[Use of properties and regulation peculiarities of enzymes of glycogenolysis in fish skeletal muscle depending on peculiarities of motor activity of species].

Levels of activity, properties, and peculiarities of activation of glycogen phosphorylase (GP; EC 2.4.1.1) and glycogen phosphorylase kinase (GPK; EC 2.7.1.38) were studied in the white skeletal muscle of fish differing in motor behavior. No differences in the GP and GPK activity levels were revealed in laskir Diplodus annularis (L.), horse mackerel Trachurus mediterraneus ponticus, salmon Salmo trutta morphario, scorpena Scorpaena porcus, Scophtalnus maeoticus, and carp Cyprinus carpio; however, properties of the isolated enzymes and peculiarities of formation of their activated forms during swimming in a hydrodynamic tube are determined by functional peculiarities of the muscle tissue and are associated with the motor activity character of the species. In fish capable for the spurt type of swimming (scorpena, salmon) the more rapid ion regulation plays the predominant role. In other species, the glycogenolysis hormonal regulation leading to a change of the GPK activity index has been found.

Cooperative self-association of phosphorylase kinase from rabbit skeletal muscle.

Ca(2+)- and Mg(2+)-induced association of phosphorylase kinase (PhK) from rabbit skeletal muscle has been studied at the magnitudes of the ionic strength close to the physiological values (40 mM Hepes, pH 6.8, containing 0.1 M NaCl, 0.1 mM Ca(2+), 10 mM Mg(2+); 25 degrees C) and under the molecular crowding conditions produced by high concentrations (1 M) of the natural osmolyte, trimethylamine N-oxide (TMAO). In the presence of 0.1 M NaCl two forms of PhK were registered, namely the "basic form" and "highly associated form", suggesting that PhK association may be treated as an example of cooperative association. According to the data on dynamic light scattering the average hydrodynamic radii of these forms were 16 and 144 nm. The addition of 1 M TMAO produces the time dependent increase in the light scattering intensity caused by the conversion of the basic form into the highly associated form. According to the data of the sedimentation analysis the basic form of PhK comprises a hexadecamer (M(r)=1320 kDa) and its small associates. The removal of Ca(2+) by addition of EGTA results in the reverse conversion of the highly associated form into the basic form suggesting reversibility of self-association of PhK. FAD, the ligand that is specifically bound to PhK, blocks the conversion of the basic form of PhK into the highly associated form.

Baculovirus-mediated overexpression of the phosphorylase b kinase holoenzyme and alpha gamma delta and gamma delta subcomplexes.

Recombinant baculoviruses were created and used to coexpress rat phosphorylase kinase (Phk) alpha, gamma, and delta subunits and rabbit beta subunit in insect cells. Coexpression allowed creation of the (alphabetagammadelta)4 hexadecamer, the alphagammadelta heterotrimer, and the gammadelta heterodimeric subcomplexes. Neither the individual alpha, beta, or gamma subunit nor any complex containing the beta subunit other than the hexadecameric holoenzyme was obtained in soluble form. The expressed complexes exhibited pH- and [Ca2+]-dependent specific activities that were similar to those of the Phk holoenzyme purified from rabbit skeletal muscle (SkM Phk). SkM Phk, expressed Phk, and the alphagammadelta subcomplex were activated by exogenous calmodulin and underwent Ca(2+)-dependent autophosphorylation. In some of these features there were subtle differences that could likely be attributed to differences in the covalent modification state of the baculovirus-driven expressed protein. Our results provide an important avenue to probe the detailed characterization of the structure of Phk and the function of the individual domains of the subunits using baculovirus-mediated expression of Phk and Phk subcomplexes.

Direct visualization of the calmodulin subunit of phosphorylase kinase via electron microscopy following subunit exchange.

Calmodulin is a tightly bound, intrinsic subunit (delta) of the hexadecameric phosphorylase-b kinase holoenzyme, (alphabetagammadelta)4. To introduce specifically labeled calmodulin into the phosphorylase-b kinase complex for its eventual visualization by electron microscopy, we have developed a method for rapidly exchanging exogenous calmodulin for the intrinsic delta subunit. This method exploits previous findings that low concentrations of urea in the absence of Ca(2+) ions cause the specific dissociation of only the delta subunit from the holoenzyme [Paudel, H. K., and Carlson, G. M. (1990) Biochem. J. 268, 393-399]. In the current study, phosphorylase-b kinase was incubated with excess exogenous calmodulin and a threshold concentration of urea to promote exchange of its delta subunit with the exogenous calmodulin. Size exclusion HPLC was then used to remove the excess calmodulin from the holoenzyme containing exchanged delta subunits. Using metabolically labeled [35S]calmodulin to allow quantification and optimization of exchange conditions, we achieved exchange of approximately 10% of all delta subunits within 1 h, with the exchanged holoenzyme retaining full catalytic activity. Calmodulins derivatized with Nanogold for visualization by scanning transmission electron microscopy were then exchanged for delta, which for the first time allowed localization of the delta subunit within the bridged, bilobal phosphorylase b kinase holoenzyme complex. The delta subunits were determined to be near the edge of the lobes, just distal to the interlobal bridges and proximal to a previously identified region of the enzyme's catalytic gamma subunit.

Muscle phosphorylase kinase is not a substrate of AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (cAMPK) have been reported to phosphorylate sites on phosphorylase kinase (PhK). Their target residues Ser 1018 and Ser 1020, respectively, are located in the so-called multi-phosphorylation domain in the PhK alpha subunit. In PhK preparations, only one of these serines is phosphorylated, but never both of them. The aim of this study was to determine whether phosphorylation by cAMPK or AMPK would influence subsequent phosphorylation by the other kinase. Surprisingly, employing four different PhK substrates, it could be demonstrated that, in contradiction to previous reports, PhK is not phosphorylated by AMPK.

A recombinant form of the catalytic subunit of phosphorylase kinase that is soluble, monomeric, and includes key C-terminal residues.

Residues 302-326 of the catalytic (gamma) subunit of phosphorylase kinase (PhK) may comprise an autoinhibitory, pseudosubstrate domain that binds calmodulin. To study this, the cDNA corresponding to rabbit muscle PhKgamma was expressed using Escherichia coli. This yielded two stable, high-activity PhKgamma forms (35 and 42 kDa by SDS-PAGE) that were smaller than an authentic sample of rabbit muscle PhKgamma (45 kDa by SDS-PAGE). Each recombinant form was purified to homogeneity. The N-terminal sequence of the larger, 42-kDa form (pk42) matched that of the rabbit muscle enzyme. This suggested that pk42 consisted of PhKgamma residues 1-362, including the putative calmodulin-binding, autoinhibitory domain. Kinetic parameters obtained for pk42 were like those previously reported for the intact gamma subunit. This implied that the lack of 25 PhKgamma C-terminal residues did not affect phosphorylase kinase activity, but greatly improved enzyme stability. An additional 60 residues were removed from the C-terminus of pk42 using the protease m-calpain. This increased the kinase activity 1.5-fold. Consistent with this, the activity of a mutant PhKgamma that consisted of residues 1-300, denoted gamma1-300, was like that of the m-calpain-treated enzyme. Therefore, although the effect was small, some influence by the C-terminus of pk42 was noted. Moreover, when pk42 was incubated with ATP alone, a C-terminal threonine residue became phosphorylated. Although the influence of this autophosphorylation cannot be inferred from this data, it was evidence that the C-terminus accessed the enzyme's active site. Taken together, these data imply that pk42 will be useful to study phosphorylase kinase structure/activity relationships.

Zero-length crosslinking of the beta subunit of phosphorylase kinase to the N-terminal half of its regulatory alpha subunit.

Phosphorylase kinase, a regulatory enzyme of glycogenolysis in skeletal muscle, is a hexadecameric oligomer containing four copies each of four distinct subunits: alpha, beta, gamma, and delta. By intramolecular zero-length crosslinking with transglutaminase, we have previously demonstrated that the regulatory alpha and beta subunits abut one another in the holoenzyme [Nadeau, O. W., and Carlson, G. M. (1994) J. Biol. Chem. 269, 29670-29676]. Selective partial proteolysis of the 138 kDa alpha subunit in holoenzyme that had been crosslinked by transglutaminase has revealed a high molecular weight conjugate corresponding to full-length beta subunit crosslinked to a 60 kDa N-terminal fragment of alpha (determined by SDS-PAGE, Western blotting and N-terminal sequencing). This conjugate was also observed when the enzyme was first activated by partial proteolysis of alpha and then crosslinked by transglutaminase. Both forms of the kinase, generated by either sequential crosslinking and proteolysis or the reverse, coeluted with non-crosslinked hexadecameric control enzyme in size exclusion chromatography, indicating that the crosslinking was intramolecular, i.e., within hexadecamers. This is the first demonstration of any intersubunit interaction involving the N-terminal domain of the alpha subunit and the first region of any subunit shown to interact with the beta subunit. The results are consistent with the predicted path of the polypeptide backbone of the alpha subunits within the holoenzyme and with the proposed location of the beta subunits.

The inhibition of the insulin receptor by the receptor protein PC-1 is not specific and results from the hydrolysis of ATP.

The membrane protein plasma cell differentiation antigen 1 (PC-1) has been purified as an inhibitor of insulin receptor tyrosine kinase activity and has been implicated in the pathogenesis of NIDDM. However, we show here that PC-1 is a general protein kinase inhibitor in vitro and that this inhibition results from the hydrolysis of ATP by the intrinsic nucleotide pyrophosphatase activity of PC-1. Thus, the inhibition diminished with increasing ATP concentrations, and it was nullified when the ATP concentration was kept constant with a regenerating system or when ATP was added repetitively. When care was taken to avoid ATP depletion, PC-1 did not affect the insulin sensitivity of insulin receptor autophosphorylation. We conclude that the reported inhibition of insulin signaling by PC-1 does not result from a direct inhibition of the insulin receptor kinase activity.

The association of phosphorylase kinase with membranes of rat liver smooth endoplasmic reticulum.

Upon fractionation of a post mitochondrial supernatant from rat liver, phosphorylase kinase activity was largely recovered in the cytosol and the smooth endoplasmic reticulum (SER) fraction. The presence of phosphorylase kinase in SER vesicles was not due to an interaction of the enzyme with glycogen particles, since previous elimination of SER glycogen either by 48 h animal starvation or by treatment of the membrane fraction with alpha-amylase did not significantly alter phosphorylase kinase activity content. Washing of the initial pellet of SER fraction (crude SER) by dilution and recentrifugation, released in the supernatant an amount of phosphorylase kinase activity, which is dependent on: i) the degree of dilution, ii) the number of washes, iii) the ionic strength of the washing solution and iii) the presence or absence of Ca2+. Crude SER-associated phosphorylase kinase was marginally affected by increased concentrations of antibody against rabbit skeletal muscle holoenzyme which nevertheless drastically inhibited cytosolic enzyme activity, while it showed a higher resistance to partial proteolysis and a different Western blotting profile with anti-phosphorylase kinase when compared with the soluble kinase. A small but significant fraction of SER phosphorylase kinase was strongly associated with the microsomal fraction being partly extractable only in presence of detergents. This membrane-bound enzyme form exhibited an alkaline pH optimum, in contrast to the neutral pH optima of both soluble and weakly associated phosphorylase kinase.

[Association of rabbit skeletal muscle phosphorylase kinase with sarcoplasmic reticulum membranes]. / Assotsiatsiia kinazy fosforilazy skeletnykh myshts krolika s membranami sarkoplazmaticheskogo retikuluma.

The binding of phosphorylase kinase to sarcoplasmic reticulum has been studied using gel chromatography. The presence of Ca2+, Mg2+ and glycogen was found to be necessary for the maximal binding. The phosphorylase kinase adsorbed on sarcoplasmic reticulum is capable of phosphorylating exogenous phosphorylase b. Phosphorylase kinase was shown to take no part in the phosphorylation of sarcoplasmic reticulum proteins. Exogenous calmodulin initiates the incorporation of [gamma-32P] of ATP into sarcoplasmic reticulum proteins. The data obtained point to a possibility that another Ca(2+)-calmodulin-dependent protein kinase may participate in the phosphorylation of sarcoplasmic proteins.

Identification of the substrate and pseudosubstrate binding sites of phosphorylase kinase gamma-subunit.

Using site-directed mutagenesis, we proposed that an autoinhibitory domain(s) is located at the C-terminal region (301-386) of the phosphorylase kinase gamma-subunit (Huang, C.-Y.F., Yuan C.-J., Livanova, N.B., and Graves, D.J. (1993) Mol. Cell. Biochem. 127/128, 7-18). Removal of the putative inhibitory domain(s) by truncation results in the generation of a constitutively active and calmodulin-independent form, gamma 1-300. To probe the structural basis of autoinhibition of gamma-subunit activity, two synthetic peptides, PhK13 (gamma 303-327) and PhK5 (gamma 343-367), corresponding to the two calmodulin-binding regions, were assayed for their ability to inhibit gamma 1-300. Competitive inhibition of gamma 1-300 by PhK13 was found versus phosphorylase b (Ki = 1.8 microM) and noncompetitive inhibition versus ATP. PhK5 showed noncompetitive inhibition with respect to both phosphorylase b and ATP. Calmodulin released the inhibition caused by both peptides. These results indicate that there are two distinct auto-inhibitory domains within the C terminus of the gamma-subunit and that these two domains overlap with the calmodulin-binding regions. Two mutant forms of gamma 1-300, E111K and E154R, were used to probe the enzyme-substrate-binding region using peptide substrate analogs corresponding to residues 9-18 of phosphorylase b (KRK11Q12ISVRGL). The data suggest that Glu111 interacts with the P-3 position of the substrate (Lys11) and Glu154 interacts with the P-2 site (Gln12). Both E111K and E154R were competitively inhibited with respect to phosphorylase b by PhK13, with 14- and 8-fold higher Ki values, respectively, than that observed with the wild-type enzyme. These data are consistent with a model for the regulation of the gamma-subunit of phosphorylase kinase in which PhK13 acts as a competitive pseudosubstrate that directly binds the substrate binding site of the gamma-subunit (Glu111 and Glu154).

Expression, purification and crystallisation of phosphorylase kinase catalytic domain.

The catalytic subunit of phosphorylase kinase is composed of a kinase catalytic core domain (residues 1 to 298), which has a 33% identity with the kinase core of the cyclic AMP-dependent protein kinase, and a C-terminal calmodulin binding domain. The kinase domain of the catalytic subunit has been expressed in Escherichia coli, purified and crystallised in the presence of ATP and magnesium from 5% (w/v) polyethylene glycol 8000, 10% (v/v) glycerol, 50 mM Hepes/NaOH (pH 6.9). A three-fold excess of magnesium to ATP was used for crystal growth. The inclusion of glycerol in the crystallization medium produced a marked reduction in mosaic spread of the diffraction spots from greater than 1 degree to 0.3 degree. The crystals are orthorhombic, space group P2(1)2(1)2(1) with unit cell dimensions a = 47.1 A, b = 69.1 A, c = 112.9 A and one molecule per asymmetric unit. Data to 3 A resolution have been collected and structure determination is in progress.

Phosphorylation/activation of phosphorylase b kinase by cAMP/Ca2(+)-independent, autophosphorylation-dependent protein kinase.

Phosphorylase b kinase from rabbit skeletal muscle can be phosphorylated and activated by a cyclic nucleotide- and Ca2(+)-independent protein kinase previously identified as an autophosphorylation-dependent multifunctional protein kinase (auto-kinase) from brain and liver (Yang et al., J. Biol. Chem. 262, 7034-7040 (1987) and Yang et al. J. Biol. Chem. 262, 9421-9427 (1987)). This independent kinase phosphorylates both alpha and beta subunits of phosphorylase b kinase and results in a approximately 5-fold activation of the kinase when 0.55 and 0.5 mol of phosphate are incorporated into the alpha and beta subunits, respectively. Activation of phosphorylase b kinase catalyzed by auto-kinase is about 70% of that observed with cAMP-dependent protein kinase. Analysis of phosphopeptide maps of alpha and beta subunits further reveals that both kinases phosphorylate almost the same sites on both alpha and beta subunits, suggesting that activation of phosphorylase b kinase by the two kinases may be through a common molecular action mechanism. Taken together with the previous result that auto-kinase can inactivate glycogen synthase, the present study provides initial evidence that a coordinate control mechanism for simultaneous regulation of glycogenolysis and glycogenesis can be modulated by autophosphorylation-dependent protein kinase in a cAMP- and Ca2(+)-independent pathway, representing a new mode of control mechanism for the regulation of glycogen metabolism in cells.

Baculovirus-directed expression of the gamma-subunit of phosphorylase kinase: purification and calmodulin dependence.

A recombinant baculovirus containing a cDNA encoding the gamma-subunit of phosphorylase kinase from mouse skeletal muscle was constructed. Cultures of Sf-9 insect cells infected with the gamma-baculovirus produce an intact and soluble gamma-protein. A purification procedure is presented that yields a sample of gamma-protein which is devoid of interfering enzyme activity and which is not associated with calmodulin from the insect cells. The isolated gamma sample has a Km for phosphorylase b of 36 (+/- 6, S.E.M) microM at pH 8.2 and 140 (+/- 25) microM at pH 6.8. These values are similar to those reported for the activated phosphorylase kinase holoenzyme isolated from skeletal muscle tissue. However, the Vmax. of the baculovirus-expressed gamma is 65 and 80% of that of the activated holoenzyme at pH 6.8 and 8.2 respectively. These results indicate that one or more of the regulatory subunits alpha, beta, or calmodulin stimulate the activity of the catalytic subunit gamma in the activated holoenzyme. Addition of calmodulin to the baculovirus-expressed gamma stimulates its activity 1.5-2.0 fold at pH 6.8 in both the presence and absence of calcium. At pH 8.2, calmodulin has only minor stimulatory affects. The stimulation by calmodulin at pH 6.8 results from an increase in the Vmax of gamma with little effect on its Km. This result is unlike that for most calmodulin-stimulated kinases which bind calmodulin only in the presence of calcium and exhibit a decrease in their Km upon binding calmodulin. The change in Vmax. of gamma in the presence of calmodulin and in the absence of calcium presents a novel mechanism for the regulation of a calmodulin-stimulated kinase.

Preparation and functional characterization of a catalytically active fragment of phosphorylase kinase.

Limited proteolysis of rabbit muscle phosphorylase kinase catalyzed by chymotrypsin generates a 33 kD product whose kinase activity is independent of both calcium and pH over the range of 6.8 to 8.3 (Malencik, D.A. & Fischer, E.H. Calcium and Cell Function III: 161-188, 1982). This active preparation consists of three related species containing residues 1-290, 1-296, and 1-298 of the 44.7 kD gamma-subunit of phosphorylase kinase (Harris, W.R., Malencik, D.A., Johnson, C.M., Carr, S.A., Roberts, G.D., Byles, C.E., Anderson, S.R., Heilmeyer, L.M.G., Fischer, E.H. & Crabb, J.W.J. Biol. Chem. 265:11740-11745, 1991). Good recoveries of catalytic activity--with varying degrees of calcium dependence--result upon the digestion of phosphorylase kinase with assorted proteases. However, especially high yields of the chymotryptic fragment are obtainable, with purification on an Ultrogel-34 column and a DEAE Sepharose CL-6B column giving 23% of the maximum possible protein. Physical characterization shows that the 33 kD chymotryptic fragment is globular, with S20,w = 2.9S, and that it has an isoelectric point of 5.3. Our continuous catalytic assay, based on differences in the binding of the fluorescent dye 1-anilinonaphthalene-8-sulfonate by phosphorylase a and b, shows that, on a molar basis, the activity of the fragment is 2.8 fold greater than that of phosphorylase kinase (Malencik, D.A., Zhao, Z. and Anderson, S.R. Biochem. Biophys. Res. Comm. 174: 344-350, 1991). The active fragment also undergoes autophosphorylation. Incubation with Mg[gamma-P32] ATP results in the reaction of 0.7 mol 32P/mol fragment. When the catalytic subunit of the cAMP-dependent protein kinase is also present, the amount of 32P incorporated increases to 1.1 mol/mol. In the former case, phosphorylation occurs primarily at Ser30 while in the latter an additional reaction takes place at Ser81. The phosphopeptides correspond to sequences occurring in the gamma-subunit of phosphorylase kinase.

Expression, purification, characterization, and deletion mutations of phosphorylase kinase gamma subunit: identification of an inhibitory domain in the gamma subunit.

A catalytic fragment, gamma 1-298, derived from limited chymotryptic digestion of phosphorylase b kinase (Harris, W.R. et al., J. Biol. Chem., 265: 11740-11745, 1990), is reported to have about six-fold greater specific activity than does the gamma subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the gamma subunit, full-length wild-type and seven truncated forms of gamma were expressed in E. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar (gamma 1-353 and gamma 1-341) or less (gamma 1-331 and gamma 1-276) specific activity than does the full-length wild-type gamma, gamma 1-386. Three truncated forms, gamma 1-316, gamma 1-300, and gamma 1-290 have molar specific activities approximately twice as great as those of the full-length wild-type gamma and the nonactivated holoenzyme. All recombinant gamma s exhibit similar Km values for both substrates, i.e., about 18 microM for phosphorylase b and about 75 microM for MgATP. Three truncated gamma s, gamma 1-316, gamma 1-300, and gamma 1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (Vmax/Km) than that of the full-length wild-type gamma and a 3.5- to 4.5-fold greater efficiency than that of the truncated gamma 1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of gamma, which is located at gamma 301-331. gamma 1-290, but not gamma 1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the gamma subunit to exhibit phosphotransferase activity. Both gamma 1-290 and gamma 1-300 have several properties similar to full-length wild-type gamma, including metal ion responses (activation by free Mg2+ and inhibition by free Mn2+), pH dependency, and substrate specificities.

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.

The binding of phosphorylase kinase to immobilized calmodulin.

The binding of phosphorylase kinase to calmodulin-Sepharose 4B was studied by column and batch methods. It was found that the Ca2+ dependence of the interaction strongly depended on the degree of substitution of agarose with calmodulin. Equilibrium adsorption isotherms (i.e., bulk ligand binding functions and lattice site binding functions) of phosphorylase kinase were measured on calmodulin-Sepharose. Sigmoidal bulk ligand binding functions (bulk adsorption coefficients: 1.5-5.8) were found which indicate intermolecular attraction during binding. Hyperbolic lattice site binding functions (lattice adsorption coefficients: 1.0) were obtained thus excluding the existence of a critical surface concentration of immobilized calmodulin and indicating single independent binding sites on the gel surface and on phosphorylase kinase. These findings were combined to optimize the adsorption of phosphorylase kinase on calmodulin-Sepharose, for purification procedures at low Ca2+ concentrations (5-10 microM) minimizing proteolysis by calpains. With this novel method phosphorylase kinase from rabbit and frog skeletal muscle could be purified ca 100- and 200-fold, respectively, in two steps.

Phosphorylase kinase, a metal ion-dependent dual specificity kinase.

Phosphorylase kinase is shown to be a dual specificity kinase. The specificity of phosphorylation is determined by divalent cation. Mg2+ causes seryl phosphorylation of phosphorylase b, but Mn2+ activates tyrosine phosphorylation of angiotensin II. In contrast to seryl phosphorylation, the tyrosine kinase activity of holoenzyme is not regulated by Ca2+. Preincubation of the holoenzyme with Ca2+, Mg2+ and ATP that causes autophosphorylation activates tyrosine kinase activity. The tyrosyl kinase activity is a property of the gamma subunit. Addition of varying amounts of Mn2+ to a truncated form of the gamma subunit of phosphorylase kinase containing MgATP inhibits serine kinase but activates tyrosine kinase activity. This result along with an oxidative reaction caused by Cu2+ and site-directed mutagenesis of the putative catalytic base inhibiting both serine and tyrosine kinase activity suggest that one active site is involved in both activities. Kinetic studies with Mn2+ and ATP show that Km for nucleotide is not changed with a seryl or tyrosyl substrate. The Vm values are different, and the value for tyrosyl phosphorylation is similar to other tyrosyl kinases. We propose two conformations for the active site; one favors seryl phosphorylation, and the second tyrosyl phosphorylation is caused by the binding of divalent cation at a second metal ion binding site.

Isolation of an autoinhibitory region from the regulatory beta-subunit of phosphorylase kinase.

An equimolar mixture of the regulatory alpha- and beta-subunits of phosphorylase kinase has been shown to inhibit its catalytic gamma-subunit (Paudel, H.K., and Carlson, G.M. (1987) J. Biol. Chem. 262, 11912-11915). The possible presence of an autoinhibitory sequence within those regulatory subunits has been evaluated by peptide isolation and characterization following chemical and proteolytic cleavage of an isolated equimolar mixture of those subunits; the peptides generated were tested for their ability to inhibit the activity of a complex of the gamma-subunit and calmodulin. An isolated inhibitory fragment, hereafter referred to as I-peptide, was sequenced and found to correspond to residues 420-436 of the beta-subunit (KRNPGSQKRFPSNCGRD). This sequence showed homology with the kinase's natural substrate, phosphorylase b. A synthetic peptide based on this sequence was constructed and used to study the mechanism of inhibition. Kinetic analysis of the inhibition of the gamma-subunit-calmodulin complex by the I-peptide revealed a competitive pattern versus the homologous substrate phosphorylase b and an uncompetitive pattern versus MgATP, suggesting an ordered binding of substrates, with the nucleotide binding first. In addition to its ability to inhibit, the I-peptide was also a substrate for the gamma-subunit-calmodulin complex, with a relatively good Km but poor Vmax. The parallel inhibition of free gamma-subunit and the gamma-subunit-calmodulin complex by progressively increasing concentrations of I-peptide provided further evidence that the peptide inhibits by interacting directly with the catalytic subunit and not with the stimulatory calmodulin molecule. The results of this study are consistent with previous findings from this laboratory showing that the conformation of the beta-subunit changes following activation of phosphorylase kinase through a variety of mechanisms.