Skeletal Muscle Phosphorylase Kinase Research Articles

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)(4) complex that regulates glycogenolysis in skeletal muscle. Activity of the catalytic γ subunit is regulated by allosteric activators targeting the regulatory α, β, and δ subunits. Three-dimensional EM reconstructions of PhK show it to be two large (αβγδ)(2) lobes joined with D(2) symmetry through interconnecting bridges. The subunit composition of these bridges was unknown, although indirect evidence suggested the β subunits may be involved in their formation. We have used biochemical, biophysical, and computational approaches to not only address the quaternary structure of the β subunits within the PhK complex, i.e. whether they compose the bridges, but also their secondary and tertiary structures. The secondary structure of β was determined to be predominantly helical by comparing the CD spectrum of an αγδ subcomplex with that of the native (αβγδ)(4) complex. An atomic model displaying tertiary structure for the entire β subunit was constructed using chemical cross-linking, MS, threading, and ab initio approaches. Nearly all this model is covered by two templates corresponding to glycosyl hydrolase 15 family members and the A subunit of protein phosphatase 2A. Regarding the quaternary structure of the β subunits, they were directly determined to compose the four interconnecting bridges in the (αβγδ)(4) kinase core, because a β(4) subcomplex was observed through both chemical cross-linking and top-down MS of PhK. The predicted model of the β subunit was docked within the bridges of a cryoelectron microscopic density envelope of PhK utilizing known surface features of the subunit.

Interaction of Hsp27 with Native Phosphorylase Kinase under Crowding Conditions

Interaction of the wild type (wt) heat shock protein Hsp27 and its three-dimensional (3D) mutant (mimicking phosphorylation at Ser15, 78, and 82) with rabbit skeletal muscle phosphorylase kinase (PhK) has been studied under crowding conditions modeled by addition of 1 M trimethylamine N-oxide (TMAO). According to the data of sedimentation velocity and dynamic light scattering, crowding provokes the formation of large-sized associates of both PhK and Hsp27. Under crowding conditions, small associates of PhK and Hsp27 interact with each other thus leading to dissociation of large homooligomers of each protein. Taking into account high concentrations of PhK in the cell, we speculate that native PhK might modulate the oligomeric state and chaperone-like activity of Hsp27.

The Regulatory β Subunit of Phosphorylase Kinase Interacts with Glyceraldehyde-3-phosphate Dehydrogenase

Skeletal muscle phosphorylase kinase (PhK) is an (alphabetagammadelta) 4 hetero-oligomeric enzyme complex that phosphorylates and activates glycogen phosphorylase b (GP b) in a Ca (2+)-dependent reaction that couples muscle contraction with glycogen breakdown. GP b is PhK's only known in vivo substrate; however, given the great size and multiple subunits of the PhK complex, we screened muscle extracts for other potential targets. Extracts of P/J (control) and I/lnJ (PhK deficient) mice were incubated with [gamma- (32)P]ATP with or without Ca (2+) and compared to identify potential substrates. Candidate targets were resolved by two-dimensional polyacrylamide gel electrophoresis, and phosphorylated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified by matrix-assisted laser desorption ionization mass spectroscopy. In vitro studies showed GAPDH to be a Ca (2+)-dependent substrate of PhK, although the rate of phosphorylation is very slow. GAPDH does, however, bind tightly to PhK, inhibiting at low concentrations (IC 50 approximately 0.45 microM) PhK's conversion of GP b. When a short synthetic peptide substrate was substituted for GP b, the inhibition was negligible, suggesting that GAPDH may inhibit predominantly by binding to the PhK complex at a locus distinct from its active site on the gamma subunit. To test this notion, the PhK-GAPDH complex was incubated with a chemical cross-linker, and a dimer between the regulatory beta subunit of PhK and GAPDH was formed. This interaction was confirmed by the fact that a subcomplex of PhK missing the beta subunit, specifically an alphagammadelta subcomplex, was unable to phosphorylate GAPDH, even though it is catalytically active toward GP b. Moreover, GAPDH had no effect on the conversion of GP b by the alphagammadelta subcomplex. The interactions described herein between the beta subunit of PhK and GAPDH provide a possible mechanism for the direct linkage of glycogenolysis and glycolysis in skeletal muscle.

Structural Evidence for Co-Evolution of the Regulation of Contraction and Energy Production in Skeletal Muscle

Skeletal muscle phosphorylase kinase (PhK) is a Ca2+-dependent enzyme complex, (αβγδ)4, with the δ subunit being tightly bound endogenous calmodulin (CaM). The Ca2+-dependent activation of glycogen phosphorylase by PhK couples muscle contraction with glycogen breakdown in the “excitation–contraction–energy production triad.” Although the Ca2+-dependent protein–protein interactions among the relevant contractile components of muscle are well characterized, such interactions have not been previously examined in the intact PhK complex. Here we show that zero-length cross-linking of the PhK complex produces a covalent dimer of its catalytic γ and CaM subunits. Utilizing mass spectrometry, we determined the residues cross-linked to be in an EF hand of CaM and in a region of the γ subunit sharing high sequence similarity with the Ca2+-sensitive molecular switch of troponin I that is known to bind actin and troponin C, a homolog of CaM. Our findings represent an unusual binding of CaM to a target protein and supply an explanation for the low Ca2+ stoichiometry of PhK that has been reported. They also provide direct structural evidence supporting co-evolution of the coordinate regulation by Ca2+ of contraction and energy production in muscle through the sharing of a common structural motif in troponin I and the catalytic subunit of PhK for their respective interactions with the homologous Ca2+-binding proteins troponin C and CaM.

The Regulatory β Subunit of Phosphorylase Kinase Interacts with Glyceraldehyde‐3‐phosphate Dehydrogenase

Skeletal muscle phosphorylase kinase (PhK), a Ca2+-dependent (αβγδ)4 complex, phosphorylates glycogen phosphorylase (GP), its only known in vivo substrate. Given PhK's size and complexity, we screened muscle extracts for other potential targets. Extracts of P/J (control) and I/lnJ (PhK deficient) mice were incubated with [γ-32P]ATP ± Ca2+ and compared to identify potential substrates. Candidate targets were resolved by 2D-PAGE and phosphorylated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified by MALDI-MS. In vitro studies showed GAPDH to be a Ca2+-dependent substrate of PhK, although the rate and stoichiometry of phosphorylation were low. GAPDH did, however, inhibit at low concentrations (IC50 = 0.45μM) PhK's conversion of GP. When a short synthetic peptide substrate was substituted for GP, the inhibition was negligible, suggesting that GAPDH may inhibit predominantly by binding to PhK at a locus distinct from the active site on the γ subunit. To test this notion, the PhK/GAPDH complex was incubated with chemical crosslinkers and crosslinking of the regulatory β subunit of PhK to GAPDH was shown. This interaction was confirmed by the finding that GAPDH had no effect on GP conversion by a subcomplex of PhK missing the β subunit, an αγδ trimer. Interactions between PhK and GAPDH provide a possible mechanism for linking glycogenolysis and glycolysis in muscle. (Supported by NIH grant DK32953).

Electrostatic changes in phosphorylase kinase induced by its obligatory allosteric activator Ca2+

Skeletal muscle phosphorylase kinase (PhK) is a 1.3-MDa hexadecameric complex that catalyzes the phosphorylation and activation of glycogen phosphorylase b. PhK has an absolute requirement for Ca(2+) ions, which couples the cascade activation of glycogenolysis with muscle contraction. Ca(2+) activates PhK by binding to its nondissociable calmodulin subunits; however, specific changes in the structure of the PhK complex associated with its activation by Ca(2+) have been poorly understood. We present herein the first comparative investigation of the physical characteristics of highly purified hexadecameric PhK in the absence and presence of Ca(2+) ions using a battery of biophysical probes as a function of temperature. Ca(2+)-induced differences in the tertiary and secondary structure of PhK measured by fluorescence, UV absorption, FTIR, and CD spectroscopies as low resolution probes of PhK's structure were subtle. In contrast, the surface electrostatic properties of solvent accessible charged and polar groups were altered upon the binding of Ca(2+) ions to PhK, which substantially affected both its diffusion rate and electrophoretic mobility, as measured by dynamic light scattering and zeta potential analyses, respectively. Overall, the observed physicochemical effects of Ca(2+) binding to PhK were numerous, including a decrease in its electrostatic surface charge that reduced particle mobility without inducing a large alteration in secondary structure content or hydrophobic tertiary interactions. Without exception, for all analyses in which the temperature was varied, the presence of Ca(2+) rendered the enzyme increasingly labile to thermal perturbation.

Interaction of phosphorylase kinase from rabbit skeletal muscle with flavin adenine dinucleotide

The interaction of flavin adenine dinucleotide (FAD) with rabbit skeletal muscle phosphorylase kinase has been studied. Direct evidence of binding of phosphorylase kinase with FAD has been obtained using analytical ultracentrifugation. It has been shown that FAD prevents the formation of the enzyme-glycogen complex, but exerts practically no effect on the phosphorylase kinase activity. The dependence of the relative rate of phosphorylase kinase-glycogen complex formation on the concentration of FAD has cooperative character (the Hill coefficient is 1.3). Under crowding conditions in the presence of 1 M trimethylamine-N-oxide (TMAO), FAD has an inhibitory effect on self-association of phosphorylase kinase. The data suggest that the complex of glycogen metabolism enzymes in protein-glycogen particles may function as a flavin depot in skeletal muscle.

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 α 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 PhKα.

Differentiation-dependent mechanisms of transcriptional regulation of the catalytic subunit of phosphorylase kinase

The amount of phosphorylase kinase in skeletal muscle is exquisitely sensitive to developmental signals such as differentiation and innervation, and is clearly regulated in such a manner so as to always maintain the γ catalytic subunit under the control of its regulatory α, β and γ subunits. To identify how the transcription of the γ subunit is regulated, we have analysed 3.8kb of the upstream regulatory region using a luciferase reporter system. A complex sequence of interdependent regulations is evident. The γ catalytic subunit gene contains two inhibitory controls with very dominant features. Also evident are an array of multiple positive regulatory elements, prominent amongst which are four E-boxes, of which two are downstream, one is upstream and one is in the middle of the CAAT-TATA core promoter. Differentiation-dependent positive regulation arises as a consequence of both E-box regulation and the activation of at least one other regulatory element. The primary mode of transcriptional regulation of the γ catalytic subunit gene appears to occur by the relief of regulation of an otherwise default inhibitory status. It is noteworthy that such a mode of regulation mirrors the regulation of the enzymic activity of many protein kinases, including phosphorylase kinase. With phosphorylase kinase, both its transcriptional regulation as well as the regulation of the protein itself, are primed to maintain the γ catalytic subunit either unexpressed or inactivate respectively, until a positive signal occurs to override an otherwise dominant default inhibitory condition.

Differentiation-dependent mechanisms of transcriptional regulation of the catalytic subunit of phosphorylase kinase

The amount of phosphorylase kinase in skeletal muscle is exquisitely sensitive to developmental signals such as differentiation and innervation, and is clearly regulated in such a manner so as to always maintain the gamma catalytic subunit under the control of its regulatory alpha, beta and gamma subunits. To identify how the transcription of the gamma subunit is regulated, we have analysed 3.8 kb of the upstream regulatory region using a luciferase reporter system. A complex sequence of interdependent regulations is evident. The gamma catalytic subunit gene contains two inhibitory controls with very dominant features. Also evident are an array of multiple positive regulatory elements, prominent amongst which are four E-boxes, of which two are downstream, one is upstream and one is in the middle of the CAAT-TATA core promoter. Differentiation-dependent positive regulation arises as a consequence of both E-box regulation and the activation of at least one other regulatory element. The primary mode of transcriptional regulation of the gamma catalytic subunit gene appears to occur by the relief of regulation of an otherwise default inhibitory status. It is noteworthy that such a mode of regulation mirrors the regulation of the enzymic activity of many protein kinases, including phosphorylase kinase. With phosphorylase kinase, both its transcriptional regulation as well as the regulation of the protein itself, are primed to maintain the gamma catalytic subunit either unexpressed or inactivate respectively, until a positive signal occurs to override an otherwise dominant default inhibitory condition.

A tentative mechanism of the ternary complex formation between phosphorylase kinase, glycogen phosphorylase b and glycogen

The kinetics of rabbit skeletal muscle phosphorylase kinase interaction with glycogen has been studied. At pH 6.8 the binding of phosphorylase kinase to glycogen proceeds only in the presence of Mg 2+, whereas at pH 8.2 formation of the complex occurs even in the absence of Mg 2+. On the other hand, the interaction of phosphorylase kinase with glycogen requires Ca 2+ at both pH values. The initial rate of the complex formation is proportional to the enzyme and glycogen concentrations, suggesting the formation of the complex with stoichiometry 1:1 at the initial step of phosphorylase kinase binding by glycogen. According to the kinetic and sedimentation data, the substrate of the phosphorylase kinase reaction, glycogen phosphorylase b, favors the binding of phosphorylase kinase with glycogen. We suggest a model for the ordered binding of phosphorylase b and phosphorylase kinase to the glycogen particle that explains the increase in the tightness of phosphorylase kinase binding with glycogen in the presence of phosphorylase b.

Neural regulation of the formation of skeletal muscle phosphorylase kinase holoenzyme in adult and developing rat muscle.

Neural influences on the co-ordination of expression of the multiple subunits of skeletal muscle phosphorylase kinase and their assembly to form the holoenzyme complex, alpha4beta4gamma4delta4, have been examined during denervation and re-innervation of adult skeletal muscle and during neonatal muscle development. Denervation of the tibialis anterior and extensor digitorum longus muscles of the rat hindlimb was associated with a rapid decline in the mRNA for the gamma subunit, and an abrupt decrease in gamma-subunit protein. The levels of the alpha- and beta-subunit proteins in the denervated muscles also declined rapidly, their time course of reduction being similar to that for the gamma-subunit protein, but they did not decrease to the same extent. In contrast with the rapid decline in gamma-subunit mRNA upon denervation, alpha- and beta-subunit mRNAs stayed at control innervated levels for approx. 8-10 days, but then decreased rapidly. Their decline coincided very closely with the onset of re-innervation. Re-innervation of the denervated muscles, which occurs rapidly and uniformly after the sciatic nerve crush injury, produced an eventual slow and prolonged recovery of the mRNA for all three subunits and parallel increases in each of the subunit proteins. A similar co-ordinated increase of both subunit mRNA and subunit proteins of the phosphorylase kinase holoenzyme was observed during neonatal muscle development, during the period when the muscles were attaining their adult pattern of motor activity. The phosphorylase kinase holoenzyme remains in a non-activated form during all of these physiological changes, as is compatible with the presence of the full complement of the regulatory subunits. These data are consistent with a model whereby the transcriptional and translational expression of phosphorylase kinase gamma subunit occurs only with concomitant expression of the alpha and beta subunits. This would ensure that free and unregulated, activated gamma subunit alone, which would give rise to unregulated glycogenolysis, is not produced. The data also suggest that control of phosphorylase kinase subunit expression and the formation of the holoenzyme in skeletal muscle is provided by the motor nerve, probably through imposed levels or patterns of muscle activity.

Continuous Enzymatic Assay for Phosphorylase Kinase in a Monocascade Enzyme System

A turbidimetric method for continuous monitoring of the enzymatic reaction catalyzed by rabbit skeletal muscle phosphorylase kinase has been developed. The reaction mixture contained the substrates of glycogen phosphorylasea,i.e., glycogen and glucose 1-phosphate (orPi), in addition to the usual components of the kinase reaction. The kinetics of the cascade enzyme system were followed by the change in glycogen concentration over time, as measured by the absorbance of the reaction medium at 360 nm. The reliability of this turbidimetric method for measuring phosphorylase kinase activity was proven by comparison with a commonly used radiochemical assay. We present here a newly developed method for calculating the initial rate of phosphorylase kinase reaction in our conjugated system. We demonstrate that our procedure is applicable for investigating the hysteretic properties of phosphorylase kinase.

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.

Hysteretic properties of rabbit skeletal muscle phosphorylase kinase: synergistic activation by phosphorylase b, Ca2+, and Mg2+.

To study the hysteretic properties of rabbit skeletal muscle phosphorylase kinase the method of continuous registration of the kinetics of the kinase reaction developed by us earlier has been used. It was shown that duration of the lag period on the kinetic curves is independent of the phosphorylase kinase concentration and the simultaneous presence of phosphorylase b, Ca2+, and Mg2+ is required for the complete transition of the enzyme into the activated state.

Subcellular distribution of phosphorylase kinase in rat brain. Association of the enzyme with mitochondria and membranes

The evaluation of glycogen phosphorylase kinase in rat brain subcellular fractions was undertaken in order to get further insight into the association of this kinase with specific neuronal cell compartments. The enzyme was found to be primarily soluble, but considerable latent specific activities were observed in particulate fractions, especially in microsomes, mitochondria and synaptosomes, which could be unmasked by treatment with Triton X-100. The submitochondrial and subsynaptic distribution patterns of phosphorylase kinase revealed high overt activity in the mitochondrial intermembrane space and high latent activities in mitochondrial membranes, and synaptic vesicles, membranes and mitochondria. The Ca 2+-dependency of soluble phosphorylase kinase was similar to that of microsomal enzyme but higher than that of other particulate enzyme forms. Mitochondrial phosphorylase kinase showed a higher pH 6.8:8.2 activity ratio than the soluble and the microsomal enzyme. The rate of inactivation of cytosolic phosphorylase kinase by proteinase K was higher than that of microsomal and mitochondrial enzymes. Antibodies against rabbit skeletal muscle phosphorylase kinase effectively inhibited both cytosolic and microsomal enzyme but failed to significantly affect the kinase activity present in intact mitochondria and intermembrane space. Western blotting with anti-phosphorylase kinase showed that rat brain mitochondria exhibited a significantly lower immunoreactivity compared to soluble cytosol. In conclusion, the presence of phosphorylase kinase activity in a variety of particulate fractions of rat brain suggests a multiplicity of actions of this kinase in neuronal tissues.

Analysis by mutagenesis of the ATP binding site of the gamma subunit of skeletal muscle phosphorylase kinase expressed using a baculovirus system.

Active gamma subunit of skeletal muscle phosphorylase kinase has been obtained by expression of the rat soleus cDNA in a baculovirus system. The protein exhibited the expected pH 6.8/8.2 activity ratio of 0.6, and its activity was insensitive to Ca2+ addition, indicating that it was free gamma subunit and not a gamma subunit-calmodulin complex. It was stimulated approximately 2-fold by Ca(2+)-calmodulin addition, demonstrating that it had retained high-affinity calmodulin binding. By site-directed mutagenesis, we have examined the role of six of the amino acids that constitute the consensus ATP binding site of the protein kinase, which in the gamma subunit is represented by the sequence 26Gly.Arg.Gly.Val.Ser.Ser.Val.Val33. Changes were evaluated by the kinetic determination of the dissociation constants of gamma-ATP, gamma-ADP, gamma-AMP.PCP, and gamma-phosphorylase and the maximum catalytic activity. The mutants Ser26-gamma, Ser29-gamma, Phe30-gamma, and Gly31-gamma each exhibited an essentially identical dissociation constant for gamma subunit phosphorylase, indicating that these mutations had not caused a global alteration in the protein structure but were limited to changes in the nucleotide binding site domain. Substitution of either Val33 (by Gly) or Gly28 (by Ser), two of the most conserved residues in all protein kinases, resulted in enzyme with marginally detectable activity. In noted contrast, the Ser26 mutant, which substituted the first glycine of the consensus glycine trio motif, and which is also very highly conserved, retained at least 25% of the enzymatic activity. The Gly31 substitution, which restored a glycine to a position characteristic for most protein kinases, had little overall effect upon the maximum rate of catalysis. Restoration of Ser30 to the more typical phenylalanine, which is present in most protein kinases, had minimal effect on catalysis. These data provide the first direct evaluation of the roles that different residues play within this consensus glycine trio/valine motif of the protein kinases, which up to now have only been surmised to be of importance because of their conservation. Two unexpected findings are that for one residue that is very conserved (Gly26) there is some flexibility of substitution not apparent from the evolutionary conservation and that a second quite conserved residue in protein kinases (equivalent to Gly at position 31) does not produce a protein optimized for nucleotide binding.

Farnesylcysteine, a constituent of the alpha and beta subunits of rabbit skeletal muscle phosphorylase kinase: localization by conversion to S-ethylcysteine and by tandem mass spectrometry.

The primary structure of the alpha and beta subunits of phosphorylase kinase reveals that both proteins contain a carboxyl-terminal CA1A2X motif (where C is cysteine, A1 and A2 are aliphatic amino acids, and X is an uncharged amino acid), the recognition signal for a protein polyisoprenyltransferase. The product, a polyisoprenylated cysteine, can be detected by phenylthiocarbamoylamino acid analysis and by microsequencing following conversion to S-ethylcysteine. Mass spectrometry confirms a covalently linked farnesyl residue in both subunits. Tandem mass spectrometry localizes these modifications at the cysteine residues present in the carboxyl-terminal CAMQ and CLVS sequences of the alpha and beta subunits, respectively. Membrane association of phosphorylase kinase, probably mediated by these farnesyl residues, is discussed.

Coordinated expression of phosphorylase kinase subunits in regenerating skeletal muscle.

The developmental expression of the alpha, beta, and gamma subunits of skeletal muscle phosphorylase kinase has been examined in regenerating muscle. Rat extensor digitorum longus (EDL) muscles, treated with bupivacaine, promptly undergo a rapid degeneration of the muscle, followed by regeneration and recovery of essentially normal morphology and physiology by 3-4 weeks post-treatment (Hall-Craggs, E. C. B., and Seyan, H. S. (1975) Exp. Neurol. 46, 345-354). Phosphorylase kinase activity dropped to approximately 10% of control within 3 days of bupivacaine treatment and remained at this low level for several days but had attained at least 60% of normal levels by day 21. The pH 6.8/8.2 activity ratio was unusually high during the period of low activity, suggesting that the catalytic activity was not under normal regulation at this time. The subunit mRNAs were readily detected in control EDL but were undetectable at day 3 post-bupivacaine treatment. Very small amounts of message for all three subunits were evident by day 6 and began to approach normal levels by day 12-15. The mRNA for both the alpha and alpha' subunits of phosphorylase kinase exhibited a similar pattern of recovery, as did also the mRNA for phosphorylase. In contrast to both phosphorylase kinase and phosphorylase, actin mRNA exhibited a quite a different pattern, with a nearly full recovery of message levels by day 6 post-bupivacaine. These data indicate that synthesis of phosphorylase and the alpha, beta, and gamma subunits of phosphorylase kinase appears to be coordinately regulated at the level of message accumulation and that the expression of phosphorylase kinase activity is likely to be also regulated post-transcriptionally.

A kinetic re-interpretation of the regulation of rabbit skeletal-muscle phosphorylase kinase activity by Ca2+ and phosphorylation.

The regulation of phosphorylase kinase has been proposed to occur physiologically under conditions of zero-order ultrasensitivity [Meinke & Edstrom (1991) J. Biol. Chem. 266, 2259-2266]. This is also one of the conditions that recent theoretical approaches have indicated to be essential in order for an interconvertible enzyme cascade to generate a sensitive response to an effector [Cardenas & Cornish-Bowden (1989) Biochem. J. 257, 339-345]. In contrast, all published kinetic data to date have strongly suggested that activation of phosphorylase kinase by Ca2+ or phosphorylation is attributable solely to a change in affinity for phosphorylase, with no effect on the Vmax. of the reaction. In this study an attempt is made to resolve this conflict. Findings suggest that changes in Vmax. can fully account for the activation of phosphorylase kinase by the physiological mechanisms of cyclic AMP-dependent phosphorylation and increase in Ca2+ concentration.