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PubMed1983

Calcium--calmodulin-dependent activation of porcine liver phosphorylase kinase.

Porcine liver phosphorylase kinase was activated about 1.5-fold by calmodulin in a calcium-dependent manner. Half-maximal stimulation was observed at about 80 nM calmodulin and the activation was almost pH-independent. The specific binding of porcine liver phosphorylase kinase to calmodulin--Sepharose affinity column exhibited an absolute dependence upon the presence of calcium. The physiological role of the calmodulin-dependent activation for liver phosphorylase kinase is discussed.

Animals↗

PubMed1981

Calmodulin ligands. The interaction of muscle phosphorylase kinase with phosphodiesterase. Comparison of calmodulin ligands in muscle extracts from normal and phosphorylase kinase-deficient mice.

Interactions between phosphorylase kinase (ATP:phosphorylase-b phosphotransferase, EC 2.7.1.38) and calmodulin were studied with pure preparations of muscle phosphorylase kinase, and with crude extracts from muscles of control (C57 Black) and deficient (ICR/IAn) mice, which lack muscle phosphorylase kinase activity. Calmodulin was determined by its ability to stimulate a calmodulin-dependent phosphodiesterase. The amount of calmodulin bound to phosphorylase kinase in muscle extract was estimated to a maximum of 30% of the total amount of calmodulin. In the muscle of the deficient strain a decrease of 35% in the total amount of calmodulin was observed. This correlates with the absence of the calmodulin fraction specifically bound to phosphorylase kinase. From sucrose gradient studies we demonstrated that in the presence of Ca2+ the amount of calmodulin bound to phosphorylase kinase was enhanced, compared to the control in the presence of EGTA. This observation was made both in crude extracts and in pure phosphorylase kinase preparations. Sucrose gradient also showed that muscle phosphorylase kinase can be dissociated to low molecular species when extracts are made in the presence of Ca+; this dissociation was found to be related to a Ca2+-dependent proteolytic effect.

Animals↗

PubMed1975

Glycogen phosphorylase and its converter enzymes in haemolysates of normal human subjects and of patients with type VI glycogen-storage disease. A study of phosphorylase kinase deficiency.

1. The properties of phosphorylase a, phosphorylase b, phosphorylase kinase and phosphorylase phosphatase present in a human haemolysate were investigated. The two forms of phosphorylase have the same affinity for glucose 1-phosphate but greatly differ in Vmax. Phosphorylase b is only partially stimulated by AMP, since, in the presence of the nucleotide, it is about tenfold less active than phosphorylase a. In a fresh human haemolysate phosphorylase is mostly in the b form; it is converted into phosphorylase a by incubation at 20degreesC, and this reaction is stimulated by glycogen and cyclic AMP. Once activated, the enzyme can be inactivated after filtration of the haemolysate on Sephadex G-25. This inactivation is stimulated by caffeine and glucose and inhibited by AMP and fluoride. The phosphorylase kinase present in the haemolysate can also be measured by the rate of activation of added muscle phosphorylase b, on addition of ATP and Mg2+. 2. The activity of phosphorylase kinase was measured in haemolysates obtained from a series of patients who had been classified as suffering from type VI glycogenosis. In nine patients, all boys, an almost complete deficiency of phosphorylase kinase was observed in the haemolysate and, when it could be assayed, in the liver. A residual activity, about 20% of normal, was found in the leucocyte fraction, whereas the enzyme activity was normal in the muscle. These patients suffer from the sex-linked phosphorylase kinase deficiency previously described by others. Two pairs of siblings, each time brother and sister, displayed a partial deficiency of phosphorylase kinase in the haemolysate and leucocytes and an almost complete deficiency in the liver. This is considered as being the autosomal form of phosphorylase kinase deficiency. Other patients were characterized by a low activity of total (a+b) phosphorylase and a normal or high activity of phosphorylase kinase in their haemolysate.

Adenosine Monophosphate↗

PubMed1981

Phosphorylation of calf thymus H1 histone by muscle glycogen phosphorylase kinase.

Muscle glycogen phosphorylase kinase [EC 2.7.1.38] has the ability to phosphorylate five fractions of calf thymus histone. H1 histone is the most preferable substrate, and maximally about 1.3 mol of phosphate is incorporated into every mole of this histone. This reaction absolutely depends on CA2+, and the molecular activity is about one third of that of cyclic AMP-dependent protein kinase (protein kinase A). The affinity of phosphorylase kinase for H1 histone is higher than that of protein kinase A. Calmodulin stimulates this histone phosphorylation. Analysis of the N-bromosuccinimide-bisected fragments of fully phosphorylated H1 histone has revealed that the enzyme phosphorylates mostly seryl residues in both amino- and carboxyl-terminal portions, although phosphorylation of the carboxyl-terminal portion is twice as much as that of the amino-terminal portion. Fingerprint analysis indicates that the phosphorylation sites in H1 histone for this enzyme are different from the sites phosphorylated by protein kinase A. This catalytic activity also differs from that of a newly found multifunctional protein kinase which may be activated by the simultaneous presence of Ca2+ and phospholipid.

Animals↗

PubMed1997

The regulatory Ser262 of microtubule-associated protein tau is phosphorylated by phosphorylase kinase.

Abnormally phosphorylated tau is the major component of paired helical filaments found in the brains of patients suffering from Alzheimer's disease. Therefore, the identification of kinases that phosphorylate tau is of considerable interest. A DEAE-Sepharose column resolved porcine brain extract into five tau kinase activity peaks. Among these peaks, two were completely inhibited by EGTA, indicating that these two activity peaks contained Ca2+-dependent tau kinases. One of the above two Ca2+-dependent tau kinase activity peaks also contained phosphorylase kinase activity. The tau kinase and phosphorylase kinase activities associated with this peak could not be separated from each other by Superose 12 gel filtration, hydroxylapatite, and calmodulin-agarose affinity chromatographies. Phosphorylase kinase, purified from rabbit skeletal muscle, phosphorylated tau to a stoichiometry of 2.1 mol of phosphate/mol of tau and converted tau to a species with a retarded mobility on SDS-polyacrylamide gel electrophoresis. The apparent Km and kcat values for tau phosphorylation by muscle phosphorylase kinase were 6.9 microM and 47.4 min-1, respectively. As a substrate of muscle phosphorylase kinase, phosphorylase was eight times better than tau. Sequence analyses of tryptic and thermolytic phosphopeptides derived from tau phosphorylated by muscle phosphorylase kinase revealed five phosphorylation sites, Ser237, Ser262, Ser285, Ser305, and Ser352. Among these sites, Ser262 was previously shown to be phosphorylated in human tau from fetal, adult, and Alzheimer's diseased brains (Seubert, P., Mawal-Dewan, M., Barbour, R., Jakes, R., Goedert, M., Johnson, G. V. W., Litersky, J. M., Schenk, D., Lieberburg, I., Trojanowski, J. Q., and Lee, V. M. Y. (1995) J. Biol. Chem. 270, 18917-18922); and its phosphorylation abolished tau's binding to microtubules (Drewes, G., Trinczek, B., Illenberger, S., Biernat, J., Schmitt-Ulms, G., Meyer, H. E., Mandelkow, E.-M., and Mandelkow, E. (1995) J. Biol. Chem. 270, 7679-7688). Slot-blot analysis using a monoclonal antibody against muscle phosphorylase kinase and an activity assay using phosphorylase revealed that phosphorylase kinase was present in microtubules extensively purified by repeated cycles of polymerization and depolymerization. Taken together, these results suggest that in neurons, phosphorylase kinase may be one of the kinases that participate in the phosphorylation of tau.

Amino Acid Sequence↗

PubMed1976

Ca2+-dependent activation of phosphorylase by phosphorylase kinase in adipose tissue.

Phosphorylase kinase (EC 2.7.1.38) activity in crude cytosol preparations of chicken adipose tissue was assayed using as substrate either the endogenous phosphorylase b in the preparation or added purified rabbit skeletal muscle phosphorylase b. The results obtained with the two substrates were similar. The phosphorylase kinase reaction was markedly inhibited by ethyleneglycol-bis(beta-aminoethylether)-N,N',-tetraacetic acid (EGTA), maximum inhibition (about 90%) occurring at approx. 0.2 mM. This inhibition was readily reversed by addition of Ca2+. Full reversal was achieved with 0.3 mM Ca2+ in the presence of 0.5 mM EGTA; the estimated free Ca2+ concentration required was 4 muM. The activation of phosphorylase b was blocked immediately and completely by EGTA added during the course of the assay; reversal was achieved without a time lag by the addition of Ca2+. The Ca2+ requirement was also demonstrated directly by preparing an enzyme fraction from which Ca2+ had been removed and by using Ca2+-free reagents. Under these conditions the Ca2+ concentration needed for half maximum activation was 10 muM and maximum activation was obtained at about 100 muM. The possibility that the effects of EGTA and Ca2+ might be related to changes in phosphorylase phosphatase activity rather than phosphorylase kinase was considered unlikely since the phosphorylase phosphatase activity was inhibited during the phosphorylase kinase assay step by the inclusion of fluoride and beta-glycerophosphate. Phosphorylase kinase activity in rat adipocytes, using endogenous phosphorylase as substrate, was also inhibited EGTA but, whereas the activity in chicken adipose tissue was inhibited by 90%, the activity in rat adipose tissue was inhibited only 60%. These data indicate that adipose tissue phosphorylase kinase has a Ca2+ requirement for optimal activity and is thus qualitatively similar to the enzyme in contractile tissues.

Adenosine Triphosphate↗

PubMed1994

Curcumin is a non-competitive and selective inhibitor of phosphorylase kinase.

Recently, we reported that curcumin (diferuloylmethane) inhibits the growth of several different kinds of tumor cells. In order to investigate the mechanism of this inhibition, we examined the effects of curcumin on different protein kinases: highly purified protein kinase A (PkA), protein kinase C (PkC), protamine kinase (cPK), phosphorylase kinase (PhK), autophosphorylation-activated protein kinase (AK) and pp60c-src tyrosine kinase. While all kinases tested were inhibited by curcumin, only PhK was completely inhibited at relatively lower concentrations. At around 0.1 mM curcumin, PhK, pp60c-src, PkC, PkA, AK, and cPK were inhibited by 98%, 40%, 15%, 10%, 1%, and 0.5%, respectively. Lineweaver-Burk plot analysis indicated that curcumin is a non-competitive inhibitor of PhK with a Ki of 0.075 mM. Overall, our results indicate that curcumin is a potent and selective inhibitor of phosphorylase kinase, a key regulatory enzyme involved in the metabolism of glycogen. This has important implications for the anti-proliferative effects of curcumin.

Binding, Competitive↗

PubMed1996

A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1.

We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.

Amino Acid Sequence↗

PubMed1990

Kinetic analysis of the separate phosphorylation events in the phosphorylase kinase reaction.

Glycogen phosphorylase, a dimer of identical subunits, is activated by phosphorylase kinase-catalyzed phosphorylation of one serine residue in each subunit. In this paper, the effect of the phosphorylation of one subunit on the phosphorylation of the other subunit was examined. The three forms of phosphorylase, phosphorylase b (nonphosphorylated), phosphorylase ab (one subunit phosphorylated), and phosphorylase a (both subunits phosphorylated), were separated by anion-exchange high-performance liquid chromatography (HPLC). Purified phosphorylase ab was found to be stable under the conditions of the phosphorylase kinase assay. Initial rate kinetics showed that phosphorylase kinase had a lower KM for phosphorylase ab (3.9 +/- 0.24 microM) than for phosphorylase b (14.9 +/- 2.6 microM). Using the HPLC separation as a simultaneous assay for the three forms of phosphorylase during the phosphorylase kinase reaction, it was found that the pseudo-first-order rate constant for the second phosphorylation step (k2) was 3.7 times greater than that for the first step (k1). The activator AMP reduced the ratio k2/k1 from 3.7 without AMP to 1.4. When the monomeric gamma delta complex of phosphorylase kinase subunits was used as the enzyme, the ratio k2/k1 was 2.1, compared to 3.7 with the multimeric holophosphorylase kinase. One explanation for these data is that phosphorylation of one subunit of phosphorylase b causes conformational changes that make the other subunit a better substrate for the kinase. In this context, the effect of AMP is to reduce the conformational differences between phosphorylases b and ab, and the gamma delta complex is less sensitive to the conformational differences between the two forms of phosphorylase.

Animals↗

PubMed1982

Identification of a calmodulin-dependent glycogen synthase kinase in rabbit skeletal muscle, distinct from phosphorylase kinase.

A glycogen synthase kinase that is completely dependent on Ca2+ and calmodulin has been identified in mammalian skeletal muscle, and purified approximately 3000-fold by chromatography on phosphocellulose and calmodulin--Sepharose. The presence of 50 mM NaCl in the homogenisation buffer was critical for extraction of the enzyme. The calmodulin-dependent glycogen synthase kinase (app. Mr 850 000) is distinct from myosin light-chain kinase and phosphorylase kinase, but phosphorylates the same serine residue on glycogen synthase as phosphorylase kinase. The physiological role of the enzyme is discussed.

Animals↗

PubMed1976

Thiophosphate-activated phosphorylase kinase as a probe in the regulation of phosphorylase phosphatase.

Rabbit muscle nonactivated phosphorylase kinase (EC 2.7.1.38) is converted to thiophosphate-activated phosphorylase kinase by cyclic AMP dependent protein kinase, Mg2+ and ATP-gamma-S/adenosine-5'-O-(s-thiotriphosphate)/. The formation of thiophosphate-activated phosphorylase kinase wal also observed in the protein-glycogen complex from skeletal muscle. This new form of kinase is resistant to the action of phosphatase and behaves as a competitive inhibitor in the dephosphorylation of phosphorylase alpha by phosphorylase phosphatase (Ki = 0.04 mg per ml). The fact that the inhibitory effect of thiophosphate-activated phosphorylase kinase is 3 times higher than in the case of nonactivated kinase, may explain the transient inhibition of phosphorylase phosphatase in the protein-glycogen complex. The use of activated (phosphorylated) phosphorylase kinase supports this assumption since it causes a delay in the dephosphorylation of phosphorylase alpha, i.e. the conversion of phosphorylase alpha into beta could start only after the dephosphorylation of activated phosphorylase kinase.

Adenosine Triphosphate↗

PubMed1976

Stimulation of phosphorylase kinase autophosphorylation by peptide analogs of phosphorylase.

Autoactivation of phosphorylase kinase in the presence of substrates has been studied to determine the cause of the hysteresis, or lag, in the phosphorylase kinase reaction. Peptide analogs corresponding to the convertible serine region of phosphorylase have been used as low molecular weight alternative substrates. Autophosphorylation of the kinase molecule was measured under conditions that favored autoactivation. Phosphorylase b and a tetradecapeptide, which was found to be a good model of phosphorylase, stimulated autoactivation by 86- and 37-fold, respectively. The tetradecapeptide also stimulated autophosphorylation of subunits A and B of the kinase molecule. This increased autophosphorylation coincided with an increased ability to convert phosphorylase. This finding supports the hypothesis that autophosphorylation is responsible for the lag in the phosphorylase kinase reaction. No evidence was obtained to suggest that the lag could be due to dissociation of the kinase. The stoichiometry of phosphate incorporation into phosphorylase kinase subunits by autophosphorylation was much greater than that reported to occur by protein kinase phosphorylation. Multiple phosphorylation sites in subunit A accounted for most of the phosphate incorporation during autophosphorylation. Saturating levels of hexa- and octapeptide analogs also caused stimulation of autophosphorylation. Possible mechanisms and experimental implications of substrate-stimulated autophosphorylation are discussed. Consideration also is given to the possible role of effectors in autophosphorylation in vivo.

Animals↗

PubMedDate not supplied

Effect of molecular crowding on self-association of phosphorylase kinase and its interaction with phosphorylase b and glycogen.

Self-association of phosphorylase kinase (PhK) and its interaction with glycogen (M=5500 kDa) and phosphorylase b (Phb) has been studied using analytical ultracentrifugation and turbidimetry under the conditions of molecular crowding arising from the presence of high concentrations of osmolytes. In accordance with the predictions of the molecular crowding theory, trimethylamine N-oxide (TMAO) and betaine greatly favor self-association of PhK induced by Mg2+ and Ca2+ and PhK interaction with glycogen. In contrast, proline suppresses these processes, probably, due to its specific interaction with PhK. All osmolytes tested prevented the complex formation between PhK and its physiological substrate, Phb. The specific interactions of PhK and Phb with glycogen, in the living cell, presumably is a factor allowing the negative effect of crowding on the recognition of Phb by PhK to be overcome.

Animals↗

PubMed1985

Stimulating effect of phosphatidic acid on autophosphorylation of phosphorylase kinase.

Autophosphorylation of phosphorylase kinase from rabbit skeletal muscle was stimulated by acidic phospholipids such as phosphatidic acid (PA), phosphatidylinositol, and phosphatidyl-serine. PA stimulated an initial velocity of autophosphorylation 3.8-fold. When fully autophosphorylated, about 11 mol of phosphate per tetramer (alpha beta gamma delta) were incorporated in the presence of PA and about 6.5 mol in the absence of PA. In the presence of PA (100 micrograms/ml), there was a concomitant enhancement of its kinase activity about 25-fold at pH 6.8. PA (100 micrograms/ml) sharply decreased an apparent Ka for Ca2+ on autophosphorylation from 4.0 X 10(-5) M to 1.0 X 10(-6) M. Available evidence indicates that the Ca2+-activated, PA-dependent autophosphorylation of phosphorylase kinase shows an ability to stimulate glycogen breakdown.

Animals↗

PubMed1989

The substrate and sequence specificity of the AMP-activated protein kinase. Phosphorylation of glycogen synthase and phosphorylase kinase.

In addition to acetyl-CoA carboxylase and HMG-CoA reductase, the AMP-activated protein kinase phosphorylates glycogen synthase, phosphorylase kinase, hormone-sensitive lipase and casein. A number of other substrates for the cyclic AMP-dependent protein kinase, e.g., L-pyruvate kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, are not phosphorylated at significant rates. Examination of the sites phosphorylated on acetyl-CoA carboxylase, hormone-sensitive lipase, glycogen synthase and phosphorylase kinase suggests a consensus recognition sequence in which the serine residue phosphorylated by the AMP-activated protein kinase has a hydrophobic residue on the N-terminal side (i.e., at -1) and at least one arginine residue at -2, -3 or -4. Substrates for cyclic AMP-dependent protein kinase which lack the hydrophobic residue at -1 are not substrates for the AMP-activated protein kinase.

Acetyl-CoA Carboxylase↗

PubMed1987

Substrate specificity of phosphorylase kinase: effects of heparin and calcium.

Phosphorylase b and two peptides with sequences homologous to phosphorylation site 2 (syntide 2) and site 3 (syntide 3) of glycogen synthase were compared as substrates for purified muscle phosphorylase kinase. The substrate specificity of phosphorylase kinase varied according to whether heparin (at pH 6.5) or Ca2+ (at pH 8.2) was used as a stimulator of its activity. Phosphorylase b was preferentially phosphorylated in the presence of Ca2+; the rate of syntide 2 phosphorylation was the same for both stimulators; and the phosphorylation of syntide 3 was completely dependent on the presence of heparin. A kinetic analysis confirmed this stimulator-dependent substrate specificity since both the Vmax and Km for these substrates were affected diversely by heparin and Ca2+. Heparin stimulated phosphorylase kinase maximally at pH 6.5, whereas the effect of Ca2+ was optimal at a pH above 8. However, the stimulator-related substrate specificity could not be explained by the different pH values at which the effects of the stimulators were assessed. Nor did substrate-directed effects by heparin or Ca2+ apparently play a role. No indications were found for a stimulator-dependent specificity in the phosphorylation of sites in protein substrates of phosphorylase kinase (phosphorylase b, the alpha- and beta-subunits of phosphorylase kinase, or glycogen synthase). The diverse substrate specificity of the calcium- and heparin-dependent activities of phosphorylase kinase could be explained in two ways: either by the existence of separate calcium- and heparin-stimulated catalytic sites, or by just one catalytic site with two active conformations. The second possibility is favored by the observation that both calcium and heparin stimulated the isolated gamma-subunit (gamma X calmodulin complex) of phosphorylase kinase.

Adenosine Diphosphate↗

PubMed1993

Characterization of the gene for rat phosphorylase kinase catalytic subunit.

Phosphorylase kinase, a key enzyme in glycogen metabolism, has a subunit composition of (alpha beta gamma delta)4, in which the alpha and beta subunits are regulatory, delta is calmodulin, and the gamma subunit is catalytic. As one segment of our studies on the regulation of the expression of phosphorylase kinase subunits, we present in this report the structure of the gene for the catalytic gamma subunit. The gene extends over 16 kilobase pairs (kb) of DNA, and contains eight introns within the coding region plus one 3.3-kb intron upstream in the 5'-untranslated region. Within this first intron, and also upstream of the transcription start site, are sequences homologous to defined regulatory elements, including some found in other muscle-specific genes. The positions of intron splice junctions for this gene have been compared with similar data for other protein kinase genes. A somewhat unexpected finding for the gamma subunit is that two of the splice junctions fall in the midst of highly conserved strings of amino acids, both of which have been nominally defined as functional domains for the protein kinases and appear to make key contributions to substrate binding and phosphotransferase catalysis.

Amino Acid Sequence↗

PubMed1990

K252a is a potent and selective inhibitor of phosphorylase kinase.

The inhibition of phosphorylase kinase by a number of protein kinase inhibitors was examined. Both K252a and staurosporine are potent inhibitors of phosphorylase kinase with IC50 values of 1.7 nM and 0.5 nM respectively. K252a shows a 300-fold selectivity for this enzyme over protein kinase C whereas staurosporine shows only a 20-fold selectivity for phosphorylase kinase. In contrast, the Roche bis-indolyl maleimides inhibit phosphorylase kinase with IC50 values of approximately 1 microM and are highly selective for protein kinase C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗