Phosphorylation and inactivation of rabbit skeletal muscle glycogen synthase: distinction between kinase Fa-, phosphorylase kinase-, and glycogen synthase (casein) kinase-1-catalyzed reactions

Rabbit skeletal muscle glycogen synthase was phosphorylated by kinase Fa, phosphorylase kinase, and cAMP-independent synthase (casein) kinase-1 to determine the differences among these kinase-catalyzed reactions. The stoichiometry of phosphate incorporation, the extent of inactivation, and the sites of phosphorylation were compared. Synthase (casein) kinase-1 catalyzes the highest level of synthase phosphorylation (4 mol/subunit) and inactivation (reduction of the activity ratio to below 0.05). The sites, defined by characteristic tryptic peptides, phosphorylated by synthase (casein) kinase-1 are distinguishable from those by kinase Fa and phosphorylase kinase. In addition, synthase (casein) kinase-1, unlike kinase Fa, does not activate ATP X Mg2+-dependent protein phosphatase. These results demonstrate that synthase (casein) kinase-1 is a distinct glycogen synthase kinase.     

Comparison of the phosphorylation of rabbit skeletal muscle phosphorylase kinase by cAMP-dependent protein kinase and cAMP-independent glycogen synthase (casein) kinase-1

We have previously reported that rabbit skeletal muscle phosphorylase kinase is phosphorylated by glycogen synthase (casein) kinase-1 (CK-1) primarily on the beta subunit (beta = 1 mol of PO4; alpha = 0.2 mol of PO4) when the reaction was carried out in beta-glycerophosphate. The resultant enzyme activation was 16-fold (Singh, T. J., Akatsuka, A., and Huang, K.-P. (1982) J. Biol. Chem. 257, 13379-13384). In the present study we found that in Tris-Cl buffer CK-1 catalyzes the incorporation of greater than 2 mol of PO4/monomer into each of the alpha and beta subunits. Phosphorylase kinase activation resulting from the higher level of phosphorylation remained 16-fold. 32P-Labeled tryptic peptides from the alpha and beta subunits were analyzed by isoelectric focusing. Cyclic AMP-dependent protein kinase (A-kinase) phosphorylates a single major site in each of the alpha and beta subunits at 1.5 mM Mg2+. In addition to these two sites, A-kinase phosphorylates at least three other sites in the alpha subunit at 10 mM Mg2+. CK-1 also catalyzes the phosphorylation of multiple sites in both the alpha and beta subunits. Of the two major sites phosphorylated by CK-1 in the beta subunit, one of these sites is also recognized by A-kinase. At least three sites are phosphorylated by CK-1 in the alpha subunit. One of these sites is recognized by CK-1 only after a prior phosphorylation of phosphorylase kinase by A-kinase at a single site in each of the alpha and beta subunits at 1.5 mM Mg2+. The roles of the different phosphorylation sites in phosphorylase kinase activation are discussed.     

Autophosphorylation of phosphorylase kinase. Divalent metal cation and nucleotide dependency

This study reports on the divalent metal ion specificity for phosphorylase kinase autophosphorylation and, in particular, provides a comparison between the efficacy of Mg2+ and Mn2+ in this role. As well as requiring Ca2+ plus divalent metal ion-ATP2- as substrate, both phosphorylase kinase autoactivation and phosphorylase conversion are additionally modulated by divalent cations. However, these reactions are affected differently by different ions. Phosphorylase kinase-catalyzed phosphorylase conversion is maximally enhanced by a 4- to 10-fold lower concentration of Mg2+ than is autocatalysis and, whereas both reactions are stimulated by Mg2+, autophosphorylation is activated by Mn2+, Co2+, and Ni2+ while phosphorylase a formation is inhibited. This difference may be due to an effect of free Mn2+ on phosphorylase rather than the inability of phosphorylase kinase to use MnATP as a substrate when catalyzing phosphorylase conversion since Mn2+, when added at a level which minimally decreases [MgATP], greatly inhibits phosphorylase phosphorylation. The interactions of Mn2+ with phosphorylase kinase are different from those of Mg2+. Not only are the effects of these ions on phosphorylase activation opposite, but they also provoke different patterns of subunit phosphorylation during phosphorylase kinase autocatalysis. With Mn2+, the time lag of phosphorylation of both the alpha and beta subunits of phosphorylase kinase in autocatalysis is diminished in comparison to what is observed with Mg2+, and the beta subunit is only phosphorylated to a maximum of 1 mol/mol of subunit. With both Mg2+ and Mn2+ the alpha subunit is phosphorylated to a level in excess of 3 mol/mol, a level similar to that obtained for beta subunit phosphorylation in the presence of Mg2+. The support of autophosphorylation by both Co2+ and Ni2+ has characteristics similar to those observed with Mn2+. Although Mn2+ stimulation of autophosphorylation occurs at levels much higher than normal physiological levels, the possible potential of phosphorylase kinase autophosphorylation as a control mechanism is illustrated by the 80- to 100-fold activation that occurs in the presence of Mn2+, a level far in excess of the enzyme activity change normally seen with covalent modification. Autophosphorylation of phosphorylase kinase demonstrates a Km for Mg X ATP2- of 27.7 microM and a Ka for Mg2+ of 3.1 mM. The reaction mechanism of autophosphorylation is intramolecular. This latter observation may indicate that phosphorylase kinase autocatalysis could be of potential physiological relevance and could occur with equal facility in cells containing either constitutively high or low levels of this enzyme.     

Subunit phosphorylation and activation of skeletal muscle phosphorylase kinase by the cAMP-dependent protein kinase. Divalent metal ion, ATP, and protein concentration dependence

This report provides a characterization of the effects of varying the concentrations of Mg2+, ATP, phosphorylase kinase, and the cAMP-dependent protein kinase on the activation and phosphorylation of phosphorylase kinase. The results show the following. (a) The Km for MgATP2- for the cAMP-dependent protein kinase-catalyzed phosphorylation is decreased by increasing Mg2+, probably as a consequence of decreasing the free ATP:MgATP2- ratio and increasing free Mg2+. (b) Whereas beta subunit phosphorylation of phosphorylase kinase plays a prominent role in determining its activity, alpha subunit phosphorylation can also modulate activity. (c) The phosphorylation of the alpha subunit, which occurs following the initial cAMP-dependent phosphorylation of the beta subunit, is catalyzed by the cAMP-dependent protein kinase and is not a consequence of EGTA-insensitive (or EGTA-sensitive) autophosphorylation occurring as a result of the enhanced phosphorylase kinase activity. (d) The relationship between subunit phosphorylation and phosphorylase kinase activation is complex and particularly dependent upon concentrations of cAMP-dependent protein kinase and phosphorylase kinase in the activation reaction. The data suggest the possibilities that the pathway of phospho-intermediates involved in the activation process probably varies with the activation conditions, that the efficacy of a specific site to be covalently modified is dependent upon the phosphorylation status of other sites, and that the effect of phosphorylation in regulating activity may also be dependent on the phosphorylation status of other sites. It is clear from the data that the activation process for phosphorylase kinase can be very complex, and it is possible that this complexity might have significant physiological ramifications.     

Phosphorylation and activation of the cardiac isoenzyme of phosphorylase kinase by the cAMP-dependent protein kinase

The cAMP-dependent protein kinase catalyzes the phosphorylation of the alpha- and beta-subunits of the cardiac isozyme of phosphorylase kinase. beta-Subunit phosphorylation achieves a maximum level of between 1 to 2 mol of phosphate/mol of phosphorylase kinase, a value less than the stoichiometric content of beta-subunits in the enzyme. This, less than stoichiometric incorporation, is not a result of the presence of endogenous phosphate in equivalent sites in the remaining beta-subunit moieties. Pretreatment of phosphorylase kinase with phosphoprotein phosphatase, under conditions proven to dephosphorylate such sites, does not modify the observed extent of beta-subunit phosphorylation. alpha'-Subunit phosphorylation is initiated at a slower rate than beta but achieves a higher maximum level of incorporation. alpha'-Subunit phosphorylation, but not the extent of beta-subunit phosphorylation, is stimulated by MnCl2 and partially inhibited by NaF; neither is effected by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The activation of cardiac phosphorylase kinase that occurs concomitantly with phosphorylation appears to be dependent upon phosphate incorporation into both the alpha- and beta-subunits. At low levels of activation a close correlation is observed between activation and either alpha-subunit phosphorylation, beta-subunit phosphorylation, or total phosphorylation. However, the cAMP-dependent catalyzed phosphorylation of alpha, at a time after which beta-subunit phosphorylation is already maximal, also results in activation of cardiac phosphorylase kinase.     

The role of the cyclic AMP-dependent protein kinase in the glucagon-stimulated phosphorylation of ATP-citrate lyase

We have examined the mechanism whereby glucagon stimulates the phosphorylation of ATP-citrate lyase in intact rat hepatocytes. Purified ATP-citrate lyase is phosphorylated in vitro by the catalytic subunit of the cyclic AMP-dependent protein kinase, in a reaction wherein 2-3 mol phosphate/mol lyase are incorporated, at an initial rate that approaches that observed for mixed histone. This reaction is completely abolished by the protein kinase inhibitor protein. Limited tryptic digestion of ATP-citrate lyase phosphorylated in vitro by the cyclic AMP-dependent protein kinase yields a pattern of 32P-labeled peptides, indistinguishable from those observed in parallel digests of lyase isolated from 32P-labeled, glucagon-stimulated hepatocytes. Phosphorylase b kinase catalyzes the incorporation of 1 mol phosphate/mol lyase, albeit at less than 1/160 the rate observed for phosphorylase b. The phosphorylation of purified ATP-citrate lyase is also catalyzed by homogenates of hepatocytes. This reaction is stimulated by cyclic AMP. At 30 degrees C, in the presence of maximally stimulating concentrations of cyclic AMP, the addition of excess protein kinase inhibitor protein inhibits the phosphorylation of ATP-citrate lyase by 67%. Thus, hepatocytes contain both cyclic AMP-dependent and cyclic AMP-independent ATP-citrate lyase kinase activities. Pretreatment of hepatocytes with glucagon (10(-8) M for 2 min) prior to homogenization results in activation of an endogenous hepatocyte ATP-citrate lyase kinase, as well as histone kinase and phosphorylase b kinase; the glucagon-stimulated increment in lyase kinase (and histone kinase) is observed only when homogenates are assayed in the absence of added cyclic AMP, and is completely abolished by an excess of the protein kinase inhibitor protein. We conclude that the glucagon-stimulated phosphorylation of ATP-citrate lyase in intact hepatocytes is catalyzed directly by the cyclic AMP-dependent protein kinase.     

Phosphorylation of calf thymus H1 histone by muscle glycogen phosphorylase kinase

Muscle glycogen phosphorylase kinase [EC 2.7.1.38] has the ability to phosphorylate five fractions of calf thymus histone. H1 histone is the most preferable substrate, and maximally about 1.3 mol of phosphate is incorporated into every mole of this histone. This reaction absolutely depends on CA2+, and the molecular activity is about one third of that of cyclic AMP-dependent protein kinase (protein kinase A). The affinity of phosphorylase kinase for H1 histone is higher than that of protein kinase A. Calmodulin stimulates this histone phosphorylation. Analysis of the N-bromosuccinimide-bisected fragments of fully phosphorylated H1 histone has revealed that the enzyme phosphorylates mostly seryl residues in both amino- and carboxyl-terminal portions, although phosphorylation of the carboxyl-terminal portion is twice as much as that of the amino-terminal portion. Fingerprint analysis indicates that the phosphorylation sites in H1 histone for this enzyme are different from the sites phosphorylated by protein kinase A. This catalytic activity also differs from that of a newly found multifunctional protein kinase which may be activated by the simultaneous presence of Ca2+ and phospholipid.     

Phosphorylation of ribosomal protein S6 at multiple sites by a cyclic AMP-independent protein kinase from lymphoid cells

Ribosomes prepared from murine lymphosarcoma cells were phosphorylated by a cyclic AMP-independent protein kinase designated H4P kinase. H4P kinase was isolated as an inactive enzyme which was activated by Mg2+-ATP and an endogenous converting enzyme. In the absence of preactivation by Mg2+-ATP and an endogenous converting enzyme, H4P kinase catalyzed phosphorylation of 80, 60, and 40 S ribosomal subunits at a low rate. After activation, the H4P kinase selectively catalyzed phosphorylation of the S 6 protein in the 40 S ribosomal subunit. Under the assay conditions selected, at least 90% of the [32P]phosphate transferred to the 40 S ribosomal preparation was incorporated into S 6. The apparent Km for 40 S subunits phosphorylated by H4P kinase was 7.2 microM. The calculated Vmax was 50 nmol of Pi transferred per min/mg. Exhaustive phosphorylation of 40 S subunits resulted in incorporation of 3 mol of phosphate/mol of S 6, in contrast to results reported previously which indicated 0.3 mol of phosphate was transferred by a similar enzyme from reticulocyte (Del Grande, R. W., and Traugh, J. A. (1982) Eur. J. Biochem. 123, 421-428). These data are consistent with a potential role for H4P kinase in the insulin-mediated phosphorylation of S 6 at multiple sites.     

Limited proteolysis and phosphorylation of phosphorylase kinase from chicken skeletal muscles

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

Characterization of fodrin phosphorylation by spleen protein tyrosine kinase

Fodrin, an actin and calmodulin binding and spectrin-like protein present in many nonerythrocyte tissues, could be phosphorylated up to more than 1.5 mol of phosphate/mol of protein by a highly purified non-receptor-associated protein tyrosine kinase from bovine spleen. The protein phosphorylation was not affected by Ca2+/calmodulin or by F-actin. Km and Vmax values of the reaction were 91 nM and 0.35 nmol of P2 min-1 (mg of kinase)-1, respectively. Both subunits A and B of fodrin were phosphorylated, with the rate of subunit A phosphorylation much greater than that of subunit B phosphorylation. Tryptic phosphopeptide mapping of the phosphorylated subunits suggested that there were three major phosphorylation sites in subunit A and one in subunit B. Phosphotyrosylfodrin could be dephosphorylated by the calmodulin-stimulated phosphatase (calcineurin) in the presence of activating metal ions; Ni2+ was a much more effective activator than Mn2+ for this reaction. Fodrin phosphorylation by the spleen protein tyrosine kinase did not appear to alter the actin and calmodulin binding properties of the protein. On the other hand, the calmodulin-dependent stimulation of smooth muscle actomyosin Mg2+-ATPase by fodrin was enhanced by 101% +/- 3% (n = 3) upon fodrin phosphorylation. Ni2+-calcineurin, which was shown to effectively dephosphorylate the phosphotyrosyl residues on fodrin, could reverse the phosphorylation-enhanced Mg2+-ATPase stimulatory activity of fodrin.     

Phosphorylation of L-type pyruvate kinase by a Ca2+/calmodulin-dependent protein kinase

Rat liver L-type pyruvate kinase was phosphorylated in vitro by a Ca2+/calmodulin-dependent protein kinase purified from rabbit liver. The calmodulin (CaM)-dependent kinase catalyzed incorporation of up to 1.7 mol of 32P/mol of pyruvate kinase subunit; maximum phosphorylation was associated with a 3.0-fold increase in the K0.5 for P-enolpyruvate. This compares to incorporation of 0.7 to 1.0 mol of 32P/mol catalyzed by the cAMP-dependent protein kinase with a 2-fold increase in K0.5 for P-enolpyruvate. When [32P]pyruvate kinase, phosphorylated by the CaM-dependent protein kinase, was subsequently incubated with 5 mM ADP and cAMP-dependent protein kinase (kinase reversal conditions), 50-60% of the 32PO4 was removed from pyruvate kinase, but the K0.5 for P-enolpyruvate decreased only 20-30%. Identification of 32P-amino acids after partial acid hydrolysis showed that the CaM-dependent protein kinase phosphorylated both threonyl and seryl residues (ratio of 1:2, respectively) whereas the cAMP-dependent protein kinase phosphorylated only seryl groups. The two phosphorylation sites were present in the same 3-4-kDa CNBr fragment located near the amino terminus of the enzyme subunit. These results indicate that the CaM-dependent protein kinase catalyzed phosphorylation of L-type pyruvate kinase at two discrete sites. One site is apparently the same serine which is phosphorylated by the cAMP-dependent protein kinase. The second site is a unique threonine residue whose phosphorylation also inactivates pyruvate kinase by elevating the K0.5 for P-enolpyruvate. These results may account for the Ca2+-dependent phosphorylation of pyruvate kinase observed in isolated hepatocytes.     

Activation of bovine heart ATP-MG2+-dependent phosphoprotein phosphatase: isolation of a phosphoenzyme intermediate and its conversion to the active form via a Mg2+-dependent autodephosphorylation reaction

The ATP-Mg2+-dependent protein phosphatase, a holoenzyme form of type I protein phosphatase (phosphatase-1) requires the action of phosphatase-1 kinase (FA) for activation. The enzyme (75 kDa) purified from bovine heart consists of a catalytic (C) and a regulatory (R) subunit of 40 kDa and 34 kDa, respectively, and activation is associated with phosphorylation of the R-sub-unit. A procedure has been developed for isolation of [32P]phosphatase-1 ( [32P]E-P) in non-denatured form. In the absence of divalent cation, [32P]E-P is catalytically inactive and the phosphorylation is stable. Addition of Mg2+ triggers autodephosphorylation of [32P]E-P with concomitant generation of phosphorylase phosphatase activity. The autodephosphorylation/activation process is dependent on Mg2+ concentration. The KA value for Mg2+ is 0.6 mM. The phosphorylase phosphatase activity generated from the release of 1 mol. 32P is 1.1 X 10(12) units which is equivalent to 15,000 units per mg enzyme protein. The present findings provide direct evidence that the phosphorylated phosphatase-1 is not the active form (Ea). Instead, Ea is directly produced from the intermediate by a Mg2+-dependent autodephosphorylation reaction.     

Identification of three phosphorylation sites on each heavy chain of Acanthamoeba myosin II

It has been previously demonstrated that the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II is inhibited by phosphorylation of its two heavy chains (Collins, J. H., and Korn, E. D. (1980) J. Biol. Chem. 255, 8011-8014). In this paper, it is shown that a partially purified kinase preparation from Acanthamoeba catalyzes the incorporation of 3 mol of phosphate into each mole of myosin II heavy chain. Tryptic digestion of the 32P-myosin, followed by two-dimensional peptide mapping, indicates that two of the three sites phosphorylated by the kinase in vitro correspond to the two major phosphorylation sites on the myosin heavy chain in vivo. Phosphorylation of myosin II in vitro by the kinase fraction completely inhibits the actin-activated Mg2+-ATPase activity of myosin II. Myosin II can be isolated in a highly phosphorylated, enzymatically inactive form, then dephosphorylated to an active form, and finally rephosphorylated to an inactive form. The Acanthamoeba kinase fraction catalyzes the phosphorylation of all three sites on the heavy chain of myosin II at virtually the same rate. From a comparison of the decrease in actin-activated Mg2+-ATPase activity with the amount of phosphate incorporated into myosin II, and from the results obtained previously by dephosphorylating myosin II (Collins, J. H., and Korn, E. D., (1980) J. Biol. Chem. 255, 8011-8014), it can be inferred that two of the sites phosphorylated in vitro act in a synergistic manner to inhibit the actin-activated myosin II Mg2+-ATPase.     

Autophosphorylation of the alpha subunit of phosphorylase kinase from rabbit skeletal muscle

The autophosphorylation of the alpha subunit of phosphorylase kinase occurs simultaneously at multiple sites during incorporation of the first mol of phosphate. The predominant and initial autophosphorylation site on this subunit is different than the major site phosphorylated by cAMP-dependent protein kinase, which also phosphorylates multiple sites, as evidenced by two-dimensional phosphopeptide maps. All of the sites on the alpha subunit phosphorylated by cAMP-dependent protein kinase comigrate on peptide maps with autophosphorylation phosphopeptides; however, several phosphopeptides observed after autophosphorylation are not evident following phosphorylation by cAMP-dependent protein kinase. The phosphopeptide maps of the alpha subunit are the same whether autophosphorylation is carried out at pH 6.8 or 8.2 or whether MnATP is used instead of MgATP; there is only a slight difference in the maps brought about by EGTA-insensitive autophosphorylation. The autophosphorylation is shown to be an intrinsic activity of the phosphorylase kinase molecule; this conclusion is based on the observed copurification of the autophosphorylation activity with activities toward phosphorylase b and kappa-casein and the unaltered influence of various effectors on these activities throughout different sequential adsorption chromatography purification steps. Additional support to that already in the literature that the initial autophosphorylation events are predominantly intramolecular is gained by showing that previously autophosphorylated enzyme has little ability to catalyze the phosphorylation of nonphosphorylated enzyme.     

The role of Ca2+ and cyclic AMP in the phosphorylation of rat-liver soluble proteins by endogenous protein kinases

Rat liver soluble proteins were phosphorylated by endogenous protein kinase with [gamma-32P]ATP. Proteins were separated in dodecyl sulphate slab gels and detected with the aid of autoradiography. The relative role of cAMP-dependent, cAMP-independent and Ca2+-activated protein kinases in the phosphorylation of soluble proteins was investigated. Heat-stable inhibitor of cAMP-dependent protein kinase inhibits nearly completed the phosphorylation of seven proteins, including L-type pyruvate kinase. The phosphorylation of eight proteins is not influenced by protein kinase inhibitor. The phosphorylation of six proteins, including phosphorylase, is partially inhibited by protein kinase inhibitor. These results indicate that phosphoproteins of rat liver can be subdivided into three groups: phosphoproteins that are phosphorylated by (a) cAMP-dependent protein kinase or (b) cAMP-independent protein kinase; (c) phosphoproteins in which both cAMP-dependent and cAMP-independent protein kinase play a role in the phosphorylation. The relative phosphorylation rate of substrates for cAMP-dependent protein kinase is about 15-fold the phosphorylation rate of substrates for cAMP-independent protein kinase. The Km for ATP of cAMP-dependent protein kinase and phosphorylase kinase is 8 microM and 38 microM, respectively. Ca2+ in the micromolare range stimulates the phosphorylation of (a) phosphorylase, (b) a protein with molecular weight of 130 000 and (c) a protein with molecular weight of 15 000. The phosphate incorporation into a protein with molecular weight of 115 000 is inhibited by Ca2+. Phosphorylation of phosphorylase and the 15 000-Mr protein in the presence of 100 microM Ca2+ could be completely inhibited by trifluoperazine. It can be concluded that calmodulin is involved in the phosphorylation of at least two soluble proteins. No evidence for Ca2+-stimulated phosphorylation of subunits of glycolytic or gluconeogenic enzymes, including pyruvate kinase, was found. This indicates that it is unlikely that direct phosphorylation by Ca2+-dependent protein kinases is involved in the stimulation of gluconeogenesis by hormones that act through a cAMP-independent, Ca2+-dependent mechanism.     

Purification and properties of cyclic AMP-independent glycogen synthase kinase 1 from rabbit skeletal muscle.

A cyclic AMP-independent casein (phosvitin) kinase eluted from a phosphocellulose column with 0.35 M KCl also possesses glycogen synthase kinase activity. This kinase, designated synthase kinase 1, is separable from other cyclic AMP-independent protein kinases, which also contain glycogen synthase kinase activity, by chromatography on a phosphocellulose column. This kinase was purified 15,000-fold from the crude extract. Synthase kinase activity co-purifies with casein and phosvitin kinase activities. Heat inactivation of these three kinase activities follow similar kinetics. It is suggested that these three kinase activities reside in a single protein. This kinase has a molecular weight of approximately 34,000 as determined by glycerol density gradient centrifugation and by gel filtration. The Km values for the synthase kinase-catalyzed reaction are 0.12 mg/ml (0.35 micronM) for synthase, 12 micronM for ATP, and 0.15 mM for Mg2+. The phosphorylation of glycogen synthase by the kinase results in the incorporation of 4 mol of phosphate/85,000 subunit; however, only two of the phosphate sites predominantly determine the glucose-6-P dependency of the synthase. Synthase kinase activity is sensitive to inhibition by NaCl or KCl at concentrations encountered during purification. Synthase kinase activity is insensitive to the allosteric effector (glucose-6-P) or substrate (UDP-glucose) of glycogen synthase at concentrations usually found under physiological condition.     

Amino acid sequence of the phosphorylation site of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase from rat liver was phosphorylated by cyclic AMP-dependent protein kinase and [gamma-32P]ATP. Treatment of the 32P-labeled enzyme with thermolysin removed all of the radioactivity from the enzyme core and produced a single labeled peptide. The phosphopeptide was purified by ion exchange chromatography, gel filtration, and reverse phase high pressure liquid chromatography. The sequence of the 12-amino acid peptide was found to be Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser(P)-Ser-Ile-Pro-Gln. Correlation of the extent of phosphorylation with activity showed that a 50% decrease in the ratio of kinase activity to bisphosphate activity occurred when only 0.25 mol of phosphate was incorporated per mol of enzyme subunit, and maximal changes occurred with 0.7 mol incorporated. The kinetics of cyclic AMP-dependent protein kinase-catalyzed phosphorylation of the native bifunctional enzyme was compared with that of other rat liver protein substrates. The Km for 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (10 microM) was less than that for rat liver pyruvate kinase (39 microM), fructose-1,6-bisphosphatase (222 microM), and 6- phosphofructose -1-kinase (230 microM). Comparison of the initial rate of phosphorylation of a number of protein substrates of the cyclic AMP-dependent protein kinase revealed that only skeletal muscle phosphorylase kinase was phosphorylated more rapidly than the bifunctional enzyme. Skeletal muscle glycogen synthase, heart regulatory subunit of cyclic AMP-dependent protein kinase, and liver pyruvate kinase were phosphorylated at rates nearly equal to that of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, while phosphorylation of fructose-1,6-bisphosphatase and 6-phosphofructo-1-kinase was barely detectable. Phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was not catalyzed by any other protein kinase tested. These results are consistent with a primary role of the cyclic AMP-dependent protein kinase in regulation of the enzyme in intact liver.     

A specific substrate from rabbit cerebellum for guanosine 3':5'-monophosphate-dependent protein kinase. II. Kinetic studies on its phosphorylation by guanosine 3':5'-monophosphate-dependent and adenosine 3':5'-monophosphate-dependent protein kinases

Kinetic studies on the activity of purified cGMP-dependent protein kinase and catalytic subunit of cAMP-dependent protein kinase have been carried out using a protein termed G-substrate (see preceding paper) as the phosphate acceptor. Each enzyme catalyzed the phosphorylation of 2.0-2.1 mol of 32P/mol of G-substrate, with phosphorylation occurring primarily at threonine residues. When phosphorylation was carried out in the simultaneous presence of the two enzymes, the stoichiometry increased only slightly, to a value of 2.4, suggesting that both enzymes phosphorylated the same two sites. Initial rate studies on the phosphorylation of G-substrate by cGMP-dependent protein kinase yielded a Km of 0.21 microM and a Vmax of 2.2 mumol/min/mg. Similar studies with the cAMP-dependent protein kinase yielded a Km of 5.8 microM and a Vmax of 2.3 mumol/min/mg. cGMP-dependent protein kinase thus exhibited a high degree of specificity towards this substrate which was apparently based on selective substrate binding rather than catalytic efficacy. The activity of cGMP-dependent protein kinase towards G-substrate was maximal at pH 7.5-8.0 and a Mg2+ concentration of 1-3 mM. Activity declined sharply at high ionic strength (greater than 20 mM KCl).     

Protein kinase and specific phosphate acceptor proteins associated with tupaia herpesvirus.

A protein kinase activity was found to be associated with tree shrew (tupaia) herpesvirus. The protein kinase was characterized with respect to its requirements for enzymatic activity. A divalent cation such as Mg2+ or Mn2+ was necessary as well as ATP as the phosphate donor. Distinct tupaia herpesvirus polypeptides (molecular weights of 100,000 [100K], 82K, and 53K) were found to be phosphate acceptor proteins when 5 mM Mg2+ was used. At a higher Mg2+ concentration (20 mM), additional viral proteins (220K, 71K, 31K, and 20K) were phosphorylated. The viral phosphoproteins were analyzed by chemical and enzymatic hydrolyses. The predominant sites of phosphorylation were the beta-OH groups of the serine and threonine residues of these tupaia herpesvirus proteins. Kinase activity was not stimulated by cyclic nucleoside monophosphates. Endogenously added proteins did not enhance protein kinase activity. Protein kinase activity was inhibited by 5'-p-fluorosulfonylbenzoyladenosine.     

The phosphorylation of purified phospholamban by cyclic AMP-dependent protein kinase is stimulated by phosphatidylinositol

A pure bovine phospholamban sample was phosphorylated by cyclic AMP-dependent protein kinase maximally to about 1 mol of phosphate/mol of protein (Mr 25,000), whereas phospholamban purified from bovine cardiac SR (sarcoplasmic reticulum) vesicle prephosphorylated by the protein kinase was found to contain 4.6 mol of phosphate/mol of phospholamban. The decrease in phospholamban phosphorylation occurred during the protein purification at the immunoaffinity chromatography step. The protein phosphorylation could be restored by the addition of the affinity column flow-through fraction to the phosphorylation reaction. The phosphorylation-stimulating activity of the flow-through fraction was resistant to boiling and trypsin treatment and extractable by organic solvent, suggesting that the endogenous factor(s) is lipid. Various phospholipids were found capable of stimulating the phosphorylation of phospholamban by cyclic AMP-dependent protein kinase, but only phosphatidylinositol could stimulate the protein phosphorylation to a level achieved by the phosphorylation of SR membrane-bound phospholamban, about 5 mol of phosphate/mol. Phospholamban phosphorylated in the presence of phosphatidylinositol showed similar sites of phosphorylation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility shifts as the phospholamban isolated from phosphorylated SR vesicles. Results of the present study suggest that phospholamban in SR is embedded in a phosphatidylinositol-rich microenvironment, and that this specific environment may be important for the regulation of Ca2+ pump by phospholamban.