An evaluation of indirubin analogues as phosphorylase kinase inhibitors.

Phosphorylase kinase (PhK) has been linked with a number of conditions such as glycogen storage diseases, psoriasis, type 2 diabetes and more recently, cancer (Camus et al., 2012 [6]). However, with few reported structural studies on PhK inhibitors, this hinders a structure based drug design approach. In this study, the inhibitory potential of 38 indirubin analogues have been investigated. 11 of these ligands had IC50 values in the range 0.170-0.360µM, with indirubin-3'-acetoxime (1c) the most potent. 7-Bromoindirubin-3'-oxime (13b), an antitumor compound which induces caspase-independent cell-death (Ribas et al., 2006 [20]) is revealed as a specific inhibitor of PhK (IC50=1.8µM). Binding assay experiments performed using both PhK-holo and PhK-γtrnc confirmed the inhibitory effects to arise from binding at the kinase domain (γ subunit). High level computations using QM/MM-PBSA binding free energy calculations were in good agreement with experimental binding data, as determined using statistical analysis, and support binding at the ATP-binding site. The value of a QM description for the binding of halogenated ligands exhibiting σ-hole effects is highlighted. A new statistical metric, the 'sum of the modified logarithm of ranks' (SMLR), has been defined which measures performance of a model for both the "early recognition" (ranking earlier/higher) of active compounds and their relative ordering by potency. Through a detailed structure activity relationship analysis considering other kinases (CDK2, CDK5 and GSK-3α/ß), 6'(Z) and 7(L) indirubin substitutions have been identified to achieve selective PhK inhibition. The key PhK binding site residues involved can also be targeted using other ligand scaffolds in future work.

Identification of phosphorylase kinase as a novel therapeutic target through high-throughput screening for anti-angiogenesis compounds in zebrafish.

Angiogenesis is essential for development and tumor progression. With the aim of identifying new compound inhibitors of the angiogenesis process, we used an established enhanced green fluorescent protein-transgenic zebrafish line to develop an automated assay that enables high-throughput screening of compound libraries in a whole-organism setting. Using this system, we have identified novel kinase inhibitor compounds that show anti-angiogenic properties in both zebrafish in-vivo system and in human endothelial cell in-vitro angiogenesis models. Furthermore, we have determined the kinase target of these compounds and have identified and validated a previously uncharacterized involvement of phosphorylase kinase subunit G1 (PhKG1) in angiogenesis in vivo. In addition, we have found that PhKG1 is upregulated in human tumor samples and that aberrations in gene copy number of PhK subunits are a common feature of human tumors. Our results provide a novel insight into the angiogenesis process, as well as identify new potential targets for anti-angiogenic therapies.

Kinetics, in silico docking, molecular dynamics, and MM-GBSA binding studies on prototype indirubins, KT5720, and staurosporine as phosphorylase kinase ATP-binding site inhibitors: the role of water molecules examined.

With an aim toward glycogenolysis control in Type 2 diabetes, we have investigated via kinetic experiments and computation the potential of indirubin (IC50 > 50 µM), indirubin-3'-oxime (IC50 = 144 nM), KT5720 (K(i) = 18.4 nM) and staurosporine (K(i) = 0.37 nM) as phosphorylase kinase (PhKγtrnc) ATP-binding site inhibitors, with the latter two revealed as potent inhibitors in the low nM range. Because of lack of structural information, we have exploited information from homologous kinase complexes to direct in silico calculations (docking, molecular dynamics, and MMGBSA) to predict the binding characteristics of the four ligands. All inhibitors are predicted to bind in the same active site area as the ATP adenine ring, with binding dominated by hinge region hydrogen bonds to Asp104:O and Met106:O (all four ligands) and also Met106:NH (for the indirubins). The PhKγtrnc-staurosporine complex has the greatest number of receptor-ligand hydrogen bonds, while for the indirubin-3'-oxime and KT5720 complexes there is an important network of interchanging water molecules bridging inhibitor-enzyme contacts. The MM-GBSA results revealed the source of staurosporine's low nM potency to be favorable electrostatic interactions, while KT5720 has strong van der Waals contributions. KT5720 interacts with the greatest number of protein residues either by direct or 1-water bridged hydrogen bond interactions, and the potential for more selective PhK inhibition based on a KT5720 analogue has been established. Including receptor flexibility in Schrödinger induced-fit docking calculations in most cases correctly predicted the binding modes as compared with the molecular dynamics structures; the algorithm was less effective when there were key structural waters bridging receptor-ligand contacts.

The regulatory beta subunit of phosphorylase kinase interacts with glyceraldehyde-3-phosphate dehydrogenase.

Skeletal muscle phosphorylase kinase (PhK) is an (alphabetagammadelta) 4 hetero-oligomeric enzyme complex that phosphorylates and activates glycogen phosphorylase b (GP b) in a Ca (2+)-dependent reaction that couples muscle contraction with glycogen breakdown. GP b is PhK's only known in vivo substrate; however, given the great size and multiple subunits of the PhK complex, we screened muscle extracts for other potential targets. Extracts of P/J (control) and I/lnJ (PhK deficient) mice were incubated with [gamma- (32)P]ATP with or without Ca (2+) and compared to identify potential substrates. Candidate targets were resolved by two-dimensional polyacrylamide gel electrophoresis, and phosphorylated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified by matrix-assisted laser desorption ionization mass spectroscopy. In vitro studies showed GAPDH to be a Ca (2+)-dependent substrate of PhK, although the rate of phosphorylation is very slow. GAPDH does, however, bind tightly to PhK, inhibiting at low concentrations (IC 50 approximately 0.45 microM) PhK's conversion of GP b. When a short synthetic peptide substrate was substituted for GP b, the inhibition was negligible, suggesting that GAPDH may inhibit predominantly by binding to the PhK complex at a locus distinct from its active site on the gamma subunit. To test this notion, the PhK-GAPDH complex was incubated with a chemical cross-linker, and a dimer between the regulatory beta subunit of PhK and GAPDH was formed. This interaction was confirmed by the fact that a subcomplex of PhK missing the beta subunit, specifically an alphagammadelta subcomplex, was unable to phosphorylate GAPDH, even though it is catalytically active toward GP b. Moreover, GAPDH had no effect on the conversion of GP b by the alphagammadelta subcomplex. The interactions described herein between the beta subunit of PhK and GAPDH provide a possible mechanism for the direct linkage of glycogenolysis and glycolysis in skeletal muscle.

Caspase-3 dependent cleavage and activation of skeletal muscle phosphorylase b kinase.

Phosphorylase b kinase (PhK) is a key enzyme involved in the conversion of glycogen to glucose in skeletal muscle and ultimately an increase in intracellular ATP. Since apoptosis is an ATP-dependent event, we investigated the regulation of skeletal muscle PhK during apoptosis. Incubation of PhK with purified caspase-3 in vitro resulted in the highly selective cleavage of the regulatory alpha subunit and resulted in a 2-fold increase in PhK activity. Edman protein sequencing of a stable 72 kD amino-terminal fragment and a 66 kD carboxy-terminal fragment revealed a specific caspase-3 cleavage site within the alpha subunit at residue 646 (DWMD G). Treatment of differentiated C2C12 mouse muscle myoblasts with the inducers of apoptosis staurosporine, TPEN, doxorubicin, or UV irradiation resulted in the disappearance of the alpha subunit of PhK as determined by immunoblotting, as well as a concurrent increase in caspase-3 activity. Moreover, induction of apoptosis by TPEN resulted in increased phosphorylase activity and sustained ATP levels throughout a 7 h time course. However, induction of apoptosis with staurosporine, also a potent PhK inhibitor, led to a rapid loss in phosphorylase activity and intracellular ATP, suggesting that PhK inhibition by staurosporine impairs the ability of apoptotic muscle cells to generate ATP. Thus, these studies indicate that PhK may be a substrate for caspase regulation during apoptosis and suggest that activation of this enzyme may be important for the generation of ATP during programmed cell death.

The calmodulin-binding domain of the catalytic gamma subunit of phosphorylase kinase interacts with its inhibitory alpha subunit: evidence for a Ca2+ sensitive network of quaternary interactions.

Chemical cross-linking as a probe of conformation has consistently shown that activators, including Ca(2+) ions, of the (alphabetagammadelta)(4) phosphorylase kinase holoenzyme (PhK) alter the interactions between its regulatory alpha and catalytic gamma subunits. The gamma subunit is also known to interact with the delta subunit, an endogenous molecule of calmodulin that mediates the activation of PhK by Ca(2+) ions. In this study, we have used two-hybrid screening and chemical cross-linking to dissect the regulatory quaternary interactions involving these subunits. The yeast two-hybrid system indicated that regions near the C termini of the gamma (residues 343-386) and alpha (residues 1060-1237) subunits interact. The association of this region of alpha with gamma was corroborated by the isolation of a cross-linked fragment of alpha containing residues 1015-1237 from an alpha-gamma dimer that had been formed within the PhK holoenzyme by formaldehyde, a nearly zero-length cross-linker. Because the region of gamma that we found to interact with alpha has previously been shown to contain a high affinity binding site for calmodulin (Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1989) J. Biol. Chem. 264, 17156-17163), we tested the influence of Ca(2+) on the conformation of the alpha subunit and found that the region of alpha that interacts with gamma was, in fact, perturbed by Ca(2+). The results herein support the existence of a Ca(2+)-sensitive communication network among the delta, gamma, and alpha subunits, with the regulatory domain of gamma being the primary mediator. The similarity of such a Ca(2+)-dependent network to the interactions among troponin C, troponin I, and actin is discussed in light of the known structural and functional similarities between troponin I and the gamma subunit of PhK.

An inhibitory segment of the catalytic subunit of phosphorylase kinase does not act as a pseudosubstrate.

The C terminus of the catalytic gamma subunit of phosphorylase kinase contains two autoinhibitory calmodulin binding domains designated PhK13 and PhK5. These peptides inhibit truncated gamma(1-300). Previous data show that PhK13 (residues 302-326) is a competitive inhibitor with respect to phosphorylase b, with a K(i) of 1.8 microm. This result suggests that PhK13 may bind to the active site of truncated gamma(1-300). Variants of PhK13 were prepared to localize the determinants for interaction with the catalytic fragment gamma(1-300). PhK13-1, containing residues 302-312, was found to be a competitive inhibitor with respect to phosphorylase b with a K(i) of 6.0 microm. PhK13 has been proposed to function as a pseudosubstrate inhibitor with Cys-308 occupying the site that normally accommodates the phosphorylatable serine in phosphorylase b. A PhK13-1 variant, C308S, was synthesized. Kinetic characterization of this peptide reveals that it does not serve as a substrate but is a competitive inhibitor. Additional variants were designed based on previous knowledge of phosphorylase kinase substrate determinants. Variants were analyzed as substrates and as inhibitors for truncated gamma(1-300). Although PhK13-1 does not appear to function as a pseudosubstrate, several specificity determinants employed in the recognition of phosphorylase b as substrate are utilized in the recognition of PhK13-1 as an inhibitor.

Drug-induced suppression of phosphorylase kinase activity correlates with resolution of psoriasis as assessed by clinical, histological and immunohistochemical parameters.

BACKGROUND: Phosphorylase kinase (PhK), also known as adenosine triphosphate (ATP)-phosphorylase b phosphotransferase, integrates multiple calcium/calmodulin-dependent signalling pathways, including those involved in cell migration and cell proliferation, while coupling these pathways to glycogenolysis and ATP-dependent phosphorylation, thus ensuring continuing energy supply for these activities. OBJECTIVES: Our laboratory recently reported correlation of elevated PhK activity with psoriatic activity. This study further evaluates the significance of drug-induced suppression of PhK activity on psoriatic activity. PATIENTS AND METHODS: PhK activity was assayed in four groups, each with 10 patients: (i) active untreated psoriasis; (ii) resolving psoriasis treated by calcipotriol (Dovonex(R), Bristol Myers Squibb, Princeton, NJ, U.S.A. ), a vitamin D3 analogue and an indirect inhibitor of PhK; (iii) curcumin (diferuloylmethane), a selective PhK inhibitor; and (iv) 10 normal non-psoriatic subjects. RESULTS: PhK activity in units mg-1 protein was highest in active untreated psoriasis (1204 +/- 804.3; mean +/- SD), lower in the calcipotriol-treated group (550.7 +/- 192. 9), lower in curcumin-treated group (207.2 +/- 97.6), and lowest in normal skin (105.4 +/- 44.6). One-way analysis of variance performed on log-transformed PhK activity measure showed significant differences among the four groups, F3,36 = 48.79, P < 0.0001. Decreased PhK activity in curcumin-and calcipotriol-treated psoriasis was associated with corresponding decreases in keratinocyte transferrin receptor (TRR) expression, severity of parakeratosis and density of epidermal CD8+ T cells. CONCLUSIONS: Our results demonstrate that drug-induced suppression of PhK activity is associated with resolution of psoriatic activity as assessed by clinical, histological and immunohistochemical criteria, and support the hypothesis that effective antipsoriatic activity may be achieved through modulation of PhK activity.

In vitro inhibition of the activity of phosphorylase kinase, protein kinase C and protein kinase A by caffeic acid and a procyanidin-rich pine bark (Pinus marittima) extract.

Caffeic acid (CA) is a common constituent of human diet while pine bark extract (PBE) is utilized either as nutritional supplement or as phytochemical remedy for different diseases. CA and PBE, are reported as efficient antioxidants and more recently have been described to modulate cellular response to oxidative challenge and to possess many other biological activities, i.e. anti-inflammatory, antimutagenic, antitumoral effects. In order to investigate in depth the mechanism of action of these polyphenols, the effects of CA and PBE on the activity of some protein kinases involved in the regulation of fundamental cellular processes were studied in vitro: phosphorylase kinase (PhK), protein kinase A (PKA), protein kinase C (PKC). PBE at the concentration of 20 microg/ml (corresponding to 69 microM catechin equivalents) inhibited PKA, PhK and PKC by about 90, 59, 57%, respectively, while 100 microM CA inhibited by 37, 52 and 54%, respectively. Considerable inhibitions have been still observed at even lower concentrations of CA and PBE. For PhK and PKA, the inhibition follows a non-competitive mechanism. CA also inhibits PKC activity in a partially purified cellular extract. The results suggest a possible involvement of CA and PBE in modulation of cellular functions.

Substrate and inhibitor recognition of protein kinases: what is known about the catalytic subunit of phosphorylase kinase?

Although much can be learned about the specificity of protein kinases from studies with peptide substrates, the question remains, how do kinases recognize their three-dimensional protein substrates? Information derived from such studies provides further understanding of substrate recognition and can facilitate the design of specific protein kinase inhibitors. Phosphorylase kinase (PhK) catalyzes the phosphorylation of phosphorylase b (phos. b) to form the active phosphorylase a. No other protein kinase can duplicate this reaction. Why? To probe this question and establish what features in the protein are important for substrate binding and product release, mutants of phos. b have been studied. This report shows how mutations change the properties of the protein substrate and the ability of these mutants to be phosphorylated by PhK and other kinases. Action of protein kinases on their substrates is often regulated by autoinhibitory segments. The C-terminus of the catalytic gamma-subunit of PhK contains two inhibitory sites overlapping two calmodulin-binding regions. These two peptide segments resemble sequences in phos. b and may explain why peptides of these regions are potent inhibitors of PhK. We will show results with peptide inhibitors, using various expressed forms of the catalytic subunit, which describe their modes of interaction and mechanisms of inhibition. Metal ions can change molecular interactions. With PhK, Mn2+ facilitates the use of GTP as a phosphoryl group donor and greatly increases phosphorylation of a tyrosine residue in angiotensin II. This implies that the spatial arrangement of specificity determinants can be manipulated so that PhK can utilize other substrates.

Mg2+ induces conformational changes in the catalytic subunit of phosphorylase kinase, whether by itself or as part of the holoenzyme complex.

Phosphorylase kinase (PhK) from skeletal muscle is a structurally complex, highly regulated, hexadecameric enzyme of subunit composition (alpha beta gamma delta)4. Previous studies have revealed that the activity of its catalytic gamma subunit is controlled by alterations in quaternary structure initiated at allosteric and covalent modification sites on PhK's three regulatory subunits; however, changes in the conformation of the holoenzyme initiated by the catalytic subunit have been more difficult to document. In this study a monoclonal antibody (mAb gamma79) has been generated against isolated gamma subunit and used as a conformational probe of that subunit. The epitope recognized by this antibody is within the catalytic core of the gamma subunit, between residues 100 and 240, and monovalent fragments of the antibody inhibit the catalytic activity of the holoenzyme, the gamma-calmodulin binary complex, and the free gamma subunit. Activation of PhK by a variety of mechanisms known or thought to act through its regulatory subunits (phosphorylation, ADP binding, or alkaline pH) increased the binding of the holoenzyme to immobilized mAb gamma79, indicating that activation by any of these distinct mechanisms involves repositioning of the portion of the catalytic domain of the gamma subunit containing the epitope for mAb gamma79. The activating ligand Mg2+ also stimulated the binding of the PhK holoenzyme to immobilized mAb gamma79, as well as the binding of mAb gamma79 to immobilized gamma subunit. Thus, Mg2+ increases the accessibility of the mAb gamma79 epitope in both the isolated gamma subunit and in the holoenzyme. Our results suggest that previously reported influences of Mg2+ on the quaternary structure of the PhK holoenzyme are directly mediated by the gamma subunit.

Differential affinity cross-linking of phosphorylase kinase conformers by the geometric isomers of phenylenedimaleimide.

Phosphorylase b kinase (PbK) from skeletal muscle is a highly regulated oligomer consisting of four copies of four distinct subunits (alphabetagamma)delta4. The gamma subunit is catalytic, and the remaining subunits are regulatory. To characterize effector-induced changes in the quaternary structure of the enzyme, we utilized the ortho-, meta, and para-isomers of phenylenedimaleimide (PDM), which in addition to having different geometries, also vary 2.5-fold in their cross-linking spans. Even at concentrations equivalent to the alphabetagammadelta protomers of PbK, all three isomers caused specific, rapid, and extensive cross-linking of the holoenzyme to form primarily alphabeta dimers, plus smaller amounts of betagammagamma and alphagammagamma trimers. The formation of these three conjugates was nearly totally inhibited by a 10-fold molar excess over PDM of N-(o- and p-tolyl)succinimide, which are chemically inert structural analogs of PDM. This inhibition suggests that PbK has binding sites for PDM and that PDM acts as an affinity cross-linker in binding to these sites prior to forming cross-linked conjugates. The largest effect on cross-linking in progressing from o- to p-PDM was on the alphagammagamma trimer, which is preferentially formed by the p-isomer. Activation of the enzyme by either phosphorylation or the allosteric activators ADP and GDP resulted in large increases in the amount of alphagammagamma formed, small increases in betagammagamma, and little change in alphabeta. When cross-linked in the presence of the reversibly activating nucleoside diphosphates, PbK remained activated after their removal, indicating that cross-linking had locked it in the active conformation. Our results provide direct evidence for perturbations in the interactions of the catalytic gamma subunit with the regulatory alpha and beta subunits upon activation of PbK.

Activation and inhibition of phosphorylase kinase by monospecific antibodies raised against peptides from the regulatory domain of the gamma-subunit.

The C terminus of the catalytic gamma-subunit of phosphorylase kinase comprises a regulatory domain that contains regions important for subunit interactions and autoinhibitory functions. Monospecific antibodies raised against four synthetic peptides from this region, PhK1 (362-386), PhK5 (342-366), PhK9 (322-346), and PhK13 (302-326), were found to have significant effects on the catalytic activities of phosphorylase kinase holoenzyme and the gamma delta complex. Antibodies raised against the very C terminus of the gamma-subunit, anti-PhK1 and anti-PhK5, markedly activated both holoenzyme and the gamma delta complex, in the presence and absence of Ca2+. In the presence of Ca2+ at pH 8.2, anti-PhK1 activated the holoenzyme more than 11-fold and activated the gamma delta complex 2.5-fold. Activation of the holoenzyme and the gamma delta complex by anti-PhK5 was 50-70% of that observed with anti-PhK1. Prior phosphorylation of the holoenzyme by the cAMP-dependent protein kinase blocked activation by both anti-PhK1 and anti-PhK5. Antibodies raised against the peptides from the N terminus of the regulatory domain, anti-PhK9 and anti-PhK13, were inhibitory, with their greatest effects on the gamma delta complex. These data demonstrate that the binding of antibodies to specific regions within the regulatory domain of the gamma-subunit can augment or inhibit structural changes and subunit interactions important in regulating phosphorylase kinase activity.

Subcellular distribution of phosphorylase kinase in rat brain. Association of the enzyme with mitochondria and membranes.

The evaluation of glycogen phosphorylase kinase in rat brain subcellular fractions was undertaken in order to get further insight into the association of this kinase with specific neuronal cell compartments. The enzyme was found to be primarily soluble, but considerable latent specific activities were observed in particulate fractions, especially in microsomes, mitochondria and synaptosomes, which could be unmasked by treatment with Triton-X-100. The submitochondrial and subsynaptic distribution patterns of phosphorylase kinase revealed high overt activity in the mitochondrial intermembrane space and high latent activities in mitochondrial membranes, and synaptic vesicles, membranes and mitochondria. The Ca(2+)-dependency of soluble phosphorylase kinase was similar to that of microsomal enzyme but higher than that of other particulate enzyme forms. Mitochondrial phosphorylase kinase showed a higher pH 6.8:8.2 activity ratio than the soluble and the microsomal enzyme. The rate of inactivation of cytosolic phosphorylase kinase by proteinase K was higher than that of microsomal and mitochondrial enzymes. Antibodies against rabbit skeletal muscle phosphorylase kinase effectively inhibited both cytosolic and microsomal enzyme but failed to significantly affect the kinase activity present in intact mitochondria and intermembrane space. Western blotting with anti-phosphorylase kinase showed that rat brain mitochondria exhibited a significantly lower immunoreactivity compared to soluble cytosol. In conclusion, the presence of phosphorylase kinase activity in a variety of particulate fractions of rat brain suggests a multiplicity of actions of this kinase in neuronal tissues.

Involvement of phosphorylase kinase inhibition in the effect of resorcinol and proglycosyn on glycogen metabolism in the liver.

The purpose of this study was to identify the mechanism by which proglycosyn and resorcinol decrease the phosphorylase a content and the fructose 2,6-bisphosphate concentration in isolated hepatocytes. The intracellular concentrations of the glucuronide derivatives of proglycosyn and resorcinol have been measured by HPLC in hepatocytes incubated for 5 min or 30 min with different concentrations of these agents. At both times, there was a reciprocal relationship between the phosphorylase a content and the intracellular concentration of the glucuronidated metabolites, half-maximal inactivation being observed at about 2 mumol/g protein and 0.25 mumol/g protein for resorcinylglucuronide and proglycosyn-glucuronide, respectively. Glycogen synthase was not significantly activated by these agents after 5 min but was well activated after 30 min. Preincubation of hepatocytes with 1 mM resorcinol or with 100 microM proglycosyn resulted in a decrease in the rate at which phosphorylase was activated following the addition of glucagon, vasopressin, the protein phosphatase inhibitor calyculin A or the calcium ionophore A 23187, but did not reduce the rate of synthase inactivation. Proglycosynglucuronide and resorcinylglucuronide inhibited phosphorylase kinase in liver Sephadex filtrates, with Ki values of about 0.75 mM and 4 mM, respectively. Preincubation of the filtrates with ATP and cAMP decreased the sensitivity of phosphorylase kinase to resorcinylglucuronide by about fourfold. It is concluded that the effect of resorcinol and proglycosyn on the phosphorylase a content is due, at least partly, to an inhibition of phosphorylase kinase by their glucuronidated metabolites. Resorcinol and proglycosyn caused a parallel decrease in the concentration of fructose 2,6-bisphosphate and of hexose 6-phosphates, without significantly changing the activity of 6-phosphofructo-2-kinase. The decrease in the fructose 2,6-bisphosphate concentration appears therefore to be secondary to the decrease in the hexose 6-phosphate concentration.

Characterization of the regulatory domain of the gamma-subunit of phosphorylase kinase. The two noncontiguous calmodulin-binding subdomains are also autoinhibitory.

Phosphorylase kinase is a multimeric protein kinase (alpha 4 beta 4 gamma 4 delta 4) whose enzymatic activity is conferred by its gamma-subunit. A library of 18 overlapping synthetic peptides spanning residues 277-386 of the gamma-subunit has been prepared to use in identifying important regulatory structures in the protein. In the present study, the library was screened to identify regions that might function as autoinhibitory domains. Peptides from two distinct regions were found to inhibit the Ca2(+)-activated holoenzyme. The same regions were previously found to bind calmodulin (i.e. the delta-subunit; Dasgupta, M. Honeycutt, T., and Blumenthal, D. K. (1989) J. Biol. Chem. 264, 17156-17163). The most potent substrate antagonist peptides were PhK13 (residues 302-326; Ki = 300 nM) and PhK5 (residues 342-366; Ki = 20 microM). Both peptides inhibited the holoenzyme competitively with respect to phosphorylase b and noncompetitively with respect to Mg.ATP. When the pattern of inhibition with both peptides present was analyzed, inhibition was observed to be synergistic and modestly cooperative indicating that the two peptides can simultaneously occupy the protein substrate-binding site(s). These data are consistent with a model in which the regions of the gamma-subunit represented by PhK5 and PhK13 work in concert as regulatory subdomains that transduce Ca2(+)-induced conformational changes in the delta-subunit to the catalytic gamma-subunit through a pseudosubstrate autoinhibitory mechanism.

The gamma subunit of phosphorylase kinase contains a pseudosubstrate sequence.

The catalytic subunit, gamma, of phosphorylase kinase is regulated by a complex set of interactions involving the calcium-binding protein calmodulin and two other subunits designated alpha and beta. These interactions regulate gamma activity that, at least for the calmodulin interactions, involves the regulatory domain in gamma spanning residues 302-366. Within this regulatory domain, we report the identification of a sequence (residues 326-334) that resembles the phosphorylation site in gamma substrates with the exception that a V residue (V332) occurs at the analogous position of the phosphorylated S/T residue. The inhibitory properties of the sequence were assayed with a 10-amino-acid peptide of the sequence. This peptide inhibits a truncated version of gamma, residues 1-300, which is missing the regulatory domain, more potently than it inhibits full-length gamma, and it is a better inhibitor of the full-length gamma at pH 8.2 than at pH 6.8. A similar peptide of the same sequence, except for a S substitution of the V residue, is a good substrate with a comparable Km and better Vmax than peptides of similar length that represent the phosphorylation site in the substrate of the enzyme, glycogen phosphorylase. A mutant gamma protein, with a S for V332 substitution ([V332S]gamma), was prepared using the baculovirus expression system. [V332S]gamma autophosphorylates by an intramolecular mechanism. This demonstrates that this sequence can occupy the catalytic site in the protein. Development of [V332S]gamma affords an experimental model in which the effects of the regulatory factors on autophosphorylation can be determined.

Oxidation and site-directed mutagenesis of the sulfhydryl groups of a truncated gamma catalytic subunit of phosphorylase kinase. Functional and structural effects.

A truncated form of the gamma subunit of phosphorylase kinase is inactivated by Cu2+ with the formation of two intra-molecular disulfide bonds. The formation of a disulfide bond between Cys-36 and Cys-172 (semioxidized form) results in approximately 50% loss of specific activity because the Km for MgATP is about 10-fold higher. The second disulfide bond is between Cys-184 and Cys-197 and causes further loss of activity. Eight Cys mutants, i.e. C36S, C36A, C42S, C138S, C172S, C184S, C184A, and C197S, were expressed and purified. Kinetic studies suggest that Cys-36 is important for interaction at the nucleotide site because of its hydrophobicity. With Cys-184 mutants, C184S and C184A, tyrosyl phosphorylation of angiotensin II is affected much more than serine kinase activity. The loss of tyrosine kinase activity is related to a lowered activity with Mn2+. With Mn2+, angiotensin II is a competitive inhibitor with respect to seryl kinase activity of C184S. With Mg2+, however, angiotensin II is a noncompetitive inhibitor. We suggest that metal ions influence the conformation of truncated gamma and that the protein substrate binding region containing Cys-184 is important for the dual specificity of this kinase.

Characterization of natural toxins with inhibitory activity against serine/threonine protein phosphatases.

Recent studies suggest that the ability to inhibit the activity of certain serine/threonine protein phosphatases underlies the toxicity of several natural compounds including: okadaic acid, microcystin-LR, nodularin, calyculin A and tautomycin. To characterize further the actions of these toxins, this study compares the inhibitory effects of okadaic acid, chemical derivatives of okadaic acid, microcystin-LR, microcystin-LA, nodularin, calyculin A and tautomycin on the activity of serine/threonine protein phosphatases types 1 (PP1), 2A (PP2A) and a recently identified protein phosphatase purified from bovine brain (PP3). This study shows that, like PP1 and PP2A, the activity of PP3 is potently inhibited by okadaic acid, both microcystins, nodularin, calyculin A and tautomycin. Further characterization of the toxins employing the purified catalytic subunits of PP1, PP2A and PP3 under identical experimental conditions indicates that: (a) okadaic acid, microcystin-LR, and microcystin-LA inhibit PP2A and PP3 more potently than PP1 (order of potency PP2A > PP3 > PP1); (b) nodularin inhibits PP1 and PP3 at a similar concentration that is slightly higher than that which affects PP2A, and (c) both calyculin A and tautomycin show little selectivity among the phosphatases tested. This study also shows that the chemical modification of the (C1) carboxyl group of okadaic acid can have a profound influence on the inhibitory activity of this toxin. Esterification of okadaic acid, producing methyl okadaate, or reduction, producing okadaol, greatly decreases the inhibitory effects against all three enzymes tested. Further reduction, producing 1-nor-okadaone, or acetylation, producing okadaic acid tetraacetate, results in compounds with no inhibitory activity. In contrast, the substitution of alanine (-LA) for arginine (-LR) in microcystin has no apparent effect on the inhibitory activity against PP1, PP2A or PP3.

Curcumin is a non-competitive and selective inhibitor of phosphorylase kinase.

Recently, we reported that curcumin (diferuloylmethane) inhibits the growth of several different kinds of tumor cells. In order to investigate the mechanism of this inhibition, we examined the effects of curcumin on different protein kinases: highly purified protein kinase A (PkA), protein kinase C (PkC), protamine kinase (cPK), phosphorylase kinase (PhK), autophosphorylation-activated protein kinase (AK) and pp60c-src tyrosine kinase. While all kinases tested were inhibited by curcumin, only PhK was completely inhibited at relatively lower concentrations. At around 0.1 mM curcumin, PhK, pp60c-src, PkC, PkA, AK, and cPK were inhibited by 98%, 40%, 15%, 10%, 1%, and 0.5%, respectively. Lineweaver-Burk plot analysis indicated that curcumin is a non-competitive inhibitor of PhK with a Ki of 0.075 mM. Overall, our results indicate that curcumin is a potent and selective inhibitor of phosphorylase kinase, a key regulatory enzyme involved in the metabolism of glycogen. This has important implications for the anti-proliferative effects of curcumin.