Antischistosomal and liver protective effects of Curcuma longa extract in Schistosoma mansoni infected mice

With a view to clarify the induction of the "Crabtree consequence" in liver cells of S. mansoni infected mice, the curative effect of oil extract of C. longa was tested and compared to praziquantel (PZQ) the effective drug against all schistosome species occurring in man. Protein, glucose, glucose-6-phopsphatase, AMP-deaminase, adensoine deaminase, urea concentration, pyravate kinase (PK), phosphoenol pyruvate carboxykinase (PEPCK) and PK/PEPCK ratio were estimated. In addition, worm burden and ova count in mice infected with S. mansoni were elucidated. The result showed that C. longa normalized the concentration of protein, glucose, AMP-deaminase and adenosine deaminase, which were changed by infection. Moreover, it lowered pyruvate kinase level, while PZQ-treatment induced more elevation of this enzyme. PZQ was more effective in lowering worm burden while C. longa extract was more potent in reducing egg count.     

Differential phosphorylation of multiple sites in purified protein I by cyclic AMP-dependent and calcium-dependent protein kinases

Protein I, a specific neuronal phosphoprotein, has previously been shown, using rat brain synaptosome preparations, to contain multiple sites of phosphorylation which were differentially regulated by cAMP and calcium. In the present study, Protein I was purified to homogeneity from rat brain and its phosphorylation was investigated using homogeneous cAMP-dependent protein kinase and a partially purified calcium-calmodulin-dependent protein kinase from rat brain. Employing various peptide mapping techniques, a minimum of three phosphorylation sites could be distinguished in Protein I; the phosphorylated amino acid of each site was serine. One phosphorylation site was located in the collagenase-resistant portion of Protein I and was the principal target for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase. This site was also phosphorylated by calcium-calmodulin-dependent protein kinase. The other two phosphorylation sites were located in the collagenase-sensitive portion of Protein I. These latter sites were markedly phosphorylated by calcium-calmodulin-dependent protein kinase, but not by cAMP-dependent protein kinase in concentrations sufficient to phosphorylate maximally the site in the collagenase-resistant portion. Thus, the phosphorylation of purified Protein I by purified cAMP-dependent and calcium-calmodulin-dependent protein kinases provides an enzymological explanation for the regulation of phosphorylation of endogenous Protein I in synaptosome preparations by cAMP and by calcium observed previously. The studies suggest that certain of the synaptic actions of two distinct second messengers, cAMP and calcium, are expressed through the distinct specificities of cAMP- and calcium-dependent protein kinases for the multiple phosphorylation sites in one neuron-specific protein, Protein I.     

Role of adenylate kinase, AMP deaminase and 5'-nucleotidase in the metabolism of adenylic nucleotides

The activities of adenylate kinase, AMP-deaminase and 5'-nucleotidase in various tissues of the rat were studied. The activity of the forward adenylate kinase reaction (ATP + AMP----2 ADP) against the back one (2 ADP----ATP + AMP) was predominant. The liver was shown to contain two, while the blood serum--three adenylate kinase isoenzymes. In the skeletal muscles, the catabolism of adenylic acid involving AMP-deaminase and 5'-nucleotidase predominantly occurred via deamination, in the liver--via dephosphorylation, while in the leucocytes, erythrocytes and blood serum the activity of these processes was essentially the same. In vitro, ATP enhanced the activity of AMP-deaminase in the liver, leucocytes and erythrocytes and decreased it in the blood serum. Under effects of ATP, the activity of 5'-nucleotidase in the leucocytes and blood serum was markedly elevated, that in the liver and erythrocytes was unaffected.     

Enzymes in thick filaments of vertebrate cross-striated muscles]

Contemporary data on three enzymes of vertebrate cross-striated muscle thick filaments, such as creatine kinase, AMP-deaminase and phosphofructokinase, are reviewed. The physico-chemical, enzymatic and regulatory properties and localization of these enzymes in different zones of the thick filament are considered. The functional relevance of localization of creatine kinase, AMP-deaminase and phosphofructokinase on thick filaments is discussed in terms of the possible role of the enzyme adsorption on subcellular structures in regulation of metabolic processes.     

Phosphorylation of caldesmon by protein kinase C

Protein kinase C catalyzes phosphorylation of caldesmon, an F-actin binding protein of smooth muscle, in the presence of Ca2+ and phospholipid. Protein kinase C incorporates about 8 mol of phosphate/mol of chicken gizzard caldesmon. When calmodulin was added in the medium, there was an inhibition of phosphorylation. The fully phosphorylated, but not unphosphorylated, caldesmon inhibited myosin light chain kinase activity. The possibility that protein kinase C plays some role in smooth muscle contractile system through caldesmon, warrants further attention.     

Protein kinase Cbeta and delta selectively phosphorylate odorant and metabotropic glutamate receptors.

Recombinant protein segments from a metabotropic glutamate receptor and from an odorant receptor were used as substrates in protein kinase C phosphorylation assays. Protein kinase Cbeta and delta phosphorylated an intracellular consensus phosphorylation site in the metabotropic glutamate receptor. Only protein kinase Cdelta phosphorylated a novel extracellular consensus phosphorylation site in the odorant receptor. These results suggest differential regulation of these receptors by protein kinase C isotypes.     

Protein Kinase C-Mediated Phosphorylation of Kvβ2 in Adult Rat Brain

The phosphorylation of Kvβ2 was investigated by different protein kinases. Protein kinase A catalytic subunit (PKA-CS) yielded the greatest phosphorylation of recombinant Kvβ2 (rKvβ2), with limited phosphorylation by protein kinase C catalytic subunit (PKC-CS) and no detectable phosphorylation by casein kinase II (CKII). Protein kinase(s) from adult rat brain lysate phosphorylated both rKvβ2 and endogenous Kvβ. The PKA inhibitor, PKI 6-22, fully inhibited PKA-mediated phophorylation of rKvβ2 yet showed minimal inhibition of kinase activity present in rat brain. The inhibitor Gö 6983, that blocks PKCα, PKCβ, PKCγ, PKCδ and PKCζ activities, inhibited rKvβ2 phosphorylation by rat brain kinases, with no inhibition by Gö 6976 which blocks PKCα and PKCβΙ activities. Dose-response analysis of Gö 6983 inhibitory activity indicates that at least two PKC isozymes account for the kinase activity present in rat brain. Τhus, while PKA was the most active protein kinase to phosphorylate rKvβ2 in vitro, Kvβ2 phosphorylation in the rat brain is mainly mediated by PKC isozymes.     

Involvement of protein kinase C in the phosphorylation of an insulin-granule membrane protein.

The involvement of protein kinase C in the Ca2+-dependent phosphorylation of a 29 000-Mr insulin-granule membrane protein prepared from a rat insulinoma was investigated. Protein kinase C activity towards exogenous lysine-rich histone was detected in a cytosolic fraction prepared from an insulinoma homogenate in the presence of EGTA. This activity bound reversibly to insulin granules in a Ca2+-dependent manner. Phosphatidylserine liposomes removed both protein kinase C activity and the 29 000-Mr protein-phosphorylating activity from the cytosolic fraction in a Ca2+-dependent fashion. Protein kinase C activity and the enzymic activity responsible for the phosphorylation of the 29 000-Mr granule protein behaved identically on sucrose-density-gradient centrifugation, ion-exchange chromatography, (NH4)2SO4 fractionation and gel filtration of the cytosolic fraction. These results are consistent with protein kinase C being the enzyme responsible for the phosphorylation of the 29 000-Mr insulin-granule membrane protein.     

Protein kinase C catalyses the phosphorylation and activation of rat liver phospholipid methyltransferase.

When a partially purified rat liver phospholipid methyltransferase is incubated with [gamma-32P]ATP and rat brain protein kinase C, phospholipid methyltransferase (Mr 50,000, pI 4.75) becomes phosphorylated. Phosphorylation of the enzyme showed Ca2+/lipid-dependency. Protein kinase C-dependent phosphorylation of phospholipid methyltransferase was accompanied by an approx. 2-fold activation of the enzyme activity. Activity changes and enzyme phosphorylation showed the same time course. Activation of the enzyme also showed Ca2+/lipid-dependency. Protein kinase C mediates phosphorylation of predominantly serine residues of the methyltransferase. One major peak of phosphorylation was identified by analysis of tryptic phosphopeptides by isoelectrofocusing. This peak (pI 5.2) differs from that phosphorylated by the cyclic AMP-dependent protein kinase (pI 7.2), demonstrating the specificity of phosphorylation of protein kinase C. Tryptic-peptide mapping by h.p.l.c. of the methyltransferase phosphorylated by protein kinase C revealed one major peak of radioactivity, which could be resolved into two labelled phosphopeptides by t.l.c. The significance of protein kinase C-mediated phosphorylation of phospholipid methyltransferase is discussed.     

Modulation of protein 4.1 binding to inside-out membrane vesicles by phosphorylation

The effect of phosphorylation on the binding of protein 4.1 to erythrocyte inside-out vesicles was investigated. Protein 4.1 was phosphorylated with casein kinase A, protein kinase C, and cAMP-dependent protein kinase. An analysis of the phosphopeptides generated by alpha-chymotryptic and tryptic digestion indicates these kinases phosphorylate similar as well as distinct domains within protein 4.1. All three enzymes catalyze the phosphorylation to varying degrees of the 46-, 16-, and 8-10-kDa fragments derived from limited chymotryptic cleavage. In addition, casein kinase A phosphorylates a 24-kDa domain, whereas protein kinase C phosphorylates a 30-kDa domain. Protein 4.1 phosphorylated by casein kinase A and protein kinase C, but not cAMP-dependent protein kinase, exhibits a reduced binding to KI-extracted inside-out vesicles. On the other hand, phosphorylation of inside-out vesicles by casein kinase A does not affect their ability to bind protein 4.1. The inside-out vesicles, however, inhibit the phosphorylation of protein 4.1 by casein kinase A and protein kinase C, but not by cAMP-dependent protein kinase. These results suggest that casein kinase A and protein kinase C may modulate the binding of protein 4.1 to the membrane by phosphorylation of specific domains of the cytoskeletal protein. Since the 30-kDa domain has been suggested as a membrane-binding site, that phosphorylation by protein kinase C reduces the binding of protein 4.1 to inside-out vesicles is perhaps not surprising. On the other hand, the role of the casein kinase A substrate 24-kDa domain in membrane binding has not been established and needs to be examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin antagonizes ischemia-induced Thr172 phosphorylation of AMP-activated protein kinase alpha-subunits in heart via hierarchical phosphorylation of Ser485/491

Previous studies showed that insulin antagonizes AMP-activated protein kinase activation by ischemia and that protein kinase B might be implicated. Here we investigated whether the direct phosphorylation of AMP-activated protein kinase by protein kinase B might participate in this effect. Protein kinase B phosphorylated recombinant bacterially expressed AMP-activated protein kinase heterotrimers at Ser(485) of the alpha1-subunits. In perfused rat hearts, phosphorylation of the alpha1/alpha2 AMP-activated protein kinase subunits on Ser(485)/Ser(491) was increased by insulin and insulin pretreatment decreased the phosphorylation of the alpha-subunits at Thr(172) in a subsequent ischemic episode. It is proposed that the effect of insulin to antagonize AMP-activated protein kinase activation involves a hierarchical mechanism whereby Ser(485)/Ser(491) phosphorylation by protein kinase B reduces subsequent phosphorylation of Thr(172) by LKB1 and the resulting activation of AMP-activated protein kinase.     

Identification of the phosphoserine residue in histone H1 phosphorylated by protein kinase C

The site-specific phosphorylation of bovine histone H1 by protein kinase C was investigated in order to further elucidate the substrate specificity of protein kinase C. Protein kinase C was found to phosphorylate histone H1 to 1 mol per mol. Using N-bromosuccinimide and thrombin digestions, the phosphorylation site was localized to the globular region of the protein, containing residues 71-122. A tryptic peptide containing the phosphorylation site was purified. Modification of the phosphoserine followed by amino acid sequence analysis demonstrated that protein kinase C phosphorylated histone H1 on serine 103. This sequence, Gly97-Thr-Gly-Ala-Ser-Gly-Ser(PO4)-Phe-Lys105, supports the contention that basic amino acid residues C-terminal to the phosphorylation site are sufficient determinants for phosphorylation by protein kinase C.     

The effect of Ca2+-phospholipid dependent phosphorylation on passive calcium transport in the myocardial sarcolemma

Protein kinase C in vesicular preparations of the myocardium sarcolemma is shown to phosphorylate proteins with the molecular weight of 250, 140, 67, 58, 24 and 11 kD. The exogenic protein kinase C catalyzed phosphorylation of the sarcolemma preparations lowers the initial rate of the passive calcium transport from 0.56 down to 0.18 mmol per 1 mg second. Activation of endogenic protein kinase C by 4 beta-phorbol-12 beta-myristate-13 alpha-acetate is also accompanied by phosphorylation of vesicular preparations of sarcolemma and by inhibition of the passive calcium transport. Polymyxin B, being an inhibitor of protein kinase C, suppresses the phosphorylation and thus prevents the inhibitory action of phosphorylation on the passive calcium transport.     

Protein phosphorylation in rat cardiac microsomes: Effects of inhibitors of protein kinase A and of phosphatases

The phosphorylation of rat cardiac microsomal proteins was investigated with special attention to the effects of okadaic acid (an inhibitor of protein phosphatases), inhibitor 2 of protein phosphatase 1 and inhibitor of cyclic AMP-dependent protein kinase (protein kinase A). The results showed that okadaic acid (5 µM) modestly but reproducibly augmented the protein kinase A-catalyzed phospholamban (PLN) phosphorylation, although exerted little effect on the calcium/calmodulin kinase-catalyzed PLN phosphorylation. Microsomes contained three other substrates (M_r 23, 19 and 17 kDa) that were phosphorylated by protein kinase A but not by calcium/calmodulin kinase. The protein kinase A-catalyzed phosphorylation of these three substrates was markedly (2-3 fold) increased by 5 µM okadaic acid. Calmodulin was found to antagonize the action of okadaic acid on such phosphorylation. Protein kinase A inhibitor was found to decrease the protein kinase A-catalyzed phosphorylation of microsomal polyp eptides. Unexpectedly, inhibitor 2 was also found to markedly decrease protein kinase A-catalyzed phosphorylation of phospholamban as well these other microsomal substrates. These results are consistent with the views that protein phosphatase 1 is capable of dephosphorylating membrane-associated phospholamban when it is phosphorylated by protein kinase A, but not by calcium/calmodulin kinase, and that under certain conditions, calcium/calmodulin-stimulated protein phosphatase (protein phosphatase 2B) is also able to dephosphorylate PLN phosphorylated by protein kinase A. Additionally, the observations show that protein phosphatase 1 is extremely active against the three protein kinase A substrates (M_r 23, 19 and 17 kDa) that were present in the isolated microsomes and whose state of phosphorylation was particularly affected in the presence of dimethylsulfoxide. Protein phosphatase 2B is also capable of dephosphorylating these three substrates. (Mol Cell Biochem 175: 109–115, 1997     

Quantitative methods for the analysis of protein phosphorylation in drug development

Most signal transduction and cell signaling pathways are mediated by protein kinases. Protein kinases have emerged as important cellular regulatory proteins in many aspects of neoplasia. Protein kinase inhibitors offer the opportunity to target diseases such as cancer with chemotherapeutic agents specific for the causative molecular defect. In order to identify possible targets and assess kinase inhibitors, quantitative methods for analyzing protein phosphorylation have been developed. This review examines some of the current formats used for quantifying kinase function for drug development.     

Myeloid cell leukemia 1 is phosphorylated through two distinct pathways, one associated with extracellular signal-regulated kinase activation and the other with G2/M accumulation or protein phosphatase 1/2A inhibition

Protein kinase C activators and microtubule-damaging drugs stimulate BCL2 phosphorylation, which has been associated with either enhancement or inhibition of cell viability. In a Burkitt lymphoma cell line, both types of agents likewise stimulated phosphorylation of myeloid cell leukemia 1 (MCL1), another viability-promoting BCL2 family member. However, while MCL1 phosphorylation induced by the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), did not affect its electrophoretic mobility, microtubule-damaging agents, such as taxol, induced MCL1 phosphorylation associated with a band shift to decreased mobility. Inhibitors of extracellular signal-regulated kinase (ERK) activation blocked TPA-induced MCL1 phosphorylation but not the taxol-induced band shift. TPA-induced MCL1 phosphorylation occurred rapidly and was not associated with decreased viability, while the taxol-induced band shift occurred upon extended exposure as cells accumulated in G(2)/M followed by cell death. Protein phosphatase 1/2A inhibitors also induced the MCL1 band shift/phosphorylation. Thus, MCL1 undergoes two distinct types of phosphorylation: (i) TPA-induced, ERK-associated phosphorylation, which does not alter the electrophoretic mobility of MCL1, and (ii) ERK-independent phosphorylation, which results in an MCL1 band shift and is induced by events in G(2)/M or protein phosphatase 1/2A inhibitors.     

Quantitative methods for the analysis of protein phosphorylation in drug development

Most signal transduction and cell signaling pathways are mediated by protein kinases. Protein kinases have emerged as important cellular regulatory proteins in many aspects of neoplasia. Protein kinase inhibitors offer the opportunity to target diseases such as cancer with chemotherapeutic agents specific for the causative molecular defect. In order to identify possible targets and assess kinase inhibitors, quantitative methods for analyzing protein phosphorylation have been developed. This review examines some of the current formats used for quantifying kinase function for drug development.     

Phosphorylation of smooth muscle myosin light chain kinase by Ca2+-activated, phospholipid-dependent protein kinase

Protein kinase C incorporates phosphate into two sites of myosin light chain kinase (MLC-kinase) in the absence of calmodulin. Phosphorylation is all but abolished in the presence of Ca2+ and calmodulin, suggesting that both sites of phosphorylation are close to the calmodulin binding site. The phosphorylation of MLC-kinase results in an approximately 10-fold increase in the dissociation constant of MLC-kinase for calmodulin. Following phosphorylation (2 mol/mol of enzyme) of MLC-kinase by protein kinase C, an additional 2 mol of phosphate can be incorporated into the MLC-kinase apoenzyme by the cAMP-dependent protein kinase. Different maps of phosphopeptides were obtained by tryptic hydrolysis from MLC-kinase preparations phosphorylated by each kinase. The phosphorylation sites for the cAMP-dependent kinase were located in a fragment of approximately 25,000 daltons. In contrast the phosphorylation sites for protein kinase C are found in a much smaller tryptic peptide. These results suggest that the phosphorylation sites on MLC-kinase are different for protein kinase C and for cAMP-dependent protein kinase. However, phosphorylation in both regions results in a reduced affinity for calmodulin.     

Control of glycogen synthase by hierarchal protein phosphorylation

Protein phosphorylation is one of the most common mechanisms for controlling protein function. We now know that most phosphoproteins contain multiple phosphorylation sites and that these sites are often located in clusters. From the study of the enzyme glycogen synthase, one mechanism for the formation of phosphorylation clusters has been discovered that involves the concerted action of two or more protein kinases. One protein kinase, the primary kinase, introduces a phosphate group that is a requirement for the action of another, secondary, protein kinase. Thus the multiple phosphorylation occurs in a hierarchal fashion. This mechanism, which is critical for the phosphorylation of glycogen synthase, is likely to be a much more widespread phenomenon.     

In Vivo and In Vitro Protein Phosphorylation Studies on Ochromonas danica, an Alga with a Chlorophyll a/c/Fucoxanthin Binding Protein

The phosphorylation of thylakoid membranes in the Chromophyte alga Ochromonas danica was studied in whole cells and in vitro. Protein kinase activity was observed in the thylakoid fraction, and several membrane-bound polypeptides were found to be phosphorylated. The thylakoid protein kinase demonstrated several unusual regulatory properties. Both the polypeptides that were phosphorylated and the rate of protein phosphorylation were independent of illumination. Protein kinase activity was also unaffected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron. The kinase activity was inhibited under strong reducing conditions. Whole cells labeled with ³²PO₄³⁻ were converted to light states I and II by pre-illumination favoring photosystem I or photosystem II, respectively. Analysis of the phosphoproteins from cells in state I and state II showed that no changes in phosphorylation accompanied the change in energy redistribution.