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PubMed2002

Three-dimensional structure of phosphorylase kinase at 22 A resolution and its complex with glycogen phosphorylase b.

Phosphorylase kinase (PhK) integrates hormonal and neuronal signals and is a key enzyme in the control of glycogen metabolism. PhK is one of the largest of the protein kinases and is composed of four types of subunit, with stoichiometry (alphabetagammadelta)(4) and a total MW of 1.3 x 10(6). PhK catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a (GPa) and the stimulation of glycogenolysis. We have determined the three-dimensional structure of PhK at 22 A resolution by electron microscopy with the random conical tilt method. We have also determined the structure of PhK decorated with GPb at 28 A resolution. GPb is bound toward the ends of each of the lobes with an apparent stoichiometry of four GPb dimers per (alphabetagammadelta)(4) PhK. The PhK/GPb model provides an explanation for the formation of hybrid GPab intermediates in the PhK-catalyzed phosphorylation of GPb.

Animals↗
PubMed1982

Rabbit skeletal muscle phosphorylase kinase. Interactions between subunits and influence of calmodulin on different complexes.

Aspects of the molecular interaction and subunit structure of rabbit skeletal muscle phosphorylase kinase, (alpha beta gamma delta)4, were investigated. Exogenous addition of the delta subunit (calmodulin) stimulated the activities of nonactivated phosphorylase kinase and the alpha gamma delta complex, but not the gamma delta complex. This stimulatory effect does not seem to affect the activity-pH profile of the native kinase and is expressed at least partly through an interaction of the calmodulin with the alpha subunit, resulting in an increase in the apparent Vm parameters. Low concentrations of trifluoperazine had only slight effect on the activities of the three forms of kinase, whereas high concentrations caused nonspecific inactivation. The effects of trifluoperazine and EGTA on the activities of the alpha gamma delta and gamma delta complexes were additive; together, they inactivated the two complexes to about 10% of their original activities. The delta subunit remained tightly bound to phosphorylase kinase and the alpha gamma delta complex, even in the presence of 8 M urea, but less so with the gamma delta complex. The nonactivated kinase is more stable towards heat inactivation at 37 degrees C than the alpha gamma delta complex, whereas the gamma delta complex is least stable. Similar to the holoenzyme, limited trypsin digestion activated the alpha gamma delta complex. The pH 6.8/8.2 activity ratio of this complex increased from 0.5 to 0.9, with concomitant degradation of the alpha subunit. In contrast, the gamma delta complex is fairly inert to limited proteolysis. By using sucrose density gradient ultracentrifugation and pretreatment of the gamma delta complex with EGTA, results indicated that the gamma subunit may be a Ca2+-independent kinase, which has about 30% of the original phosphorylase kinase activity.

Animals↗
PubMed1989

[The role of phosphorylase kinase subunits in interaction with glycogen].

The interaction of rabbit skeletal muscle phosphorylase kinase with CNBr-activated glycogen results in the formation of a covalent complex. The non-bound kinase was removed by chromatography on DEAE-cellulose and phenyl-Sepharose. The amount of the bound protein increased with an increase in the number of activated groups in the glycogen molecule; the enzyme activity was thereby decreased. The kinase covalently and non-covalently bound to glycogen exhibited a higher affinity for the protein substrate (phosphorylase b) as well as for Mg2+ and Ca2+ than did the kinase in the absence of glycogen. Electrophoresis performed under denaturating conditions showed that the gamma-subunit of phosphorylase kinase is responsible for the enzyme binding to CNBr-glycogen. The effect of cross-linking reagents (glutaric aldehyde, 1.5-difluoro-2.4-dinitrobenzene) on the binding of phosphorylase kinase subunits was studied. Glycogen afforded protection of the gamma-subunit from the cross-linking to other enzyme subunits. An analysis of the subunit composition of phosphorylase kinase covalently bound to CNBr-glycogen and of the enzyme treated with cross-linking reagents in the presence of glycogen-revealed that the gamma-subunit is involved in the specific binding of phosphorylase kinase to glycogen.

Animals↗
PubMed1993

The model calmodulin-binding peptide melittin inhibits phosphorylase kinase by interacting with its catalytic center.

The inhibition by melittin, a model calmodulin-binding peptide, of phosphorylase kinase, which contains an intrinsic calmodulin subunit, has been characterized in detail. The inhibition was competitive with respect to phosphorylase b for both the phosphorylase kinase holoenzyme and its isolated catalytic gamma-subunit (minus calmodulin), and the ratios of the Km for phosphorylase to the Ki for melittin were similar for both forms of the kinase. These findings indicate that inhibition of the phosphorylase kinase holoenzyme by melittin is caused predominantly by its interaction with the catalytic subunit of the enzyme, and not with the endogenous calmodulin subunit. Further proof that melittin interacts directly with the catalytic site was obtained when it was observed that melittin was also a substrate for phosphorylase kinase, with a Km that was less than that for phosphorylase b, although the kcat/Km specificity constant was only 1/200th of that for phosphorylase. The apparent tight binding of melittin to the kinase active site could not be readily rationalized by conventional comparison of sequence similarity between melittin and phosphorylase; however, considerable sequence similarity, centered around the convertible seryl residue of phosphorylase, was observed when the sequences were aligned in reversed polarity. The possible regulatory significance of the direct interaction of the catalytic site of this Ca(2+)-dependent kinase with a calmodulin-binding peptide is discussed.

Amino Acid Sequence↗
PubMed1980

Differential interaction of rabbit skeletal muscle phosphorylase kinase isozymes with calmodulin.

Rabbit skeletal muscle contains two phosphorylase kinase isozymes arising from the two different muscle types, the white and the red muscle (Jennissen, H. P., and Heilmeyer, L. M. G. (1974) FEBS Lett. 42, 77-80). The two phosphorylase kinase isozymes could be separated by affinity chromatography on a calmodulin-Sepharose 4B column. In media containing high concentrations of Ca2+, about 2 mM, both isozymes were bound to the affinity column. When the column was eluted with a buffer containing 0.2 mM Ca2+, the red muscle isozyme was eluted, whereas white muscle isozyme was eluted from the column by an ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid. The purified white muscle isozyme can be distinguished from the red muscle isozyme by its ability to inhibit calmodulin-dependent cyclic nucleotide phosphodiesterase. The two isozymes are also regulated differently by calmodulin. Both isozymes contain tightly bound calmodulin as a subunit (Cohen, P., Burchell, A., Foulkes, J. G., Cohen, P. T. W., Vanaman, T. C., and Nairn, A. C. (1978) FEBS Lett. 92, 287-293), which renders the enzyme sensitive to Ca2+. Only the white muscle isozyme can be activated by exogenous calmodulin.

Animals↗
PubMed1982

Infantile glycogen storage myopathy in a girl with phosphorylase kinase deficiency.

A 19-month-old girl with moderate hypotonia was studied. Histochemical and electronmicroscopic findings revealed that many skeletal muscle fibers contained an excess amount of glycogen. The phosphorylase reaction was normalized only after activation with 5' AMP. Biochemical studies showed an increased glycogen content and decreased activities of phosphorylase "a" and an active form of phosphorylase kinase, whereas activities of total phosphorylase, total phosphorylase kinase, and cyclic AMP-dependent protein kinase were all in the normal range. Thus, phosphorylase kinase in the patient's muscle seemed to be a variant form, which was activated partially under the physiologic condition. This condition may be inherited as an X-linked recessive trait.

Acid Phosphatase↗
PubMed1989

Electron microscopical examination of different aggregation and decomposition states of phosphorylase kinase. Identification and computer averaging of the alpha gamma delta fragment.

A sample of phosphorylase kinase aged by storage was subjected to chromatography on a size exclusion column. Samples from the four major peaks were analysed by electron microscopy. The first major peak consisted of oligomers of the enzyme monomers which showed no structural order. The second peak mainly consisted of dimers of phosphorylase kinase connected to each other by the outer tipes of the "wings", thus identifying a binding locus through cross-labelling. The third peak showed the already known typical forms of phosphorylase kinase ("butterflies", "chalice form"). The last peak yielded structures identical to the alpha gamma delta fragments obtained by lithium bromide cleavage of the intact enzyme. These structures correspond to the curved outer parts of the cup substructures of the chalice and butterfly forms. The result could be verified by computer averaging of the alpha gamma delta fragments. This finding confirms reports (Chan, K.-F. J. & Graves, D.J. (1984) Calcium Cell Funct. 5, 1-31) that the beta-subunits (missing in the distally situated fragment) are located in the central parts of the molecules. The good quality of the averages of the low molecular mass fragment (about 200 kDa) recommends computer averaging of electron micrographs of partial complexes as suitable method for the study of protein complexes.

Bromides↗
PubMed1986

Analyses of phosphorylase kinase by transmission and scanning transmission electron microscopy.

Under conventional electron microscopy negatively stained phosphorylase kinase exhibits a bilobal structure resembling two bridged opposing parentheses. In this predominant particle orientation, usually only one bridge is observed; however, in many particles two bridges can be seen. Scanning transmission electron microscopy of unstained phosphorylase kinase shows very similar structures, with a particle mass equivalent to that of the hexadecameric holoenzyme. Partial digestion of the enzyme with chymotrypsin, which preferentially hydrolyzes the alpha-subunits, causes no significant changes in the structure; however, when both the alpha and beta subunits are degraded by trypsin, single lobed particles appear, i.e. the connecting bridges are missing. Mass analysis of scanning transmission electron microscopy images of trypsinized enzyme indicates that the protease does, in fact, split the particle into halves. Transmission electron microscopy of an alpha gamma delta complex isolated after incubation of the holoenzyme with LiBr shows only small particles approximately one-fourth the size of the holoenzyme. Thus, integrity of the beta subunit may be necessary in order for the two lobes of phosphorylase kinase to be bridged. These data also indicate that the subunits are arranged as a bridged dimer of octamers 2 (alpha 2 beta 2 gamma 2 delta 2).

Animals↗
PubMed1987

[Limited proteolysis and phosphorylation of phosphorylase kinase from chicken skeletal muscles].

The changes in the quaternary structure of chicken skeletal muscle phosphorylase kinase during limited proteolysis by trypsin and chymotrypsin were studied. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the products of phosphorylase kinase limited proteolysis revealed a similarity in the structure of the alpha'- and beta-subunits and some differences in the structure of the gamma-subunits of the chicken and rabbit enzymes. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol of 32P/mol of alpha' beta gamma' sigma monomer) and autophosphorylation (up to 8 mol of 32P/mol alpha' beta gamma' delta monomer) increased the activity of chicken phosphorylase kinase 1.5-fold and 2.0-fold, respectively. The incorporation of phosphate into the alpha' and beta-subunits in the course of the protein kinase-catalyzed reaction was demonstrated.

Animals↗
PubMed1987

Phosphorylase kinase conformers. Detection by proteases.

A variety of proteases have been evaluated as potential structural and conformational probes of nonphosphorylated and phosphorylated phosphorylase kinase. In general, the enzyme's alpha subunit is rapidly degraded, followed in most cases by hydrolysis of the beta subunit; the gamma subunit is resistant to most proteases. Trypsin clearly distinguishes between the nonactivated and activated conformers of phosphorylase kinase, in that the beta subunit in phosphorylated enzyme, as opposed to nonphosphorylated enzyme, is markedly protected from tryptic attack. In contrast, only a small difference in the rates of proteolysis of the alpha subunit in phosphorylated and nonphosphorylated enzyme is seen, even when a protease is used that is highly selective for the alpha subunit, such as chymotrypsin or endoproteinase Arg C. Incubation of nonphosphorylated phosphorylase kinase with either Mg2+ or Ca2+, which are activating cations, also protects the beta subunit from tryptic hydrolysis, whereas Mn2+, which inhibits the kinase activity, has little effect on proteolysis. The allosteric activator ADP also causes the beta subunit to become refractory to trypsin and mimics the effects of phosphorylation. Similar effector-induced conformational changes in the beta subunit are also observed with enzyme in which the alpha subunit has previously been selectively destroyed. These data indicate that activation of phosphorylase kinase by dissimilar mechanisms is associated with a conformational change in the enzyme's beta subunit that is detectable by trypsin and confirm earlier studies from this laboratory employing a chemical cross-linker as a conformational probe for activated and nonactivated conformers of the enzyme (Fitzgerald, T. J., and Carlson, G. M. (1984) J. Biol. Chem. 259, 3266-3274).

Adenosine Diphosphate↗
PubMed1987

Characterization of the isolated rat flexor digitorum brevis for the study of skeletal muscle phosphorylase kinase phosphorylation.

The flexor digitorum brevis skeletal muscle, a nearly homogeneous fast-twitch oxidative glycolytic fiber type, has been examined for its suitability to explore the regulation of phosphorylase kinase by multisite phosphorylation. A characterization of the adrenergic response of glycogenolytic enzymes, together with the previous data on contractile properties (Carlsen, R. C., Larson, D. B., and Walsh, D. A. (1985) Can. J. Physiol. Pharm. 63, 958-965), has demonstrated that this muscle is stably maintained for the several hours necessary for phosphorylation studies. The phosphorylase kinase in this muscle is primarily the alpha' isozyme, suggesting that the alpha versus alpha' isozyme distribution in muscle is related more to oxidative capacity than to fiber contractile characteristics. Using this muscle system, beta-adrenergic activation of phosphorylase kinase was observed to occur with concomitant phosphorylation of both the alpha' and beta subunits, with the total in the alpha' subunit being approximately 3-fold greater. Similarly, deactivation, following initial adrenergic activation, occurred concomitantly with the dephosphorylation of the two subunits. These results are compatible with the conclusions drawn from previous studies of the isolated enzyme and of the enzyme in perfused rat cardiac muscle, that both alpha' (or alpha) and beta subunit phosphorylation regulate phosphorylase kinase activity.

Adenosine Triphosphate↗
PubMed1999

Self-association of the alpha subunit of phosphorylase kinase as determined by two-hybrid screening.

The structural organization of the (alphabetagammadelta)(4) phosphorylase kinase complex has been studied using the yeast two-hybrid screen for the purpose of elucidating regions of alpha subunit interactions. By screening a rabbit skeletal muscle cDNA library with residues 1-1059 of the alpha subunit of phosphorylase kinase, we have isolated 16 interacting, independent, yet overlapping transcripts of the alpha subunit containing its C-terminal region. Domain mapping of binary interactions between alpha constructs revealed two regions involved in the self-association of the alpha subunit: residues 833-854, a previously unrecognized leucine zipper, and an unspecified region within residues 1015-1237. The cognate binding partner for the latter domain has been inferred to lie within the stretch from residues 864-1059. Indirect evidence from the literature suggests that the interacting domains contained within the latter two, overlapping regions may be further narrowed to the stretches from 1057 to 1237 and from 864 to 971. Cross-linking of the nonactivated holoenzyme with N-(gamma-maleimidobutyroxy)sulfosuccin-imide ester produced intramolecularly cross-linked alpha-alpha dimers, consistent with portions of two alpha subunits in the holoenyzme being in sufficient proximity to associate. This is the first report to identify potential areas of contact between the alpha subunits of phosphorylase kinase. Additionally, issues regarding the general utility of two-hybrid screening as a method for studying homodimeric interactions are discussed.

Amino Acid Sequence↗
PubMed1987

The interrelationship between cAMP-dependent alpha and beta subunit phosphorylation in the regulation of phosphorylase kinase activity. Studies using subunit specific phosphatases.

This study addresses the function of multisite phosphorylation of phosphorylase kinase catalyzed by the cAMP-dependent protein kinase. Using subunit specific protein phosphatases (the polycation-stimulated and ATP-, Mg2+-dependent enzymes), we show that the degree of phosphorylation of both the alpha and beta subunits modulates phosphorylase kinase activity. beta subunit phosphorylation is essential for activation and, independent of the degree of alpha subunit phosphorylation, enzyme fully dephosphorylated in the beta subunit is completely inactivated. alpha Subunit phosphorylation does, however, also regulate activity, and enzyme fully or partially phosphorylated in the beta subunit is inactivated as a consequence of alpha subunit dephosphorylation. The extent of inactivation caused by alpha subunit dephosphorylation is linearly dependent on the phosphorylation state of the beta subunit. Three peptide sites on the alpha subunit are phosphorylated by the cAMP-dependent protein kinase; the site primarily affecting activity is the one that is initially phosphorylated. These data provide evidence that subunit interrelationships play an important role in the regulation of phosphorylase kinase by multisite phosphorylation.

Adenosine Triphosphate↗
PubMed1978

[Inhibition of the phosphorylase kinase activity by ATP analogs and their binding to the enzyme subunits].

The interaction between phosphorylase kinase (EC 2.7.1.38), isolated from rabbit skeletal muscles, and the ATP analogs with the modified triphosphate fragment: adenosine-5'-chloromethane pyrophosphonate (1), adenosine-5'-chloroethyl phosphate (2), adenosine-5'-bromethane pyrophosphonate (3), adenosine-5'-bromoethane phosphonate (4), adenosine-5'-chloroacetylaminomethane phosphonate (5), adenosine-5'-chloroacetylaminomethane pyrophosphonate (6) and adenosine-5'-chloromethane phosphonate (7), was studied. The compounds 1, 2 and 3 irreversibly inhibit the enzyme activity. In the presence of ATP the rate of inactivation is decreased. The radioactive compounds 1, 2 and 3 are stoicheometrically incorporated into the beta- and gamma-subunits of phosphorylase kinase. A correlation is shown to exist between the degree of the beta-subunit modification by compound 1 and the enzyme inactivation. The compounds 4, 5 and 6 inhibit the enzyme reversibly: in the presence of ATP complete protection of the enzyme activity is observed. The compound 7 does not affect the kinase activity; however, it binds itself to the beta-subunit of the enzyme. The binding of analogs 1 and 7 to the beta-subunit occurs at different sites. The data obtained are indicative of the catalytic role of the beta-subunit of phosphorylase kinase.

Adenosine Triphosphate↗
PubMed1989

[Regulation of the activity of rabbit skeletal muscle phosphorylase kinase by glycogen].

The effects of glycogen on the non-activated and activated forms of phosphorylase kinase were studied. It was found that in the presence of glycogen the activity of non-activated kinase at pH 6.8 and 8.2 and that of the activated (in the course of phosphorylation) form are enhanced. The degree of activation depends on glycogen concentration. At saturating concentrations, this enzyme activity increases 2-3-fold; the enzyme affinity for the protein substrate, phosphorylase b, also shows an increase. The polysaccharide has no effect on the activity of phosphorylase kinase stimulated by limited proteolysis. In the presence of glycogen, the rate of autocatalytic phosphorylation of the enzyme is increased. Glycogen stabilizes the enzyme activity upon dilution. The experimental results suggest that the polysaccharide directly affects the phosphorylase kinase molecule. The maximal binding was shown to occur at the enzyme/polysaccharide ratio of 1:10 (w/w) in the presence of Ca2+ and Mg2+.

Animals↗
PubMed2005

Myopathy and phosphorylase kinase deficiency caused by a mutation in the PHKA1 gene.

Phosphorylase kinase (PhK) deficiency is the underlying cause of variable clinical symptoms depending on the tissues involved. Until today, only a few cases of myopathy associated with muscle PhK deficiency caused by a mutation in the gene encoding the alpha subunit of phosphorylase kinase (PHKA1) have been reported. We describe a male patient with myopathy and absent muscle PhK activity caused by a frameshift mutation in the gene encoding the alpha subunit of PhK on chromosome Xq12-q13.

Adult↗
PubMed1991

Phosphorylase kinase: development of a continuous fluorometric assay for the determination of catalytic activity.

The preferential binding of 1-anilinonaphthalene-8-sulfonate by rabbit muscle phosphorylase a is the basis of a continuous fluorometric assay for phosphorylase kinase. The maximum rate of change in fluorescence (d delta F/dt) is dependent on both the concentration of phosphorylase kinase and on conditions, such as pH and calcium ion concentration, which affect the enzyme. Parallel measurements of the increases in fluorescence and of 32P incorporation demonstrate the existence of a distinct intermediate in the conversion of phosphorylase b to a. We have used the assay to monitor the increase in calcium-independent activity which accompanies the limited chymotryptic digestion of phosphorylase kinase.

Anilino Naphthalenesulfonates↗