Site-directed mutants of glycogen phosphorylase are altered in their interaction with phosphorylase kinase

Glycogen phosphorylase is found in resting muscle as phosphorylase b, which is inactive without AMP. Phosphorylation by phosphorylase kinase (PhK) produces phosphorylase a, which is active in the absence of AMP. PhK is the only kinase that can phosphorylate phosphorylase b, which in turn is the only physiological substrate for PhK. We have explored the reasons for this specificity and how these two enzymes recognize each other by studying site-directed mutants of glycogen phosphorylase. All mutants were assayed for changes in their interaction with a truncated form of the catalytic subunit of phosphorylase kinase, gamma(1-300). Five mutations (R69K, R69E, R43E, R43E/R69E, and E501A), made at sites that interact with the amino terminus in either phosphorylase b or a, showed little difference in phosphorylation by gamma(1-300) compared to wild-type phosphorylase b. Five mutations, made at three sites in the amino-terminal tail of phosphorylase (K11A, K11E, I13G, R16A, and R16E), however, produced decreases in catalytic efficiency for gamma(1-300), compared to that for phosphorylase b. R16E was the poorest substrate for gamma(1-300), giving a 47-fold decrease in catalytic efficiency. The amino terminus, and especially Arg 16, are very important factors for recognition of phosphorylase by gamma(1-300). A specific interaction between Lys 11 of phosphorylase and Glu 110 of gamma(1-300) was also confirmed. In addition, I13G and R16A were able to be phosphorylated by protein kinase A, which does not recognize native phosphorylase.     

The interaction of muscle glycogen phosphorylase b with glycogen

Interaction of muscle glycogen phosphorylase b (EC 2.4.1.1) with glycogen was studied by sedimentation, stopped-flow and temperature-jump methods. The equilibrium enzyme concentration was determined by sedimentation in an analytical ultracentrifuge equipped with absorption optics and a photoelectric scanning system. The maximum adsorption capacity of pig liver glycogen is 3.64 mumol dimeric glycogen phosphorylase b per g glycogen, which corresponds to 20 dimeric enzyme molecules per average glycogen molecule of Mr 5.5 X 10(6). Microscopic dissociation constants were determined for the enzyme-glycogen complex within the temperature range from 12.7 to 30.0 degrees C. Enzyme-glycogen complexing is accompanied by increasing light scattering and its increment depends linearly on the concentration of the binding sites on a glycogen particle that are occupied by the enzyme. Complex formation and relaxation kinetics are in accordance with the proposed bimolecular reaction scheme. The monomolecular dissociation rate constant of the complex increases as the temperature increases from 12.7 to 30.0 degrees C, whereas the bimolecular rate constant changes slightly and is about 10(8) M-1 X S-1. These data point to the possibility of diffusional control of the complex formation.     

Kinetics of the interaction of rabbit skeletal muscle phosphorylase kinase with glycogen

The kinetics of the interaction of rabbit skeletal muscle phosphorylase kinase with glycogen was studied by the turbidimetric method at pH 6.8 and 8.2. Binding of phosphorylase kinase by glycogen occurs only in the presence of Ca2+ and Mg2+. The initial rate of complex formation is proportional to the enzyme and polysaccharide concentration; this suggests the formation of a complex with 1:1 stoichiometry in the initial step of phosphorylase kinase binding by glycogen. The kinetic data suggest that phosphorylase kinase substrate--glycogen phosphorylase b--favors the binding of phosphorylase kinase with glycogen. This conclusion is supported by direct experiments on the influence of phosphorylase b on the interaction of phosphorylase kinase with glycogen using analytical sedimentation analysis. The kinetic curves of the formation of the complex of phosphorylase kinase with glycogen obtained in the presence of ATP are characterized by a lag period. Preincubation of phosphorylase kinase with ATP in the presence of Ca2+ and Mg2+ causes the complete disappearance of the lag period. On changing the pH from 6.8 to 8.2, the rate of phosphorylase kinase binding by glycogen is appreciably increased, and complex formation becomes possible even in the absence of Mg2+. A model of phosphorylase kinase and phosphorylase b adsorption on the surface of the glycogen particle explaining the increase in the strength of phosphorylase kinase binding with glycogen in the presence of phosphorylase b is proposed.     

Effect of molecular crowding on self-association of phosphorylase kinase and its interaction with phosphorylase b and glycogen

Self-association of phosphorylase kinase (PhK) and its interaction with glycogen (M=5500 kDa) and phosphorylase b (Phb) has been studied using analytical ultracentrifugation and turbidimetry under the conditions of molecular crowding arising from the presence of high concentrations of osmolytes. In accordance with the predictions of the molecular crowding theory, trimethylamine N-oxide (TMAO) and betaine greatly favor self-association of PhK induced by Mg2+ and Ca2+ and PhK interaction with glycogen. In contrast, proline suppresses these processes, probably, due to its specific interaction with PhK. All osmolytes tested prevented the complex formation between PhK and its physiological substrate, Phb. The specific interactions of PhK and Phb with glycogen, in the living cell, presumably is a factor allowing the negative effect of crowding on the recognition of Phb by PhK to be overcome.     

Stimulation of glycogen phosphorylase kinase by phospholipids

The acidic phospholipids phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-biphosphate (PIP2) and the neutral phospholipid lysophosphatidylcholine (LPC) were found to stimulate (3 to 8-fold) the activity of nonactivated rabbit skeletal muscle phosphorylase kinase at pH 6.8, without significantly affecting the activity at pH 8.2. In this respect, phosphatidylcholine and phosphatidylethanolamine were ineffective, while the anionic detergent sodium dodecyl sulfate (SDS) and the anionic steroid dehydroisoandrosterone sulfate (DIAS) were able to mimic the action of phospholipids. SDS was also found to be a very efficient activator of the autophosphorylation of phosphorylase kinase (20-fold activation at 200 microM). The activating effect of phospholipids largely depends on the size of lipid vesicles, which is connected with the procedure of their preparation. These results suggest that phosphorylase kinase belongs to the class of Ca2+-dependent enzymes, which are sensitive to stimulation by calmodulin, limited proteolysis and anionic amphiphiles.     

The regulatory alpha subunit of phosphorylase kinase may directly participate in the binding of glycogen phosphorylase

The yeast two-hybrid screen has been used to identify potential regions of interaction of the largest regulatory subunit, , of phosphorylase kinase (PhK) with two fragments of its protein substrate, glycogen phosphorylase b (Phb). One fragment, corresponding to residues 17-484 (PhbN"), contained the regulatory domain of the protein, but in missing the first 16 residues was devoid of the sole phosphorylation site of Phb, Ser14; the second fragment corresponded to residues 485-843 (PhbC) and contained the catalytic domain of Phb. Truncation fragments of the subunit were screened for interactions against these two substrate fragments. PhbC was not found to interact with any constructs; however, PhbN" interacted with a region of (residues 864-1014) that is near the phosphorylatable region of that subunit. PhbN" was also screened for interactions against a variety of fragments of the catalytic subunit of PhK; however, no interactions were detected, even with fulllength . Our results support the idea that amino acid residues proximal to the convertible serine of Phb are important for its specific interaction with the catalytic subunit of PhK, but that regions distinct from the convertible serine residue of Phb and from the catalytic domain of PhK may also be involved in the interaction of these two proteins.     

Pest sequences present in the subunits of phosphorylase kinase

We report that the amino acid sequences of all four subunits of rabbit skeletal muscle phosphorylase kinase possess one or more regions rich in proline (P), glutamic acid (E), serine (S) and threonine (T). alpha and beta subunits contain strong PEST sequences, showing PEST scores greater than 0 (Rogers et al. (1986) Science 234, 364-368), while gamma and delta subunits contain weak PEST regions (negative PEST scores greater than -5.3). In addition to PEST sequences, alpha, beta and gamma subunits contain clusters of arginine pairs. The above sequence characteristics may serve to signal rapid turnover of phosphorylase kinase.     

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 Mg2+, whereas at pH 8.2 formation of the complex occurs even in the absence of Mg2+. On the other hand, the interaction of phosphorylase kinase with glycogen requires Ca2+ 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.     

The cyclin-dependent kinase (CDK) inhibitor flavopiridol inhibits glycogen phosphorylase

Flavopiridol has been shown to induce cell cycle arrest and apoptosis in various tumor cells in vitro and in vivo. Using immobilized flavopiridol, we identified glycogen phosphorylases (GP) from liver and brain as flavopiridol binding proteins from HeLa cell extract. Purified rabbit muscle GP also bound to the flavopiridol affinity column. GP is the rate-limiting enzyme in intracellular glycogen breakdown. Flavopiridol significantly inhibited the AMP-activated GP-b form of the purified rabbit muscle isoenzyme (IC50 of 1 microM at 0.8 mM AMP), but was less inhibitory to the active phosphorylated form of GP, GP-a (IC50 of 2.5 microM). The AMP-bound GP-a form was poorly inhibited by flavopiridol (40% at 10 microM). Increasing concentrations of the allosteric effector AMP resulted in a linear decrease in the GP-inhibitory activity of flavopiridol suggesting interference between flavopiridol and AMP. In contrast the GP inhibitor caffeine had no effect on the relative GP inhibition by flavopiridol, suggesting an additive effect of caffeine. Flavopiridol also inhibited the phosphorylase kinase-catalyzed phosphorylation of GP-b by inhibiting the kinase in vitro. Flavopiridol thus is able to interfere with both activating modifications of GP-b, AMP activation and phosphorylation. In A549 NSCLC cells flavopiridol treatment caused glycogen accumulation despite of an increase in GP activity, suggesting direct GP inhibition in vivo rather than inhibition of GP activation by phosphorylase kinase. These results suggest that the cyclin-dependent kinase inhibitor flavopiridol interferes with glycogen degradation, which may be responsible for flavopiridol's cytotoxicity and explain its resistance in some cell lines.     

Phosphorylation of myelin basic protein by glycogen phosphorylase kinase

The ability of homogeneous glycogen phosphorylase kinase (Phk) from rabbit skeletal muscle to phosphorylate bovine brain myelin basic protein (MBP) was investigated. Phk could incorporate a maximum of 1.9 mol phosphate/mol MBP. The apparent Km and Vmax for Phk phosphorylation of MBP were 27 microM and 90 nmol/min per mg enzyme, respectively. Properties of MBP phosphorylation by Phk are similar to those of phosphorylase as a substrate. Only serine residues of MBP are phosphorylated by Phk. Phosphorylation sites of MBP by Phk are not identical to those by cAMP-dependent protein kinases.     

Phosphorylation of rat liver glycogen synthase by phosphorylase kinase

Phosphorylation of rat liver glycogen synthase by rabbit skeletal muscle phosphorylase kinase results in the incorporation of approximately 0.8-1.2 mol of PO4/subunit. Analyses of the tryptic peptides by isoelectric focusing and thin layer chromatography reveal the presence of two major 32P-labeled peptides. Similar results were obtained when the synthase was phosphorylated by rat liver phosphorylase kinase. This extent of phosphorylation does not result in a significant change in the synthase activity ratio. In contrast, rabbit muscle glycogen synthase is readily inactivated by rabbit muscle phosphorylase kinase; this inactivation is further augmented by the addition of rabbit muscle cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1. Addition of cAMP-dependent protein kinase after initial phosphorylation of liver synthase with phosphorylase kinase, however, does not result in an inactivation or additional phosphorylation. The lack of additive phosphorylation under this condition appears to result from the phosphorylation of a common site by these two kinases. Partial inactivation of liver synthase can be achieved by sequential phosphorylation with phosphorylase kinase followed by synthase (casein) kinase-1. Under this assay condition, the phosphate incorporation into the synthase is additively increased and the synthase activity ratio (-glucose-6-P/+glucose-6-P) is reduced from 0.95 to 0.6. Nevertheless, if the order of the addition of these two kinases is reversed, neither additive phosphorylation nor inactivation of the synthase is observed. Prior phosphorylation of the synthase by phosphorylase kinase transforms the synthase such that it becomes a better substrate for synthase (casein) kinase-1 as evidenced by a 2- to 4-fold increase in the rate of phosphorylation. This increased rate of phosphorylation of the synthase appears to result from the rapid phosphorylation of a site neighboring that previously phosphorylated by phosphorylase kinase.     

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

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

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.     

Structural features contributing to complex formation between glycogen phosphorylase and phosphorylase kinase.

A polyclonal antibody was generated against a peptide corresponding to a region opposite the regulatory face of glycogen phosphorylase b (P-b), providing a probe for detecting and quantifying P-b when it is bound to its activating kinase, phosphorylase kinase (PhK). Using both direct and competition enzyme-linked immunosorbent assays (ELISAs), we have measured the extent of direct binding to PhK of various forms of phosphorylase, including different conformers induced by allosteric effectors as well as forms differing at the N-terminal site phosphorylated by PhK. Strong interactions with PhK were observed for both P-b', a truncated form lacking the site for phosphorylation, and P-a, the phosphorylated form of P-b. Further, the binding of P-b, P-b', and P-a was stimulated a similar amount by Mg(2+), or by Ca(2+) (both being activators of PhK). Our results suggest that the presence and conformation of P-b's N-terminal phosphorylation site do not fully account for the protein's affinity for PhK and that regions distinct from that site may also interact with PhK. Direct ELISAs detected the binding of P-b by a truncated form of the catalytic gamma subunit of PhK, consistent with the necessary interaction of PhK's catalytic subunit with its substrate P-b. In contrast, P-b' bound very poorly to the truncated gamma subunit, suggesting that the N-terminal phosphorylatable region of P-b may be critical in directing P-b to PhK's catalytic subunit and that the binding of P-b' by the PhK holoenzyme may involve more than just its catalytic core. The sum of our results suggests that structural features outside the catalytic domain of PhK and outside the phosphorylatable region of P-b may both be necessary for the maximal interaction of these two proteins.     

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.