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PubMed1976

Separation of two phosphorylase kinase phosphatases from rabbit skeletal muscle.

Cyclic-AMP-dependent protein kinase catalyses the activation of phosphorylase kinase and the phosphorylation of two serine residues on the alpha subunit and beta subunit of phosphorylase kinase [Cohen, P., Watson, D.C. and Dixon, G.H. (1975)]. The dephosphorylation of phosphorylase kinase has been shown to be catalysed by two distinct enzymes, termed alpha-phosphorylase kinase phosphatase and beta-phosphorylase kinase phosphatase. These two enzymes show essentially absolute specificity towards the alpha and beta subunits respectively. The two phosphatases copurified through ethanol fractionation, DEAE-cellulose chromatography and ammonium sulphate precipitation, but were separated from each other by a gel filtration on Sephadex G-200. alpha-Phosphorylase kinase phosphatase was purified 500-fold from the ethanol precipitation step, and beta-phosphorylase kinase phosphatase 320-fold. The molecular weights estimated by gel filtration were 170--180 000 for alpha-phosphorylase kinase phosphatase and 75--80 000 for beta-phosphorylase kinase phosphatase. Since the activity of phosphorylase kinase correlates with the state of phosphorylation of the beta subunit (Cohen, P. (1974)), beta-phosphorylase kinase phosphatase is the enzyme which reverses the activation of phosphorylase kinase. alpha-Phosphorylase kinase phosphatase is an enzyme activity that has not been recognised previously. Since the role of the alpha-subunit phosphorylation is to stimulate the rate of dephosphorylation of the beta subunit (Cohen, P. (1974)), alpha-phosphorylase kinase phosphatase can be regarded as the enzyme which inhibits the reversal of the activation of phosphorylase kinase. The implications of these findings for the hormonal control of phosphorylase kinase activity by multisite phosphorylation are discussed.

Animals↗
PubMed1977

Stimulation of cardiac sarcolemmal (Na+--K+) ATPase activity by phosphorylase kinase.

Following preincubation with phosphorylase kinase, ATPase activities of heart sarcolemmal membranes were increased: total ATPase from 9.38+/-0.65 to 15.25+/-0.90 and ouabain-sensitive (Na+--K+)ATPase from 1.67+/-0.17 to 3.12+/-0.33 micron moles Pi/mg protein/h (mean +/- S.E. of 3 experiments); (Ca2+)ATPase and (Mg2+--Ca2+)-ATPase activities were not significantly altered due to phosphorylase kinase. Under these conditions, phosphorylase kinase catalyzed phosphorylation of sarcolemmal membranes. The kinase-catalyzed phosphorylation of membranes was increased by Ca2+ ions: at pH 6.8, 30% increase in phosphorylation was observed whereas at pH 8.5, 267% increase was noted due to this action. These findings support the view that Ca2+-dependent phosphorylation of membranes regulates (Na+--K+)ATPase.

Adenosine Triphosphatases↗
PubMed1976

Site of action and biphasic effect of neutral salts in the phosphorylase kinase reaction.

The inhibition of phosphorylase kinase catalytic activity by 0.1 M neutral salts was predicted by the Hofmeister series of anions. The site of action of the salts was determined by the following evidence to be on the phosphorylase kinase molecule directly, rather than on its protein substrate. (1) Nonactivated kinase was more sensitive to salt inhibition than the activated form. (2) Ca2+ partially overcame the inhibition of nonactivated kinase. (3) Inhibition by Cl- occurred with either phosphorylase or a tetradecapeptide containing the convertible seryl residue as substrate. (4) Phosphorylation of nonactivated phosphorylase kinase by protein kinase was markedly inhibited by NaNO3, but this salt had little effect on the phosphorylation of histone by protein kinase. The influence of neutral salts on phosphorylase kinase activity was biphasic. Although activity was inhibited at low salt concentrations, it actually was stimulated as the salt concentration was increased. A similar biphasic response to various salt concentrations was observed in the velocities of autophosphorylation of phosphorylase kinase. The lag in the rate of product formation seen during the activity assay was less pronounced at inhibitory salt concentrations and was abolished at stimulatory salt concentrations. How the influence of salts relates to autophosphorylation and the lag is considered.

Animals↗
PubMed1994

DPhK-gamma, a putative Drosophila kinase with homology to vertebrate phosphorylase kinase gamma subunits: molecular characterisation of the gene and phenotypic analysis of loss of function mutants.

Partial and total loss of function mutant alleles of a putative Drosophila homologue (DPhK-gamma) of the vertebrate phosphorylase kinase gamma-subunit gene have been isolated. DPhK-gamma is required in early embryonic processes, such as gastrulation and mesoderm formation; however, defects in these processes are seen only when both the maternal and zygotic components of DPhK-gamma expression are eliminated. Loss of zygotic expression alone does not appear to affect normal embryonic and larval development; some pupal lethality is observed but the majority of mutant animals eclose as adults. Many of these adults show defects in their leg musculature (e.g. missing and degenerating muscles), in addition to exhibiting melanised "tumours" on their leg joints. Loss of only the maternal component has no obvious phenotypic consequences. The DPhK-gamma gene has been cloned and sequenced. It has an open reading frame (ORF) of 1680 bp encoding a 560 amino acid protein. The predicted amino acid sequence of DPhK-gamma has two conserved domains, the catalytic kinase and calmodulin-binding domains, separated by a linker sequence. The amino acid sequence of DPhK-gamma is homologous to that of mammalian PhK-gamma proteins but differs in the length and amino acid composition of its linker sequence. The expression of DPhK-gamma mRNA is developmentally regulated. We discuss the implications of these observations.

Amino Acid Sequence↗
PubMed1987

Autophosphorylation of phosphorylase kinase and its regulatory function in the dephosphorylation of phosphorylase A.

Autophosphorylation of phosphorylase kinase was measured under conditions that favoured autoactivation. Heparin and troponin C stimulated the autophosphorylation of phosphorylase kinase at pH 6.8 in a Ca2+-dependent manner. The concentration required for the half-maximal stimulation of autophosphorylation for calcium ions was 2 microM in the absence of effectors, whereas 0.7 microM and 0.1 microM in the presence of troponin C and heparin, respectively. Calmodulin increased the rate of autophosphorylation of the alpha subunit only, resulting in a slight increase in the rate of autoactivation of phosphorylase kinase. Troponin C, heparin and polybrene enhanced the rate of autophosphorylation of both alpha and beta subunits. The increased autophosphorylation coincided with an enhancement of kinase activity. Neither of these stimulatory macromolecules had significant influence on the total number of phosphate groups incorporated into the alpha or beta subunits by autophosphorylation. Thio-autophosphorylated form of phosphorylase kinase behaved as an inhibitor in the dephosphorylation of phosphorylase a by the catalytic subunits of phosphatase-1 or phosphatase-2A and by the latent form of phosphatase-2A. Concentration of phosphorylase kinase needed to 50% inhibition was in the range of 0.05-0.08 microM.

Calmodulin↗
PubMed2006

The cytoskeletal organizing protein Cdc42-interacting protein 4 associates with phosphorylase kinase in skeletal muscle.

Phosphorylase kinase is a key enzyme in regulating glycogenolytic flux in skeletal muscle in response to changing energy demands. In the present study, we sought to identify interacting proteins of phosphorylase kinase by yeast two-hybrid screening. Screening a rabbit skeletal muscle cDNA library with the exposed C-terminus of the alpha subunit (residues 1060-1237), we identified eight independent, yet overlapping, constructs of cdc42-interacting protein 4 (CIP4). Immunocytochemistry indicated that CIP4 colocalized with phosphorylase kinase in vivo, and the cognate binding domain on CIP4 was determined to lie between residues 398 and 545. While this region of CIP4 does contain a known src homology 3 domain, transient transfections and coimmunoprecipitation experiments showed that this domain is not responsible for the dimeric interaction. Based upon sequence analysis the association is inferred to be mediated by two proline-rich sequences in CIP4, residues 436-439 and 441-444, that bind to a cognate WW domain found between residues 1107 and 1129 of PhKalpha.

Animals↗
PubMed1980

Activation of endogenous phosphorylase kinase in liver glycogen pellet by cAMP-dependent protein kinase.

Liver glycogen phosphorylase associated with the glycogen pellet was activated by a MgATP-dependent process. This activation was reduced by 90% by ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid, not affected by the inhibitor of the cAMP-dependent protein kinase, and increased 2.5-fold by the catalytic subunit of cAMP-dependent protein kinase. Low levels of free Ca2+ (8 x 10(-8) M) completely prevented the effects of the chelator. The activation of phosphorylase by MgATP was shown not to be due to formation of AMP. DEAE-cellulose chromatography of the glycogen pellet separated phosphorylase from phosphorylase kinase. The isolated phosphorylase was no longer activated by MgATP in the presence or absence of the catalytic subunit of cAMP-dependent protein kinase. The isolated phosphorylase kinase phosphorylated and activated skeletal muscle phosphorylase b and the activation was increased 2- to 3-fold by the catalytic subunit of cAMP-dependent protein kinase. Mixing the isolated phosphorylase and phosphorylase kinase together restored the effects of MgATP and the catalytic subunit of cAMP-dependent protein kinase on phosphorylase activity. These findings demonstrate that the phosphorylase kinase associated with liver glycogen has regulatory features similar to those of muscle phosphorylase kinase.

Animals↗
PubMed1982

Some comparative aspects of regulation of muscle and liver phosphorylase kinase.

The interaction of muscle and liver phosphorylase kinase with some proteins has been studied. It was shown that muscle G-actin has a visible stimulating effect on the dephosphorylated form of muscle phosphorylase kinase. The effect of F-actin on this enzyme is very low. The interaction of phosphorylase kinase with G-actin probably is one of the additional links between glycogenolysis and muscle contraction. To answer the question what subunit(s) of phosphorylase kinase is involved in the interaction with G-actin we studied the influence of actin on the kinase preparations previously activated to a different degree by partial proteolysis with endogenous protease(s) or with trypsin. G-actin has almost no stimulating effect on the preparations of phosphorylase kinase deeply activated by proteolysis (pH 6.8-8.2 activity ratio more than 0.2). The experiments with partial proteolysis allow us to suppose that alpha-subunit is involved in the interaction of phosphorylase kinase with G-actin. Skeletal muscle G-actin activates purified preparations of liver phosphorylase kinase but to a lower degree than muscle enzyme. Brain and liver calmodulin has a low activating effect on liver phosphorylase kinase in the presence of calcium. Calcium-independent action of calmodulin on the preparations of liver phosphorylase kinase is stronger; probably it is connected with a nonspecific effect of this small acidic protein on the liver enzyme. The basic protein protamine has a strong inhibitory effect on liver phosphorylase kinase.

Actins↗
PubMed1976

A study on the autoactivation of rabbit muscle phosphorylase kinase.

Under conditions favoring its autocatalytic reaction, phosphorylase kinase may be activated and phosphorylated in 2-(N-morpholino)ethanesulfonate (Mes) buffer to a much higher level than in beta-glycerophosphate buffer. The fact that the reaction is autocatalytic is supported by several observations: (a) the progress curve of the reaction exhibits a pronounced lag phase, (b) the reaction is strongly inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetate, which inhibits phosphorylase kinase, (c) the pH profile of the reaction resembles that of the phosphorylase b to a reaction as catalyzed by nonactivated phosphorylase kinase, and (d) the reaction is not significantly affected by adenosine 3':5'-monophosphate (cAMP) nor by the heat-stable protein inhibitor of cAMP-dependent protein kinases. When fully autoactivated, phosphorylase kinase possesses an activity that is 100% higher than that of the protein kinase-activated form. The results suggest that autophosphorylation of phosphorylase kinase may be an important regulatory mechanism. The autocatalytic reaction involves phosphorylation of the two larger subunits of phosphorylase kinase, i.e. subunits A and B, with a combined total of 7 to 9 phosphates incorporated per mol of enzyme. Although the cAMP-dependent protein kinase also catalyzes the phosphorylation of subunits A and B, the two mechanisms of phosphorylation appear to involve different sites. Prior phosphorylation of phosphorylase kinase by the protein kinase has little effect on the level of autophosphorylation. Thus activation of phosphorylase kinase may be brought about by phosphorylation of the enzyme at different sites.

Adenosine Triphosphate↗
PubMed2005

Structural characterization of Ca2+/CaM in complex with the phosphorylase kinase PhK5 peptide.

Phosphorylase kinase (PhK) is a large hexadecameric enzyme consisting of four copies of four subunits: (alphabetagammadelta)4. An intrinsic calmodulin (CaM, the delta subunit) binds directly to the gamma protein kinase chain. The interaction site of CaM on gamma has been localized to a C-terminal extension of the kinase domain. Two 25-mer peptides derived from this region, PhK5 and PhK13, were identified previously as potential CaM-binding sites. Complex formation between Ca2+/CaM with these two peptides was characterized using analytical gel filtration and NMR methods. NMR chemical shift perturbation studies showed that while PhK5 forms a robust complex with Ca2+/CaM, no interactions with PhK13 were observed. 15N relaxation characteristics of Ca2+/CaM and Ca2+/CaM/PhK5 complexes were compared with the experimentally determined structures of several Ca2+/CaM/peptide complexes. Good fits were observed between Ca2+/CaM/PhK5 and three structures: Ca2+/CaM complexes with peptides from endothelial nitric oxide synthase, with smooth muscle myosin light chain kinase and CaM kinase I. We conclude that the PhK5 site is likely to have a direct role in Ca2+-regulated control of PhK activity through the formation of a classical 'compact' CaM complex.

Amino Acid Sequence↗
PubMed1994

Mutational analyses of the metal ion and substrate binding sites of phosphorylase kinase gamma subunit.

Phosphorylase kinase (PhK) and truncated gamma subunit, denoted gamma 1-300, can phosphorylate seryl and tyrosyl residues dependent on the metal ion [Yuan, C.-J., Huang, C. F., & Graves, D. J. (1993) J. Biol. Chem. 268, 17683-17686]. Recombinant gamma 1-300 was used to explore its dual specificity and the location of the metal ion binding sites by using site-directed mutagenesis. Two approaches were taken to generate 26 mutants. First, on the basis of the crystal structure of cAMP-dependent protein kinase (cAPK), the invariant Asn155 and highly conserved Asp168-Phe169-Gly170 residues were mutated. Changes included production of N155H, D168E, D168N, F169R, G170V, G170I, G170L (less than 1% of enzymatic activities were found in these mutants), F169W, and G170A mutants. Second, charge to alanine and charge reversal scanning mutations were used to probe the metal ion binding sites. Two mutants, E111K and E154R, showed very different metal ion response compared to wild-type gamma and were further characterized. The mutants F169W, G170A, E111K, and E154R had 15%, 5%, 8%, and 25% specific activity relative to wild-type gamma, respectively. The folding pattern of wild-type and mutated enzyme forms of gamma was determined by photoacoustic infrared spectroscopy. Conformational disruptions were found in G170V, G170I, and G170L mutants, but the conformation of the rest of the mutants was similar to that of wild-type gamma, suggesting that the loss of enzymatic activities of these mutants was not because of incorrect refolding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Reaction of fluorescein isothiocyanate with an ATP-binding site on the phosphorylase kinase alpha subunit.

Phosphorylase kinase can be labeled specifically on the alpha subunit with fluorescein 5'-isothiocyanate (FITC) which concomitantly inactivates the enzyme (T. G. Sotiroudis and S. Nikolaropoulus (1984) FEBS Lett. 176, 421-425). Labeled peptides have been purified and their primary structure has been determined. The amino acid sequence of the fluorescein-labeled tryptic peptide is Lys-Met-Gln-Asp-Gly-Tyr-Phe-Gly-Gly-Ala-Arg. The environment of this fluorescein-labeled lysine has been determined by sequencing peptides isolated from a Staphylococcus aureus V8 digest and two further cyanogen bromide fragments of the purified [14C]carboxymethylated alpha subunit. The partial sequences obtained have then been localized in the primary structure of the alpha subunit [Zander et al. (1988) Proc. Natl Acad. Sci. USA 85, 2929-2933]. Both the incorporation of the fluorescent label and enzymatic inactivation are inhibited by ATP only at pH 7.0; ADP and AMP do not protect. Kinetic analysis reveals a competition between ATP and FITC; a Ki for ATP of 728 +/- 100 microM has been determined.

Adenosine Triphosphate↗
PubMed1983

The effect of heart and skeletal muscle troponin complexes and calmodulin on the Ca2+-dependent reactions of phosphorylase kinase isoenzymes.

The dephosphorylated form of phosphorylase kinase was purified 700-fold from rabbit heart extract. The purified enzyme had a pH 6.8/pH 8.2 activity ratio of 0.04-0.08 and was completely dependent on Ca2+ with an apparent Ka value for Ca2+ of 2.59 microM at pH 6.8. At free Ca2+ concentrations between 0.057 microM and 400 microM, 1.5 microM rabbit heart troponin complex had no significant effect on the reaction. However, 1.5 microM rabbit skeletal muscle troponin complex stimulated the reaction 1.5-2-fold with a concomitant decrease in the Ka value for Ca2+ to 1.40 microM. No differences in the effects of these troponin complexes were observed when heart-type and skeletal muscle-type phosphorylase b isoenzymes from either rabbit or pig were used as substrate. Similar effects of heart and skeletal muscle troponin complexes were observed on the Ca2+-dependent reaction of the dephosphorylated form of phosphorylase kinase partially purified from rabbit skeletal muscle. A saturating concentration (1.36 microM) of bovine brain calmodulin stimulated 2-5-fold the Ca2+-dependent reaction of skeletal muscle phosphorylase kinase, but not the reaction of heart phosphorylase kinase. Heart troponin complex (12 microM) suppressed 80-100% the stimulatory effect of skeletal muscle troponin complex on the reactions of phosphorylase kinase isoenzymes, but had no significant effect on the stimulation by calmodulin of skeletal muscle phosphorylase kinase reaction.

Animals↗
PubMed1976

The molecular basis of skeletal muscle phosphorylase kinase deficiency.

The molecular basis of phosphorylase kinase deficiency was investigated in ICR/IAn mice, which show less than 0.2% of normal activity in skeletal muscle (Cohen, P.T.W. and Cohen, P., 1973). The genetics of the deficiency indicate it is a single gene defect on the X-chromosome (Lyon, J.B., 1970). Phosphorylase kinase was purified from skeletal muscle of a control strain, C3H/He-mg, by three different procedures. (a) Ammonium sulphate precipitation and gel filtration on Sepharose 4B. (b) Hydrophobic chromatography and affinity chromatography on Sepharose 4B to which antibody to rabbit muscle phosphorylase kinase has been linked covalently. (c) Precipitation from muscle extracts with anti-phosphorylase kinase antibody. All three procedures showed C3H/He-mg phosphorylase kinases were similar to the rabbit muscle enzymes, the structures of the two isoenzymes being (alphabetagamma)4 and (alpha'betagamma)4 respectively. The proportion of the (alpha'betagamma)4 isoenzyme relative to the (alphabetagamma)4 isoenzyme was however about 1:1 in murine muscle compared to about 1:10 in rabbit muscle. Since the alpha and alpha' subunits appear to be distinct gene products, the defect in ICR/IAn mice cannot be caused by a mutation in the genes coding for either the alpha or alpha'chains, or 50% of normal activity would be observed. All three procedures for C3H/He-mg mice failed to detect any of the four subunits alpha, alpha', beta and gamma in ICR/IAn mice, suggesting that all four chains are absent in the deficiency. An allele for the beta-subunit was identified in rabbits, and the inheritance of the allele showed that it was determined by an autosomal gene. Assuming conservation of X-linkage between mammals, the defect in ICR/IAn mice cannot be caused by a mutation in a beta-subunit gene. It is proposed that ICR/IAn mice are defective in a control gene located on the X-chromosome which is required for the expression of structural genes, at least one of which, the gene for the beta-subunit, is located on an autosome. The results imply that interchromosomal transfer of information takes place during the synthesis of phosphorylase kinase.

Amino Acids↗
PubMed1989

Expression of a cDNA for the catalytic subunit of skeletal-muscle phosphorylase kinase in transfected 3T3 cells.

Phosphorylase kinase is a multimeric enzyme of composition (alpha, beta, gamma, delta)4 whose catalytic activity resides in the gamma-subunit. As an approach to understand further its regulation, a cDNA for the gamma-subunit of phosphorylase kinase (gamma PhK) has been cloned into a mammalian expression vector behind the mouse metallothionein-1 promoter. NIH 3T3 cells were co-transfected with this construct (pEV gamma PhK) and pSV2neo, G418-resistant clones were selected, and several were found to have stably incorporated the gamma-subunit cDNA into their genomic DNA. Phosphorylase kinase activity was clearly present in extracts from cultures of pEV gamma PhK-transformed cells and increased several-fold after 24 h of incubation with Zn2+, whereas it was undetectable in the parent 3T3 cells. A significant, but variable, proportion (15-70%) of the activity was Ca2+-dependent. We conclude that the phosphorylase kinase activity expressed by the cells transformed with pEV gamma PhK is due to free gamma-subunit and gamma-subunit associated with cellular calmodulin, which replaces the delta-subunit normally associated with the gamma-subunit in the holoenzyme.

Animals↗
PubMed1979

Phosphorylase kinase from human polymorphonuclear leukocytes.

Phosphorylase kinase from human polymorphonuclear leukocytes was investigated in a gel filtered crude preparation (17,000 x g supernatant). It was found to exist in two forms, one (the phosphorylated form) more active than the other (the dephosphorylated form). Interconversion between the two forms was carried out by a cyclic AMP dependent protein kinase and phosphoprotein phosphatase, respectively. The ratio of activity measured at pH 8.0 and 6.0 was 0.36 for the non-activated and 0.83 for the activated form, which is in contrast to the behaviour of phosphorylase kinase from muscle. Km app for the substrate phosphorylase b was 650 U/ml and 85 U/ml for the non-activated and activated form, respectively, whereas Km app for ATP was 0.03 mM and identical for the two forms. The non-activated form of phosphorylase kinase was activated by Ca2+ in the range 10(-7)--5 . 10(-6) M, which may have physiological importance, whereas the activated form was insensitive to variations in Ca2+ concentration between 10(-9) and 10(-3) M.

Calcium↗