Effects of Ca2+, calmodulin and cyclic AMP on the phosphorylation of endogenous proteins by homogenates of rat 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 β-cell. To study the properties of such kinases and their endogenous protein substrates homogenates of rat islets of Langerhans were incubated with [γ-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 μM free Ca²⁺ and 0.7 μM 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 protein 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.     

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.     

Studies of kidney plasma membrane adenosine-3′,5′-monophosphate-dependent protein kinase

Porcine kidney cortex was utilized for the preparation of plasma-membrane-enriched and soluble cytoplasmic (cytosol) fractions for the purpose of examining the relative properties of cyclic [³H]AMP receptor and cyclic AMP-dependent protein kinase activities of these preparations. The affinity, specificity and reversibility of cyclic [³H]AMP interaction with renal membrane and cytosol binding sites were indicative of physiological receptors.Binding sites of cytosol and deoxycholate-solubilized membranes were half-saturated at approx. 50nM and 100 nM cyclic [³H]AMP. Native plasma membranes exhibited multiple binding sites which were not saturated up to 1 mM cyclic [³H]AMP. Modification of the cyclic phosphate configuration or 2′-hydroxyl of the ribose moiety of cyclic AMP produced a marked reduction in the effectiveness of the cyclic AMP analogue as a competitor with cyclic [³H]AMP for renal receptors. The cyclic [³H]AMP interaction with membrane and cytosol fractions was reversible and the rate and extent of dissociation of bound cyclic [³H]AMP was temperature dependent. With the plasma-membrane preparation, dissociation of cyclic [³H]AMP was enhanced by ATP or AMP.Assay of both kidney subcellular fractions for protein kinase activity revealed that cyclic AMP enhanced the phosphorylation of protamine, lysine-rich and arginine-rich histones but not casein. The potency and efficacy of activation of renal membrane and cytosol protein kinase by cyclic AMP analogues such as N⁶-butyryl-adenosine cyclic 3′,5′-monophosphate or N⁶,O²-dibutyryl-adenosine cyclic 3′,5′-monophosphate supported the observations on the effectiveness of cyclic AMP analogues as competitors with cyclic [³H]AMP in competitive binding assays.This study suggested that the membrane cyclic [³H]AMP receptors may be closely associated with the membrane-bound catalytic moiety of the cyclic AMP-dependent protein kinase system of porcine kidney.     

Characteristics of magnesium-dependent, Ca2+ -stimulated endogenous protein kinase-catalyzed phosphorylation of basic proteins in myelin isolated from rat brain stem white matter

Purified myelin fraction isolated from rat brain white matter contained Mg²⁺-dependent protein kinase capable of phosphorylation of myelin basic proteins. The Mg²⁺-supported kinase was markedly stimulated (two- to fivefold) by micromolar concentrations of free Ca²⁺ with and without Triton X-100 in the assay, the degree of stimulation being greater with the detergent present. Cyclic AMP, on the other hand, failed to show any effect on phosphorylation of myelin in the absence of Triton X-100 and in the presence of Triton caused only 25–30% stimulation. The phosphorylation reaction was temperature dependent and exhibited a pH optimum at pH 6.5. Apparent affinity toward MgATP^2? was found to be about 70 μm and Ca²⁺ had no effect on this parameter. Dependence on MgCl₂ of myelin phosphorylation indicated the presence of high- and low-affinity sites toward Mg²⁺; Ca²⁺ appeared to influence the low-affinity site. Maximal level of phosphorylation was attained by 10–15 min at 30 °C and it declined at longer incubation times due to phosphatase activity present in the preparation. Stimulatory effect of Ca²⁺ on phosphorylation was not due to inhibition of phosphatase activity. Dephosphorylation experiments showed that neither cyclic AMP nor Ca²⁺ influenced the myelin phosphatase activity. Autoradiographic analysis revealed that phosphorylation of myelin basic proteins accounted for nearly 90% of total myelin phosphorylation. This was supported by the observation that the HCl extract of myelin contained 85% of total activity and comigrated with purified myelin basic proteins. Basal and Ca²⁺-stimulated phosphorylation of basic proteins were due to phosphorylation of serines mainly, although threonine was phosphorylated to a minor extent. Within myelin, Ca²⁺ and cyclic AMP kinases are differentially bound. It appears that the myelin kinase (studied in vitro) is primarily influenced by Ca²⁺ rather than cyclic AMP. Inhibitors (Type I and Type II) of cyclic nucleotide-stimulated protein kinases had no effect on the Ca²⁺-stimulated phosphorylation although basal and cyclic AMP-stimulated phosphorylation was inhibited, indicating that the Ca²⁺ kinase is a separate and distinct enzyme from the cyclic AMP-stimulated and basal kinase(s). Also, leupeptin, a protease inhibitor, did not influence basal, cyclic AMP-stimulated, or Ca²⁺-stimulated myelin phosphorylation, indicating that under the conditions used protease(s) did not alter the myelin kinase activity. The potential significance of phosphorylation of myelin basic proteins and the stimulatory action of Ca²⁺ on this reaction are discussed.     

Calcium transport by cardiac sarcoplasmic reticulum and phosphorylation of phospholamban

Summary Active calcium transport by cardiac sarcoplasmic reticulum assumes a central role in the excitation-concentration coupling of the myocardium, in that Ca²⁺-dependent ATPase (mol.wt. 100 000) of cardiac sarcoplasmic reticulum serves as an energy transducer and a translocator of Ca²⁺ across the membrane. During the translocation of Ca²⁺, the ATPase undergoes a complex series of reactions during which the phosphorylated intermediate EP is formed. We documented how the elementary steps of the ATPase reaction are coupled with calcium translocation, and provided evidences to indicate that two key steps of ATPase correspond to the conformational change of the enzyme, and appear to alter the affinity of the enzyme for Ca²⁺.A line of evidence also indicated that Ca²⁺-dependent ATPase of cardiac sarcoplasmic reticulum is regulated by a specific protein named phospholamban (mol.wt. 22 000), which serves as a substrate for cyclic AMP-dependent protein kinase. Cyclic AMP-dependent phosphorylation of phospholamban resulted in a marked increase in the rate of turnover of the ATPase, by enhancing the rates of the key elementary steps, i.e. the steps at which the intermediate EP is formed and decomposed. Thus phospholamban is putatively thought to serve as a modulator of Cat²⁺-dependent ATPase of cardiac sarcoplasmic reticulum. A working model was proposed to interpret the mechanism. Also documented is a possibility that another protein kinase activatable by Ca²⁺ and calmodulin is functional in regulating the phospholamban-ATPase system, thus suggesting the existence of a dual control system, in which both cyclic AMP- and calmodulin-dependent phosphorylation are in control of the Cat²⁺-dependent ATPase.Such a control mechanism may provide the interpretation, at the cellular level, that catecholamines exert actions on myocardial contractility. Thus, catecholamine-mediated increases in intracellular cyclic AMP could enhance calcium fluxes across the membrane of sarcoplasmic reticulum, thus resulting in the increased rates of relaxation and, at the same time, the increased rate and extent of contraction. Such a mechanism could also be operational in the tissues, other than the myocardium, in which catecholamines and other hormones serve as the first messenger, producing intracellular cyclic AMP as the second messenger.     

Phosphorylation of cardiac sarcolemma by endogenous and exogenous protein kinases.

Sarcolemmal membranes isolated from guinea pig heart ventricles contained endogenous protein kinase activity and protein substrates for this enzyme. Phosphorylation of sarcolemma was modestly stimulated by cyclic AMP with the half-maximal stimulation at 0.5 μm cyclic AMP. The phosphorylation of sarcolemma due to endogenous kinase was dependent on Mg²⁺. The apparent affinity for Mg²⁺ was found to be 1.4 and 0.53 mm in the absence and presence of 1 μm cyclic AMP, respectively. The apparent affinity for ATP was 55 μm. Sarcolemmal membranes were also phosphorylated by exogenous (purified) cyclic AMP-dependent protein kinase(s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of phosphorylated membranes, followed by slicing and determination of the radioactivity in the gel slices, showed that endogenous protein kinase activity promoted the phosphorylation of specific protein peaks, arbitrarily designated a–g in order of increasing relative mobility (relative molecular weights 125,000, 110,000, 86,000, 58,000, 48,000, 22,000, and 16,000, respectively); peak e (48,000) was the major phosphorylated band. Exogenous protein kinase stimulated the phosphorylation of all peaks. However, the degree of stimulation of the low molecular weight peaks f and g was more marked. Results obtained after treatment of phosphorylated membranes with hydroxylamine at acid pH indicated the absence of any significant amount of acyl phosphate-type incorporation of phosphate. Purified phosphoprotein phosphatase from rabbit liver effected dephosphorylation of previously phosphorylated sarcolemma; this treatment resulted in dephosphorylation of all peaks (a–g). Pretreatment of sarcolemma with trypsin (membrane to trypsin ratio of 100) was found to markedly reduce both the total membrane phosphorylation as well as relative phosphorylation of peaks c, f, and g. On the other hand, pretreatment of sarcolemma with phospholipase c slightly stimulated total membrane phosphorylation with nondiscriminatory enhancement of the phosphorylation of all peaks. Microsomal membrane vesicles (enriched in sarcoplasmic reticulum fragments) isolated from guinea pig heart ventricle also contained endogenous protein kinase activity. Cyclic AMP modestly increased the kinase. Polypeptides of molecular weights 56,000, 22,000, and 16,000 were found to be phosphorylated. Exogenous (purified) cyclic AMP-dependent protein kinase increased the phosphorylation of microsomes and of 22,000 and 16,000 molecular weight polypeptides.     

Effects of adenosine 3′ : 5′-monophosphate and guanosine 3′ : 5′-monophosphate on calcium uptake and phosphorylation in membrane fractions of vascular smooth muscle

The effects of adenosine 3′ : 5′-monophosphate (cyclic AMP), guanosine 3′ : 5′-monophosphate (cyclic GMP) and exogenous protein kinase on Ca uptake and membrane phosphorylation were studied in subcellular fractions of vascular smooth muscle from rabbit aorta. Two functionally distinct fractions were separated on a continuous sucrose gradient: a light fraction enriched in endoplasmic reticulum (fraction E) and a heavier fraction containing mainly plasma membranes (fraction P).While cyclic AMP and cyclic GMP had no effect on Ca uptake in the absence of oxalate, both cyclic nucleotides inhibited the rate of oxalate-activated Ca uptake when used at concentrations higher than 10^?5 M. The addition of bovine heart protein kinase to either fraction produced an increase in the rate of oxalate-activated Ca uptake which was further augmented by cyclic AMP. Cyclic GMP caused smaller stimulations of protein kinase-catalyzed Ca uptake than cyclic AMP.Mg-dependent phosphorylation, attributable to endogenous protein kinase(s), was inhibited in fraction E by low concentrations (10^?8 M) of both cyclic AMP and cyclic GMP. In fraction P, an inhibition by cyclic AMP occurred also at a concentration of 10^?8 M, while with cyclic AMP a concentration of 10^?5 M was required for a similar inhibition. Bovine heart protein kinase stimulated the phosphorylation of the membrane fractions much more than Ca uptake. In fraction E, in the presence of bovine protein kinase, both cyclic AMP and cyclic GMP stimulated phosphorylation up to 200%. Under these conditions, no stimulation was observed in fraction P.These results are compatible with the hypothesis that in vascular smooth muscle soluble rather than particulate protein kinases are involved in the regulation of intracellular Ca concentration.     

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.     

Partial characterization of protein kinase-catalyzed phosphorylation of low molecular weight proteins in purified preparations of pigeon heart sarcolemma and sarcoplasmic reticulum

Pigeon heart microsomes contain three minor size protein kinase substrates of minimal molecular weights of 22 000, 15 000, and 11 500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the microsomes were partially loaded with calcium oxalate and subjected to rate zonal and isopynic centrifugations in sucrose density gradient columns, the 22 000 and the 15 000 dalton proteins settled in the heaviest fraction, which was composed mainly of vesicles of sarcoplasmic reticular membranes; the 11 500 dalton protein was concentrated in the lightest fractions, which consisted chiefly of vesicles of sarcolemmal origin. During incubation of the membrane fractions with Mg[γ-³²P]ATP significant amounts of ³²P were incorporated into all these proteins. Incorporation of ³²P into the 15 000 dalton protein was moderately and ³²P incorporation into the 22 000 dalton protein was markedly enhanced in the presence of exogenous soluble cyclic AMP-dependent protein kinase and cyclic AMP. The phosphorylation of the three proteins was virtually unaffected by CA²⁺ concentrations up to 0.1 mM and by ethyleneglycol-bis(β-aminoethylether)-N,N′-tetraacetic acid in the absence of added Ca²⁺.Phosphorylation of the 22 000 and the 11 500 dalton proteins occurred mainly at serine residues. In the 15 000 dalton protein threonine residues were the main site of endogenous phosphorylation. Nearly equal amounts of [³²P]-phosphate were incorporated into threonine and serine residues of this protein when phosphorylation was supported by exogenous cyclic AMP-dependent protein kinase and cyclic AMP.The 15 000 dalton protein could be removed from its membrane attachment by extraction with an acidic chloroform/methanol mixture. This step opens the way for the purification of this membrane-bound protein kinase substrate.     

Differential effects of cyclic adenosine-3',5'-monophosphate on phosphorylation of rat liver nuclear acidic proteins

The effects of cyclic AMP on the phosphorylation of different acidic proteins of rat liver nuclei were examined in vivo and in vitro. N⁶,O²′-dibutyryl cyclic AMP selectively stimulated in vivo phosphorylation of specific nuclear proteins more than twofold within 15 min after injection. Cyclic AMP caused only a small stimulation of phosphorylation of acidic proteins in isolated nuclei but the stimulation was selective for specific proteins. When isolated nuclear acidic proteins were incubated with a soluble cyclic AMP-dependent protein kinase, the cyclic nucleotide stimulated total phosphorylation about 1.7-fold. These results support the view that the regulatory effects of cyclic AMP may involve phosphorylation of acidic proteins associated with DNA in the chromatin.     

Control of Ca2+ efflux and cyclic AMP by 5-hydroxytryptamine and dopamine in abalone gill.

5-Hydroxytrptamine increased the rate of Ca²⁺ efflux and the concentration of endogenous cyclic AMP in abalone gill in both 10 mM and 50 mM CaCl₂ concentrations externally. Dopamine decreased the rate of Ca²⁺ efflux in 50 mM CaCl₂ but slightly increased the efflux rate in 10 mM CaCl₂. At both external Ca²⁺ concentrations, dopamine increased the endogenous cyclic AMP concentration in the gill. 5-Hydroxytryptamine but not dopamine was found to activate adenylate cyclase in broken cell preparations of abalone gill. Cyclic AMP-dependent protein kinase activity was also demonstrated in homogenate fractions of abalone gill. It is suggested that both Ca²⁺ and cyclic AMP act as second messengers in the response of abalone gill to 5-hydroxytryptamine and dopamine.     

Plasma membrane cyclic AMP-dependent protein phosphorylation system in L6 myoblasts

Plasma membranes can be isolated without disruption of cells by the plasma membrane vesiculation technique (Scott, R.E. (1976) Science 194, 743–745). A major advantage of this technique is that it avoids contamination of plasma membranes with intracellular membrane components. Using this method, we prepared plasma membranes from L₆ myoblasts grown in tissue culture and studied the characteristics of the protein phosphorylation system.We found that these plasma membrane preparations contain protein kinase which is tightly bound to the membrane and cannot be removed by washing in EDTA or in high ionic strength salt solutions. This protein kinase activity can catalyze the phosphorylation of several exogenous substrates with decreasing efficiency as acceptors of phosphate: calf thymus histones f₂b, protamine and caseine. Cyclic AMP causes a dose-dependent stimulation of protein kinase activity; the highest stimulation (4-fold) is achieved at concentration 10^?5 M cyclic AMP. Cyclic AMP-dependent stimulation can be completely inhibited by heat-stable protein kinase inhibitor isolated from rabbit skeletal muscle. On the other hand, cyclic GMP does not affect the activity of protein kinase.Plasma membrane-bound protein kinase also catalyzes the phosphorylation of endogenous membrane protein substrates and this is also stimulated by addition of cyclic AMP. Analysis of plasma membrane proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that specific polypeptides are phosphorylated by cyclic AMP-independent and by cyclic AMP-dependent protein kinase systems.The results of these studies demonstrate the presence of endogenous cyclic AMP-dependent and -independent protein phosphorylating systems (enzyme activity and substrates) in purified plasma membrane preparations. These data provide a basis for further investigations on the role of plasma membrane missing data     

Photoaffinity labeling of three renal cyclic 3′,5′-adenosine monophosphate-binding proteins

Evidence is presented for the presence of multiple cyclic AMP binding components in the plasma membrane and cytosol fractions of porcine renal cortex and medulla. N⁶-(Ethyl-2-diazomalonyl)-3′,5′-adenosine monophosphate, a photoaffinity label for cyclic AMP binding sites, exhibits non-covalent binding characteristics similar to cyclic AMP in membrane and soluble fractions. Binding data for either compound to the plasma membrane fraction yields biphasic Scatchard plots while triphasic plots are obtained with the dialyzed cytosol. When covalently labeled fractions are separated on SDS-polyacrylamide gel electrophoresis, the cyclic AMP photoaffinity label is found on 49 000 and 130 000 dalton components in each kidney fraction. DEAE-cellulose and gel filtration chromatography of the labeled cortical cytosol fraction establishes that the three components suggested by the binding data correspond to two 49 000 dalton species and a 130 000 component. The 49 000 species have higher affinities for cyclic AMP than the 130 000 component (K_a(1) = 2.0 · 10⁹, K_a(2) = 1.7 · 10⁸, K_a(3) = 1.0 · 10⁷). The 49 000 components are associated with protein kinase activity while the 130 000 component does not exhibit protein kinase, adenosine deaminase, or cyclic nucleotide phosphodiesterase activity. Immunologic results and effects of phosphorylation and cyclic GMP on cyclic AMP binding further suggest that the 49 000 components are regulatory subunits of cyclic AMP-dependent protein kinases. Cyclic AMP binding to the 130 000 component is markedly inhibited by adenosine and adenine nucleotides, but not cyclic GMP. Thus, this component may reflect an aspect of adenosine control or metabolism which may or may not be a cyclic AMP-related cellular function.     

Phosphorylation of cardiac sarcoplasmic reticulum by a calcium-activated,phospholipid-dependent protein kinase

Cardiac sarcoplasmic reticulum is phosphorylated by a cytosolic Ca²⁺-activated, phospholipid-dependent protein kinase. This phosphorylation is independent of cyclic nucleotides and enhanced by unsaturated diacylglycerols; saturated diacylglycerols, mono- and tri-glycerides are ineffective. Diacylglycerol stimulation is due to increased Ca²⁺ sensitivity of the kinase reaction. Protein kinase catalyzed phosphorylation results in enhanced Ca²⁺-transport ATPase activity and may be an important determinant of cardiac sarcoplasmic reticulum function.     

Hormonal control of uterine contraction: Characterization of cyclic AMP-dependent membrane properties in the myometrium

A mitochondria-free membrane fraction prepared from rat myometrium accumulated ⁴⁵Ca²⁺ in the presence of oxalic acid and ATP. The rate of transport of Ca²⁺ into the membranous vesicles was increased by greater than 50% in the presence of 3′,5′-cyclic AMP, but not by 2′,3′-cyclic AMP or 5′-AMP. Membrane ATPase activity was stimulated by cyclic AMP in a manner similar to Ca²⁺-transport. ATPase activity was stimulated by Mg²⁺; slight additional stimulation was obtained in the presence of Na⁺ and K⁺ but not in the presence of Ca²⁺. Despite the cyclic AMP sensitivity of membrane ATPase activity, the absence of any effect of inhibitors of Ca²⁺-transport suggest it has little to do with Ca²⁺ accumulation by the membranes.Cyclic AMP-induced increase in Ca²⁺-transport and membrane ATPase activity was duplicated in vivo by incubating uteri in 10⁻⁴ M isoproterenol prior to membrane isolation. Isoproterenol has been previously shown to increase myometrial cyclic AMP levels, and changes in Ca²⁺-transport by cell membranes in relation to intracellular cyclic AMP levels may be the mechanism through which hormones modulate uterine contractility.     

Modulation of the activity of the (Ca2+ + Mg2+)-dependent adenosine triphosphatase of the human erythrocyte

We previously reported that the activity of the (Ca2+ + Mg2+)-dependent adenosine triphosphatase (ATPase) of the human erythrocyte membrane is inhibited by micromolar or nanomolar concentrations of cyclic AMP. Our further studies have now indicated that the inhibition of (Ca2+ + Mg2+)-dependent phosphohydrolase activity requires the participation of a membrane-associated cyclic AMP-dependent protein kinase and a membrane-associated protein substrate that is distinct from the ATPase itself. We have furthermore, identified a 20 kDa membrane protein which undergoes phosphorylation that is promoted by micromolar, but not millimolar, concentrations of cyclic AMP and which, when phosphorylated, undergoes dephosphorylation that is promoted by Ca2+. We suggest that this membrane component can participate in the modulation of the activity of the (Ca2+ + Mg2+)-dependent ATPase of the human erythrocyte.     

Action of cyclic nucleotide analgoues in Chinese hamster ovary cells

Cyclic nucleotide analogues have been tested for their ability to cause the morphological conversion of Chinese hamster ovary cells in culture, as well as for effects on cyclic AMP-related enzymes. The ability of the analogues to inhibit the cyclic AMP phosphodiesterase activity and to activate the cyclic AMP-dependent protein kinase activity in cell extracts has been measured. Cell cultures were incubated with the analogues and the effects on morphology, intracellular level of cyclic AMP, and in vivo protein kinase activation were determined. All analogues which induced the morphological conversion also caused in vivo activation of the cyclic AMP-dependent protein kinase. Only N⁶,O^2′-dibutryl and N⁶-monobutyryl cyclic AMp caused caused on increase in intracellular cyclic AMP, presumably through inhibition of the intracellular cyclic AMP phosphodiesterase activity. The increase in cyclic AMP appears to cause the protein kinase activation. However, analogues such as 8-bromo and 8-benzylthio cyclic AMP do not cause any change in intracellular cyclic AMP level and appear to activate the intracellular cyclic AMP-dependent protein kinase directly.     

Phospholipid-sensitive Ca2+-dependent protein kinase and its substrates in human neutrophils

Phospholipid-sensitive Ca²⁺-dependent protein kinase (PL-Ca-PK) was found to be present at a high level in human neutrophils, with its activity localized in the particulate fraction. In contrast, cyclic AMP-dependent protein kinase (A-PK) and cyclic GMP-dependent protein kinase (G-PK), present at lower levels compared to PL-Ca-PK, were localized in the cytosolic fraction. Phosphorylation of several endogenous proteins (mol. wts. 89,000, 38,000, 34,000, 17,000 and 15,000), also localized in the particulate fraction, was stimulated specifically by a combination of phosphatidylserine and Ca²⁺, whereas no substrate proteins were observed for the calmodulin-sensitive Ca²⁺-dependent protein kinase system under the same incubation conditions. Although no substrate proteins for G-PK were detected, one substrate (mol. wt. 19,000) for A-PK was observed. Phosphorylation of substrates for PL-Ca-PK, but not that for A-PK and for enzymes independent of Ca²⁺ or cyclic AMP, was inhibited by a variety of agents, including trifluoperazine, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide], adriamycin, palmitoylcarnitine, and melittin. The present findings suggest that the $\text{phospholipid}\text{Ca}{}^{\mathrm{2+}}\text{-stimulated}$ protein phosphorylation system may be important in the membrane associated functions of human neutrophils.     

Regulation of the intracellular calcium level in human blood platelets: Cyclic adenosine 3′,5′-monophosphate dependent phosphorylation of a 22 000 dalton component in isolated Ca-accumulating vesicles

Two protein kinase activities have been separated from the supernatants of homogenized human blood platelets by DEAE cellulose chromatography. One of them (peak I enzyme) is an efficient stimulator of the uptake of Ca²⁺ into isolated membrane vesicles in the presence of cyclic AMP and ATP. The second (peak II enzyme), although equally active towards histone, exerts only about one third of the activity of the peak I enzyme. The stimulation of Ca²⁺ uptake is accompanied by the phosphorylation of a membrane protein with an apparent molecular weight of 22 000, which appears to play an essential role in the regulation of the intracellular Ca²⁺ level and hence of platelet activity.     

Regulation of the intracellular calcium level in human blood platelets: Cyclic adenosine 3′,5′-monophosphate dependent phosphorylation of a 22 000 dalton component in isolated Ca2+-accumulating vesicles

Two protein kinase activities have been separated from the supernatants of homogenized human blood platelets by DEAE cellulose chromatography. One of them (peak I enzyme) is an efficient stimulator of the uptake of Ca²⁺ into isolated membrane vesicles in the presence of cyclic AMP and ATP. The second (peak II enzyme), although equally active towards histone, exerts only about one third of the activity of the peak I enzyme. The stimulation of Ca²⁺ uptake is accompanied by the phosphorylation of a membrane protein with an apparent molecular weight of 22 000, which appears to play an essential role in the regulation of the intracellular Ca²⁺ level and hence of platelet activity.