Carrot phytoalexin inhibits Ca2+, calmodulin-dependent protein phosphorylation in carrot cells

Phosphorylation of soluble proteins obtained from cultured carrot cells was monitored by measuring the incorporation of ³²P from [³²P]ATP into the trichloroacetic acid insoluble fraction. The reaction was stimulated by Ca²⁺ and calmodulin, and inhibited by a carrot phytoalexin, 6-methoxymellein. 6-Methoxymellein also inhibited the Ca²⁺, calmodulin-dependent phosphorylative activation of NAD:quinate oxidoreductase (EC 1.1.1.24) partially purified from the carrot cells. The inhibitory effect of 6-methoxymellein was reduced when the reaction mixture contained a high concentration of calmodulin.     

The elicitation of phytoalexins by Ca2+ and cyclic AMP in carrot cells

Addition of calcium ionophore A23187 or dibutyryl cyclic AMP (dBcAMP) to carrot (Daucus carota L.) cell culture induced the production of 6-methoxymellein, a phytoalexin of carrot, in a dose-dependent manner. Several reagents known to suppress the cytoplasmic calcium concentration appreciably inhibited elicitor-promoted phytoalexin production in carrot cells. The addition of elicitor to the carrot culture caused a rapid increase in the intracellular level of cyclic AMP. Treatments of the cells with theophylline or cholera toxin stimulated the biosynthesis of 6-methoxymellein even in the absence of elicitor. These observations suggested that Ca²⁺ and cyclic AMP participate as second messengers in the regulation of 6-methoxymellein production in cultured carrot cells. Addition of verapamil to carrot cell culture markedly inhibited 6-methoxymellein production when it was added within 30 min after elicitor-treatment of the cells, but no inhibitory effect was observed after 60 min. The results suggest that these messengers function in an early stage of the elicitation process. Carrot cells which were previously treated with verapamil accumulated only small amounts of 6-methoxymellein following the addition of dBcAMP. In contrast, cells incubated initially with dBcAMP accumulated the phytoalexin at levels comparable to the control when verapamil was added to the culture.     

Inhibition of cyclic nucleotide phosphodiesterase by carrot phytoalexin

The carrot phytoalexin, 6-methoxymellein, was isolated and purified from carrot root slices infected by the fungus Chaetomium globosum. It inhibited the basal and calmodulin-promoted activity of cyclic nucleotide phosphodiesterase. The inhibition of calmodulin-promoted diesterase activity was reduced by increasing the concentration of calmodulin or calcium while the inhibition of basal diesterase activity was reversed by the addition of magnesium to the assay mixture of the enzyme.     

A synergistic effect of glutathione-depletion and elicitation on the production of 6-methoxymellein in carrot cells

The effects of buthionine sulfoximine (BSO) and yeast glucan elicitor (YE) on the production of 6-methoxymellein (6-MM) and generation of H₂O₂ in suspension-cultured carrot cells were examined. Administration of BSO and YE together affected the cells synergistically to lead to an enhanced production of 6-MM. These data indicate the significance of formation and decay of active oxygen species as a second signal of elicitation in triggering the biosynthesis of the phytoalexin.Abbreviations BSO buthionine sulfoximine - MDA malondialdehyde - 6-MM 6-methoxymellein - YE yeast glucan elicitor     

Responses of crayfish neurons and glial cells to photodynamic impact: Intracellular signaling,ultrastructural changes,and neuroglial interactions

Photodynamic therapy (PDT), an inducer of oxidative stress, is used for treatment of cancer, including brain tumors. To study the mechanisms of photodynamic injury of neurons and glial cells (GC), we used a simple model object — isolated crayfish mechanoreceptor consisting of a single sensory neuron surrounded by a multilayered glial envelope. PDT caused inhibition and elimination of neuronal activity, impairment of intracellular organelles involved in the biosynthetic, bioenergetic, and transport processes and neuroglial interactions, necrosis of neurons and glial cells, and in glial apoptosis. PDT-induced death of a neuron and GC was mediated by intercellular molecular messengers and intracellular signaling cascades. PDT-induced inhibition and elimination of neuronal activity was associated with opening of mitochondrial permeability transition pores, Ca²⁺ release into cytosol, protein kinase C and NO synthase activities. Necrosis of neurons was mediated by protein kinases B/Akt, GSK-3β and mTOR, opening of mitochondrial permeability transition pores and Ca²⁺/calmodulin/CaMKII pathway. NO and GDNF reduced neuronal necrosis. Multiple signal pathways, such as phospholipase C/Ca²⁺, Ca²⁺/calmodulin/CaMKII, Ca²⁺/PKC, Akt/mTOR, MEK/p38, and protein kinase G mediated PDT-induced necrosis both in glial cells and in neurons. NOS/NO and neurotrophic factors NGF and GDNF protected glial cells and demonstrated antinecrotic activity. Glial apoptosis was reduced by neurotrophic factors NGF and GDNF, protein kinase C, and MAP kinase JNK. In contrast, mitochondrial permeability transition pores and phospholipase C, which mobilize intracellular Ca²⁺, NOS/NO/protein kinase G, proteins GSK-3β and mTOR, stimulated apoptosis of glial cells. The schemes of involvement of various inter- and intracellular signaling processes in the responses of neurons and GC to PDT are developed.     

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent protein kinase activity in rat renal cortex cytosol

The effect of regucalcin on Ca²⁺/calmodulin-dependent protein kinase activity in the cytosol of rat renal cortex was investigated. Regucalcin is a calcium-binding protein which exists in rat liver and renal cortex. Protein kinase activity in renal cortex cytosol was markedly increased by the addition of CaCl₂ (0.5 mM) plus calmodulin (10 µg/ml) in the enzyme reaction mixture. This increase was completely prevented by the addition of trifluoperazine (25 µM), an antagonist of calmodulin. The cytosolic Ca²⁺/calmodulin- dependent protein kinase activity was clearly inhibited by the addition of regucalcin; an appreciable effect of regucalcin was seen at 0.01 µM. The cytosolic Ca²⁺/calmodulin-dependent protein kinase activity was fairly increased by increasing concentrations of added Ca²⁺ (100-1000 µM). This increase was markedly blocked by the presence of regucalcin (0.1 µM). The inhibitory effect of regucalcin on the protein kinase activity was also seen with varying concentrations of calmodulin (2-20 µg/ml). These results demonstrate that regucalcin can regulate Ca²⁺/calmodulin-dependent protein kinase activity in renal cortex cells.     

Phytoalexin production in cultured carrot cells treated with pectinolytic enzymes

6-Methoxymellein, a phytoalexin of carrot, was produced in cultured cells upon addition of partial hydrolysates of carrot cells obtained by treatment with purified endo-polygalacturonase or endo-pectin lyase. Direct addition of these enzymes to the cell culture also stimulated the accumulation of this 6-methoxymellein. When the hydrolysates obtained by these enzymes were subsequently treated within pectin esterase, the activity for the elicitation of 6-methoxymellein production decreased appreciably. These results suggest that pectinolytic enzymes release elicitor-active cell wall fragments from carrot cells and that a certain degree of esterification of the galacturonosyl moiety in these pectic polysaccharides is required for elicitor activity.     

Partial Purification and Characterization of a Ca2+-Dependent Protein Kinase from the Green Alga, Dunaliella salina

A calcium-dependent protein kinase was partially purified and characterized from the green alga Dunaliella salina. The enzyme was activated at free Ca²⁺ concentrations above 10⁻⁷ molar. and half-maximal activation was at about 3 × 10⁻⁷ molar. The optimum pH for its Ca²⁺-dependent activity was 7.5. The addition of various phospholipids and diolein had no effects on enzyme activity and did not alter the sensitivity of the enzyme toward Ca²⁺. The enzyme was inhibited by calmodulin antagonists, N-(6-aminohexyl)-1-naphthalene sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide in a dose-dependent manner while the protein kinase C inhibitor, sphingosine, had little effect on enzyme activity up to 800 micromolar. Immunoassay showed some calmodulin was present in the kinase preparations. However, it is unlikely the kinase was calmodulin regulated, since it still showed stimulation by Ca²⁺ in gel assays after being electrophoretically separted from calmodulin by two different methods. This gel method of detection of the enzyme indicated that a protein band with an apparent molecular weight of 40,000 showed protein kinase activity at each one of the several steps in the purification procedure. Gel assay analysis also showed that after native gel isoelectric focusing the partially purified kinase preparations had two bands with calcium-dependent activity, at isoelectric points 6.7 and 7.1. By molecular weight, by isoelectric point, and by a comparative immunoassay, the Dunaliella kinase appears to differ from at least some of the calcium-dependent, but calmodulin and phospholipid independent kinases described from higher plants.     

The calmodulin hypothesis of neurotransmission

Ca²⁺ plays a major role in neurotransmission and synaptic modulation. Evidence is presented to support the calmodulin hypothesis of neurotransmission developed in this laboratory stating that calmodulin, a major Ca²⁺ binding protein in brain, mediates the effects of Ca²⁺ on neurotransmission. Calmodulin was isolated from highly enriched preparations of synaptic vesicles and nerve terminal cytoplasm. Ca²⁺ and calmodulin were shown to regulate several synaptic processes in isolated and intact preparations, including endogenous synaptic Ca²⁺-calmodulin protein kinase activity, neurotransmitter release, and synaptic vesicle and synaptic membrane interactions. Ca²⁺ and calmodulin were shown to activate a synaptic tubulin kinase system which was shown to be a distinct enzyme system from the cyclic AMP protein kinase. Ca²⁺ and calmodulin stimulated phosphorylation of tubulin altered the properties of tubulin, forming insoluble tubulin fibrils. Evidence for the role of Ca²⁺-calmodulin kinase activity, especially the calmodulin-tubulin kinase, in neurotransmission are presented. The effects of several neuroactive drugs on the synaptic calmodulin system are presented. The results support the hypothesis that calmodulin mediates many of calcium's actions at the synapse, and that the effects of Ca²⁺ on synaptic protein phosphorylation, especially synaptic tubulin, may provide a biochemical mechanism for converting the Ca²⁺ signal into a motor force in the process of neurotransmission.     

Isolation of brain Ca2+-calmodulin tubulin kinase containing calmodulin binding proteins

Ca²⁺-calmodulin tubulin kinase activity was isolated from brain cytosol and separated from its substrate protein, tubulin, and Ca²⁺ regulatory protein, calmodulin. Characterization of the Ca²⁺-tubulin kinase system revealed a Km of 4 μM, 0.5 μM, 60 μM for Ca²⁺, calmodulin and ATP, respectively. The tubulin kinase system bound to a calmodulin affinity column in the presence of Ca²⁺ and was released from the column by chelation with EGTA. A major 55,000 and a minor 65,000 dalton peptide were identified as the only calmodulin binding proteins in the enzyme fraction, indicating that one or both of these peptides represent the calmodulin binding subunit of the Ca²⁺-calmodulin tubulin kinase system.     

Studies on the kinetics of cation-associated fluorescence changes in chloroplast membranes

A soluble Ca²⁺- and Ca²⁺—calmodulin-activated protein kinase was partially purified from wheat germ. The phosphorylation of histones and casein catalyzed by this enzyme is largely Ca²⁺-dependent. After repeated gel filtration of the protein kinase in the presence of 1 mM EGTA, the phosphorylation of casein and histones by the enzyme is activated 3-fold and up to 16-fold, respectively, by added calmodulin (12.5 μM). Such activation of the protein kinase by calmodulin is Ca²⁺-dependent. The protein kinase binds to calmodulin—Sepharose 4B in a Ca²⁺-dependent fashion. This type of Ca²⁺-activated protein kinase may be involved in stimulus—response coupling in plants.     

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist,also inhibits phospholipid sensitive calcium-dependent protein kinase

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), commonly regared as a calmodulin antagonist, inhibted phospholipid-sensitive Ca²⁺-dependent protein kinase and to a lesser extent cyclic GMP- and cyclic AMP-dependent protein kinases. Kinetic studies of the inhibition of the homogenous spleen phospholipid-sensitive Ca²⁺-dependent protein kinase indicated that W-7 inhibited the enzyme activity competitively with respect to phospholipid (K_i = 60 μM). N-(6-Aminohexyl)-1-naphthalenesulfonamide (W-5) was found to be musch less potent than W-7. The findings indicate that W-6 was able to inhibit a variety of protein kinases, in addition to those requiring calmodulin previously reported.     

In Vitro Stimulation of Protein Kinase C by Melatonin

It has been shown that melatonin through binding to calmodulin acts both in vitro and in vivo as a potent calmodulin antagonist. It is known that calmodulin antagonists both bind to the hydrophobic domain of Ca²⁺ activated calmodulin, and inhibit protein kinase C activity. In this work we explored the effects of melatonin on Ca²⁺ dependent protein kinase C activity in vitro using both a pure commercial rat brain protein kinase C, and a partially purified enzyme from MDCK and N1E-115 cell homogenates. The results showed that melatonin directly activated protein kinase C with a half stimulatory concentration of 1 nM. In addition the hormone augmented by 30% the phorbol ester stimulated protein kinase C activity and increased [³H] PDBu binding to the kinase. In contrast, calmodulin antagonists (500 M) and protein kinase C inhibitors (100 M) abolished the enzyme activity. Melatonin analogs tested were ineffective in increasing either protein kinase C activity or [³H] PDBu binding. Moreover, the hormone stimulated protein kinase C autophosphorylation directly and in the presence of phorbol ester and phosphatidylserine. The results show that besides the melatonin binding to calmodulin, the hormone also interacts with protein kinase C only in the presence of Ca²⁺. They also suggest that the melatonin mechanism of action may involve interactions with other intracellular hydrophobic and Ca²⁺ dependent proteins.     

Biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans

A multifunctional Ca²⁺/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca²⁺/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca²⁺/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca²⁺/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca²⁺/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca²⁺/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca²⁺/calmodulin-dependent protein kinase of Candida albicans.     

Action of linoleic acid on phospholipase C activity at the rectal level of the African locust under the control of protein kinase C

Linoleic acid (18:2n-6), linolenic acid and eicosatrienoic acid stimulated fluid reabsorption in locust rectum. Only 18:2n-6 was able to enhance phospholipase C activity, inositol_(1,4,5) trisphosphate production and to increase cytosolic free Ca²⁺ concentrations in epithelial cells via the opening of L-type Ca²⁺ channels. These effects resemble those exerted by neuroparsin, an antidiuretic neuronal hormone extracted from the storage lobes of the locust corpora cardiaca. As for neuroparsin, the effects of 18:2n-6 were abolished after pre-treatment with the protein kinase C inhibitor, polymyxin B. The results were consistent with a regulation of neuroparsin-sensitive phospholipase C activity by 18:2n-6 under control of protein kinase C, possibly by increasing membrane fluidity. Cyclooxygenase inhibitors attenuated the effects of 18:2n-6. This demonstrated that the results should be produced via the metabolites of 18:2n-6 HODEs rather than the PUFA itself.     

Calmodulin and the regulation of smooth muscle contraction

Calmodulin, the ubiquitous and multifunctional Ca²⁺-binding protein, mediates many of the regulatory effects of Ca²⁺, including the contractile state of smooth muscle. The principal function of calmodulin in smooth muscle is to activate crossbridge cycling and the development of force in response to a [Ca²⁺]_i transientvia the activation of myosin light-chain kinase and phosphorylation of myosin. A distinct calmodulin-dependent kinase, Ca²⁺/calmodulin-dependent protein kinase II, has been implicated in modulation of smooth-muscle contraction. This kinase phosphorylates myosin light-chain kinase, resulting in an increase in the calmodulin concentration required for half-maximal activation of myosin light-chain kinase, and may account for desensitization of the contractile response to Ca²⁺. In addition, the thin filament-associated proteins, caldesmon and calponin, which inhibit the actin-activated MgATPase activity of smooth-muscle myosin (the cross-bridge cycling rate), appear to be regulated by calmodulin, either by the direct binding of Ca²⁺/calmodulin or indirectly by phosphorylation catalysed by Ca²⁺/calmodulin-dependent protein kinase II. Another level at which calmodulin can regulate smooth-muscle contraction involves proteins which control the movement of Ca²⁺ across the sarcolemmal and sarcoplasmic reticulum membranes and which are regulated by Ca²⁺/calmodulin, e.g. the sarcolemmal Ca²⁺ pump and the ryanodine receptor/Ca²⁺ release channel, and other proteins which indirectly regulate [Ca²⁺]_i via cyclic nucleotide synthesis and breakdown, e.g. NO synthase and cyclic nucleotide phosphodiesterase. The interplay of such regulatory mechanisms provides the flexibility and adaptability required for the normal functioning of smooth-muscle tissues.     

Changes in protein kinase and protein phosphatase properties during the cycle of asparaginase activity in Leptosphaeria michotti

Regulation of the cyclic activity of asparaginase (obtained as a purified protein complex) by a reversible auto-phosphorylation process has been previously reported in the fungus Leptosphaeria michotii (West) Sacc. In the present study, the protein complex was purified in the presence of either a mixture of 3 protein phosphatase inhibitors (fluoride, vanadate and molybdate) or EGTA, during the cycle of asparaginase activity, and the protein kinase and protein phosphatase activities characterized. (I) At the phase of increasing asparaginase activity, a Ca²⁺/calmodulin-dependent kinase activity was identified by (a) its inhibition by calmidazolium, reversed by calmodulin, and its inhibition by EGTA, but not by poly(Glu/Tyr 4:1)n. dichloro-(ribofuranosyl)-benzimidazole or polylysine (b) an increasing level of calmodulin bound to the complex, as estimated by enzyme-linked immunosorbent assay (ELISA). (2) At the phase of decreasing asparaginase activity, the Ca²⁺-calmodulin-dependent kinase activity disappeared and a little calmodulin remained associated with the complex: phosphorylation of the complex was increased several-fold by 1 nM okadaic acid and 25 nM inhibitor-2, and was not affected by EGTA, indicating a protein phosphatase-2A-like activity. (3) When asparaginase activity was low, a little calmodulin was bound to the complex. The kinase could phosphorylate casein and phosvitin. was inhibited by poly(Glu/Tyr 4:1)n. dichloro-(ribofuranosyl)-benzimidazole and heparin, stimulated by polylysine and not affected by calmidazolium or EGTA, just as a casein kinase 2. A Ca²⁺-dependent but calmodulin-independent protein phosphatase activity, not affected by okadaic acid and inhibitor-2. was then identified. We postulate the presence in the complex, of (a) only one protein kinase and one protein phosphatase, whose properties could change during the cycle of asparaginase activity: (b) two Ca²⁺/-binding proteins: first calmodulin, which could bind to Ca²⁺ and the casein kinase-2 form to give a Ca²⁺/calmodulin-dependent kinase, which could become Ca²⁺/calmodulin-independent following an auto-phosphorylation process: second a protein homologous to calmodulin, able to bind to the protein phosphatase-2A catalytic subunit to give a protein phosphatase-2B catalytic subunit.     

Regulation of rat cardiac nuclei-associated Mg2+-NTPase by phosphorylation

A nucleoside triphosphatase (NTPase) activity appeared to be associated with a highly purified nuclear preparation from rat cardiac ventricles. Different nucleoside triphosphates (UTP > GTP > ITP > CTP) supported this enzymic activity, which was stimulated by Mg` but not by Call. The nuclear NTPase activity could be down regulated by endogenous phosphorylation of a 55,000 M_r protein. Maximal phosphorylation of the 55,000 M_r protein occurred in the presence of Mg²⁺-ATP. Addition of cAMP, cGMP, Ca²⁺, Ca²⁺/phospholipid, Ca²⁺/calmodulin, and catalytic subunit of cAMP-dependent protein kinase was not associated with any further phosphorylation of the 55,000 M_r protein. However, in the presence of Ca²⁺/calmodulin or the catalytic subunit of the cAMP-dependent protein kinase additional proteins became phosphorylated, but these had no effect on the Mg²⁺-NTPase activity. These results indicate that a protein with M_r 55,000 may be involved in the regulation the Mg²⁺-NTPase activity associated with rat cardiac nuclei.Abbreviations Hg Hemoglobin - GAR Goat Anti-Rabbit antibody - SR Sarcoplasmic Reticulum - NTP Nucleoside Triphosphate - TCA Trichloroacetic acid - PAGE Polyacrylamide gel electrophoresis     

Derailment product in NADPH-dependent synthesis of a dihydroisocoumarin 6-hydroxymellein by elicitor-treated carrot cell extracts

Synthetic activity of 6-hydroxymellein, the immediate precursor of carrot phytoalexin 6-methoxymellein, from acetyl-CoA and malonyl-CoA was induced in carrot cell extracts when the root disks were treated with CuCl2 or oligogalacturonide elicitor. These elicitors showed specific inducing activity of phytoalexin production and did not affect fatty acid synthesis in carrot tissues which may share some common properties with 6-hydroxymellein biosynthesis. 6-Hydroxymellein production was an NADPH-dependent process and, in the absence of the reagent, triacetic acid lactone was produced as a derailment product of the reaction process. This finding suggested that the reduction of the double bond at the 3,4-position of the phytoalexin takes place during the elongation of the poly(oxomethylene) chain. This NADPH-dependent reduction seems to occur at the triacetate stage before the condensation of the third malonyl-CoA as the conversion of carbonyl to hydroxyl group.     

Regulation of the Tyrosine Kinase Pyk2 by Calcium Is through Production of Reactive Oxygen Species in Cytotoxic T Lymphocytes

Pyk2 was identified as a Ca²⁺-dependent kinase, however, the regulation of Pyk2 by Ca²⁺ in T cells remains controversial. We found that Ca²⁺ mobilization preferentially induced Pyk2 phosphorylation in cytotoxic T lymphocytes (CTL). Furthermore, Pyk2 phosphorylation in CTL was not absolutely Ca²⁺ dependent but relied on the strength of T cell receptor stimulation. Ionomycin-stimulated Pyk2 phosphorylation did not require calmodulin activity, because phosphorylation was not inhibited by the calmodulin inhibitor W7, and we detected no Ca²⁺-regulated association between Pyk2 and calmodulin. Ca²⁺-stimulated Pyk2 phosphorylation was dependent on Src-family kinase activity, even at the Pyk2 autophosphorylation site. We sought to identify a Ca²⁺-regulated pathway that could trigger Pyk2 phosphorylation in T cells and found that ionomycin stimulated the production of reactive oxygen species and an H₂O₂ scavenger inhibited ionomycin-induced Pyk2 phosphorylation. Additionally, H₂O₂ induced strong Erk activation and ionomycin-stimulated Pyk2 phosphorylation was Erk dependent. These data support the conclusion that Ca²⁺ mobilization induces the production of reactive oxygen species, which in turn activate the Erk pathway, leading to Src-family kinase-dependent Pyk2 phosphorylation. Our data demonstrate that Pyk2 is not a Ca²⁺-dependent kinase in T cells but instead, increased intracellular Ca²⁺ induces Pyk2 phosphorylation through production of reactive oxygen species. These findings are consistent with the possibility that Pyk2 acts as an early sensor of numerous extracellular signals that trigger a Ca²⁺ flux and/or reactive oxygen species to amplify tyrosine phosphorylation signaling events.