogenic cells

Differences in the cyclic AMP-dependent plasma membrane phosphorylation system of undifferentiated and differentiated L6 myogenic cells have been detected. Endogenous plasma membrane protein phosphorylation in undifferentiated L6 myoblasts was stimulated more than three fold by 5 x 10(-5) M cyclic AMP, whereas no statistically significant cyclic AMP-dependent phosphorylation of endogenous plasma membrane proteins was observed in differentiated L6 cells. In undifferentiated cells cyclic AMP promoted the phosphorylation of several proteins, the most prominent of which had a molecular weight of 110,000. In differentiated cells cyclic AMP did not selectively promote the phosphorylation of specific plasma membrane proteins. Both differentiated and undifferentiated L6 cells, however, contain a cyclic AMP-dependent protein kinase capable of catalyzing the phosphorylation of exogenous substrates, such as histone f2b. Therefore, the data show that differentiation in L6 cells is associated with a selective change in the activity of a plasma membrane cyclic AMP-dependent protein kinase which employs endogenous membrane proteins as substrate.     

The effects of triethyltin bromide on red cell and brain cyclic AMP-dependent protein kinases

Triethyltin bromide activates the cyclic AMP-dependent protein kinases of human red cell membranes and of bovine brain. Additions of 25-500 microM triethyltin to red cell ghosts resulted in enhanced phosphorylation of ghost proteins. When added to partially purified cyclic AMP-dependent protein kinases from red cell ghosts or bovine brain, stimulation of the phosphorylation of calf thymus histone was observed. The enhancement of kinase activity was due to release of catalytic subunits from the intact protein kinase. Brief exposure of the partially purified enzymes to triethyltin, followed by DE52 chromatography, resulted in elution profiles for regulatory and catalytic subunits that were similar to the profile resulting after cyclic AMP activation. Triethyltin interacts with both regulatory and catalytic subunits. When it was added to the partially purified cyclic AMP-dependent protein kinases from human red cell ghosts or bovine brain, noncompetitive inhibition of cyclic AMP binding to the regulatory subunit of the enzyme was observed. It interacted with the catalytic subunit to produce slow inhibition of catalytic activity. The inhibition was non-competitive with respect to both histone and ATP. When intact red cells were subjected to brief exposure with triethyltin, enhanced phosphorylation of certain membrane proteins occurred, suggesting that the activation of the cyclic AMP protein kinases by triethyltin may be physiologically significant.     

Independent modulation of hepatic protein kinase activities

The effects of hormonal status on protein kinase activity was examined in homogenates of rat liver. Protein kinase activity was evaluated from incorporation of ³²P from [γ-³²P]ATP into protamine or histone as receptor substrates. Protamine phosphorylation in the presence or absence of cyclic AMP exceeded histone phosphorylation by at least a factor or two. Hypophysectomy markedly increased protamine phosphorylation in the presence or absence of saturating amounts of cyclic AMP. In contrast, hypophysectomy only slightly increased cyclic AMP independent phosphorylation of histone. These results could not be accounted for by differences in ATPase or protein phosphase activities. Cortisone (2 mg/day × 3) decreased total protein kinase activity in livers of hypophysectomized rats when protamine was substrate, but had no effect on the total activity toward histone. Growth hormone (100 μg/day × 3) significantly increased histone, but not protamine phosphorylation in livers of hypophysectomized rats. Administration of 5 μg of triiodothyronine/day to hypophysectomized rats also markedly increased the phosphorylation of histone, but not protamine when saturating amounts of cyclic AMP were present. These results support the hypothesis that liver may contain more than one type of protein kinase activity and that the different protein kinase activities can be separately affected by hormones. Such control distal to cyclic AMP might allow selective modulation of cyclic AMP-dependent processes in cells which carry out more than one such process.     

Adenosine 3′:5′-cyclic phosphate-dependent and -independent protein kinases of renal brush border membranes: Solubilization,separation, and characterization of multiple forms

Multiple protein kinase activities were found in the luminal segment of the renal proximal tubule cell plasma membrane (brush border membrane). Membranes were extracted with Lubrol, with no loss in activity, and the extract was chromatographed on diethylaminoethyl cellulose with a salt gradient. With protamine as substrate, activity eluted in two peaks, designated I and IIb, and was cyclic AMP independent. With histone VII-S, one peak, designated IIa, appeared, which eluted slightly ahead of IIb and was cyclic AMP dependent. The three activities eluted in their original patterns following rechromatography. Histone kinase activity in the combined IIa+b fraction was stimulated threefold by cyclic nucleotides (K_a = 0.013 and 0.94 μM for cyclic AMP and cyclic GMP, respectively) by increasing V. Cyclic AMP binding activity eluted with histone kinase activity. Rechromatography of IIa+b on diethylaminoethyl cellulose containing 1 μm cyclic AMP resulted in passage through the column of most of the histone kinase activity (IIa) prior to the salt gradient, but retention of kinase IIb, which again eluted in its original position. Characterization of the separated enzymes revealed that kinase I was highly specific for protamine and totally insensitive to cyclic AMP and a specific protein inhibitor of cyclic AMP-dependent kinases. Kinase IIa was relatively specific for histones and was completely inhibited by the protein inhibitor. Kinase IIb was nonspecific, catalyzing phosphorylation of protamine, casein, histones, and phosvitin in decreasing order of activity, and was insensitive to cyclic AMP and the protein inhibitor. Exposure of intact brush border membranes to elevated temperatures revealed that phosphorylation of intrinsic membrane proteins and protamine was thermolabile, whereas cyclic AMP-dependent histone kinase activity was relatively thermostable. These findings implicate cyclic AMP-independent protamine kinases in the cyclic AMP-independent autophosphorylation of the brush border membrane.     

Differences in the Cyclic AMP-Dependent Phosphorylation of Plasma Membrane Proteins of Differentiated and Undifferentiated L6 Myogenic Cells

Differences in the cyclic AMP-dependent plasma membrane phosphorylation system of undifferentiated and differentiated L₆ myogenic cells have been detected. Endogenous plasma membrane protein phosphorylation in undifferentiated L₆ myoblasts was stimulated more than three fold by 5 × 10⁻⁵ M cyclic AMP, whereas no statistically significant cyclic AMP-dependent phosphorylation of endogenous plasma membrane proteins was observed in differentiated L₆ cells. In undifferentiated cells cyclic AMP promoted the phosphorylation of several proteins, the most prominent of which had a molecular weight of 110,000. In differentiated cells cyclic AMP did not selectively promote the phosphorylation of specific plasma membrane proteins. Both differentiated and undifferentiated L₆ cells, however, contain a cyclic AMP-dependent protein kinase capable of catalyzing the phosphorylation of exogenous substrates, such as histone f₂b. Therefore, the data show that differentiation in L₆ cells is associated with a selective change in the activity of a plasma membrane cyclic AMP-dependent protein kinase which employs endogenous membrane proteins as substrate.     

ACTIVITY OF CYCLIC AMP-DEPENDENT MICROSOMAL PROTEIN KINASE AND PHOSPHORYLATION OF RIBOSOMAL PROTEIN IN RAT BRAIN DURING POSTNATAL DEVELOPMENT

Abstract— Microsomes from rat brain exhibited protein kinase activity which was stimulated by cyclic AMP when assayed in the presence of exogenous protein substrate, such as thymus histone. In the absence of exogenous substrate some phosphorylation of microsomal protein occurred, but no stimulation by cyclic AMP could be discerned, probably because of limitations of substrate. The maximal activity of microsomal protein kinase observed in the presence of saturating concentrations of histone and the optimal concentration (5 μ m ) of cyclic AMP remained essentially unchanged from birth to early adulthood, but the magnitude of the stimulation by cyclic AMP was significantly higher at birth than at 30 days of age. Brain ribosomal proteins could be phosphorylated by the cyclic AMP-dependent brain protein kinase. Their total capacity for acceptance of phosphate by means of this phosphorylation reaction remained unchanged throughout the postnatal development of the brain. Our results are consistent with the possibility that phosphorylation of ribosomal protein mediated by cyclic AMP-dependent protein kinase may play a a role in the postnatal regulation of cerebral protein synthesis, as a result of the changes in the levels of cyclic AMP known to occur in brain during postnatal maturation.     

Adenosine 3'':5''-cyclic monophosphate-dependence of protein kinase isoenzymes from mouse liver.

Conditions influencing the cyclic AMP-dependence of protein kinase (ATP-protein phosphotransferase, EC 2.7.1.37) during the phosphorylation of histone were studied. Protein kinase from mouse liver cytosol and the two isoenzymes [PK (protein kinase) I and PK II] isolated from the cytosol by DEAE-cellulose chromatography were tested. A relation between concentration of enzyme and cyclic AMP-dependence was observed for both isoenzymes. Moderate dilution of isoenzyme PK II decreased the stimulation of the enzyme by cyclic AMP. Isoenzyme PK I could be diluted 200 times more than isoenzyme PK II before the same decrease in cyclic AMP-dependence appeared. Long-term incubation with high concentrations of histone increased the activity in the absence of cyclic AMP relative to the activity in the presence of the nucleotide. This was more pronounced for isoenzyme PK II than for isoenzyme PK I. The cyclic AMP concentration needed to give half-maximal binding of the nucleotide was the same as the cyclic AMP concentration (Ka) at which the protein kinase had 50% of its maximal activity. The close correlation between binding and activation is also found in the presence of KCl, which increased the apparent activation constant (Ka) for cyclic AMP. With increasing [KCl], a progressively higher proportion of the histone phosphorylation observed in cytosol was due to cyclic AMP-independent (casein) kinases, leading to an overestimation of the degree of activation of the cyclic AMP-dependent protein kinases present. The relative contributions of cyclic AMP-dependent and -independent kinases to histone phosphorylation at different ionic strengths was determined by use of heat-stable inhibitor and phospho-cellulose chromatography.     

Role of dehydration in changing the permeability of erythrocyte plasma membranes by freeze-thawing

The phosphorylation of keratin polypeptides was examined in calf snout epidermis. When slices of epidermis were incubated in the medium containing 32Pi, the radioactivity was incorporated into several proteins. The predominant phosphorylated proteins migrated in SDS-polyacrylamide gels with apparent molecular weights between 49000 and 69000 and coincided with keratin polypeptides. The extent of keratin phosphorylation was not altered in the presence of dibutyryl cyclic AMP or reagents which elevate intracellular cyclic AMP. When homogenates of epidermis were incubated with [gamma-32P]ATP, keratin polypeptides were the predominant species phosphorylated as was also observed in epidermal slices. The presence of cyclic AMP or heat-stable inhibitor of cyclic AMP-dependent protein kinase in the reaction mixture did not affect the phosphorylation of keratin polypeptides, although the phosphorylation of exogenously-added histone was stimulated and inhibited, respectively, by these additions. Keratin polypeptides extracted from calf snout epidermis by 8 M urea were phosphorylated by incubation with [gamma-32P]ATP and cyclic AMP-dependent protein kinase from calf snout epidermis or bovine heart. No proteins were phosphorylated without the addition of the enzymes. The presence of cyclic AMP in the reaction mixture stimulated the keratin phosphorylation, and further addition of heat-stable protein kinase inhibitor reduced this stimulation.     

The mode of action of adenosine 3′:5′-cyclic monophosphate in mammalian islets of Langerhans. Preparation and properties of islet-cell protein phosphokinase

1. A protein was demonstrated in mammalian islets of Langerhans that after purification appeared as a single component possessing both cyclic-AMP (adenosine 3':5'-cyclic monophosphate)-binding and cyclic-AMP-dependent protein phosphokinase activities. 2. The protein had an intrinsic association constant for cyclic AMP of 1.15x10(-8)m, which was similar to the K(m) for cyclic AMP (1.11x10(-8)m) of the protein phosphokinase activity. 3. Incubation of the protein in the presence of cyclic AMP resulted in its dissociation into cyclic-AMP-independent protein phosphokinase (catalytic) and cyclic-AMP-binding (receptor) subunits, which could be separated on Sephadex G-200. 4. The cyclic-AMP-dependent protein phosphokinase was capable of phosphorylating a variety of proteins, the most readily phosphorylated being histone, casein and protein components of sub-cellular fractions prepared from islets of Langerhans. 5. The cyclic-AMP-dependent phosphorylation of histone had a K(m) for ATP of 1.1x10(-5)m. 6. The endogenous protein phosphokinase activity in rat islets incubated with agents that are known to alter the intracellular concentration of cyclic AMP was investigated. Theophylline and 3-isobutyl-1-methylxanthine, agents that raise cyclic AMP concentrations in islets, increased the activity of the protein phosphokinase, whereas adrenaline, which lowers islet cyclic AMP concentrations, decreased its activity. 7. It is suggested that cyclic AMP may exert its effects on insulin release by increasing the activity of a protein phosphokinase and may thereby promote the phosphorylation and activity of a rate-determining component of the secretory mechanism.     

PROTEIN KINASES IN MYELIN OF RAT BRAIN: SOLUBILIZATION AND CHARACTERIZATION

Myclin from rat brain contained adenosine 3′, 5′-monophosphate (cyclic AMP)-dependent protein kinase activity, which was solubilized by 0.2% Triton X-100 and required exogenous protein substrate for its activity. Also present was a protein kinase which catalysed the phosphorylation of the endogenous substrate and which was neither solubilized by Triton X-100 nor stimulated by cyclic AMP. Sodium fluoride was required to maintain the activity of the endogenous phosphorylation, probably by inhibiting ATPase activity, but had no effect on the phosphorylation of histone by the solubilized enzyme. Protamine and myelin basic protein served as well as histone as a substrate for the solubilized enzyme. A protein kinase modulator had no effect on the endogenous phosphorylation, but inhibited histone phosphorylation by the solubilized enzyme. Cyclic AMP-binding activity was observed in both the solubilized and non-solubilized preparations. The concentration of cyclic AMP required to give half-maximal binding activity of the preparations was about 2.5 nM. The results indicate that the cyclic AMP-binding site of the protein kinase in myelin may partially be accessible, whereas the catalytic site may be integrated into the membrane structure of myelin.     

Binding of cyclic AMP and cyclic GMP to renal cortical homogenates. Relationship with phosphorylation

This study examined the binding of both cyclic AMP and cyclic GMP to receptor proteins in particulate and soluble subfractions of renal cortical homogenates from the golden hamster. The binding of both nucleotides was compared to subsequent effects of both nucleotides on the phosphorylation of histone from identical fractions. Cyclic AMP binding and cyclic AMP-dependent protein kinase activity predominated in the cytosol, with some binding and enzyme activity also detected in particulate fractions. Cyclic GMP and cyclic GMP-dependent protein kinase activity could only be demonstrated in cytosolic fractions and represented only 20-30% of cyclic AMP-dependent activity in this fraction. Binding of both nucleotides was highly specific, however, cyclic AMP showed some interaction with cyclic GMP binding. Evidence suggesting that each nucleotide interacts with a specific protein kinase was as follows: both the binding activity of the cyclic nucleotides and their combined protein kinase activity show additivity; cyclic AMP and cyclic GMP binding activity could be separated on sucrose gradients; cyclic AMP and cyclic GMP protein kinase activity could be separated with Sephadex G-100 chromatography, after preincubation of homogenate supernatants with either cyclic AMP or cyclic GMP. The results demonstrate the presence of both cyclic AMP- and cyclic GMP-dependent protein kinase in renal cortex.     

Characteristics of the protein kinase activity associated with rat neurofilament preparations

Some properties of the protein kinase activity associated with neurofilaments isolated from the brain stem and spinal cord of rats have been investigated. The activity had an apparent Km for ATP of 20 microM, a pH optimum of 8.0 and phosphorylated both serine and threonine residues in neurofilament proteins. Cyclic AMP had no effect on the in vitro reaction and casein was a preferred exogenous substrate in comparison to histone. Phosphopeptide mapping of the 145 kDa subunit from neurofilaments phosphorylated in the presence and absence of microtubule proteins indicated that the neurofilament-associated activity was distinct from the microtubule-associated protein kinase. Limited proteolysis of neurofilaments with chymotrypsin indicated that the enzyme activity was not associated with a domain of the 200 kDa subunit which may form the side-arm projections on neurofilaments.     

Effects of cyclic adenosine 3': 5'-monophosphate on phosphoprotein kinase and phosphatase fractions prepared from rat liver nuclei.

A soluble rat liver nuclear extract containing total RNA polymerase activities also exhibits appreciable amounts of protein kinase activity. This unfractionated protein kinase catalyzes the phosphorylation of both endogenous proteins and exogenous lysine-rich histone in the presence of [γ-³²P]ATP and Mg²⁺. The optimal concentration of Mg²⁺ is 5 mm for histone phosphorylation and 25 mm for the phosphorylation of endogenous proteins. Cyclic AMP has no effect on the phosphorylation of lysine-rich histone by this unfractionated nuclear protein kinase. However, addition of cyclic AMP causes a reduction in the ³²P-labeling of an endogenous protein (CAI) which can be characterized by its mobility during SDS-acrylamide gel electrophoresis and elution in the unbound fraction of a DEAESephadex column. If CAI is first labeled with ³²P and then incubated with 10^?6m cyclic AMP under conditions where protein kinase activity is inhibited, the presence of the cyclic nucleotide causes a loss of the ³²P-labeling of this protein, implying the activation of a substrate-specific protein phosphatase. When rat liver RNA polymerases are purified by DEAE-Sephadex chromatography, protein kinase activity is found in the unbound fraction and in those column fractions containing RNA polymerase I and II. The fractionated protein kinases exhibit different responses to cyclic AMP, the unbound protein kinase being stimulated and the RNA polymerase-associated protein kinases being dramatically inhibited. A second protein (CAII) whose phosphorylated state is modified by cyclic AMP is found within the DEAE-Sephadex column fractions containing RNA polymerase II. The cyclic nucleotide in this case appears to reduce labeling of CAII by inhibition of the protein kinase activity which co-chromatographs with both CAII and RNA polymerase II. Based on molecular weight estimates, neither CAI nor CAII appears to be an RNA polymerase subunit. The identity of CAI as a protein factor whose phosphorylated state influences nuclear RNA synthesis is suggested by the fact that addition of fractions containing CAI to purified RNA polymerase II inhibits the activity of this enzyme, but only if CAI has been previously incubated in the presence of cyclic AMP.     

Properties and cellular distribution of cyclic AMP-dependent protein kinase from thymus

A protein kinase that catalyzes the phosphorylation of histone was partially purified from rat thymus, and the rate of histone phosphorylation was stimulated three- to fourfold by 1 × 10^?6 M adenosine 3′,5′-monophosphate (cyclic AMP). Thymic protein kinase was more active than the enzyme from spleen. Histone fractions f1, f2a, f2b, and f3 were all capable of serving as phosphate acceptors for the thymic protein kinase, and the rate of phosphorylation of each fraction was stimulated by cyclic AMP. The ability of various 3′,5′-mononucleotides to stimulate protein kinase activity was compared. Inosine 3′,5′-monophosphate (cyclic IMP) was the most effective substitute for cyclic AMP. The cellular distribution of cyclic AMP-dependent protein kinase and adenylate cyclase activities in the thymus was determined. Cyclic AMP-dependent protein kinase activity is present in both small thymocytes and residual thymic tissue. The specific activity of protein kinase from residual tissue, both for basal and cyclic AMP-stimulated enzyme, was greater than that of enzyme from small thymocytes. In contrast to this, adenylate cyclase activity is predominately localized in the thymocytes.     

Phosphorylation of keratin polypeptides

The phosphorylation of keratin polypeptides was examined in calf snout epidermis. When slices of epidermis were incubated in the medium containing ³²P_i, the radioactivity was incorporated into several proteins. The predominant phosphorylated proteins migrated in SDS-polyacrylamide gels with apparent molecular weight between 49000 and 69000 and coincided with keratin polypeptides. The extent of keratin phosphorylation was not altered in the presence of dibutyryl cyclic AMP or reagents which elevate intracellular cyclic AMP. When homogenates of epidermis were incubated with [γ-³²P]ATP, keratin polypeptides were the predominant species phosphorylated as was also observed in epidermal slices. The presence of cyclic AMP or heat-stable inhibitor of cyclic AMP-dependent protein kinase in the reaction mixture did not affect the phosphorylation of keratin polypeptides, although the phosphorylation of exogenously-added histone was stimulated and inhibited, respectively, by these additions. Keratin polypeptides extracted from calf snout epidermis by 8 M urea were phosphorylated by incubation with [γ-³²P]ATP and cyclic AMP-dependent protein kinase form calf snout epidermis or bovine heart. No proteins were phosphorylated without the addition of the enzymes. The presence of cyclic AMP in the reaction mixture stimulated the keratin phosphorylation, and further addition of heat-stable protein kinase inhibitor reduced this stimulation.     

Endogenous phosphorylation of platelet membrane proteins.

The characteristics of the phosphorylating activity of platelet membranes have been studied. Plasma membranes of human platelets isolated by the glycerol lysis technique were shown to incorporate significant amounts of [³²P]phosphate into specific membrane proteins. This activity was only partially cyclic 3′:5′-monophosphate (cyclic AMP)-dependent but had most of the other characteristics of protein kinases derived from other sources. Maximal stimulation of endogenous phosphorylation was obtained at 1 × 10^?7, m cyclic AMP and exceeded by approximately 30% the [³²P]phosphate incorporation in the absence of this cyclic nucleotide. The platelet membrane protein kinase was able to phosphorylate exogenous proteins, e.g., histone, fibrinogen etc., as well as endogenous membrane proteins. The latter solubilized by sodium dodecyl sulfate and separated by dodecyl sulfate-polyacrylamide gel electrophoresis incorporated [³²P]phosphate into three polypeptides of apparent molecular weights 52,000, 31,000, and 20,000. The phosphorylation of the polypeptide of molecular weight 52,000 was cyclic AMP-dependent.     

Modulation of protein phosphorylation by a factor purified from adipocytes.

1. A factor which modulates the activity of cyclic AMP-dependent protein kinase copurifies from rat adipocytes with an inhibitor of adenylate cyclase. Purification and stability studies suggest that both effects reside in a single factor previously referred to as a feedback regulator. 2. The magnitude and direction of the feedback regulator effect on cyclic AMP-dependent protein kinase activity was dependent on the concentration of feedback regulator and the concentration and type of protein substrate. Using histone type IIA as substrate, feedback regulator was inhibitory at low histone concentrations and stimulatory at high concentrations. Preincubation of protein kinase with feedback regulator resulted in inhibition at all histone concentrations. With some protein substrates, e.g. histone f2b and casein, inhibition was observed at all histone concentrations. 3. The stimulation of histone type IIA phosphorylation resulted from an increased V with no effect on either the apparent Ka for cyclic AMP or the Km for ATP. Time course studies suggest that feedback regulator increased the rate of phosphorylation without increasing the total number of phosphorylation sites. Increased histone phosphorylation was observed regardless of whether the cyclic AMP-dependent protein kinase was peak I or peak II (off Deae-cellulose), isolated from bovine or rabbit skeletal muscle or rat heart. A small stimulation was observed using cyclic GMP-dependent protein kinase. 4. These results indicate that feedback regulator can inhibit or stimulate protein kinase, an effect which is probably substrate directed, and depends on the reaction conditions. Whether feedback regulator modulated protein phosphorylation in vivo in addition to its inhibition of adenylate cyclase is unknown. However, stimulation of protein kinase activity in the presence of cyclic AMP is a valuable and rapid assay for monitoring feedback regulator fractions during purification procedures.     

Synaptic membrane proteins as substrates for cyclic AMP-stimulated protein phosphorylation in various regions of rat brain.

Synaptosomal plasma membranes from mammalian brain contain protein kinase activity which phosphorylates endogenous membrane proteins and is stimulated by cyclic AMP. Using polyacrylamide gel electrophoresis it was shown that at least ten proteins in the synaptosomal plasma membrane fraction could be phosphorylated by endogenous cyclic AMP-stimulated protein kinase activity. The number of proteins whose phosphorylation was stimulated by cyclic AMP was strongly influenced by the pH and Mg2+ concentration used in the phosphorylation reaction. A complex pattern of cyclic AMP-stimulated protein phosphorylation was obtained only with synaptosomal plasma membranes and a crude microsomal fraction. Mitochondrial and myelin fractions exhibited no cyclic AMP-stimulated protein kinase activity. Investigation of the distribution of substrates for cyclic AMP-stimulated phosphorylation among various brain regions failed to reveal any regional differences.