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PubMed1990

The natural history of liver glycogenosis due to phosphorylase kinase deficiency: a longitudinal study of 41 patients.

We report a longitudinal study of 41 patients with liver glycogenosis due to phosphorylase kinase deficiency. In their youth, patients displayed hepatomegaly (92%), growth retardation (68%), delayed motor development (52%), hypercholesterolaemia (76%), hypertriglyceridaemia (70%), elevation of glutamate pyruvate transaminase (56%) and fasting hyperketosis (44%). With age, these clinical and biochemical abnormalities gradually disappeared and most adult patients were asymptomatic.

Aging↗

PubMed1991

Mapping of the gene for X-linked liver glycogenosis due to phosphorylase kinase deficiency to human chromosome region Xp22.

X-linked liver glycogenosis (XLG) is a glycogenosis due to deficient activity of phosphorylase kinase (PHK) in liver. PHK consists of four different subunits, alpha, beta, gamma, and delta. Although it is unknown whether liver and muscle PHK subunits are encoded by the same genes, the muscle alpha subunit (PHKA) gene was a likely candidate gene for the mutation responsible for this X-linked liver glycogenosis as it was assigned to the X chromosome at q12-q13. Linkage analysis with X-chromosomal polymorphic DNA markers was performed in two families segregating XLG. First, multipoint linkage analysis excluded the muscle PHKA region as the site of the XLG mutation. Second, evidence was obtained for linkage between the XLG locus and DXS197, DXS43, DXS16, and DXS9 with two-point peak lod scores Zmax = 6.64, 3.75, 1.30, and 0.88, all at theta max = 0.00, respectively. Multipoint linkage results and analysis of recombinational events indicated that the mutation responsible for XLG is located in Xp22 between DXS143 and DXS41.

Chromosome Mapping↗

PubMed2001

Studies on interaction of phosphorylase kinase from rabbit skeletal muscle with glycogen in the presence of ATP and ADP.

The influence of ATP on complex formation of phosphorylase kinase (PhK) with glycogen in the presence of Ca(2+) and Mg(2+) has been studied. The initial rate of complex formation decreases with increasing ATP concentration, the dependence of the initial rate on the concentration of ATP having a cooperative character. Formation of the complex of PhK with glycogen in the presence of ATP occurs after a lag period, which increases with increasing ATP concentration. The dependence of the initial rate of complex formation (v) on the concentration of non-hydrolyzed ATP analogue, beta,gamma-methylene-ATP, follows the hyperbolic law. A correlation between PhK-glycogen complex formation and (32)P incorporation catalyzed by PhK itself and by the catalytic subunit of cAMP-dependent protein kinase has been shown. For ADP (the product and allosteric effector of the PhK reaction) the dependence of v on ADP concentration has a complicated form, probably due to the sequential binding of ADP at two allosteric sites on the beta subunit and the active site on the gamma subunit.

Adenosine Diphosphate↗

PubMed1996

A comparison of the properties of the binary and ternary complexes formed by calmodulin and troponin C with two regulatory peptides of phosphorylase kinase.

The regulatory peptides Phk13 (301-327) and a modified form of Phk5 (342-367) from the gamma-subunit of glycogen phosphorylase kinase form binary and ternary complexes with both calmodulin and the related muscle protein troponin C. Neither peptide appears to affect to a major extent a fluorescent probe linked to Cys-27 of wheat germ calmodulin. Phk13, but not Phk5, significantly modifies the properties of a probe joined to Cys-98 of troponin C. A comparison by means of radiationless energy transfer of the average separations of Trp-16 of Phk5 from specific groups in the N- and C-terminal halves of calmodulin and troponin C indicate significant changes upon going from the 1:1 binary complex to the 1:1:1 ternary complex with Phk13. A comparison of the effects of addition of Phk13 to calmodulin, troponin C, and their binary complexes with Phk5 suggests that the conformation of Phk13 is similar in the binary and ternary complexes.

Amino Acid Sequence↗

PubMed1999

Catalytic mechanism of phosphorylase kinase probed by mutational studies.

The contributions to catalysis of the conserved catalytic aspartate (Asp149) in the phosphorylase kinase catalytic subunit (PhK; residues 1-298) have been studied by kinetic and crystallographic methods. Kinetic studies in solvents of different viscosity show that PhK, like cyclic AMP dependent protein kinase, exhibits a mechanism in which the chemical step of phosphoryl transfer is fast and the rate-limiting step is release of the products, ADP and phosphoprotein, and possibly viscosity-dependent conformational changes. Site-directed mutagenesis of Asp149 to Ala and Asn resulted in enzymes with a small increase in K(m) for glycogen phosphorylase b (GPb) and ATP substrates and dramatic decreases in k(cat) (1.3 x 10(4) for Asp149Ala and 4.7 x 10(3) for Asp149Asn mutants, respectively). Viscosometric kinetic measurements with the Asp149Asn mutant showed a reduction in the rate-limiting step for release of products by 4.5 x 10(3) and a significant decrease (possibly as great as 2.2 x 10(3)) in the rate constant characterizing the chemical step. The date combined with the crystallographic evidence for the ternary PhK-AMPPNP-peptide complex [Lowe et al. (1997) EMBO J. 6, 6646-6658] provide powerful support for the role of the carboxyl of Asp149 in binding and orientation of the substrate and in catalysis of phosphoryl transfer. The constitutively active subunit PhK has a glutamate (Glu182) residue in the activation segment, in place of a phosphorylatable serine, threonine, or tyrosine residue in other protein kinases that are activated by phosphorylation. Site-directed mutagenesis of Glu182 and other residues involved in a hydrogen bond network resulted in mutant proteins (Glu182Ser, Arg148Ala, and Tyr206Phe) with decreased catalytic efficiency (approximate average decrease in k(cat)/K(m) by 20-fold). The crystal structure of the mutant Glu182Ser at 2.6 A resolution showed a phosphate dianion about 2.6 A from the position previously occupied by the carboxylate of Glu182. There was no change in tertiary structure from the native protein, but the activation segment in the region C-terminal to residue 182 showed increased disorder, indicating that correct localization of the activation segment is necessary in order to recognize and present the protein substrate for catalysis.

Catalysis↗

PubMed1984

Dephosphorylation of phosphorylase kinase by a histone H1-stimulated phosphoprotein phosphatase.

A histone H1-stimulated protein phosphatase isolated from rabbit skeletal muscle dephosphorylated [32P]phosphorylase kinase. The rate of dephosphorylation was markedly increased by 5-50 micrograms histone H1/ml. Only the alpha and alpha' subunits were dephosphorylated in the absence or presence of histone H1. This is consistent with previous results suggesting that the H1-stimulated phosphatase is a type-2 protein phosphatase. The present studies suggest that the histone H1-stimulation is the result of a direct interaction of the histone with the phosphatase.

Animals↗

PubMed1984

A new type of glycogen storage disease caused by deficiency of cardiac phosphorylase kinase.

A five-month-old Japanese boy was found to have marked glycogen accumulation only in the heart. A survey of enzymes revealed normal activities of phosphorylase, cyclic AMP-dependent protein kinase, acid maltase and amylo-1,6-glucosidase. However, the heart had capacity of activating neither rabbit muscle phosphorylase b nor endogenous phosphorylase b, which was converted to active form only when supplemented rabbit muscle phosphorylase kinase. In contrast to the heart, activities of phosphorylase kinase were found within normal levels in other organ tissues so far tested. These findings indicate that the present case of the cardiac glycogenosis is caused by deficiency of cardiac phosphorylase kinase.

Animals↗

PubMed2000

Kinetic characterization of the double mutant R148A/E182S of glycogen phosphorylase kinase catalytic subunit: the role of the activation loop.

Many protein kinases are activated by phosphorylation in a highly conserved region of their catalytic subunit, termed activation loop. Phosphorylase kinase is constitutively active without the requirement for phosphorylation of residues in the activation loop. The residue which plays an analogous role to the phosphorylatable residues in other protein kinases is Glu182, which makes contacts to a highly conserved Arg148. In turn, Arg148 adjacent to the catalytic Asp149, enabling information to be transmitted from the activation loop to the catalytic machinery. The double mutant R148A/E182S has been kinetically characterized. The mutation resulted in an approximate 16- to 22-fold decrease in the kcat/Km value of the enzyme. The kinetic data, discussed in the light of the structural data from previously determined complexes of the enzyme, lead to the suggestion that the activation loop has a major role in substrate binding but also in correct orientation of the groups participating in catalysis.

Alanine↗

PubMed1987

Isolation of cDNA clones for the catalytic gamma subunit of mouse muscle phosphorylase kinase: expression of mRNA in normal and mutant Phk mice.

We have isolated and characterized cDNA clones for the gamma subunit of mouse muscle phosphorylase kinase (gamma-Phk). These clones were isolated from a lambda gt11 mouse muscle cDNA library via screening with a synthetic oligonucleotide probe corresponding to a portion of the rabbit gamma-Phk amino acid sequence. The gamma-Phk cDNA clones code for a 387-amino acid protein that shares 93% amino acid sequence identity with the corresponding rabbit amino acid sequence. RNA gel blot analysis reveals that the muscle gamma-Phk probe hybridizes to two mRNA species (2.4 and 1.6 kilobases) in skeletal muscle, cardiac muscle, and brain, but does not hybridize to liver RNA. Phk-deficient I-strain (Phk) mouse muscle contains reduced levels of gamma-Phk mRNA as compared with control mice. Although the Phk defect is an X-linked recessive trait, hybridization to a human-rodent somatic cell hybrid mapping panel shows that the gamma-Phk gene is not located on the X chromosome. Rather, the gamma-Phk cross-hybridizing human restriction fragments map to human chromosomes 7 (multiple) and 11 (single). Reduced gamma-Phk mRNA in I-strain mice, therefore, appears to be a consequence of the Phk-mutant trait and does not stem from a mutant gamma-subunit gene.

Amino Acid Sequence↗

PubMed1984

High performance liquid chromatography purification and structural characterization of the subunits of rabbit muscle phosphorylase kinase.

A rapid reverse-phase high performance liquid chromatography (HPLC) method is presented for isolating the alpha, beta, gamma, and delta subunits of rabbit muscle phosphorylase kinase. The HPLC separation allows micropreparative purification of all the subunits with 66-88% recoveries. Relative molecular weights of the subunits as determined by sodium dodecyl sulfate gel electrophoresis in 4, 5, 7, and 10% acrylamide are alpha 132,000, alpha' 127,000, beta 113,000 and gamma 43,000. Amino acid compositions are reported for the HPLC purified subunits. alpha contains about 2 mol of endogenous phosphate/mol of protein and beta, gamma, and delta each contain about 1 mol of phosphate/mol of protein. Despite the identity of delta and calmodulin, essentially no protein-bound phosphate was found associated with bovine brain calmodulin. Holophosphorylase kinase contains about 20 mol of endogenous phosphate/mol of protein. The first NH2-terminal sequence analyses of the alpha, beta, and gamma subunits were determined by Tarr manual Edman degradation. Within the NH2-terminal 23 residues of gamma ( TRDAALPGSHSTHGFYENYESKE . . . ) there are six identities and one conservative interchange with the catalytic subunit of bovine cAMP-dependent protein kinase. The first 17 residues of the NH2-terminal sequence of alpha ( MRSRSNSGVRLDSYARL . . . ) exhibit six identities and one conservative interchange with the transforming protein from the Rous sarcoma virus (Schmidt- Rupin strain) provided a single gap is inserted in the src gene product. Further structural information is required to evaluate the significance of these sequence similarities. The beta subunit has a blocked NH2 terminus.

Amino Acid Sequence↗

PubMed1992

Regional mapping of a liver alpha-subunit gene of phosphorylase kinase (PHKA) to the distal region of human chromosome Xp.

X-linked liver glycogenosis (XLG) is a glycogen storage disorder resulting from deficient activity of phosphorylase kinase (PHK). PHK consists of four different subunits: alpha, beta, gamma, and delta. Several genes encoding PHK subunits have been cloned and localized, but only the muscle alpha-subunit (PHKA) gene has been assigned to the X chromosome, in the region Xq12----q13. However, we have previously excluded the muscle PHKA gene as a candidate gene for the XLG mutation, as linkage analysis indicated that the mutation responsible for XLG is located in Xp22 and not in Xq12----q13. We report here the chromosomal localization by in situ hybridization of a liver PHKA gene to the distal region of chromosome Xp. Strong hybridization signals were observed on the distal part of the short arm of a chromosome identified as the X chromosome by cohybridization with an X chromosome-specific centromeric probe. The localization of this gene in the same chromosomal region as the disease gene responsible for XLG suggests that the liver PHKA gene is a highly likely candidate gene for the XLG mutation.

Chromosome Mapping↗

PubMed2005

Caspase-3 dependent cleavage and activation of skeletal muscle phosphorylase b kinase.

Phosphorylase b kinase (PhK) is a key enzyme involved in the conversion of glycogen to glucose in skeletal muscle and ultimately an increase in intracellular ATP. Since apoptosis is an ATP-dependent event, we investigated the regulation of skeletal muscle PhK during apoptosis. Incubation of PhK with purified caspase-3 in vitro resulted in the highly selective cleavage of the regulatory alpha subunit and resulted in a 2-fold increase in PhK activity. Edman protein sequencing of a stable 72 kD amino-terminal fragment and a 66 kD carboxy-terminal fragment revealed a specific caspase-3 cleavage site within the alpha subunit at residue 646 (DWMD G). Treatment of differentiated C2C12 mouse muscle myoblasts with the inducers of apoptosis staurosporine, TPEN, doxorubicin, or UV irradiation resulted in the disappearance of the alpha subunit of PhK as determined by immunoblotting, as well as a concurrent increase in caspase-3 activity. Moreover, induction of apoptosis by TPEN resulted in increased phosphorylase activity and sustained ATP levels throughout a 7 h time course. However, induction of apoptosis with staurosporine, also a potent PhK inhibitor, led to a rapid loss in phosphorylase activity and intracellular ATP, suggesting that PhK inhibition by staurosporine impairs the ability of apoptotic muscle cells to generate ATP. Thus, these studies indicate that PhK may be a substrate for caspase regulation during apoptosis and suggest that activation of this enzyme may be important for the generation of ATP during programmed cell death.

Adenosine Triphosphate↗

PubMed1997

Differential affinity cross-linking of phosphorylase kinase conformers by the geometric isomers of phenylenedimaleimide.

Phosphorylase b kinase (PbK) from skeletal muscle is a highly regulated oligomer consisting of four copies of four distinct subunits (alphabetagamma)delta4. The gamma subunit is catalytic, and the remaining subunits are regulatory. To characterize effector-induced changes in the quaternary structure of the enzyme, we utilized the ortho-, meta, and para-isomers of phenylenedimaleimide (PDM), which in addition to having different geometries, also vary 2.5-fold in their cross-linking spans. Even at concentrations equivalent to the alphabetagammadelta protomers of PbK, all three isomers caused specific, rapid, and extensive cross-linking of the holoenzyme to form primarily alphabeta dimers, plus smaller amounts of betagammagamma and alphagammagamma trimers. The formation of these three conjugates was nearly totally inhibited by a 10-fold molar excess over PDM of N-(o- and p-tolyl)succinimide, which are chemically inert structural analogs of PDM. This inhibition suggests that PbK has binding sites for PDM and that PDM acts as an affinity cross-linker in binding to these sites prior to forming cross-linked conjugates. The largest effect on cross-linking in progressing from o- to p-PDM was on the alphagammagamma trimer, which is preferentially formed by the p-isomer. Activation of the enzyme by either phosphorylation or the allosteric activators ADP and GDP resulted in large increases in the amount of alphagammagamma formed, small increases in betagammagamma, and little change in alphabeta. When cross-linked in the presence of the reversibly activating nucleoside diphosphates, PbK remained activated after their removal, indicating that cross-linking had locked it in the active conformation. Our results provide direct evidence for perturbations in the interactions of the catalytic gamma subunit with the regulatory alpha and beta subunits upon activation of PbK.

Affinity Labels↗

PubMed1997

Phosphorylase-kinase-deficient liver glycogenosis with an unusual biochemical phenotype in blood cells associated with a missense mutation in the beta subunit gene (PHKB).

We have identified mutations in the phosphorylase kinase (Phk) beta subunit gene in a male patient with liver glycogenosis caused by Phk deficiency. The patient's DNA has been analyzed for mutations in the genes encoding the alpha L, beta, and gamma TL subunits of Phk, all of which can be responsible for liver glycogenosis, by a strategy primarily based on reverse transcription/polymerase chain reaction of blood RNA and complemented by analysis of genomic DNA. His alpha L and gamma TL coding sequences are normal, whereas he is compound-heterozygous for two mutations in the beta subunit gene, PHKB. The first is a splice-site mutation (IVS4 [-2A-->G]) causing the reading-frame-disrupting deletion of exon 5 in the mRNA from this allele. The second is an Ala117Pro missense mutation, also in exon 5. This is the first missense mutation identified in PHKB, as opposed to nine translation-terminating mutations described to date. It offers an explanation for the unique biochemical phenotype of this patient. In his leukocytes, low Phk activity is measured when tested with the endogenous liver isoform of phosphorylase as the protein substrate, but normal activity is observed when tested with muscle phosphorylase added in vitro. In contrast, Phk activity in his erythrocytes is low with both substrates. The missense mutation may selectively impair the interaction of Phk with one isoform of its substrate protein and may destabilize the enzyme in a cell-type-specific way. This phenotype shares some aspects with X-linked liver glycogenosis subtype 2 (XLG2), a variant of liver Phk deficiency arising from missense mutations in the alpha L subunit gene (PHKA2), but differs from XLG2 in other respects. The present case demonstrates that mutations in Phk genes other than PHKA2 can also be associated with untypically high activity in certain blood cell types. Moreover, it emphasizes that missense mutations in Phk may cause unusual patterns of tissue involvement that would not be predicted a priori from the tissue specificity of expression of the mutated gene sequences.

Blood Cells↗

PubMed2003

Muscle glycogenosis with low phosphorylase kinase activity: mutations in PHKA1, PHKG1 or six other candidate genes explain only a minority of cases.

Muscle-specific deficiency of phosphorylase kinase (Phk) causes glycogen storage disease, clinically manifesting in exercise intolerance with early fatiguability, pain, cramps and occasionally myoglobinuria. In two patients and in a mouse mutant with muscle Phk deficiency, mutations were previously found in the muscle isoform of the Phk alpha subunit, encoded by the X-chromosomal PHKA1 gene (MIM # 311870). No mutations have been identified in the muscle isoform of the Phk gamma subunit (PHKG1). In the present study, we determined Q1the structure of the PHKG1 gene and characterized its relationship to several pseudogenes. In six patients with adult- or juvenile-onset muscle glycogenosis and low Phk activity, we then searched for mutations in eight candidate genes. The coding sequences of all six genes that contribute to Phk in muscle were analysed: PHKA1, PHKB, PHKG1, CALM1, CALM2 and CALM3. We also analysed the genes of the muscle isoform of glycogen phosphorylase (PYGM), of a muscle-specific regulatory subunit of the AMP-dependent protein kinase (PRKAG3), and the promoter regions of PHKA1, PHKB and PHKG1. Only in one male patient did we find a PHKA1 missense mutation (D299V) that explains the enzyme deficiency. Two patients were heterozygous for single amino-acid replacements in PHKB that are of unclear significance (Q657K and Y770C). No sequence abnormalities were found in the other three patients. If these results can be generalized, only a fraction of cases with muscle glycogenosis and a biochemical diagnosis of low Phk activity are caused by coding, splice-site or promoter mutations in PHKA1, PHKG1 or other Phk subunit genes. Most patients with this diagnosis probably are affected either by elusive mutations of Phk subunit genes or by defects in other, unidentified genes.

Adult↗

PubMed1992

A domain of the alpha-subunit of rabbit phosphorylase kinase shows homologies with regions of rabbit alpha-tropomyosin, human EGF receptor, and the alpha chain of bovine S-100 protein.

Sequence comparison of the alpha-subunit of phosphorylase kinase with alpha-tropomyosin revealed 32% identity, and 49% similarity, between the region of alpha-tropomyosin coded by exon 5 and a 39 amino acid segment of the kinase subunit. A subsequence of the alpha-subunit segment and a sequence overlapping the same alpha-subunit region are homologous with: (a) a region of the cytoplasmic domain of EGF receptor (50% identity) and (b) a Ca(2+)-binding domain of the alpha chain of S-100 protein (50% identity) respectively. Statistical analysis shows that these homologies are significant. The biological implication of the above similarities is discussed.

Amino Acid Sequence↗

PubMed1994

Zero length conformation-dependent cross-linking of phosphorylase kinase subunits by transglutaminase.

Transglutaminase, a zero length cross-linker that catalyzes the formation of isopeptide bonds between proximal Gln and Lys side chains, was used as a structural and conformational probe of the hexadecameric phosphorylase kinase molecule (alpha beta gamma delta)4. Brief cross-linking of nonactivated kinase caused formation of alpha-beta dimers, with no cross-linking involving the gamma- and delta-subunits. When the kinase was first activated by autophosphorylation, significant amounts of alpha-alpha dimers were also observed in addition to the alpha-beta, demonstrating the occurrence of a conformational change in the alpha-subunits concomitant with activation. Both dimers resulted from intramolecular cross-linking. Because the COOH-terminal regions of the alpha-subunits are at the lobe tips of this bilobal kinase (Wilkinson D. A. Marion, T. N., Tillman, D. M., Norcum, M. T., Hainfeld, J. F., Seyer, J. M., and Carlson, G. M. (1994) J. Mol. Biol. 235, 974-982), the formation of zero length cross-linked alpha-alpha dimers indicates that the polypeptide backbones of these subunits must stretch from the lobe tips to a more central location where they abut each other. Excess putrescine, as the amine substrate in place of endogenous Lys, was incorporated by transglutaminase predominately into the alpha-subunits of the kinase, with only slight modification of the beta- and gamma-subunits. Exogenous calmodulin (delta'), an activator of the kinase with a binding site on the alpha-subunits (James, P., Cohen, P., and Carafoli, E. (1991) J. Biol. Chem. 266, 7087-7091), was a potent inhibitor of cross-linking. It also inhibited incorporation of putrescine into the alpha-subunits but stimulated incorporation into the beta- and gamma-subunits. Heparin, another activator of the kinase, had the same effects as exogenous calmodulin on cross-linking and putrescine incorporation, suggesting a commonality in the mechanism through which these two effectors activate the holoenzyme, including promoting a conformational change that increases the surface accessibility of target Gln residues on the catalytic gamma-subunit.

Animals↗

PubMed1990

Electrophoretic purification of the alpha and beta subunits of phosphorylase kinase and evidence in support of the deduced amino acid sequences.

A simple, rapid sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method is presented for isolating the alpha, alpha' and beta subunits of rabbit muscle phosphorylase kinase. The SDS-PAGE procedure can yield milligram amounts of alpha and beta from a single preparative gel and also allows isolation of the alpha' isozyme free of alpha. Notably the method provides the purified subunits in a form amenable to structural analysis. Edman degradation of alpha and alpha' reveal identical NH2-terminal structures. Amino acid analysis of the electrophoretically purified alpha and beta subunits are in good agreement with their deduced primary structures. The amino acid sequence of 488 residues in alpha and 713 residues in beta were determined by gas phase Edman degradation. The data support the recently deduced primary structures of alpha (Zander et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 9381-9385).

Amino Acid Sequence↗