Identification of two different phosphofructokinase-phosphorylating protein kinases from Ascaris suum muscle

Two different phosphofructokinase-phosphorylating protein kinases were separated from extracts of Ascaris suum muscle by chromatography on DEAE-Fractogel. They were tentatively designated phosphofructokinase kinase I and phosphofructokinase kinase II. Phosphofructokinase kinase I eluted from the chromatography column at an ionic strength of 0.07 and contained about 25% of the phosphofructokinase-phosphorylating activity assayed in crude extracts. The protein kinase activity was not stimulated by the addition of either cAMP or cGMP. It was inhibited by the heat-stable protein kinase inhibitory protein from rabbit muscle (Walsh inhibitor), by the regulatory subunit of cAMP-dependent protein kinase from beef heart, and by the cAMP-binding protein from Ascaris muscle. These properties suggest that phosphofructokinase kinase I is homologous to the catalytic subunit of cAMP-dependent protein kinases from mammals. This assumption is supported by the estimation of the Mr of 40,000 for the purified phosphofructokinase kinase I under denaturing conditions and by the fact that the presence of cAMP eliminated the inhibition by the cAMP binding proteins. The isoelectric point of the enzyme was 8.7. Phosphofructokinase kinase II was eluted from the DEAE-Fractogel column at an ionic strength of 0.16 and contained approximately 75% of the phosphofructokinase kinase activity measured in the extracts. The molecular and kinetic properties were significantly different from those of phosphofructokinase kinase I. The enzyme was not inhibited by the heat-stable inhibitor protein nor by cAMP-binding proteins. The Mr of the native enzyme was estimated as 220,000 by molecular sieve chromatography. The isoelectric point of the enzyme was pH 5.45.     

Comparison of adenylate cyclase and cAMP-dependent protein kinase in gametocytogenic and nongametocytogenic clones of Plasmodium falciparum

Adenylate cyclase and cAMP-dependent protein kinase activities in gametocytogenic (LE5) and nongametocytogenic (T9/96) clones of Plasmodium falciparum were compared to explore the role of cAMP in sexual differentiation of the parasite. Basal adenylate cyclase levels were equivalent in the 2 clones. However, cAMP-dependent histone II-A kinase activity was significantly higher in LE5 than in T9/96 over a range of cAMP concentrations. This difference was due to a decreased Vmax for the enzyme in the nongametocytogenic clone and not to an increased Ka for cAMP. Examination of parasite cAMP-binding proteins, likely to be kinase regulatory subunits, by both photoaffinity labeling with [32P]8-N3-cAMP and affinity chromatography of metabolically [35S]methionine-labeled cytosol of cAMP-agarose revealed a 53-kDa cAMP binding protein in both clones and a 49-kDa cAMP-binding protein in T9/96 that was absent in LE5. Our results suggest that T9/96 has lost the ability to undergo gametocytogenesis due to a substantial decrease in cAMP-dependent protein kinase activity rendering the parasite unable to respond to increased intracellular cAMP levels. Moreover, the reduction in cAMP-dependent protein kinase activity may be due to the presence of an alternative regulatory subunit of the kinase.     

Role of 3',5'-cyclic AMP in the control of nuclear protein kinase activity

The role of cAMP in the regulation of nuclear protein kinase activity was investigated. Acidic nuclear proteins prepared from rat liver nuclei were separated by phosphocellulose chromatography into four peaks of protein kinase activity and two peaks of cAMP-binding activity. A fraction which bound cAMP also inhibited the most active nuclear protein kinase, K IV, and the inhibition was diminished in the presence of 5 μM cAMP. Further support for the regulation of nuclear kinases by cAMP was obtained using a regulatory subunit prepared from rabbit muscle protein kinase. The muscle regulatory subunit markedly inhibited liver nuclear kinase activities. The addition of cAMP partially restored the activities.     

Cyclic AMP-binding protein and estrogen receptor: antagonism during nuclear translocation in a hormone-dependent mammary tumor

Incubation of nuclei from hormone-dependent rat mammary tumors with its cytosol activated with 5 nM 17β-estradiol resulted in a 4-fold increase of nuclear estrogen binding activity over the control nuclei. The presence of 100 nM cAMP in the activated cytosol inhibited this nuclear uptake of estrogen receptor by 50%. Conversely, incubation of the nuclei with cytosol activated with 100 nM cAMP increased nuclear cAMP binding and cAMP-dependent protein kinase activity 4-fold, while the presence of 5 nM 17β-estradiol in the activated cytosol inhibited the nuclear cAMP binding and the protein kinase activity by 50%. No competition was found between estrogen and cAMP for each other's cytoplasmic binding proteins or the nuclear acceptor sites. These data suggest that a mutual antagonism exists between the cAMP-binding protein and estrogen receptor during their nuclear translocation.     

DNA binding by cyclic adenosine 3',5'-monophosphate dependent protein kinase from calf thymus nuclei.

Cyclic adenosine 3',5'-monophosphate (cAMP) dependent protein kinase and proteins specifically binding cAMP have been extracted from calf thymus nuclei and analyzed for their abilities to bind to DNA. Approximately 70% of the cAMP-binding activity in the nucleus can be ascribed to a nuclear acidic protein with physical and biochemical characteristics of the regulatory (R) subunit of cAMP-dependent protein kinase. Several peaks of protein kinase activity and of cAMP-binding activity are resolved by affinity chromatography of nuclear acidic proteins on calf thymus DNA covalently linked to aminoethyl Sephrarose 4B. When an extensively purified protein kinase is subjected to chromatography on the DNA column in the presence of 10(-7) M cAMP, the R subunit of the kinase is eluted from the column at 0.05 M NaCl while the catalytic (C) subunit of the enzyme is eluted at 0.1-0.2 M NaCl. When chromatographed in the presence of histones, the R subunit is retained on the column and is eluted at 0.6-0.9 M NaCl. In the presence of cAMP, association of the C subunit with DNA is enhanced, as determined by sucrose density gradient centrifugation of DNA-protein kinase complexes. cAMP increases the capacity of the calf thymus cAMP-dependent protein kinase preparation to bind labeled calf thymus DNA, as determined by a technique employing filter retention of DNA-protein complexes. This protein kinase preparation binds calf thymus DNA in preference to salmon DNA, Escherichia coli DNA, or yeast RNA. Binding of protein kinases to DNA may be part of a mechanism for localizing cyclic nucleotide stimulated protein phosphorylation at specific sites in the chromatin.     

Activation of type I cyclic AMP-dependent protein kinases with defective cyclic AMP-binding sites

Two S49 mouse lymphoma cell variants hemizygous for expression of mutant regulatory (R) subunits of type I cyclic AMP-dependent protein kinase were used to investigate functional consequences of lesions in the putative cAMP-binding sites of R subunit. Kinase activation properties of wild-type and mutant enzymes were compared using cAMP and six site-selective analogs of cAMP. Kinases from both mutant sublines were relatively resistant to cyclic nucleotide-dependent activation, but they were fully activable by at least some effectors. Relative resistances of the mutant kinases varied from about 5-fold for analogs selective for their nonmutated sites to as much as 700-fold for analogs selective for their mutated sites; resistance to cAMP was intermediate. Apparent affinities of wild-type and mutant R subunits for [3H]cAMP were not appreciably different, but competition experiments with site-selective analogs of cAMP suggested that binding of cAMP to mutant R subunits was primarily to their nonmutated sites. Analyses of cooperativity in cyclic nucleotide-dependent activation of mutant kinases, synergism between site I- and site II-selective analogs in activating the mutant enzymes, and dissociation of bound cAMP from mutant R subunits provided additional evidence that the mutations in these strains selectively inactivated single classes of cAMP-binding sites: phenomena attributable in wild-type enzyme to intrachain interactions between sites I and II were always absent or severely diminished in experiments with the mutant enzymes. These results confirm that R subunit sequences implicated in cAMP binding by homology with other cyclic nucleotide-binding proteins actually correspond to functional cAMP-binding sites. Furthermore, occupation of either cAMP-binding site I or II is apparently sufficient for activation of cAMP-dependent protein kinase. The presence of four functional cAMP-binding sites in wild-type kinase enhances the cooperativity and sensitivity of cAMP-mediated activation.     

Modulation of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF) acetyltransferase in human polymorphonuclears. The role of cyclic AMP-dependent and phospholipid sensitive, calcium-dependent protein kinases

In order to characterize the mechanism of activation of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase (EC 2.3.1.67) which is the limiting step in the regulation of the synthesis of the potent inflammatory mediator 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; homogenates from human polymorphonuclear leukocytes were incubated in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase and in the presence of a partially purified phospholipid sensitive, calcium-dependent protein kinase (PrKC). The first kinase was found to enhance up to 3-fold acetyltransferase activity in a dose- and time-dependent manner. In homogenates from PMN previously stimulated with complement-coated zymosan particles, the decay of acetyltransferase activity was partially prevented by the addition of soybean trypsin inhibitor and almost completely inhibited when the homogenates were supplemented with inhibitors of alkaline phosphatase such as 50 mM KF and 100 microM paranitrophenylphosphate. Under these conditions it was possible to initiate the decay of acetyltransferase activity by adding an excess of alkaline phosphatase. Preincubation of PMN with 12-O-tetradecanoylphorbol-13-acetate previous or simultaneously to the addition of ionophore A23187 reduced the increase in acetyltransferase produced by ionophore A23187, whereas the generation of superoxide anions was enhanced. Addition of partially purified PrKC to homogenates from ionophore A23187-stimulated PMN, reduced acetyltransferase activity by 63%, whereas only a 16% inhibition was observed on homogenates from resting PMN. These data indicate the modulation of acetyltransferase activity in human polymorphonuclear leukocytes by a phosphorylation-dephosphorylation mechanism linked to cyclic AMP-dependent protein kinase. Phospholipid sensitive, calcium-dependent protein kinase seems not to be involved in the mechanism of activation, but, most probably, in the generation of negative activation signals.     

A cAMP-binding phosphoprotein in Drosophila heads is similar to the regulatory subunit of the mammalian type II cAMP-dependent protein kinase

Cyclic AMP (cAMP)-dependent regulation of in vitro phosphorylation of several proteins including a cAMP-binding protein was studied with crude membrane and cytosol fractions from Drosophila heads. Phosphorylation of at least seven distinct proteins was enhanced in the presence of cAMP. Interestingly, however, the phosphorylation of a 56 kDa protein was apparently reduced by cAMP in the membrane but not in the cytosol fraction. The following data strongly indicate that the 56 kDa phosphoprotein in both membrane and cytosol fractions is a cAMP-binding protein, very similar to the regulatory subunit (RII) of a mammalian cAMP-dependent protein kinase, and that its binding to cAMP makes this protein very susceptible to the action of phosphatases: (i) cAMP highly stimulated the dephosphorylation of the 56 kDa phosphoprotein by the endogenous phosphatase in the membrane fraction. (ii) The dephosphorylation of a similar 56 kDa phosphoprotein in the cytosol fraction by an exogenous, cAMP-independent, alkaline phosphatase was also highly stimulated by cAMP. (iii) The 56 kDa phosphoprotein was covalently bound to cAMP by u.v. irradiation. (iv) The alkaline-phosphatase treatment reversibly converted this phosphoprotein to a 53 kDa non-phosphorylated protein. (v) The 53 kDa protein was selectively bound to cAMP-agarose and subsequently eluted by cAMP and high salt. (vi) This protein served as a substrate for the catalytic subunit of a mammalian cAMP-dependent protein kinase.     

Cyclic 3',5'-adenosine monophosphate-dependent kinase and phosphoproteins in human amnion

3',5'-Cyclic adenosine monophosphate (cAMP) modulates prostaglandin production in human amnion membranes. The major effects of cAMP are presumably mediated through the phosphorylation of specific regulatory phosphoproteins following cAMP activation of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase and phosphoproteins have not previously been characterized in human amnion. Total homogenates, cytosol, and membrane fractions from human amnion were examined for [3H]cAMP binding activity and cAMP-dependent kinase activity. cAMP-dependent kinase activity was barely detectable in crude amnion fractions. Cytosol was therefore partially purified by DEAE column chromatography for further examination. Two peaks of coincident [3H]cAMP binding and cAMP-dependent kinase activity were demonstrated at 70 and 140 mM NaCl, characteristic of the Type I and Type II cAMP-dependent protein kinase isozymes. [3H]cAMP binding to the material from both peak fractions was saturable and reversible. Scatchard analysis of [3H]cAMP binding to the peak fractions was linear for peak I and curvilinear for peak II. Assuming a one-site model, [3H]cAMP binding to the Type I isozyme showed a KD = 4.17 x 10(-8) M and Bmax = 73 pmole/mg protein; using a two-site model, [3H]cAMP binding to the high-affinity site for the Type II isozyme had a KD = 3.94 x 10(-8) M and Bmax = 6.3 pmole/mg protein. Other cyclic nucleotides competed for these [3H]cAMP binding sites with a potency order of cAMP much greater than cGMP greater than (BU)2cAMP.cAMP caused a dose-dependent increase in cAMP-dependent kinase activity in the peak fractions; half-maximal activation was observed with 5.0 x 10(-8) M cAMP. The ability of cAMP to increase phosphorylation of endogenous proteins in both crude amnion cytosol and cytosol from cultures of amnion epithelial cells was assessed using [32P]ATP, SDS-polyacrylamide gel electrophoresis and autoradiography. cAMP stimulated 32P incorporation into three proteins having Mr = 80,000, 54,000, and 43,000 (P less than .01). Half-maximal 32P incorporation into these proteins occurred at 1.0 x 10(-7) M cAMP. cAMP-dependent kinase is present in human amnion; specific cAMP-enhanced phosphoproteins are also present. Hormones elevating cAMP levels in amnion may exert their effects by activating cAMP-dependent kinase and phosphorylating these phosphoproteins.     

Regulation of protein kinase C by cyclic adenosine 3':5'-monophosphate and a tumor promoter in skeletal myoblasts

The specific activity of protein kinase C in rat skeletal myoblasts decreased when they were exposed for very short periods to isoproterenol, forskolin, dibutyryl cyclic AMP (Bt2cAMP), or the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). In the presence of Bt2cAMP or forskolin only the cytosolic but not the membrane-bound kinase activity was found to decrease. Treatment with TPA, however, led to a decrease in the activity of the enzyme both in the cytosolic as well as the membrane fractions. The effects observed in vivo could be duplicated in crude extracts of myoblasts incubated with cAMP analogues or TPA. In the presence of ATP, protein kinase C activity decreased considerably in crude cytosolic fractions treated with the cAMP analogues, but a requirement for ATP was not evident for the decrease in activity brought about by TPA. For the cAMP analogues the decrease in protein kinase C was also prevented by incubation of the extracts with an inhibitor of cAMP-dependent protein kinase. The regulation of protein kinase C by Bt2cAMP (but not by TPA) was altered in Rous sarcoma virus-transformed myoblasts. It is considered likely that a component affected by cAMP (probably a substrate for cAMP-dependent protein kinase) participates in the regulation of protein kinase C activity, and it is altered in unknown ways in transformed myoblasts.     

Purification and characterization of a membrane-associated cAMP-binding protein from developing Dictyostelium discoideum

Plasma membranes of 6-h differentiated Dictyostelium discoideum cells contain a cAMP-binding protein with the properties ascribed to the chemotaxis receptor present on these cells. We have purified this cAMP-binding protein using DEAE-Sephadex chromatography, hydrophobic chromatography on decylagarose and preparative polyacrylamide gel electrophoresis in nonionic detergent. Photoaffinity labeling of the DEAE-purified material with 8-azido-[32P] cAMP shows that only an Mr = 70,000 species on sodium dodecyl sulfate gels contains a cAMP-binding site. Two-dimensional polyacrylamide gel electrophoresis of material eluted from decyl-agarose and photoaffinity labeled indicates that the cAMP-binding protein is the most acidic of many Mr = 70,000 proteins present. This method is readily scaled up to process up to 10(11) cells which yield from 25 to 100 micrograms of cAMP-binding protein. Nucleotide specificity studies established that the cAMP-binding site of the protein is similar to that of the cAMP receptor assayed on intact cells and membranes. The rates of association and dissociation of the cAMP-binding protein are extremely rapid as found for the receptor, and its affinity for cAMP is comparable. The cAMP-binding protein is a concanavalin A binding glycoprotein, and is resistant to proteolysis by trypsin, but not chymotrypsin. Like the cAMP receptor in membranes and crude detergent extracts, this cAMP-binding protein is inhibited by phenylmethylsulfonyl fluoride. The purified binding protein exists in solution largely as a monomeric species, with some dimer being detected on gel filtration. Based on these criteria, we conclude that this cAMP binding protein represents the binding subunit of the cAMP chemotaxis receptor.     

Inhibitory action of certain cyclophosphate derivatives of cAMP on cAMP-dependent protein kinases

A series cAMP derivatives with modifications in the adenine, ribose and cyclophosphate moiety were screened for their binding affinity for the two types of cAMP-binding sites in mammalian protein kinase type 1. In addition, the activation of the kinase by these analogs was monitored. The binding data indicate that cAMP is bound to both sites in a comparable manner: the adenine appears to have no hydrogen-bond interactions with the binding sites, whereas the ribose may be bound by three hydrogen bonds involving the 2', 3' and 5' positions of cAMP. The binding data are not conclusive about the nature of the interaction with the exocyclic oxygen atoms on phosphorus, though a charge interaction seems to be absent. The cAMP molecule seems to be bound in the syn conformation. The results of activation experiments show that modifications in the adenine and ribose moiety do not affect the maximal activation level, while alteration of the two exocyclic oxygen atoms may result in a reduced maximal activation level and in one case, (Rp)-adenosine 3', 5'-monophosphorothioate [Rp-cAMPS], in total absence of activation even at concentrations at which the analog saturates both binding sites. Since occupancy of the cAMP-binding sites by this derivative apparently did not lead to activation of the enzyme, we examined whether this compound could antagonize the activation by cAMP. Indeed (Rp)-cAMPS was found to inhibit cAMP stimulated kinase activity at concentrations compatible to its binding affinity. Also with mammalian protein kinase type II (Rp)-cAMPS showed antagonistic activity, while with a cAMP-dependent protein kinase from Dictyostelium discoideum partial agonistic activity was observed. Previously a mechanism for activation of protein kinase type I was proposed involving a charge interaction between the equatorial exocyclic oxygen atom and the binding site [De Wit et. al. (1982) Eur. J. Biochem 122, 95-99]. This was based on measurements with impure preparations of (Rp)-cAMPS and the Rp and Sp isomers adenosine 3', 5'-monophosphodimethylamidate. cAMPN(CH3)2. The present work using highly purified compounds suggests the absence of a charge interaction, since the uncharged analog (Sp)-cAMPN(CH3)2 activates the kinase effectively. The data seem compatible with an activation model involving the formation of a covalent bond with phosphorus in both cAMP binding sites.     

Cyclic AMP-binding proteins: inverse relationship with estrogen-receptors in hormone-dependent mammary tumor regression.

Dimethylbenzanthracene-induced rat carcinomas possess activities binding cyclic adenosine 3':5'-monophosphate (cAMP) and estrogen. When dimethylbenzanthracene-induced tumors regress after ovariectomy of the host, a change in the specific binding of cAMP and estrogen occurs in the tumors. Six days after ovariectomy, cAMP binding increases 5-fold in the nuclei and 2-fold in the cytosol of tumors, while nuclear and cytoplasmic estrogen binding decreases by 80% and 50%, respectively. These changes in activities binding cAMP and estrogen are detectable within 1 day after ovariectomy and the changes are reversed when resumption of tumor growth is induced by the injection of 17beta-estradiol. When dimethylbenzanthracene-induced tumors fail to regress after ovariectomy, the change in activities binding cAMP and estrogen does not occur. Significant increases in the cAMP level as well as in adenylate cyclase and cAMP-phosphodiesterase activities are also found in the regressing tumors. Concomitant with the increase of cAMP-binding activity is an increase in histone kinase activity in the regressing tumor. These data suggest the involvement of cAMP in the growth control of a hormone-dependent mammary rumor.     

Expression and characterization of mutant forms of the type I regulatory subunit of cAMP-dependent protein kinase. The effect of defective cAMP binding on holoenzyme activation

The mouse wild type and four mutant regulatory type I (RI) subunits were expressed in Escherichia coli and subjected to kinetic analyses. The defective RI subunits had point mutations in either cAMP-binding site A (G200/E), site B (G324/D, R332/H), or in both binding sites. In addition, a truncated form of RI which lacked the entire cAMP-binding site B was generated. All of the mutant RI subunits which bound [3H]cAMP demonstrated more rapid rates of cAMP dissociation compared to the wild type RI subunit. Dissociation profiles showed only a single dissociation component, suggesting that a single nonmutated binding site was functional. The mutant RI subunits associated with purified native catalytic subunit to form chromatographically separable holoenzyme complexes in which catalytic activity was suppressed. Each of these holoenzymes could be activated but showed varying degrees of cAMP responsiveness with apparent Ka values ranging from 40 nM to greater than 5 microM. The extent to which the mutated cAMP-binding sites were defective was also shown by the resistance of the respective holoenzymes to activation by cAMP analogs selective for the mutated binding sites. Kinetic results support the conclusions that 1) Gly-200 of cAMP-binding site A and Gly-324 or Arg-332 of site B are essential to normal conformation and function, 2) activation of type I cAMP-dependent protein kinase requires that only one of the cAMP-binding sites be functional, 3) mutational inactivation of site B (slow exchange) has a much more drastic effect than that of site A on increasing the Ka of the holoenzyme for cAMP, as well as in altering the rate of cAMP dissociation from the remaining site of the free RI subunit. The strong dependence of one cAMP-binding site on the integrity of the other site suggests a tight association between the two sites.     

The effect of theophylline on adenosine 3′, 5′-monophosphate-dependent protein kinase and ACTH1–24 stimulated steroidogenesis in bovine adrenal cortical cells

Theophylline (theo), a known phosphodiesterase (PDE) inhibitor, was tested for its effects on ACTH_1–24 regulated steroidogenesis in isolated bovine adrenal cortical cells. Theo produced a dose related inhibition of ACTH_1–24 stimulated cortisol synthesis with half maximal inhibition occuring at 7 mM. Theo enhanced ACTH_1–24 stimulated cellular adenosine 3′, 5′-monophosphate (cAMP) levels above that produced by ACTH_1–24 alone confirming its inhibition of cAMP PDE. When tested on cAMP binding protein and cAMP-dependent protein kinase activities in cytosol prepared from bovine adrenal cortex, theo displaced ³H-cAMP binding to cAMP binding protein and inhibited cAMP-stimulated protein kinase activity. The half maximal inhibition of cAMP binding and protein kinase activity was observed at 10 and 5 mM, respectively. Inhibition of cAMP-dependent protein kinase by theo provides a possible explanation of its inhibitory effects on adrenal steroidogenesis and further implicates cAMP-dependent protein kinase in mediating ACTH stimulated steroidogenesis. Furthermore these studies suggest a novel mechanism of action for theo in addition to its known action on cAMP PDE.     

Isolation of stimulatory modulator of guanosine 3':5'-monophosphate-dependent protein kinase from mammalian heart devoid of inhibitory modulator of adenosine 3':5'-monophosphate-dependent protein kinase.

The stimulatory and inhibitory activities in the crude preparation of protein kinase modulator from dog heart were separated by Sephadex G-100 gel filtration, and the stimulatory modulator was further purified by DEAE-cellulose chromatography. The isolated stimulatory modulator, as the crude modulator preparation, stimulated the activity of the purified guanosine 3':5'-monophosphate (cGMP)-dependent protein kinases of both mammalian and arthropod origins in the presence of cGMP. The cGMP-dependent protein kinases were not activated by cGMP in the absence of either the isolated stimulatory modulator or the crude modulator. The stimulatory modulator, unlike the crude modulator had no effect on the activity of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase. The stimulatory modulator was a protein since its activity was destroyed by trypsin but was resistant to hydrolysis by DNase, RNase, phospholipase C, and lysozyme. The isolated inhibitory modulator, presumably the same as the protein inhibitor of cAMP-dependent protein kinase reported by Walsh et al. (Wash. D.A., Ashby, C.D., Gonzalez, C., Calkins, D., Fischer. E.H., and Krebs, E.G. (1971) J. Biol. Chem. 246, 1977-1985), depressed the cAMP-stimulated activity of cAMP-dependent protein kinase as did the crude preparation of protein kinase modulator. The isolated inhibitory modulator, unlike the crude preparation, was without effect on cGMP-dependent protein kinase. The present findings provide evidence to support that in mammals there are separate proteins for the stimulatory and the inhibitory activities of protein kinase modulator, in contrast to the modulator from an arthropod tissue (lobster tail muscle, Donnelly et al. (Donnelly, T.E., Jr., Kuo, J.F., Reyes, P.L., Liu, Y.P., and Greengard, P. (1973) J. Biol. Chem. 248, 190-198) which has been shown to possess both activities.     

Mutations that prevent cyclic nucleotide binding to binding sites A or B of type I cyclic AMP-dependent protein kinase

Cyclic nucleotide binding and activation properties of cAMP-dependent protein kinases from five independent mutants of S49 mouse lymphoma cells were studied. These mutants were all hemizygous for expression of mutant regulatory (R) subunits of the type I kinase with lesions that altered the electrostatic charge of R subunit: lesions in three of the mutants mapped to cAMP-binding site A, and those in two of the mutants mapped to cAMP-binding site B. A nucleotide mismatch assay using 32P-labeled cRNA and ribonuclease A confirmed and refined localization of the mutations to single amino acid residues implicated in cAMP binding. R subunits from all mutants retained the ability to bind cAMP, but binding behaved as if it were entirely to nonmutated sites: 1) relative affinities of 11 adenine-modified derivatives of cAMP for mutant enzymes were identical to their relative affinities for the site of wild-type kinase that corresponded to the nonmutated site of the mutant; 2) the potencies of these analogs as activators of mutant kinases were strictly correlated with their binding affinities (for wild-type enzyme activation potencies were correlated with mean affinities of the analogs for cAMP-binding sites A and B); 3) combinations of analogs with strong preferences for opposite cAMP-binding sites in wild-type kinase showed no synergism in activating mutant kinases; 4) dissociation of cAMP from mutant kinases was monophasic; and 5) high salt accelerated dissociation of cAMP from kinases with site B lesions but retarded dissociation from those with site A lesions.     

Limited proteolysis alters the photoaffinity labeling of adenosine 3',5'-monophosphate dependent protein kinase II with 8-azidoadenosine 3',5'-monophosphate

Photoaffinity labeling of the regulatory subunits of cAMP-dependent protein kinase with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) has proved to be a very specific method for identifying amino acid residues that are in close proximity to the cAMP-binding sites. Each regulatory subunit contains two tandem cAMP-binding sites. The type II regulatory subunit (RII) from porcine heart was modified at a single site, Tyr-381 [Kerlavage, A., & Taylor, S.S. (1980) J. Biol. Chem. 255, 8483-8488]. When a proteolytic fragment of this RII subunit was photolabeled with 8-N3cAMP, two sites were covalently modified. One site corresponded to Tyr-381 and, thus, was analogous to the native RII. The other site of modification was identified as Tyr-196, which is not labeled in the native protein. Photoaffinity labeling was carried out in the presence of various analogues of cAMP that show a preference for one of the two tandem cAMP-binding sites. These studies established that the covalent modification of Tyr-381 was derived from 8-N3cAMP that was bound to the second cAMP-binding site (domain B) and that covalent modification to Tyr-196 was due to 8-N3cAMP that was bound to the first cAMP-binding site (domain A). These sites of covalent modification have been correlated with a model of each cAMP-binding site on the basis of the crystal structure of the catabolite gene activator protein (CAP), which is the major cAMP-binding protein in Escherichia coli.     

Two different intrachain cAMP sites in the cAMP-dependent protein kinase of the dimorphic fungus Mucor rouxii

cAMP sites of the cAMP-dependent protein kinase from the fungus Mucor rouxii have been characterized through the study of the effects of cAMP and of cAMP analogs on the phosphotransferase activity and through binding kinetics. The tetrameric holoenzyme, which contains two regulatory (R) and two catalytic (C) subunits, exhibited positive cooperativity in activation by cAMP, suggesting multiple cAMP-binding sites. Several other results indicated that the Mucor kinase contained two different cooperative cAMP-binding sites on each R subunit, with properties similar to those of the mammalian cAMP-dependent protein kinase. Under optimum binding conditions, the [3H]cAMP dissociation behavior indicated equal amounts of two components which had dissociation rate constants of 0.09 min-1 (site 1) and 0.90 min-1 (site 2) at 30 degrees C. Two cAMP-binding sites could also be distinguished by C-8 cAMP analogs (site-1-selective) and C-6 cAMP analogs (site-2-selective); combinations of site-1- and site-2-selective analogs were synergistic in protein kinase activation. The two different cooperative binding sites were probably located on the same R subunit, since the proteolytically derived dimeric form of the enzyme, which contained one R and one C component, retained the salient properties of the untreated tetrameric enzyme. Unlike any of the mammalian cyclic-nucleotide-dependent isozymes described thus far, the Mucor kinase was much more potently activated by C-6 cAMP analogs than by C-8 cAMP analogs. In the ternary complex formed by the native Mucor tetramer and cAMP, only the two sites 1 contained bound cAMP, a feature which has also not yet been demonstrated for the mammalian cAMP-dependent protein kinase.     

Hormonal regulation of cyclic AMP-dependent protein kinase in cultured ovarian granulosa cells. Effects of follicle-stimulating hormone and gonadotropin-releasing hormone

The hormonal regulation of cAMP-dependent protein kinase was examined in granulosa cells from diethylstilbestrol-implanted immature rats. Follicle-stimulating hormone (FSH) increased the number of available cAMP-binding sites in a dose- and time-dependent manner, with a maximum 4-6-fold increase at 50-100 ng/ml between 6 and 48 h of culture after a transient decrease in available sites during the first 6 h. The potent gonadotropin-releasing hormone (GnRH) agonist [D - Ala6]des - Gly10 - GnRH - N - ethylamide (GnRHa) reduced the FSH-induced increase in cAMP-binding sites by approximately 50% at 24 and 48 h of culture. Photoaffinity labeling with 8-azido-[32P] cAMP revealed the existence of one major cAMP-binding protein (Mr = 55,000 +/- 400) which appeared to be the regulatory (R) subunit of type II cAMP-dependent protein kinase. While FSH induced a 5-10-fold increase in the labeling of R II both in vivo and in vitro, GnRHa reduced the amount of R II induced by FSH in granulosa cells cultured for 48 h. The large increase in R II subunit was not accompanied by a corresponding increase in protein kinase activity, which was only enhanced by 50% after 48 h of culture with FSH. Fractionation of granulosa cell cytosol from FSH-treated ovaries on DEAE-cellulose showed a single peak of cAMP-dependent phosphokinase activity with the elution properties of a type II protein kinase. However, the peak of cAMP binding activity (eluted at 0.20 M KCl) was not coincident with the protein kinase activity. FSH transiently stimulated cAMP-dependent protein kinase activity during the first 10-30 min of culture. GnRHa impaired the FSH-induced early increase in protein kinase activity, causing a delay in activation until 60 min. These findings suggest that a large dose- and time-dependent increase in the content of cAMP-binding sites may be a major factor in cAMP-mediated differentiation of granulosa cells. The inhibitory effect of GnRHa on both FSH-induced protein kinase activation during the first minutes of culture and on FSH-induced R II synthesis during the subsequent 48 h of culture could be crucial events in the prevention of granulosa cell maturation by GnRH agonists.