The phosphorylation of substrate peptides derived from PKI, the heat-stable inhibitor protein of the cAMP-dependent protein kinase (PKA), has been studied with both PKA and the cGMP-dependent protein kinase (PKG) using a variety of substitution and deletion analogs. On the basis of Km, kcat and kcat/Km values, (Ser21)PKI alpha(14-22) amide (numbering based upon native PKI alpha) is the most effective peptide substrate yet discovered for either kinase, although other peptides, while phosphorylated considerably less efficiently by PKG, are more specific. Although the inhibitory peptide corresponding to this sequence (i.e., with an Ala at position 21) is a much more potent inhibitor of PKA than of PKG (approximately 250-fold), PKG actually exhibits a 60% higher kcat than does PKA with the (Ser21)PKI alpha(14-22) amide substrate peptide, with only a 20-fold higher Km value. The two key PKI residues within this peptide which were found to be essential for substrate activity with both kinases were Arg18 (P-3) and Ile22 (P+1). The Arg19 (P-2) residue, which contributes significantly to both PKI-based peptide inhibitors and substrates of PKA, was only a more minor contributor to PKG substrate efficacy. Of particular note, the Phe10 (P-11) residue, which contributes very substantially to high affinity binding of both PKI and longer PKI peptide inhibitors, neither positively nor negatively affects the kinetics of either PKA or PKG with PKI-based substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylase kinase, a key enzyme in glycogen metabolism, has a subunit composition of (alphabetagammadelta)4, in which the alpha and beta subunits are regulatory, delta is calmodulin, and the gamma subunit is catalytic. As one segment of our studies on the regulation of the expression of phosphorylase kinase subunits, we present in this report the structure of the gene for the catalytic gamma subunit. The gene extends over 16 kilobase pairs (kb) of DNA, and contains eight introns within the coding region plus one 3.3-kb intron upstream in the 5'-untranslated region. Within this first intron, and also upstream of the transcription start site, are sequences homologous to defined regulatory elements, including some found in other muscle-specific genes. The positions of intron splice junctions for this gene have been compared with similar data for other protein kinase genes. A somewhat unexpected finding for the gamma subunit is that two of the splice junctions fall in the midst of highly conserved strings of amino acids, both of which have been nominally defined as functional domains for the protein kinases and appear to make key contributions to substrate binding and phosphotransferase catalysis.

Phosphorylase kinase is a calcium-regulated multimeric enzyme of composition (alpha-beta-gamma-delta)4, which contains calmodulin as the integral delta-subunit and also is activated further by addition of extrinsic calmodulin. Previous studies by Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. ((1989) J. Biol. Chem. 264, 1715617163) have identified gamma-302-326 and gamma-342-366 as two calmodulin binding regions. Using peptides that were synthesized based on alpha and beta-primary structure and that were predicted to contain the basic amphiphilic alpha-helix motif thought important for calmodulin binding, four additional potential calmodulin binding domains have now been identified: one of high affinity, beta-770-794; two of intermediate affinity, beta-5-28 and beta-920-946; and one with marginally low affinity, alpha-1070-1093. Peptide beta-770-794 was of higher calmodulin affinity than either gamma-302-326 or gamma-342-366; it was of higher affinity than the model synthetic peptide IV defined by O' Neil, K. T., and DeGrado, W. F. ((1990) Trends Biochem. Sci. 15, 59-64); and it is currently the most potent calmodulin-binding peptide so far described. Correlated with their affinity for calmodulin, all six phosphorylase kinase-derived peptides and several other established calmodulin-binding peptides inhibited phosphorylase kinase previously activated by cAMP-dependent phosphorylation, reducing its activity to the level of the nonactivated enzyme. However, these peptides did not inhibit (and some peptides slightly activated) the nonphosphorylated enzyme. Even in the presence of these peptides both activated and nonactivated enzyme remained fully Ca2+-dependent. The beta-770-794 peptide has at least a 5-fold greater calmodulin binding affinity than the holo-phosphorylase kinase. This, and its higher affinity for calmodulin than either of the sites on the gamma-subunit, raises the possibility that in the native enzyme it may be involved in binding the intrinsic delta-subunit. Further, inhibition of activated but not nonactivated enzyme by calmodulin-binding peptides would suggest that the phosphorylation-dependent activation of phosphorylase kinase may be mediated by changes in the binding interactions of the intrinsic calmodulin delta-subunit.

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

articleMotifs of protein phosphorylation and mechanisms of reversible covalent regulationD. A. Walsh, P. Newsholme, K. C. Cawley, S. M. van Patten, and K. L. AngelosD. A. Walsh, P. Newsholme, K. C. Cawley, S. M. van Patten, and K. L. AngelosPublished Online:01 Jan 1991https://doi.org/10.1152/physrev.1991.71.1.285MoreSectionsPDF (6 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformationCited ByProtein phosphatase inhibitors arrest cell cycle and reduce branching morphogenesis in fetal rat lung culturesB. Keith Taylor, Tamara D. Stoops, and Allen D. Everett1 May 2000 | American Journal of Physiology-Lung Cellular and Molecular Physiology, Vol. 278, No. 5Specific phosphorylation of Torpedo 43K rapsyn by endogenous kinase(s) with thiamine triphosphate as the phosphate donorThe FASEB Journal, Vol. 14, No. 3Bradykinin induces tubulin phosphorylation and nuclear translocation of MAP kinase in mesangial cellsAyad A. Jaffa, Bradley S. Miller, Steven A. Rosenzweig, Padma S. Naidu, Victoria Velarde, and Ronald K. Mayfield1 December 1997 | American Journal of Physiology-Renal Physiology, Vol. 273, No. 6Activation of ion transport pathways by changes in cell volumeBiochimica et Biophysica Acta (BBA) - Reviews on Biomembranes, Vol. 1071, No. 4An hypothesis: Phosphorylation fields as the source of positional information and cell differentiation—(cAMP, ATP) as the universal morphogenetic turing coupleProgress in Biophysics and Molecular Biology, Vol. 56, No. 2 More from this issue > Volume 71Issue 1January 1991Pages 285-304 Copyright & PermissionsCopyright © 1991 the American Physiological Societyhttps://doi.org/10.1152/physrev.1991.71.1.285PubMed1986390History Published online 1 January 1991 Published in print 1 January 1991 Metrics

The form of inhibitor protein of the cAMP-dependent protein kinase (PKI) that has been most thoroughly studied is a protein purified from rabbit skeletal muscle. Beale et al. previously isolated a species of PKI from rat testis that appeared from its amino acid composition to be quite distinct from the rabbit skeletal muscle protein [Beale, E. G., Dedman, J. R. & Means, A. R. (1977) J. Biol. Chem. 252, 6322-6327]. The amino acid sequence of a form of rat testis PKI has now been determined both by sequencing overlapping peptide fragments for 95% of the protein and by the isolation of a cDNA clone containing the coding region for the 70-amino acid protein. The sequence of the 70-amino acid testis PKI displays a maximum of only 41% sequence identity with the previously sequenced 75-amino acid rabbit skeletal muscle PKI. However, the two forms have identical potency as inhibitors and the key amino acids of the pseudosubstrate site, shown to be critical for maximal inhibition with the rabbit skeletal muscle PKI, have been conserved in the testis protein. The rabbit skeletal muscle and rat testis PKIs most likely represent distinct isoforms. The nucleotide sequence of the rat testis PKI cDNA suggests that a second form of testis PKI, longer by 8 additional amino-terminal amino acids, might also be produced.

The potential correlations between phosphorylase kinase subunit phosphorylation and activation have been examined using 32P-perfused rat hearts exposed to a variety of hormonal stimuli. Phosphate incorporation was measured after isolation of the enzyme by immunoprecipitation from heart extracts. Time courses of catecholamine or glucagon treatment produced a rapid rise in both the activity and the beta subunit phosphorylation of the enzyme, and a slightly slower increase in alpha' subunit phosphorylation. For short durations of catecholamine stimulation, the ratio of phosphate in the alpha' versus beta subunit was dependent upon hormone dose. After removal of hormone, both inactivation and alpha' subunit dephosphorylation were fairly slow, while the beta subunit was dephosphorylated more rapidly. For all of the above conditions, activation correlated with both alpha' and beta subunit phosphorylation. The maximum level of phosphate incorporation observed in response to hormonal stimulation is estimated to be approximately 1.3-1.7 mol of [32P]phosphate/mol of (alpha' beta gamma delta)4, divided about equally between the alpha' and beta subunits. When hearts were treated with hormone either in the absence of added calcium or in the presence of a calcium channel blocker, the time courses of subunit phosphorylation and activation were similar to those seen with standard perfusion conditions, suggesting that if any Ca2+-dependent autophosphorylation of phosphorylase kinase were occurring it does not make a major contribution to the observed hormonal responses. The complicated relationships observed here between phosphorylase kinase subunit phosphorylation and activation for the most part provide physiological affirmation of the patterns observed in vitro, but they also show some possible differences of potential interest.

The flexor digitorum brevis skeletal muscle, a nearly homogeneous fast-twitch oxidative glycolytic fiber type, has been examined for its suitability to explore the regulation of phosphorylase kinase by multisite phosphorylation. A characterization of the adrenergic response of glycogenolytic enzymes, together with the previous data on contractile properties (Carlsen, R. C., Larson, D. B., and Walsh, D. A. (1985) Can. J. Physiol. Pharm. 63, 958-965), has demonstrated that this muscle is stably maintained for the several hours necessary for phosphorylation studies. The phosphorylase kinase in this muscle is primarily the alpha' isozyme, suggesting that the alpha versus alpha' isozyme distribution in muscle is related more to oxidative capacity than to fiber contractile characteristics. Using this muscle system, beta-adrenergic activation of phosphorylase kinase was observed to occur with concomitant phosphorylation of both the alpha' and beta subunits, with the total in the alpha' subunit being approximately 3-fold greater. Similarly, deactivation, following initial adrenergic activation, occurred concomitantly with the dephosphorylation of the two subunits. These results are compatible with the conclusions drawn from previous studies of the isolated enzyme and of the enzyme in perfused rat cardiac muscle, that both alpha' (or alpha) and beta subunit phosphorylation regulate phosphorylase kinase activity.

Two daughters of a propositus with documented McArdle's disease were shown by enzyme assay, gel electrophoresis, and immunoblotting to be partially deficient in skeletal muscle phosphorylase and, presumably, heterozygous for the trait. Both exhibited only the adult form of the skeletal muscle isozyme. By 31P-nuclear magnetic resonance, both heterozygotes showed a greater production of acid during fully aerobic exercise than when blood flow was occluded in ischemic exercise. This pattern is in contrast to that of control subjects, where there is significantly greater acid production in ischemic versus aerobic exercise, and distinct from that of phosphorylase-negative patients in which no acid is produced in either circumstance. We suggest that these heterozygotes may have adapted to their diminished phosphorylase by enhancing utilization of plasma glucose. If so, this mechanism could account for the observation that most of the symptoms of McArdle's disease are often manifest only in adulthood. These studies also show that although there are very high concentrations of phosphorylase in skeletal muscle (approximately 2% of the soluble protein), such a high level is essential for normal muscle glycogenolysis.

In perfused rat hearts insulin can activate, and catecholamines can inactivate, glycogen synthase (EC 2.4.1.11); the magnitude of each hormonal response is magnified if tissue glycogen levels are depleted. Both beta-adrenergic and alpha-adrenergic agonists inactivate insulin-stimulated and basal glycogen synthase, with each promoting the same extent of inactivation in both circumstances. In this system beta-adrenergic agonists act via cyclic AMP (cAMP), and alpha-adrenergic agonists via Ca2+, whereas insulin action appears to be independent of either cAMP or Ca2+. The action on cardiac glycogen synthase by the physiological catecholamine epinephrine is apparently mediated by the concomitant interaction with both alpha and beta receptors; interaction with each is mediated by their separate second messenger systems, which combine to produce the end physiological response.

32P-labeled perfused rat hearts were used to study the hormonal regulation of glycogen synthase. Following equilibration of perfused hearts with inorganic [32P]phosphate for 30 min, there was an incorporation of approximately 200 pmol of [32P]phosphate/unit of enzyme activity that arose from an exchange of [32P] phosphate with the endogenous [31P]phosphate. Maximum insulin-induced activation (10 milliunits/ml for 5 min), which promoted an I/D activity ratio change from 25% I to 40% I, was associated with a 22% decrease in phosphate content of the enzyme. With hearts from alloxan-induced diabetic animals, there was a 17% higher level of phosphate incorporation and a 4-fold decrease in % I glycogen synthase activity compared to normal animals, but with the diabetic tissue, insulin added to the perfusate had no effect on either the phosphate content or the activity ratio of the enzyme. In perfused hearts from normal animals, DL-isoproterenol and glucagon caused an increase in glycogen synthase phosphorylation of 85-100 pmol/unit of enzyme activity, while L-phenylephrine increased the phosphate content by only 20-35 pmol, but all three hormones caused the same degree of inactivation. The increase in cardiac glycogen synthase phosphorylation induced by DL-isoproterenol, glucagon, and L-phenylephrine was identical, with or without insulin pretreatment; this despite the fact that these three hormones promoted a 3- to 4-fold larger decrease in the enzyme activity ratio with the insulin-treated tissue. In perfused diabetic hearts, DL-isoproterenol, glucagon, and L-phenylephrine caused increased phosphorylation of glycogen synthase without affecting the albeit already low activity ratio of the enzyme. These results show that in the intact perfused heart the same degree of glycogen synthase inactivation can occur as a consequence of differing degrees of phosphorylation, presumably due to phosphorylation at different sites promoted by different second messengers. Conversely, the data indicate that the same extent of phosphorylation, as stimulated by the same second messenger, can inactivate glycogen synthase by different amounts depending upon the prior phosphorylation state of other sites in the protein.

This chapter discusses the method of purification and properties of the enzyme—cardiac phosphorylase kinase—isolated from bovine cardiac muscle. It also discusses the procedure to measure the activation state of the cardiac enzyme in intact tissue. Phosphorylase kinase is a complex enzyme of molecular weight 1.3 × 106 and a subunit composition position of α4 ·β4 · γ4 ·δ4. In muscle, two major isozymes exist, that can be distinguished by the size of the α subunit. The α subunit in fast-twitch glycolytic muscle is distinct and slightly larger than that from oxidative red skeletal and cardiac muscle and this subunit in the latter two is designated α'. The γ-subunit of phosphorylase kinase contains the catalytic site although there may be more than one type of catalytic site. The δ-subunit is identical to calmodulin and is the site of Ca2+-dependent allosteric regulation, The fast-twitch skeletal isozyme (α), but not the a' isozyme, has a second site of Ca 2+ sensitivity as a consequence of a readily reversible interaction with either additional calmodulin or troponin C subunit (TNC); the latter two have also been referred to as “phosphorylase kinase δ subunit.”

The hormonal regulation of glycogen synthase has been studied with isolated perfused hearts that were depleted of 85% of their endogenous glycogen. Glycogen depletion alone promoted a 3-fold activation of glycogen synthase and magnified by 3-fold the response to insulin. Glycogen depletion also facilitated the detection of epinephrine-promoted glycogen synthase inactivation. Hormonal effects on glycogen synthase have been correlated with changes in phosphorylase, phosphorylase kinase, and tissue cAMP levels. Insulin activation of glycogen synthase was observed within 90 s of hormone addition and was maximal by 4 min. A half-maximum effect was obtained at an insulin concentration of 100 microunits/ml. Insulin-dependent activation is reversed by beta-adrenergic agonists, alpha-adrenergic agonists, and glucagon. Each promote the same degree of inactivation and the maximum extent of inactivation produced by each is independent of whether or not the tissue has been stimulated with insulin. beta-Adrenergic agonists and glucagon act via cAMP, alpha-agonists most likely act via intracellular Ca2+ translocation, and insulin action would appear to be independent of either cAMP or Ca2+. The action of epinephrine on cardiac glycogen synthase is mediated by interaction with both alpha- and beta-receptors. As indicated by dose-response curves, receptor occupancy of each occurs to an almost equal extent at suboptimal epinephrine concentrations. Regulation of cardiac glycogen synthase by epinephrine thus is mediated by two second messenger systems which converge to produce the end physiological response.