ATP sensitive bi-quinoline activator of the AMP-activated protein kinase

The AMP-activated protein kinase (AMPK) regulates cellular and whole-body energy balance in response to changes in adenylate charge and hormonal signals. Activation of AMPK in tissues such as skeletal muscle and liver reverses many of the metabolic defects associated with obesity and Type 2 diabetes. Here we report a bi-quinoline (JJO-1) that allosterically activates all AMPK αβγ isoforms in vitro except complexes containing the γ3 subunit. JJO-1 does not directly activate the autoinhibited α subunit kinase domain and differs among other known direct activators of AMPK in that allosteric activation occurs only at low ATP concentrations, and is not influenced by either mutation of the γ subunit adenylate-nucleotide binding sites or deletion of the β subunit carbohydrate-binding module. Our findings indicate that AMPK has multiple modes of allosteric activation that may be exploited to design isoform-specific activators as potential therapeutics for metabolic diseases.     

The relationship between the effects of drugs on bilayer stability and on protein kinase C activity

A variety of substances of diverse structure have been shown to affect the activity of protein kinase C. Many of the agents which affect protein kinase C activity also markedly shift the bilayer to hexagonal phase transition temperature of phosphatidylethanolamines. Although one of the more potent activators of protein kinase C, diacylglycerols, are effective destabilizers of the bilayer phase of membranes, this is not a general property of all protein kinase C activators nor are inhibitors of this enzyme bilayer stabilizing agents. However, if we consider only compounds which are uncharged or are zwitterionic, then those which promote the conversion of phospholipid bilayers to the hexagonal phase are all activators of protein kinase C, while those which stabilize the bilayer phase are protein kinase C inhibitors. Among charged substances, all of those which are negatively charged are activators of protein kinase C, while all of the positively charged compounds are protein kinase C inhibitors.     

Constitutive activation of the Saccharomyces cerevislae mating response pathway by a MAP kinase kinase from Candida albicans

The HST7 gene of Candida albicans encodes a protein with structural similarity to MAP kinase kinases. Expression of this gene in Saccharomyces cerevisiae complements disruption of the Ste7 MAP kinase kinase required for both mating in haploid cells and pseudohyphal growth in diploids. However, Hst7 expression does not complement loss of either the Pbs2 (Hog4) MAP kinase kinase required for response to high osmolarity, or loss of the Mkk1 and Mkk2 MAP kinase kinases required for proper cell wall biosynthesis. Intriguingly, HST7 acts as a hyperactive allele of STE7; expression of Hst7 activates the mating pathway even in the absence of upstream signaling components including the Ste7 regulator Ste11, elevates the basal level of the pheromone-inducible FUS1 gene, and amplifies the pseudohyphal growth response in diploid cells. Thus Hst7 appears to be at least partially independent of upstream activators or regulators, but selective in its activity on downstream target MAP kinases. Creation of Hst7/Ste7 hybrid proteins revealed that the C-terminal two-thirds of Hst7, which contains the protein kinase domain, is sufficient to confer this partial independence of upstream activators.     

Use of docking peptides to design modular substrates with high efficiency for mitogen-activated protein kinase extracellular signal-regulated kinase.

The mitogen-activated protein kinase extracellular regulated kinase (ERK) plays a key role in the regulation of cellular proliferation. Mutations in the ERK cascade occur in 30% of malignant tumors. Thus understanding how the kinase identifies its cognate substrates as well as monitoring the activity of ERK is central to cancer research and therapeutic development. ERK binds to its protein targets, both downstream substrates and upstream activators, via a binding site distinct from the catalytic site of ERK. The substrate sequences that bind, or dock, to these sites on ERK influence the efficiency of phosphorylation. For this reason, simple peptide substrates containing only phosphorylation sequences typically possess low efficiencies for ERK. Appending short docking peptides derived from full-length protein substrates and activators of ERK to a phosphorylation sequence increased the affinity of ERK for the phosphorylation sequence by as much as 200-fold while only slightly diminishing the maximal velocity of the reaction. The efficiency of the phosphorylation reaction was increased by up to 150-fold, while the specificity of the substrate for ERK was preserved. Simple modular peptide substrates, which can be easily tailored to possess high phosphorylation efficiencies, will enhance our understanding of the regulation of ERK and provide a tool for the development of new kinase assays.     

Rapid dephosphorylation of protein kinase C substrates by protein kinase A activators results from inhibition of diacylglycerol release

The biochemical events encompassing the dephosphorylation of protein kinase C substrates by protein kinase A activators have been investigated in a neurotumor cell line, NCB-20. Treatment of [32P]orthophosphate-labeled cells with protein kinase A activators (e.g. forskolin, dibutyryl cAMP, prostaglandin E1) resulted in an inhibition of protein kinase C activity due to a failure of the protein kinase C complex to translocate into the membrane. Phospholipase C activity, as measured by the synchronous release of diacylglycerol and inositol phosphates (inositol 1,4,5-trisphosphate, inositol 1,4-bisphosphate, and inositol 1-phosphate) in response to bradykinin, was inhibited up to 50% following exposure to protein kinase A activators. At the same time, phospholipase C-specific inositol phospholipid substrates (phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate) were found to accumulate in NCB-20 cells following treatment with protein kinase A activators. This suggests that phospholipase C may be altered through protein kinase A-mediated protein phosphorylation. Second messenger generation (inositol phosphates, diacylglycerol, and Ca2+) is therefore inhibited through cyclic AMP-mediated shutdown of the inositol lipid cycle at the level of phospholipase C.     

Constitutive activation of the Saccharomyces cerevislae mating response pathway by a MAP kinase kinase from Candida albicans

The HST7 gene of Candida albicans encodes a protein with structural similarity to MAP kinase kinases. Expression of this gene in Saccharomyces cerevisiae complements disruption of the Ste7 MAP kinase kinase required for both mating in haploid cells and pseudohyphal growth in diploids. However, Hst7 expression does not complement loss of either the Pbs2 (Hog4) MAP kinase kinase required for response to high osmolarity, or loss of the Mkk1 and Mkk2 MAP kinase kinases required for proper cell wall biosynthesis. Intriguingly, HST7 acts as a hyperactive allele of STE7; expression of Hst7 activates the mating pathway even in the absence of upstream signaling components including the Ste7 regulator Ste11, elevates the basal level of the pheromone-inducible FUS1 gene, and amplifies the pseudohyphal growth response in diploid cells. Thus Hst7 appears to be at least partially independent of upstream activators or regulators, but selective in its activity on downstream target MAP kinases. Creation of Hst7/Ste7 hybrid proteins revealed that the C-terminal two-thirds of Hst7, which contains the protein kinase domain, is sufficient to confer this partial independence of upstream activators.Communicated by C. P. Hollenberg     

Nucleoside Transport in Neurons. Regulation by Secretagogues and Effectors of Protein Kinases A and C

Abstract

In chromaffin cells, secretagogues and direct activators of protein kinase C and protein kinase A inhibited the nucleoside transport with a parallel decrease in the high affinity binding sites.     

Modulation of the Na–;H antiport by insulin: Interplay between protein kinase C,tyrosine kinase,and protein phosphatases

The insulin modulation of Na-H antiport in rat hepatocytes was studied using the fluorescent, pH-sensitive intracellular probe, 2′,7′ bis (carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Our data show that insulin stimulates the Na? H antiport. The dose-response of insulin effect shows a behavior typical of other insulin responses: a maximum in the physiological range (1 nM) and smaller effects at higher and lower hormone concentrations. The time-course of activation is very fast at high hormone concentrations and slow, but reaching a higher value, for the physiological concentrations (0.26± 0.05 and 0.18 ± 0.022 pH units for 1 nM and 1 μM insulin respectively). The use of phorbol, 12-myristate, 13-acetate (PMA), a potent activator of protein kinase C and its inhibitor staurosporine, and the inhibitor of tyrosine kinase erbstatin analog, suggests that both protein kinase C and tyrosine kinase could be involved in the mechanism leading to Na? H antiport activation by insulin. We suggest that the activation of the antiport involves the two pathways depending on the hormone concentration. In particular, protein kinase C would mediate the effects of high hormone concentrations, acting as a growth factor, since staurosporine fully inhibited insulin 1 μM, but only partially 1 nM effects, and tyrosine kinase would mediate the effect of insulin 1 nM and only partially 1 μM. Okadaic acid 1 μM, a potent inhibitor of protein phosphatases, mimicked the hormone effects on the antiport and abolished the different time-course due to hormone concentration, suggesting a role of kinases and phosphatases in the signal transduction. The effect of all activators was abolished by amiloride analog, 5-(N-ethyl-N-isopropyl) amiloride (EIPA), confirming the specificity of these effects. © 1994 wiley-Liss, Inc.     

Reduction of β-Amyloid Levels by Novel Protein Kinase C? Activators

Isoform-specific protein kinase C (PKC) activators may be useful as therapeutic agents for the treatment of Alzheimer disease. Three new ϵ-specific PKC activators, made by cyclopropanation of polyunsaturated fatty acids, have been developed. These activators, AA-CP4, EPA-CP5, and DHA-CP6, activate PKCϵ in a dose-dependent manner. Unlike PKC activators that bind to the 1,2-diacylglycerol-binding site, such as bryostatin and phorbol esters, which produce prolonged down-regulation, the new activators produced sustained activation of PKC. When applied to cells expressing human APPSwe/PS1δ, which produce large quantities of β-amyloid peptide (Aβ), DCP-LA and DHA-CP6 reduced the intracellular and secreted levels of Aβ by 60–70%. In contrast to the marked activation of α-secretase produced by PKC activators in fibroblasts, the PKC activators produced only a moderate and transient activation of α-secretase in neuronal cells. However, they activated endothelin-converting enzyme to 180% of control levels, suggesting that the Aβ-lowering ability of these PKCϵ activators is caused by increasing the rate of Aβ degradation by endothelin-converting enzyme and not by activating nonamyloidogenic amyloid precursor protein metabolism.     

Biphasic regulation of macrophage attachment by activators of cyclic adenosine monophosphate-dependent kinase and protein kinase C

A method is described that enabled us to study the adhesiveness of J-774 murine macrophages. Cell attachment was stimulated by activators of kinase C (i.e., phorbol esters) as well as kinase A (cyclic adenosine monophosphate; cAMP). This novel effect of cAMP was observed when its levels were increased via receptor triggering (prostaglandin E1, beta-adrenergic agonists), activation of Ns (cholera toxin), or inhibition of phosphodiesterase (Ro 20-1724) or when the kinase was directly activated by Br8-cAMP. The simultaneous treatment with kinase A and kinase C activators at the time of attachment resulted in a partially additive response. On the other hand, preincubation of the cells in suspension with one of the activators rendered them refractory to subsequent stimulation at the onset of the adhesion assay, whatever agent was used. Such a refractoriness was also observed in cells preincubated with oleoyl-acetyl-glycerol (OAG). On the other hand, when added at the time of attachment, this near-physiological activator of kinase C evoked a biphasic response: the early stimulation of cell attachment was followed by an accelerated rate of "detachment." In conclusion, kinase C and kinase A play a role in the sequence of events leading to cell adhesion. The cross desensitization observed is distal and takes place at or beyond the kinase step.     

Phorbol ester and 1,2-diolein are not fully equivalent activators of protein kinase C in respect to phosphorylation of membrane proteins in vitro

Phosphorylation of liver plasma proteins by protein kinase C was studied by using the two best known activators of the enzyme, 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1,2-diolein. While the effects of TPA and diolein were almost identical on two proteins and similar in magnitude on four proteins, the phosphorylation of an additional four proteins was increased only by TPA. We conclude that in respect to phosphorylation of membrane proteins, TPA and diglycerides are not fully equivalent activators of kinase C.     

Protein kinase C phosphorylates pp60src at a novel site

The transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homolog (pp60c-src) are demonstrated to be phosphorylated at serine 12 in vivo under certain conditions. We propose that protein kinase C is responsible for this modification based on the following evidence. First, the tumor promoters, 12-O-tetradecanoylphorbol-13-acetate and teleocidin, and synthetic diacylglycerol, known activators of protein kinase C in vivo, cause nearly complete phosphorylation of pp60src at serine 12. Second, among five purified serine/threonine-specific protein kinases tested, only protein kinase C phosphorylates pp60c-src and pp60v-src in vitro at serine 12. Third, purified protein kinase C phosphorylates a synthetic peptide corresponding to the N-terminal 20 amino acids of pp60c-src at serine 12. The physiological significance of this novel phosphorylation is discussed.     

Protein kinase C activators and bradykinin selectively inhibit vasopressin-stimulated cAMP synthesis in MDCK cells

To evaluate a possible modulation by protein kinase C of hormonal, cAMP-mediated effects on renal epithelial cells, we studied the effect of protein kinase C activators and of bradykinin on intracellular cAMP accumulation in MDCK cells. A 15-min pretreatment of cells with phorbol 12-myristate 13-acetate or 1-oleoyl-2-acetylglycerol induced a dose-dependent inhibition of vasopressin-stimulated cAMP synthesis, but not of basal or glucagon-, prostaglandin E2-, and forskolin-stimulated cAMP generation. 4 alpha-Phorbol 12,13-didecanoate, inactive on protein kinase C, did not affect cAMP accumulation. Bradykinin (0.1-10 microM) also inhibited the stimulatory effect of vasopressin on cAMP synthesis in a concentration-dependent manner, but affected neither basal cAMP content, nor its stimulation by glucagon, prostaglandin E2 and forskolin. The effect of activators of protein kinase C and of bradykinin occurred while renal prostaglandin synthesis was blocked with indomethacin. The inhibitory effect of protein kinase C activators and bradykinin on cAMP generation was reversed by the protein kinase C inhibitor H7, was enhanced by monensin, one effect of which is to block the recycling of membrane receptors, and persisted when the GTP-binding protein N1 was blocked with 1 mM Mn2+. Our data suggest that: protein kinase C can modulate the tubular effects of vasopressin by inhibiting cAMP generation; this effect is not mediated by renal prostaglandins, and might result from a direct action on the vasopressin receptor, or on its coupling with Ns; the modulation by bradykinin of vasopressin effects are likely to be exerted, at least partly, through activation of protein kinase C.     

Activation of protein kinase isozymes by cyclic nucleotide analogs used singly or in combination. Principles for optimizing the isozyme specificity of analog combinations

104 cAMP analogs, most of them modified in the adenine moiety, were tested as activators of cAMP-dependent protein kinase I (from rabbit or rat skeletal muscle) and kinase II (from bovine heart or rat skeletal muscle). When tested singly, only 2-phenyl-1,N6-etheno-cAMP showed a considerably (sevenfold) higher potency as an activator of kinase II than of kinase I. Analogs containing an 8-amino modification preferentially activated kinase I, some being more than 10-fold more potent as activators of kinase I than kinase II. When two analogs were combined, the concentration of one (complementary) analog required to half-maximally activate each isozyme was determined in the presence of a fixed concentration of another (priming) analog. Analogs tested in combination had been analyzed for their affinity for the intrasubunit binding sites (A, B) of isozyme I and II. The degree to which complementary analogs preferentially activated one isozyme was plotted against the mean site selectivity, i.e. (affinity A/B isozyme I X affinity A/B isozyme II) 1/2. This plot produced a straight line, the slope of which reflected the ability of the priming analog to discriminate homologous sites on the isozymes. This means that the isozyme discriminating power of an analog pair can be quantitatively predicted from the affinity of the analogs for site A and B of the two enzymes. It also means that a systematic analysis of those features of analogs imparting a high mean site selectivity or the ability to discriminate between homologous isozyme sites will facilitate the synthesis of new even more isozyme-selective analogs.     

Allosteric activation of rat liver microsomal [hydroxymethylglutaryl-CoA reductase (NADPH)]kinase by nucleoside phosphates

Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase kinase activity is enhanced about 5 fold by 2 mM of either AMP or ADP. Activation constants, Ka, for AMP and ADP are 17 microM and 430 microM respectively, showing that AMP is a more potent activator than ADP. This property is expressed by increasing not only the rate of reductase inactivation but also the rate of reductase phosphorylation from [gamma-32P]ATP. GTP can replace ATP as substrate of reductase kinase but GMP and GDP cannot replace AMP as activators. Kinetic studies show that ATP can only act as a substrate. Nucleoside mono or diphosphates and nucleoside triphosphates, thus, appear to bind to different sites on microsomal HMG-CoA reductase kinase. Nucleoside mono or diphosphates act as allosteric activators of reductase kinase. The adenosyl moiety and the unaltered phosphate ester at the 5' position are two essential features of the activator molecule. Phosphorylation of reductase either by microsomal or cytosolic AMP-activated reductase kinase produces an 80% inactivation, with a concomitant incorporation of 0.8 mol of 32P per mol of reductase (Mr 55,000). In both cases exhaustive tryptic digestion of 32P-labeled HMG-CoA reductase, which had been denatured in 2M urea, yields two major phosphopeptides, the phosphoryl group being bound to serine residues.     

Activation of casein kinase II by sphingosine

Sphingosine activates casein kinase II in the presence of endogenous substrates as well as a synthetic peptide substrate. The activation response occurred between 12 and 25 micrograms/ml sphingosine and exhibited positive cooperativity with a Hill coefficient of 3.0. Sphingosine not only increased the Vmax of casein kinase II but decreased the Km(app) for the peptide substrate from 0.5 to 0.08 mM. In contrast, the Km(app) for MgCl2 was increased from 0.12 to 0.7 mM. Consequently, sphingosine altered significantly several parameters which determine casein kinase II activity. The effect of sphingosine was relatively specific, inasmuch as related lipids were less potent activators or largely ineffective in stimulating casein kinase II. On the other hand, the effect of sphingosine itself could be potentiated or inhibited by other lipids. Ceramide and sphingosylphosphorylcholine augmented the sphingosine effect. Phospholipids alone did not alter the activity of casein kinase II significantly, but abolished enzyme activation by sphingosine with different potencies (phosphatidylserine greater than phosphatidylethanolamine greater than phosphatidylinositol greater than phosphatidylcholine). Moreover, the sphingosine effect could be abrogated by KCI and NaCl, which alone are known to induce enzyme activation and dissociation of aggregated casein kinase II protein; LiCl and NH4Cl also inhibited the sphingosine effect. Polyamines, known activators of casein kinase II, partially mimicked the effect of sphingosine on endogenous polypeptide phosphorylation but failed to do so with the peptide substrate. These observations demonstrate that sphingosine is a potent activator of casein kinase II. The potential pharmacological and physiological modulation of casein kinase II by sphingoid bases is discussed.     

Activation of protein kinase C by lipoxin A and other eicosanoids. Intracellular action of oxygenation products of arachidonic acid

Arachidonic acid, linolenic acid and 14 different oxygenated fatty acid derivatives were tested as activators of human protein kinase C in vitro using histone as substrate. Lipoxin A (5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic activated the kinase in the presence of calcium at 30 fold lower concentration (1 microM) than did arachidonic acid or 1,3-dioleoylglycerol. The methyl ester of lipoxin A and the free acids of leukotriene B4 as well as two lipoxin B isomers were without effect. In contrast, linolenic acid, leukotriene C4, certain mono- and dihydroxylated eicosanoids and one lipoxin B isomer had stimulatory effects, albeit at higher concentrations. The substrate specificity of protein kinase C activated by lipoxin A proved to be different from that of the phosphatidylserine or phorbol ester activated kinase. Results of the present study suggest that arachidonic acid derived oxygenation products, in particular lipoxin A, may serve as intracellular activators of protein kinase C.     

The interplay between covalent and non-covalent regulation of glycogen phosphorylase. The role of different effectors of phosphorylase b on the phosphorylase b to a conversion rate.

Glycogen phosphorylase b is converted to glycogen phosphorylase a, the covalently activated form of the enzyme, by phosphorylase kinase. Glc-6-P, which is an allosteric inhibitor of phosphorylase b, and glycogen, which is a substrate of this enzyme, are already known to have respectively an inhibiting and activating effect upon the rate of conversion from phosphorylase b to phosphorylase a by phosphorylase kinase. In the former case, this effect is due to the binding of glucose-6-phosphate to glycogen phosphorylase b. In order to investigate whether or not the rate of conversion of glycogen phosphorylase b to phosphorylase a depends on the conformational state of the b substrate, we have tested the action of the most specific effectors of glycogen phosphorylase b activity upon the rate of conversion from phosphorylase b to phosphorylase a at 0 degrees C and 22 degrees C : AMP and other strong activators, IMP and weak activators, Glc-6-P, glycogen. Glc-1-P and phosphate. AMP and strong activators have a very important inhibitory effect at low temperature, but not at room temperature, whereas the weak activators have always a very weak, if even existing, inhibitory effect at both temperatures. We confirmed the very strong inhibiting effect of Glc-6-P at both temperatures, and the strong activating effect of glycogen. We have shown that phosphate has a very strong inhibitory effect, whereas Glc-1-P has an activating effect only at room temperature and at non-physiological concentrations. The concomitant effects of substrates and nucleotides have also been studied. The observed effects of all these ligands may be either direct ones on phosphorylase kinase, or indirect ones, the ligand modifying the conformation of phosphorylase b and its interaction with phosphorylase kinase. Since we have no control experiments with a peptidic fragment of phosphorylase b, the interpretation of our results remains putative. However, the differential effects observed with different nucleotides are in agreement with the simple conformational scheme proposed earlier. Therefore, it is suggested that phosphorylase kinase recognizes differently the different conformations of glycogen phosphorylase b. In agreement with such an explanation, it is shown that the inhibiting effect of AMP is mediated by a slow isomerisation which has been previously ascribed to a quaternary conformational change of glycogen phosphorylase b. The results presented here (in particular, the important effect of glycogen and phosphate) are also discussed in correlation with the physiological role of the different ligands as regulatory signals in the in vivo situation where phosphorylase is inserted into the glycogen particle.     

Activation of Protein Kinase C in Permeabilized Human Neuroblastoma SH-SY5Y Cells

The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.     

Protein kinase C modulates actin conformation in human T lymphocytes

We studied the effect of activators and inhibitors of protein kinase C on actin conformation in human blood lymphocytes by flow cytometry and gel electrophoresis. PMA, 1-oleyl-2-acetyl-glycerol, and mezerein, activators of protein kinase C, caused an increase in lymphocyte F-actin within 2 to 5 min. After stimulation with PMA, lymphocytes formed pseudopods containing an increased concentration of F-actin and had an increase of actin in the Triton-insoluble cytoskeletal fraction. Sphingosine and H-7, inhibitors of protein kinase C activation, inhibited the increase in F-actin induced by PMA. The increase in F-actin in response to PMA was striking in Th and Ts lymphocytes (2- to 3-fold increase), but B lymphocytes had only a slight increase (1.15-fold). Thus, activation of protein kinase C modulates actin conformation specifically in T lymphocytes.