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

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

Effect of Mg2+ concentrations on phosphorylation/activation of phosphorylase b kinase by cAMP/Ca2+-independent,autophosphorylation-dependent protein kinase

In a previous report (Yu and Yang,Biochem. Biophys. Res. Commun. 207, 140–147 (1995)], phosphorylase b kinase from rabbit skeletal muscle was found to be phosphorylated and activated by a cyclic nucleotide- and Ca²⁺-independent protein kinase previously identified as an autophosphorylation-dependent multifunctional protein kinase (autokinase) from brain and liver (Yanget al, J. Biol. Chem. 262, 7034–7040, 9421–9427 (1987)]. In this report, the effect of Mg²⁺ ion concentration on the auto-kinase-catalyzed activation of phosphorylase b kinase is investigated. The levels of phosphorylation and activation of phosphorylase b kinase catalyzed by auto-kinase are found to be dependent on the concentration of Mg²⁺ ion used. Phosphorylation of phosphorylase b kinase at high Mg²⁺ ion (>9 mM) is 2–3 times higher than that observed at low Mg²⁺ ion (1 mM) and this results in a further 2- to 3-fold activation of the enzyme activity at high Mg²⁺ ion. Analysis of the phosphorylation stoichiometry ofα andβ subunits of phosphorylase b kinase at different Mg²⁺ ion concentrations further reveals that the phosphorylation level of theβ subunit remains almost unchanged, whereas the phosphorylation level of theα subunit increases dramatically and correlates with the increased enzyme activity. In similarity with theβ subunit, phosphorylations of myelin basic protein and histone 2A by auto-kinase are also unaffected by Mg²⁺ ion. Taken together, the results provide initial evidence that Mg²⁺ ion may specifically render thea subunit a better substrate for auto-kinase to cause further phosphorylation/activation of phosphorylase b kinase, representing a new mode of control mechanism for the regulation of auto-kinase involved in the phosphorylation and concurrent activation of phosphorylase b kinase.     

Ca2+- and Mg2+-dependent association of phosphorylase kinase with human erythrocyte membranes

The interaction of rabbit muscle phosphorylase kinase (EC 2.7.1.38) with human erythrocyte membranes was investigated. It was found that at pH 7.0 the kinase binds to the inner face of the erythrocyte membrane (inside-out vesicles) and that this binding is Ca2+- and Mg2+-dependent. The sharpest increase in the binding reaction occurs at concentrations between 70 and 550 nM free Ca2+. Erythrocyte ghost or right-side out erythrocyte vesicles showed a significantly lower capacity to interact with phosphorylase kinase. Autophosphorylated phosphorylase kinase shows a similar Ca2+-dependent binding profile, while trypsin activation of the kinase and calmodulin decrease the original binding capacity by about 50%. Heparin (200 micrograms/ml) and high ionic strength (50 mM NaCl) almost completely blocks enzyme-membrane interaction; glycogen does not affect the interaction.     

The effect of Mg2+ on the Ca2+-binding properties of non-activated phosphorylase kinase.

The calcium binding properties of non-activated phosphorylase kinase at pH 6.8 have been studied by the gel filtration technique at calcium concentrations from 50 nM to 50 muM. Taking into account the subunit structure alpha4beta4gamma4 the enzyme binds 12 mol Ca2+ per mol with an association constant of 6.0 X 10(7) M-1, 4 mol with an association constant of 1.7 X 10(6) M-1 and 36 mol with a binding constant of 3.9 X 10(4) M-1 at low ionic strength. In buffer of high ionic strength, i.e. 180 mM NH4Cl or 60 mM (NH4)2SO4, only a single set of eight binding sites with a binding constant of 5.5 X 10(7) M-1 is left. In a buffer containing 155 mM NH4Cl and 10 mM MgCl2, the calcium affinity of these sites is reduced to a KCa of 3.0 X 10(6) M-1, indicating competition between Ca2+ and Mg2+. From these measurements, the binding constant of Mg2+ for these sites is calculated to be 1.7 X 10(3) M-1 is left. In a buffer containing 155 mM NH4Cl and 10 mM MgCl2, the calcium affinity of these sites is reduced to a KCa of 3.0 X 10(6) M-1, indicating competition between Ca2+ and Mg2+. from these measurements, the binding constant of Mg2+ for these sites is calculated to be 1.7 X 10(3) M-1. Additionally, 10 mM Mg2+ induces a set of four new Ca2+ binding sites which show positive cooperativity. Their half-saturation constant under the conditions described is 3.5 X 10(5) M-1, and they, too, exhibit competition between Ca2+ and Mg2+. Since this set of sites is induced by Mg2+ a third group of binding sites for the latter metal must be postulated.     

Control of phosphorylase kinase in the isolated glycogen particle by Ca2+-Mg2+ synergistic activation and cAMP-dependent phosphorylation

The isolated glycogen particle provides a means to examine the regulation of glycogen metabolism with the components organized in a functional cellular complex. With this system, we have studied the control of phosphorylase kinase activation by Ca2+ and cAMP. Contrary to a previous report (Heilmeyer, L. M. G., Jr., Meyer, F., Haschke, R. H., and Fisher, E. H. (1980) J. Biol. Chem. 245, 6649-6656), phosphorylase kinase became activated during incubation of the glycogen particle with MgATP2- and Ca2+. Part of this activation could be attributed to the action of the cAMP-dependent protein kinase; however, it was not possible to quantitatively correlate activation with phosphorylation in the presence of Ca2+ and Mg2+ due to a large, but uncertain, contribution of synergistic activation caused by these ions. This latter activation had properties similar to those described by King and Carlson (King, M. M., and Carlson, G. M. (1980) Arch. Biochem. Biophys. 209, 517-523) with the purified enzyme, and its occurrence also explains why phosphorylase kinase activation in the glycogen particle was not observed previously. The cAMP-dependent activation of phosphorylase kinase in the glycogen particle has been characterized. It occurred in a similar manner when either the cAMP-dependent protein kinase or cAMP was added, thus indicating that the phosphorylation sites of phosphorylase kinase complexed in the glycogen particle were accessible to endogenous or exogenous enzyme. In the glycogen particle, both the alpha and beta subunits were phosphorylated by the cAMP-dependent protein kinase, but the alpha subunit dephosphorylation appeared to be preferentially regulated by Ca2+. The activity of phosphorylase kinase in the glycogen particle is regulated by the phosphorylation of both the alpha and beta subunits.     

Physicochemical changes in phosphorylase kinase induced by its cationic activator Mg2+

For over four decades free Mg²⁺ ions, that is, those in excess of MgATP, have been reported to affect a wide variety of properties of phosphorylase kinase (PhK), including its affinity for other molecules, proteolysis, chemical crosslinking, phosphorylation, binding to certain monoclonal antibodies, and activity, which is stimulated. Additionally, for over three decades Mg²⁺ has been known to act synergistically with Ca²⁺, another divalent activator of PhK, to affect even more properties of the enzyme. During all of this time, however, no study has been performed to determine the overall effects of free Mg²⁺ ions on the physical properties of PhK, even though the effects of Ca²⁺ ions on PhK's properties are well documented. In this study, changes in the physicochemical properties of PhK induced by Mg²⁺ under nonactivating (pH 6.8) and activating (pH 8.2) conditions were investigated by circular dichroism spectroscopy, zeta potential analyses, dynamic light scattering, second derivative UV absorption, negative stain electron microscopy, and differential chemical crosslinking. The effects of the activator Mg²⁺ on some of the properties of PhK measured by these techniques were found to be quite different at the two pH values, and displayed both differences and similarities with the effects previously reported to be induced by the activator Ca²⁺ (Liu et al., Protein Sci 2008;17:2111–2119). The similarities may reflect the fact that both cations are activators, and foremost among their similarities is the dramatically less negative zeta potential induced by their binding to PhK.     

Ca2+-dependent stimulation of muscle and liver phosphorylase kinase by heparin

• 1.1. Heparin stimulates the activity of nonactivated and activated skeletal muscle phosphorylase kinase in a Ca²⁺-dependent manner.
• 2.2. The stimulatory effect of heparin on the activity of nonactivated phosphorylase kinase is also expressed in the presence of calmodulin and glycogen. Heparin acted in synergism with glycogen.
• 3.3. Heparin increases the affinity of phosphorylase kinase to Ca²⁺ 5–12 fold depending upon the activation conditions.
• 4.4. Ca²⁺ influences the stimulation of liver phosphorylase kinase by heparin in a similar way.

     

Effect of free Mg2+ on liver phosphorylase kinase activity

When pig liver phosphorylase kinase was assayed at various concentrations of Mg2+, about 2-fold stimulation was observed around 2-3 mM Mg2+ (Mg2+/ATP ratio, 20-30) compared with the activity at 0.3 mM Mg2+ (Mg2+/ATP ratio, 3). This stimulation was specific for Mg2+ among the divalent cations tested and the process was reversible. Km values for ATP and phosphorylase b were decreased 3.6- and 9.5-fold, respectively, at 3 mM Mg2+ compared with those obtained at 0.3 mM Mg2+. These results indicate that the activity of liver phosphorylase kinase is influenced by free Mg2+.     

Synergistic activation of the Arabidopsis NADPH oxidase AtrbohD by Ca2+ and phosphorylation

Plant respiratory burst oxidase homolog (rboh) proteins, which are homologous to the mammalian 91-kDa glycoprotein subunit of the phagocyte oxidase (gp91(phox)) or NADPH oxidase 2 (NOX2), have been implicated in the production of reactive oxygen species (ROS) both in stress responses and during development. Unlike mammalian gp91(phox)/NOX2 protein, plant rboh proteins have hydrophilic N-terminal regions containing two EF-hand motifs, suggesting that their activation is dependent on Ca(2+). However, the significance of Ca(2+) binding to the EF-hand motifs on ROS production has been unclear. By employing a heterologous expression system, we showed that ROS production by Arabidopsis thaliana rbohD (AtrbohD) was induced by ionomycin, which is a Ca(2+) ionophore that induces Ca(2+) influx into the cell. This activation required a conformational change in the EF-hand region, as a result of Ca(2+) binding to the EF-hand motifs. We also showed that AtrbohD was directly phosphorylated in vivo, and that this was enhanced by the protein phosphatase inhibitor calyculin A (CA). Moreover, CA itself induced ROS production and dramatically enhanced the ionomycin-induced ROS production of AtrbohD. Our results suggest that Ca(2+) binding and phosphorylation synergistically activate the ROS-producing enzyme activity of AtrbohD.     

Ca2+-induced structural changes in phosphorylase kinase detected by small-angle X-ray scattering

Phosphorylase kinase (PhK), a 1.3-MDa (alphabetagammadelta)(4) hexadecameric complex, is a Ca(2+)-dependent regulatory enzyme in the cascade activation of glycogenolysis. PhK comprises two arched (alphabetagammadelta)(2) octameric lobes that are oriented back-to-back with overall D(2) symmetry and joined by connecting bridges. From chemical cross-linking and electron microscopy, it is known that the binding of Ca(2+) by PhK perturbs the structure of all its subunits and promotes redistribution of density throughout both its lobes and bridges; however, little is known concerning the interrelationship of these effects. To measure structural changes induced by Ca(2+) in the PhK complex in solution, small-angle X-ray scattering was performed on nonactivated and Ca(2+)-activated PhK. Although the overall dimensions of the complex were not affected by Ca(2+), the cation did promote a shift in the distribution of the scattering density within the hydrated volume occupied by the PhK molecule, indicating a Ca(2+)-induced conformational change. Computer-generated models, based on elements of the known structure of PhK from electron microscopy, were constructed to aid in the interpretation of the scattering data. Models containing two ellipsoids and four cylinders to represent, respectively, the lobes and bridges of the PhK complex provided theoretical scattering profiles that accurately fit the experimental data. Structural differences between the models representing the nonactivated and Ca(2+)-activated conformers of PhK are consistent with Ca(2+)-induced conformational changes in both the lobes and the interlobal bridges.     

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.     

Lanthanide ions and Cd2+ are able to substitute for Ca2+ in regulating phosphorylase kinase

Trivalent lanthanide ions and Cd2+ were found to mimic effectively the stimulatory action of Ca2+ on rabbit muscle phosphorylase kinase. In the range of concentrations tested, Cd2+ and lanthanides (Tb3+, Gd3+, Pr3+, Ce3+) could substitute for Ca2+ in activating the enzyme to about 60% and 70% respectively of the maximal level seen with Ca2+, at pH 8.2. The effect induced by Cd2+ was biphasic (stimulation followed by inhibition with increasing metal cation concentration). Similar results were obtained at pH 6.8. Cd2+ and Tb3+ were also able to replace Ca2+ required for the stimulation of phosphorylase kinase activity at pH 8.2 by exogenous calmodulin. Maximal stimulation induced by calmodulin in presence of Cd2+ was significantly higher than that in presence of Ca2+ or Tb3+.     

The effect of Mg2+ and Ca2+ on urea-catalyzed phosphorylation reactions

Summary When struvite (MgNH₄PO₄ 6H₂O) is heated with urea at 65–100°C, inorganic pyrophosphate is formed in good yield. Under similar conditions pyro-phosphate is formed much more slowly from ammonium phosphate or hydroxylapatite. The major products formed by the reaction of nucleotides with urea and either ammonium phosphate or hydroxylapatite are derivatives phosphorylated on the 2 or 3 position. With struvite, on the other hand, the main reaction is pyrophosphate bond formation. Yields of up to 25% of uridine diphosphate can be obtained at temperatures as low as 65°C.     

Effect of Mg2+ concentration on the cAMP-dependent protein kinase-catalyzed activation of rabbit skeletal muscle phosphorylase kinase.

Phosphorylase kinase was found to be activated and phosphorylated at 10mM Mg2+ by the cAMP-dependent protein kinase-catalyzed reaction ot much higher levels than observed previously when reactions were carried out in 1 to 2 mM Mg2+ (Cohen, P. (1973) Eur. J. Biochem. 34, 1; Hayakawa, T., Perkin, J.P., and Krebs, E.G. (1973) Biochemistry 12, 574). That the reaction at 10 mM Mg2+ is protein kinase-catalyzed is supported by several observations: (a) the reaction is facilitated by the addition of protein kinase; (b) the reaction depends on cAMP when protein kinase holoenzyme is uded; (c) the reaction is not inhibited by 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetate which is known to inhibit autoactivation and autophosphorylation of phosphorylase kinase; and (d) the protein inhibitor of protein kinase inhibits this reaction. The phosphorylation and activation of phosphorylase kinase seem to occur in two phases. At low Mg2+ only the first phase is manifested and involves the incorporation of 2 mol of phosphate, 1 mol into each of Subunits A and B. At high Mg2+ additional sites are phosphorylated almost exclusively on Subunit A, with phosphate incorporation approaching the final level of 7 to 9 mol. Enzyme activity at high Mg2+ is 2 to 3 times higher than that observed when activation is studied at low Mg2+. The observation that both casein and type II histone are phosphorylated to the same extent at 1 mM and 10 mM Mg2+ suggested that high Mg2+ may be altering the conformation of phosphorylase kinase thus rendering more phosphorylation sites accessible to protein kinase. Since the phosphorylation of phosphorylase kinase by either the protein kinase-catalyzed or autocatalytic reaction can result in the incorporation of 7 to 9 mol of phosphate, the finding that only about seven sites become phosphorylated by both mechanisms acting together suggest that activation by these two mechanisms may involve common phosphorylation sites.     

Inhibition of Ca2+-dependent protein kinase and Ca2+/Mg2+-ATPase in cardiac sarcolemma by the anti-calmodulin drug calmidazolium

Sarcolemma (SL) vesicles, isolated from pig heart, contain both a Ca2+-calmodulin-dependent protein kinase (CaM-PK) and a Ca2+-dependent Mg2+-ATPase (Ca2+/Mg2+)-ATPase). Some of their properties have been compared: their affinity for Ca2+ ions, dependence on exogenous calmodulin (CaM) and sensitivity to the anti-CaM drug calmidazolium (R24571). The properties of Ca2+-CaM-dependent brain phosphodiesterase (PDE) have also been examined. R24571 appeared to be the most potent inhibitor from brain PDE. For the three enzymes studied, exogenously added CaM was able to antagonize the R24571 inhibition, although the efficiency to counteract was rather low in the case of the SL Ca2+/Mg2+-ATPase. R24571 decreased the affinity of the Ca2+/Mg2+-ATPase for Ca2+ ions (KCa 0.35 versus 0.75 microM) and exerted an inhibition non-competitive with Ca2+ ions on the other CaM-dependent enzymes. Membrane-bound CaM, which is involved in controlling the Ca2+/Mg2+-ATPase, appeared to be present in a stoichiometry varying from 1:1 to 1:4 compared to the 32P-intermediate of the ATPase. R24571 treatment of SL vesicles selectively solubilized a number of proteins in the molecular range of 13-20 kD, which may include CaM. The results suggest that different mechanisms are involved in the CaM control of the two SL enzymes studied.     

Ca2+-stimulated phosphorylation of muscle glycogen synthase by phosphorylase b kinase.

Phosphorylase b kinase from rabbit muscle phosphorylates glycogen synthase purified from the same tissue. The reaction is markedly stimulated by Ca2+ and results in a decrease in the synthase %I activity. Phosphorylase b kinase action leads to the incorporation of phosphate (0.6 to 0.8 mol/mol of subunit) preferentially into a single cyanogen bromide fragment of synthase (fragment III). Cyclic AMP-independent synthase kinase also shows a specificity for the site(s) contained in fragment III whereas the cyclic AMP-dependent protein kinase exerts a preference for the site(s) located in a distinct cyanogen bromide fragment (fragment II). A Ca2+-stimulated endogenous kinase also results in the phosphorylation of fragment III and can be attributed to the presence of phosphorylase b kinase. The finding of a Ca2+-stimulated phosphorylation of glycogen synthase has important implications for the regulation of glycogen metabolism and particularly those processes thought to be controlled by cytoplasmic Ca2+ concentration.     

Does protein kinase C require Ca2+ for activation?

Ca2+ requirement for protein kinase C activation is a matter of controversy. In this report we have examined Ca2+ dependency of the reaction in different assay systems and shown that the enzyme response to Ca2+, as well as diacylglycerol, depends upon phospholipid species, protein substrate and lipid conformation (micelles or sonicates). These results emphasize that the enzyme characteristics as defined in reconstituted membrane systems may not have a physiological relevance.     

Sustained activation of the tyrosine kinase Syk by antigen in mast cells requires local Ca2+ influx through Ca2+ release-activated Ca2+ channels

Mast cell activation involves cross-linking of IgE receptors followed by phosphorylation of the non-receptor tyrosine kinase Syk. This results in activation of the plasma membrane-bound enzyme phospholipase Cgamma1, which hydrolyzes the minor membrane phospholipid phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol trisphosphate. Inositol trisphosphate raises cytoplasmic Ca2+ concentration by releasing Ca2+ from intracellular stores. This Ca2+ release phase is accompanied by sustained Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. Here, we find that engagement of IgE receptors activates Syk, and this leads to Ca2+ release from stores followed by Ca2+ influx. The Ca2+ influx phase then sustains Syk activity. The Ca2+ influx pathway activated by these receptors was identified as the CRAC channel, because pharmacological block of the channels with either a low concentration of Gd3+ or exposure to the novel CRAC channel blocker 3-fluoropyridine-4-carboxylic acid (2',5'-dimethoxybiphenyl-4-yl)amide or RNA interference knockdown of Orai1, which encodes the CRAC channel pore, all prevented the increase in Syk activity triggered by Ca2+ entry. CRAC channels and Syk are spatially close together, because increasing cytoplasmic Ca2+ buffering with the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis failed to prevent activation of Syk by Ca2+ entry. Our results reveal a positive feedback step in mast cell activation where receptor-triggered Syk activation and subsequent Ca2+ release opens CRAC channels, and the ensuing local Ca2+ entry then maintains Syk activity. Ca2+ entry through CRAC channels therefore provides a means whereby the Ca2+ and tyrosine kinase signaling pathways can interact with one another.