Phosphorylation of acetyl-CoA-carboxylase in the chicken liver during intensification of lipogenesis and treatment with nicotinic acid

Sites of cAMP and ATP binding which regulate acetyl-CoA-carboxylase phosphorylation rate characterized under conditions of lipogenesis intensification and nicotinic acid action on this enzyme 1500 fold purified and containing proteinkinase activity. The acetyl-CoA-carboxylase preparation contains only one type of the cAMP binding sites which possess higher capacity under the action of nicotinic acid in vivo. A decrease of the cAMP binding under the conditions of lipogenesis intensification is induced by diminution of the cAMP binding site capacity without changing the binding constant value. It is established that [gamma-32P]ATP is incorporated in enzyme with Km value equal for two states under study. It this case the [gamma-32P]ATP incorporation rate is much higher for acetyl-CoA-carboxylase produced from chicken liver under the action of nicotinic acid.     

Structure, properties and regulation of acetyl-CoA-carboxylase activity

The paper deals with the analysis of data available in literature and those of the authors' own investigations concerning the structure, properties and regulation of acetyl-CoA-carboxylase. Nicotinic acid is one of the factors regulating the enzyme activity in the animal liver. It inhibits the acetyl-CoA-carboxylase activity through two mechanisms--allosteric regulation and covalent modification. A comparative characteristic of the studied enzyme preparations has shown that administration of nicotinic acid to animals leads to phosphorylation of acetyl-CoA-carboxylase which affects its structure. A complex with homogenic acetyl-CoA-carboxylase is found to contain cAMP-independent and cAMP-dependent protein kinase. The phosphorylation is controlled by citrate competing with nicotinic acid for the coupling sites.     

Citrate influence on acetyl-CoA-carboxylase activation and phosphorylation in chicken liver with lipogenesis inhibited by nicotinic acid

Nicotinic acid in vivo affects the citrate demand for acetyl-CoA-carboxylase activation in the chicken liver under conditions of alimentary lipogenesis stimulation. Stoichiometry of the citrate binding with the dissociation constant of the enzyme-allosteric activator complex is determined under experimental conditions. Endogenic phosphorylation of acetyl-CoA-carboxylase completely correlates with its inactivation and depends on the citrate level. cAMP is established to have an activating effect on phosphorylation of acetyl-CoA-carboxylase of test animals.     

Inhibition of activity of acetyl-CoA-carboxylase from chicken liver by nicotinic acid and its derivatives]

Nicotinic acid and nicotinamide inhibit in vitro the acetyl-CoA-carboxylase activity of partially purified enzyme from chicken liver. The incorporation of 10, 20, 50 and 100 mkmoles of nicotinic acid or nicotinamide into the incubation medium (0,9 ml) leads to the inhibition of the enzyme activity by 19, 45, 70 and 100% and by 39, 51, 60 and 78%, respectively. NADH+ and NADP+ at concentrations by one order of magnitude lower than those of nicotinic acid and nicotinamide decrease the enzyme activity in a similar manner. The constants of inhibition by the above-mentioned compounds were calculated with respect to ATP, acetyl-CoA and citrate.     

Nicotinic acid effect on acetyl-CoA-carboxylic activity in chicken liver

Changes of acetyl-CoA-carboxylase (EC 6.4.1.2) activity and the NAD content in the liver tissue were studied in dynamics after excessive administration of nicotinic acid to chickens. It is established that in chickens, which were given a high-carbohydrate diet after fasting, administration of nicotinic acid at first causes a fall of the acetyl-CoA-activity in the liver tissue, followed by its gradual rise against a background of the NAD content drop and by the 24th hour its level approaches the initial values. The maxima of NAD accumulation and of the acetyl-CoA-carboxylase activity decrease coincide in time. The administration of nicotinic acid to these chickens causes both a decrease in the intensity of 2-14C acetate incorporation into free fatty acids and a drop in their content.     

Effect of nicotinic acid on chicken liver protein kinase activity and cAMP content

The biosynthesis of fatty acids in the chicken liver was stimulated by feeding up chickens with high-carbon products. After fasting the cAMP content and protein kinase activity in chicken fall considerably as compared to the control. After administration of nicotinic acid to chicken under experiment the content of cAMP and the protein kinase activity in the liver tissue rise to the highest extent, returning to initial values by the end of the day. The maximal increase in the cAMP content and protein kinase activity coincides in time with the maximum of the acetyl-CoA-carboxylase activity decrease. An assumption is advanced that biosynthesis of fatty acids in the liver tissue of chickens is regulated by a change in the degree of acetyl-CoA-carboxylase phosphorylation with the participation of adenylate cyclase system.     

Physico-chemical properties of specific protein kinases during intensification of lipogenesis and treatment with nicotinic acid

Protein kinase strong-associated with acetyl-CoA-carboxylase is isolated from the liver of chicken and 300-fold purified with alimentary intensification of lipogenesis and under the effect of nicotinic acid against this background. The obtained enzymes are studied comparatively. It is found that their preparations are phosphorylated with different rate, have two pH optima and differ in the sensitivity to cAMP and to thermostable protein inhibitor. The hydrophobic chromatography was used to separate components of the acetyl-CoA-carboxylase-protein kinase complex and to reveal in the chicken liver cAMP-dependent and cAMP-independent protein kinases highly specific to acetyl-CoA-carboxylase and strongly bound with it.     

Specific epidermal growth factor receptors on porcine thyroid cell membranes

The influence of islet-activating protein (IAP), a Bordetella pertussis toxin, was studied on adenylate cyclase and GTPase activities in rat adipocyte membranes. Pretreatment of rats or intact rat adipocytes with IAP did not affect adenylate cyclase inhibition by the stable GTP analog, GTP gamma S, whereas inhibition by GTP was abolished. Concomitantly, activation of the adipocyte enzyme by sodium and its inhibition by nicotinic acid were prevented. Furthermore, IAP treatment of adipocyte membranes prevented nicotinic acid-induced stimulation of a high affinity GTPase. The data suggest that a GTP-hydrolyzing system involved in the inhibitory regulation of adenylate cyclase is the target of IAP's action.     

Cholinergic Receptor-Mediated Phosphorylation and Activation of Tyrosine Hydroxylase in Cultured Bovine Adrenal Chromaffin Cells

We have identified a 56-kilodalton protein in cultured bovine adrenal chromaffin cells that is phosphorylated when catecholamine secretion is stimulated. Immunodetection on Western blots from both one- and two-dimensional polyacrylamide gels indicated that this protein was tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. Two-dimensional polyacrylamide gel electrophoresis of proteins from unstimulated cells revealed small amounts of phosphorylated protein with a molecular weight of 56K and pI values of 6.37 and 6.27 which were subunits of tyrosine hydroxylase. Nicotinic stimulation of chromaffin cells caused the phosphorylation of three proteins of 56 kilodaltons with pI values of approximately 6.37, 6.27, and 6.15 which were tyrosine hydroxylase. The immunochemical analysis also revealed that there was unphosphorylated tyrosine hydroxylase 56 kilodaltons with a pI of 6.5 which may have decreased on nicotinic stimulation. The phosphorylation of tyrosine hydroxylase was associated with an increase in in situ conversion of [3H]tyrosine to [3H]dihydroxyphenylalanine ([3H]DOPA). Muscarinic stimulation also caused phosphorylation of tyrosine hydroxylase, but to a smaller extent than did nicotinic stimulation. The secretagogues, elevated K+ and Ba2+, stimulated phosphorylation of tyrosine hydroxylase and [3H]DOPA production. The effects of nicotinic stimulation and elevated K+ on tyrosine hydroxylase phosphorylation and [3H]DOPA production were Ca2+-dependent. Nicotinic agonists also raised cyclic AMP levels in chromaffin cells after 2 min. Dibutyryl cyclic AMP and forskolin, which have little effect on catecholamine secretion, also caused phosphorylation of tyrosine hydroxylase. These stimulators of cyclic AMP-dependent processes caused the appearance of two phosphorylated subunits of tyrosine hydroxylase with pI values of 6.37 and 6.27. There was also a small amount of phosphorylated subunit with a pI of 6.15. Both agents stimulated [3H]DOPA production. The experiments indicate that tyrosine hydroxylase is phosphorylated and activated when chromaffin cells are stimulated to secrete. The data suggest that the earliest phosphorylation of tyrosine hydroxylase induced by a nicotinic agonist occurs through stimulation of a Ca2+-dependent protein kinase. After 2 min phosphorylation by a cyclic AMP-dependent protein kinase may also occur. Phosphorylation of tyrosine hydroxylase is associated with an increase in in situ tyrosine hydroxylase activity.     

Activation of Retinal Tyrosine Hydroxylase In Vitro by Cyclic AMP-Dependent Protein Kinase: Characterization and Comparison to Activation In Vivo by Photic Stimulation

Abstract: Tyrosine hydroxylase in rat retina is activated in vivo as a consequence of photic stimulation. Tyrosine hydroxylase in crude extracts of dark-adapted retinas is activated in vitro by incubation under conditions that stimulate protein phosphorylation by cyclic AMP-dependent protein kinase. Comparison of the activations of the enzyme by photic stimulation in vivo and protein phosphorylation in vitro demonstrated several similarities. Both treatments decreased the apparent K _m of the enzyme for the synthetic pterin cofactor 6MPH₄. Both treatments also produced the same change in the relationships of tyrosine hydroxylase activity to assay pH. When retinal extracts containing tyrosine hydroxylase activated either in vivo by photic stimulation or in vitro by protein phosphorylation were incubated at 25°C, the enzyme was inactivated in a time-dependent manner. The inactivation of the enzyme following both activation in vivo and activation in vitro was partially inhibited by sodium pyrophosphate, an inhibitor of phosphoprotein phosphatase. In addition to these similarities, the activation of tyrosine hydroxylase in vivo by photic stimulation was not additive to the activation in vitro by protein phosphorylation. These data indicate that the mechanism for the activation of tyrosine hydroxylase that occurs as a consequence of light-induced increases of neuronal activity is similar to the mechanism for activation of the enzyme in vitro by protein phosphorylation. This observation suggests that the activation of retinal tyrosine hydroxylase in vivo may be mediated by phosphorylation of tyrosine hydroxylase or some effector molecule associated with the enzyme.     

Glucose-induced stimulation of the Ras-cAMP pathway in yeast leads to multiple phosphorylations and activation of 6-phosphofructo-2-kinase

Yeast cells respond to changes of the environment by complex modifications of the metabolism. An increase of the extracellular glucose concentration activates the Ras-cAMP pathway. Via a production of cAMP this pathway stimulates the cAMP-dependent protein kinase (PKA) which is involved in the posttranslational regulation of the key enzymes of gluconeogenesis and glycolysis. 6-Phosphofructo-2-kinase (PFK2) catalyzes the synthesis of fructose 2,6-bisphosphate, the most potent activator of the glycolytic key enzyme 6-phosphofructo-1-kinase. We investigated the molecular mechanism of the glucose-induced phosphorylation and activation of PFK2 in Saccharomyces cerevisiae. After an incubation of PFK2 with ATP and PKA in vitro, two amino acid residues, Thr157 and Ser644, are phosphorylated and the enzyme is activated. A stimulation of the Ras-cAMP pathway by glucose addition to cultivated yeast cells leads to an in vivo activation of PFK2 which is accompanied by a more complex phosphorylation pattern of the enzyme. The phosphorylation of the protein on Ser644 is the result of PKA stimulation while the protein kinase(s) catalyzing the 5-fold phosphorylation of the peptide fragment T(67)(-)(101) is (are) still unknown. The functional significance of T(67)(-)(101) and its phosphorylation is supported by the finding that PFK2 lacking this peptide is inactive.     

Possibility of correcting brain bioenergetics in neuroses using nicotinic acid derivatives

Experiments were made to study the effect of the animals' neurosis status on the synthesis of macroergs according to the oxidative phosphorylation data and macroerg utilization via the ATPase system. It was demonstrated that in the stage of metabolic deadaptation , neurosis leads to dramatic inhibition of oxidative phosphorylation and its dissociation, decreases the content of macroergs , discoordinates the ATPase system. The prophylactic administration of nicotinic acid derivatives stimulated oxidative phosphorylation, making return to normal the content of macroergs and the ATPase activity. According to some data, the action of chlorodiazepoxide aggravated adverse effects of neurosis. The possibility of correcting brain bioenergetics with nicotinic acid derivatives has been shown.     

The CLK family kinases, CLK1 and CLK2, phosphorylate and activate the tyrosine phosphatase, PTP-1B.

The protein-tyrosine phosphatase PTP-1B is an important regulator of intracellular protein tyrosine phosphorylation, and is itself regulated by phosphorylation. We report that PTP-1B and its yeast analog, YPTP, are phosphorylated and activated by members of the CLK family of dual specificity kinases. CLK1 and CLK2 phosphorylation of PTP-1B in vitro activated the phosphatase activity approximately 3-5-fold using either p-nitrophenol phosphate, or tyrosine-phosphorylated myelin basic protein as substrates. Co-expression of CLK1 or CLK2 with PTP-1B in HEK 293 cells led to a 2-fold stimulation of phosphatase activity in vivo. Phosphorylation of PTP-1B at Ser(50) by CLK1 or CLK2 is responsible for its enzymatic activation. These findings suggest that phosphorylation at Ser(50) by serine threonine kinases may regulate the activation of PTP-1B in vivo. We also show that CLK1 and CLK2 phosphorylate and activate the S. cerevisiae PTP-1B family member, YPTP1. CLK1 phosphorylation of YPTP1 led to a 3-fold stimulation of phosphatase activity in vitro. We demonstrate that CLK phosphorylation of Ser(83) on YPTP1 is responsible for the activation of this enzyme. These findings demonstrate that the CLK kinases activate PTP-1B family members, and this phosphatase may be an important cellular target for CLK action.     

Determination of the sites of cAMP-dependent phosphorylation on the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor is a substrate for cAMP-dependent protein kinase both in vitro and in vivo. Recently, it has been demonstrated that phosphorylation of the nicotinic receptor by this kinase increases its rate of rapid desensitization. We now report the identification of the cAMP-dependent phosphorylation sites on the gamma and delta subunits. Two-dimensional phosphopeptide mapping of the phosphorylated gamma and delta subunits, after limit proteolysis with thermolysin, indicated that each subunit is phosphorylated on a single site. Phosphoamino acid analysis of the 32P-labeled subunits demonstrates that phosphorylation had occurred exclusively on serine residues. Purified phosphorylated subunits were cleaved with cyanogen bromide and the resultant phosphopeptides were purified by reverse-phase high performance liquid chromatography. Shorter phosphopeptides, obtained by secondary digestion with trypsin, were purified and subjected to both automated gas-phase sequencing and manual Edman degradation. The results demonstrate that the gamma subunit was phosphorylated at Ser-353, contained within the sequence Arg-Arg-Ser(P)-Ser-Phe-Ile and that the delta subunit was phosphorylated at Ser-361, contained within the sequence Arg-Ser-Ser(P)-Ser-Val-Gay-Tyr-Ser-Lys. Determination of the sites phosphorylated within the structure of the gamma and delta subunits should contribute to the molecular characterization of the regulation of desensitization of the nicotinic acetylcholine receptor by protein phosphorylation.     

Nicotinamide riboside kinase structures reveal new pathways to NAD+

The eukaryotic nicotinamide riboside kinase (Nrk) pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD⁺) by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD⁺ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD⁺.     

Nicotinamide Riboside Kinase Structures Reveal New Pathways to NAD+

The eukaryotic nicotinamide riboside kinase (Nrk) pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD⁺) by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD⁺ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD⁺.     

Regulation by nicotinic acid of malate dehydrogenase activity in tissues of Black Sea mussels

Nicotinic acid was studied for its effect on the malate dehydrogenase activity from mussels' tissues and on its ability to link substrate and coenzyme. NADH and nicotinic acid in high concentrations are shown to produce an inhibiting effect on the reverse malate dehydrogenase reaction which is determined by the nonspecific action either of the vitamin or its metabolites. When pH of the medium is shifted toward the acid zone the affinity of the enzyme to the coenzyme decreases. This phenomenon may be one of the mechanisms of the mussel organism adaptation to anaerobiosis.     

Acanthamoeba myosin I heavy chain kinase is activated by phosphatidylserine-enhanced phosphorylation

The actin-activated Mg2(+)-ATPase activities of myosins I from Acanthamoeba castellanii are fully expressed only when a single amino acid on their heavy chain is phosphorylated by myosin I heavy chain kinase. Here we show that kinase isolated by a procedure designed to minimize its phosphorylation during purification can incorporate up to 7.5 mol of phosphate/mol of enzyme when incubated with ATP, possibly by autophosphorylation. The rate of phosphorylation is enhanced about 20-fold by phosphatidylserine but is unaffected by calcium ions. Phosphorylation increases the rate at which the kinase phosphorylates the regulatory site of myosin I by about 50-fold. These results suggest that (auto?)phosphorylation may regulate the activity of myosin I heavy chain kinase in vivo. The stimulation of kinase phosphorylation by phosphatidylserine (other phospholipids have not yet been tested) is of particular interest because myosin I has been shown to be tightly associated with membranes, especially the plasma membrane.     

Regulation of phospholipase D

Phospholipase D (PLD) is a widely distributed enzyme that is under elaborate control by hormones, neurotransmitters, growth factors and cytokines in mammalian cells. Protein kinase C (PKC) plays a major role in the regulation of the PLD1 isozyme through interaction with its N-terminus. PKC activates this isozyme by a non-phosphorylation mechanism in vitro, but phosphorylation plays a role in the action of PKC on the enzyme in vivo. Although PLD1 can be phosphorylated by PKC in vitro, it is unclear that this occurs in vivo. Small GTPases of the ADP-ribosylation factor (ARF) and Rho families directly activate PLD1 in vitro and there is evidence that Rho proteins are involved in agonist regulation of PLD1 in vivo. ARF proteins stimulate PLD activity in the Golgi apparatus, but the role of these proteins in agonist regulation of the enzyme is less clear. PLD1 undergoes tyrosine phosphorylation in response to H₂O₂ treatment of cells. The functional consequence of this phosphorylation and soluble tyrosine kinase(s) involved are presently unknown.