cyclic-gmp and kemptide

The cGMP-dependent protein kinase (PKG) of Eimeria tenella and Toxoplasma gondii is the target of a novel coccidiostat that is effective against coccidiosis and toxoplasmosis in animal models. Preparations of native PKG enzyme from Toxoplasma and Eimeria contain a membrane-associated polypeptide (isoform-I) of about 110 kDa and a slightly smaller soluble polypeptide (isoform-II). Expression of T. gondii and E. tenella PKG cDNA clones in Toxoplasma yield similarly sized recombinant polypeptides, which co-migrate on SDS-polyacrylamide gels with the corresponding native isoforms. Results of targeted mutagenesis of potential translational initiation sites suggest that parasite isoform-II is a product of alternative translational initiation from an internal initiator methionine codon. Exclusive expression of isoform-II or isoform-I can be achieved by preventing initiation at the respective primary or secondary sites. Immunofluorescence analysis indicates that recombinant isoform-I localizes primarily to the parasite plasma membrane, while isoform-II remains cytosolic. Mutagenesis and metabolic labeling studies reveal that the observed membrane-association of full-length recombinant PKG is mediated by N-terminal myristoylation and palmitoylation at amino acids G2 and C4. We also confirm the functional significance of a putative third PKG allosteric site, common to apicomplexan PKGs but absent from vertebrate or insect PKGs. In assays with transiently transfected parasites, constructs harboring a mutation at this site express markedly lower levels of cGMP-dependent PKG activity, while a triple mutant bearing mutations in all three sites reduces kinase activity to background levels.

Topics: Acylation; Adenosine Triphosphate; Allosteric Site; Amino Acid Sequence; Animals; Animals, Genetically Modified; Base Sequence; Blotting, Western; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Eimeria tenella; Fibroblasts; Humans; Kinetics; Molecular Sequence Data; Molecular Weight; Mutation; Oligopeptides; Protein Isoforms; Recombinant Proteins; Species Specificity; Toxoplasma

In the present study, the inhibitory effect of the cGMP analog (Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate ((Rp)-8-pCPT-cGMPS) on the cGMP-dependent protein kinase-mediated protein phosphorylation in intact human platelets was investigated. In vitro phosphorylation experiments with the substrate kemptide demonstrated an inhibition of the cGMP-dependent protein kinase by (Rp)-8-pCPT-cGMPS with a Ki of 0.5 microM. In intact human platelets, (Rp)-8-pCPT-cGMPS antagonized the activation of the cGMP-dependent protein kinase by 8-pCPT-cGMP without affecting cAMP-dependent protein kinase or cGMP-regulated phosphodiesterases. The data obtained suggest that (Rp)-8-pCPT-cGMPS may be a useful tool for studying the role of cGMP in vitro and in intact cells.

Topics: Animals; Blood Platelets; Blotting, Western; Cattle; Chemical Phenomena; Chemistry, Physical; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Activation; Humans; In Vitro Techniques; Oligopeptides; Phosphorylation; Platelet Aggregation Inhibitors; Thionucleotides

Affinities of the catalytic subunit (C1) of Saccharomyces cerevisiae cAMP-dependent protein kinase and of mammalian cGMP-dependent protein kinase were determined for the protein kinase inhibitor (PKI) peptide PKI(6-22)amide and seven analogues. These analogues contained structural alterations in the N-terminal alpha-helix, the C-terminal pseudosubstrate portion, or the central connecting region of the PKI peptide. In all cases, the PKI peptides were appreciably less active as inhibitors of yeast C1 than of mammalian C alpha subunit. Ki values ranged from 5- to 290-fold higher for the yeast enzyme than for its mammalian counterpart. Consistent with these results, yeast C1 exhibited a higher Km for the peptide substrate Kemptide. All of the PKI peptides were even less active against the mammalian cGMP-dependent protein kinase than toward yeast cAMP-dependent protein kinase, and Kemptide was a poorer substrate for the former enzyme. Alignment of amino acid sequences of these homologous protein kinases around residues in the active site of mammalian C alpha subunit known to interact with determinants in the PKI peptide [Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N-h, Taylor, S. S., & Sowadski, J. M. (1991) Science 253, 414-420] provides a structural basis for the inherently lower affinities of yeast C1 and cGMP-dependent protein kinase for binding peptide inhibitors and substrates. Both yeast cAMP-dependent and mammalian cGMP-dependent protein kinases are missing two of the three acidic residues that interact with arginine-18 in the pseudosubstrate portion of PKI. Further, the cGMP-dependent protein kinase appears to completely lack the hydrophobic/aromatic pocket that recognizes the important phenylalanine-10 residue in the N-terminus of the PKI peptide, and binding of the inhibitor by the yeast protein kinase at this site appears to be partially compromised.

Topics: Amino Acid Sequence; Animals; Binding Sites; Cattle; Cyclic AMP; Cyclic GMP; Kinetics; Molecular Sequence Data; Myocardium; Oligopeptides; Peptide Fragments; Peptides; Protein Kinase Inhibitors; Protein Kinases; Saccharomyces cerevisiae; Substrate Specificity

The effectiveness of the cGMP-dependent protein kinase inhibitor, KT5823, was investigated in human neutrophils. KT5823 did not inhibit the cGMP-dependent protein kinase mediated in vitro, or in situ phosphorylation of vimentin, a known substrate for this enzyme in activated neutrophils. In addition, KT5823 was shown to induce dramatic shape changes in neutrophils, suggesting it has an activating effect upon the cells.

Topics: Alkaloids; Carbazoles; Cyclic GMP; Electrophoresis; Fluorescent Antibody Technique; Histones; Humans; Indoles; Neutrophils; Oligopeptides; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; Vimentin

This paper describes the partial characterization of soluble cyclic AMP-dependent protein kinase (A-kinase) in guinea-pig lung using Kemptide, a synthetic serine-containing heptapeptide, and examines the sensitivity of this enzyme to drugs which are reported to increase and to decrease the intracellular concentration of cyclic AMP. Differential centrifugation of lung homogenates revealed that 78% of A-kinase was present in the 31,000 gmax x 15 min supernatant fraction. Both basal and cyclic AMP-stimulated phosphotransferase activity of this 'soluble' enzyme were abolished by the heat-stable inhibitor of A-kinase. Soluble A-kinase was Mg2(+)-dependent (apparent Km and and Kact 8.6 and 2.6 mM, respectively) and was stimulated nine-fold by saturating concentrations of both cyclic AMP (Kact: 131 nM) and cyclic GMP (Kact: 28.7 microM) at a protein (enzyme) concentration of 1.3 micrograms. Kinetic analysis of the effect of Kemptide and ATP revealed linear, Hanes plots with Michealis constants of ca. 12 and 13 microM, respectively. Chromatography of the soluble enzyme over DEAE-cellulose resolved three peaks of catalytic activity when fractions were assayed in the presence of cyclic AMP (10 microM): (i) free catalytic subunits (5%), (ii) Type I isoenzyme (5%) and (iii) Type II isoenzyme (90%). The A-kinase activity ratio was markedly increased in lung pre-treated with the smooth muscle relaxants isoprenaline and forskolin. This biochemical effect was both time- and concentration-dependent and was temporally associated with the ability of these drugs to reduce lung parenchymal tone. In contrast, the contractile agonists, methacholine (Mch) and leukotriene (LT) D4 exerted opposite effects on A-kinase activity. Thus, Mch significantly reduced cyclic AMP levels and lowered basal A-kinase activity whilst the converse was true for LTD4. For both drugs this biochemical effect accompanied contraction of the lung. Pre-treatment of lung tissue with flurbiprofen, an irreversible inhibitor of cyclo-oxygenase in vitro, abolished the ability of LTD4 to increase the A-kinase activity ratio suggesting that this biochemical response was mediated indirectly through the stimulated biosynthesis and release of a prostanoid(s) able to activate adenylyl cyclase; the increase in tension induced by LTD4, however, was not significantly affected by flurbiprofen pre-treatment. Collectively, these data support the concept that soluble A-kinase activity in guinea-pig lung can be regulated by

Topics: Adenosine Triphosphate; Animals; Carrier Proteins; Chromatography, DEAE-Cellulose; Colforsin; Cyclic AMP; Cyclic GMP; Enzyme Activation; Guinea Pigs; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Isoenzymes; Isoproterenol; Lung; Magnesium; Male; Methacholine Chloride; Methacholine Compounds; Oligopeptides; Protein Kinase Inhibitors; Protein Kinases; Solubility; SRS-A

The allosteric regulation of binding to and the activation of cGMP-dependent protein kinase (cGMP kinase) was studied under identical conditions at 30 degrees C using three forms of cGMP-kinase which differed in the amino-terminal segment, e.g. native cGMP kinase, phosphorylated cGMP kinase which contained 1.4 +/- 0.4 mol phosphate/subunit and constitutively active cGMP kinase which lacked the amino-terminal dimerization domain. These three enzyme forms have identical kinetic constants, e.g. number of cGMP-binding sites, Km values for MgATP and the heptapeptide kemptide, and Vmax values. In the native enzyme, MgATP decreases the affinity for binding site 1. This effect is abolished by 1 M NaCl. In contrast, high concentrations of Kemptide increase the affinity of binding site 2 about fivefold. Under the latter conditions, identical Kd values of 0.2 microM were obtained for sites 1 and 2. Salt, MgATP and Kemptide do not affect the binding kinetics of the phosphorylated or the constitutively active enzyme, suggesting that allosteric regulation depends solely on the presence of a native amino-terminal segment. Cyclic GMP activates the native enzyme at Ka values which are identical with the Kd values for both binding sites. The activation of cGMP-dependent protein kinase is noncooperative but the Ka value depends on the substrate peptide concentration. These results show that the activity of cGMP kinase is primarily regulated by conformational changes within the amino-terminal domain.

Topics: Adenosine Triphosphate; Allosteric Regulation; Allosteric Site; Cyclic GMP; Enzyme Activation; Kinetics; Oligopeptides; Phosphorylation; Protein Conformation; Protein Kinases; Sodium Chloride; Temperature