Protein kinase C–catalyzed calponin phosphorylation in swine carotid arterial homogenate

Calponin, a thin filament–associated protein, inhibits actin-activated myosin ATPase activity, and this inhibition is reversed by phosphorylation. Calponin phosphorylation by protein kinase C and Ca²⁺/calmodulin-dependent protein kinase II has been shown in purified protein systems but has been difficult to demonstrate in more physiological preparations. We have previously shown that calponin is phosphorylated in a cell-free homogenate of swine carotid artery. The goal of this study was to determine whether protein kinase C and/or Ca²⁺/calmodulin-dependent protein kinase II catalyzes calponin phosphorylation. Ca²⁺-dependent calponin phosphorylation was not inhibited by calmodulin antagonists. In contrast, both Ca²⁺- and phorbol dibutyrate/1-oleoyl-2-acetyl-sn-glycerol–dependent calponin phosphorylation were inhibited by the pseudosubstrate inhibitor of protein kinase C and staurosporine. Our results also demonstrate that stimulation with either Ca²⁺, phorbol dibutyrate, or 1-oleoyl-2-acetyl-sn-glycerol activates endogenous protein kinase C. We interpret our results as clearly demonstrating that the physiological kinase for calponin phosphorylation is protein kinase C and not Ca²⁺/calmodulin-dependent protein kinase II. We also present data showing that the direct measurement of ³²P incorporation into calponin and the indirect measurement of calponin phosphorylation using nonequilibrium pH gradient gel electrophoresis provide similar quantitative values of calponin phosphorylation. J. Cell. Physiol. 176:545–552, 1998. © 1998 Wiley-Liss, Inc.     

Multifunctional Ca2+/calmodulin-dependent protein kinase

Multifunctional Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) is a prominent mediator of neurotransmitters which elevate Ca²⁺. It coordinates cellular responses to external stimuli by phosphorylating proteins involved in neurotransmitter synthesis, neurotransmitter release, carbohydrate metabolism, ion flux and neuronal plasticity. Structure/function studies of CaM kinase have provided insights into how it decodes Ca²⁺ signals. The kinase is kept relatively inactive in its basal state by the presence of an autoinhibitory domain. Binding of Ca²⁺/calmodulin eliminates this inhibitory constraint and allows the kinase to phosphorylate its substrates, as well as itself. This autophosphorylation significantly slows dissociation of calmodulin, thereby trapping calmodulin even when Ca²⁺ levels are subthreshold. The kinase may respond particularly wel to multiple Ca²⁺ spikes since trapping may enable a spike frequency-dependent recruitment of calmodulin with each successive Ca²⁺ spike leading to increased activation of the kinase. Once calmodulin dissociates, CaM kinase remains partially active until it is dephosphorylated, providing for an additional period in which its response to brief Ca²⁺ transients is potentiated.Special issue dedicated to Dr. Paul Greengard.     

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.     

Intracellular calcium signals regulate growth of hepatic stellate cells via specific effects on cell cycle progression

Hepatic stellate cells (HSC) are important mediators of liver fibrosis. Hormones linked to downstream intracellular Ca²⁺ signals upregulate HSC proliferation, but the mechanisms by which this occurs are unknown. Nuclear and cytosolic Ca²⁺ signals may have distinct effects on cell proliferation, so we expressed plasmid and adenoviral constructs containing the Ca²⁺ chelator parvalbumin (PV) linked to either a nuclear localization sequence (NLS) or a nuclear export sequence (NES) to block Ca²⁺ signals in distinct compartments within LX-2 immortalized human HSC and primary rat HSC. PV-NLS and PV-NES constructs each targeted to the appropriate intracellular compartment and blocked Ca²⁺ signals only within that compartment. PV-NLS and PV-NES constructs inhibited HSC growth. Furthermore, blockade of nuclear or cytosolic Ca²⁺ signals arrested growth at the G2/mitosis (G2/M) cell-cycle interface and prevented the onset of mitosis. Blockade of nuclear or cytosolic Ca²⁺ signals downregulated phosphorylation of the G2/M checkpoint phosphatase Cdc25C. Inhibition of calmodulin kinase II (CaMK II) had identical effects on LX-2 growth and Cdc25C phosphorylation. We propose that nuclear and cytosolic Ca²⁺ are critical signals that regulate HSC growth at the G2/M checkpoint via CaMK II-mediated regulation of Cdc25C phosphorylation. These data provide a new logical target for pharmacological therapy directed against progression of liver fibrosis.     

Calmodulin can induce and control damped oscillations in plasma membrane Ca<Superscript>2+</Superscript>-ATPase activity: A kinetic model

Plasma membrane Ca²⁺-ATPase is the pump that extrudes calcium ions from cells using ATP hydrolysis to maintain low Ca²⁺ concentrations in the cell. Calmodulin stimulates Ca²⁺-ATPase by binding to the autoinhibitory enzyme domain, which allows the access of cytoplasmic ATP and Ca²⁺ to the catalytic and transport sites. Our kinetic model predicts damped oscillations of the enzyme activity and interprets the known nonmonotonic kinetic behavior of the enzyme in the presence of calmodulin. For parameters close to experimental data, the kinetic model explains the dependence of the frequency and damping factor of the oscillatory enzyme activity on the calmodulin concentration. The calculated pre-steady-state curves fit well to known experimental data. Kinetic analysis allows us to assign Ca²⁺-ATPase to hysteretic enzymes exhibiting activity oscillations in open systems.     

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.     

Effects of Calcium-BAPTA Buffers and the Calmodulin Antagonist W-7 on Mouse Egg Activation

Results of numerous experiments indicate that the transient rise in intracellular Ca²⁺following sperm–egg fusion is essential for the subsequent events that constitute egg activation. Some events of egg activation, e.g., cortical granule exocytosis, however, appear more sensitive to intracellular Ca²⁺than other events, e.g., cell cycle resumption. To examine if specific events of egg activation have different thresholds for Ca²⁺, we manipulated buffered intracellular Ca²⁺concentrations by microinjecting Ca²⁺-BAPTA buffers and then examined the effect on the cortical granule exocytosis, recruitment of maternal mRNAs, and cell cycle resumption. We find that whereas cortical granule exocytosis occurs over a narrow threshold range of injected free Ca²⁺concentrations between 0.5 and 1.0 μM,recruitment of maternal mRNAs is only partially stimulated at injected free Ca²⁺concentrations of 2.5 μM,and no evidence for cell cycle resumption was observed (up to 2.5 μMCa²⁺). Although the Ca²⁺- and phospholipid-dependent protein kinase, protein kinase C, is implicated in aspects of egg activation, calmodulin is also a potential target for the transient increase in Ca²⁺that occurs following fertilization. Whereas incubation of eggs in the presence of the calmodulin antagonist W-7 followed by insemination does not block cortical granule exocytosis, cell cycle resumption, as assessed by the metaphase-to-anaphase transition, a decrease in histone H1 kinase activity and the time course for the emission of the second polar body are significantly delayed/inhibited.     

Purification and characterization of a Ca<Superscript>2+</Superscript>-dependent/calmodulin-stimulated protein kinase from moss chloronema cells

We have demonstrated the presence of a Ca²⁺-dependent/calmodulin-stimulated protein kinase (PK) in chloronema cells of the mossFunaria hygrometrica. The kinase, with a molecular mass of 70,000 daltons (PK70), was purified to homogeneity using ammonium sulphate fractionation, DEAE-cellulose chromatography, and calmodulin (CaM)-agarose affinity chromatography. The kinase activity was stimulated at a concentration of 50 (AM free Ca²⁺, and was further enhanced 3–5-fold with exogenously added 3–1000 nm moss calmodulin (CaM). Autophosphorylation was also stimulated with Ca²⁺ and CaM. Underin vitro conditions, PK70 phosphorylated preferentially lysine-rich substrates such as HIIIS and HVS. This PK shares epitopes with the maize Ca²⁺-dependent/calmodulin-stimulated PK (CCaMK) and also exhibits biochemical properties similar to the maize, lily, and tobacco CCaMK. We have characterized it as a moss CCaMK.     

Decoding of calcium oscillations by phosphorylation cycles: analytic results

Experimental studies have demonstrated that Ca²⁺-regulated proteins are sensitive to the frequency of Ca²⁺ oscillations, and several mathematical models for specific proteins have provided insight into the mechanisms involved. Because of the large number of Ca²⁺-regulated proteins in signal transduction, metabolism and gene expression, it is desirable to establish in general terms which molecular properties shape the response to oscillatory Ca²⁺ signals. Here we address this question by analyzing in detail a model of a prototypical Ca²⁺-decoding module, consisting of a target protein whose activity is controlled by a Ca²⁺-activated kinase and the counteracting phosphatase. We show that this module can decode the frequency of Ca²⁺ oscillations, at constant average Ca²⁺ signal, provided that the Ca²⁺ spikes are narrow and the oscillation frequency is sufficiently low—of the order of the phosphatase rate constant or below. Moreover, Ca²⁺ oscillations activate the target more efficiently than a constant signal when Ca²⁺ is bound cooperatively and with low affinity. Thus, the rate constants and the Ca²⁺ affinities of the target-modifying enzymes can be tuned in such a way that the module responds optimally to Ca²⁺ spikes of a certain amplitude and frequency. Frequency sensitivity is further enhanced when the limited duration of the external stimulus driving Ca²⁺ signaling is accounted for. Thus, our study identifies molecular parameters that may be involved in establishing the specificity of cellular responses downstream of Ca²⁺ oscillations.     

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 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.     

CaMKIIα interacts with M4 muscarinic receptors to control receptor and psychomotor function

Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the mammalian brain and are essential for neuronal functions. These receptors are believed to be actively regulated by intracellular signals, although the underlying mechanisms are largely unknown. In this study, we show that Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) binds directly and selectively to one of five mAChR subtypes, M4 receptors (M4Rs), at their C‐terminal regions of second intracellular loops. This binding relies on Ca²⁺ activation of the kinase and leads to the phosphorylation of M4Rs at a specific threonine site (Thr145). Complementary in vivo studies in rat striatal neurons enriched with M4Rs confirm that rising Ca²⁺ recruits CaMKIIα to M4Rs to potentiate receptor signalling, which controls behavioural sensitivity to dopamine stimulation in an activity‐dependent manner. Our data identify a new model of protein–protein interactions. In a Ca²⁺‐sensitive manner, CaMKIIα regulates M4R efficacy and controls the acetylcholine–dopamine balance in the basal ganglia and also the dynamics of movement.     

Ca2+-Independent Autophosphorylation of Postsynaptic Density-Associated Ca2+/Calmodulin-Dependent Protein Kinase

A major protein in the postsynaptic density fraction is -CAM kinase II, the -subunit of the Ca²⁺/calmodulin-dependent protein kinase. Autophosphorylation of the postsynaptic density-associated CaM kinase II is likely to be a crucial event in the induction of activity-dependent synaptic modification. This study focuses on the regulation and consequences of Ca²⁺-independent autophosphorylation of the enzyme. In isolated postsynaptic densities, a sub-stochiometric level of autophosphorylation in the presence of Ca²⁺ is sufficient to trigger maximal Ca²⁺-independent autophosphorylation of -CaM Kinase II. A major fraction of the sites phosphorylated in the absence of Ca²⁺ can be dephosphorylated by the endogenous phosphatase activity in the preparation. Ca²⁺-independent autophosphorylation is correlated with a drastic decrease in calmodulin binding to postsynaptic densities. This may represent a physiological mechanism that lowers the calmodulin trapping capacity of the organelle, thus increasing the availability of calmodulin to other elements within a spine.     

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.     

Intracellular calcium mobilization and neurite outgrowth in mammalian neurons

Cultured adult rat dorsal root ganglion (DRG) neurons were used to study depolarization-induced Ca²⁺ mobilization and the effects of intracellular Ca²⁺ depletion on neurite outgrowth. Cytoplasmic and nuclear Ca²⁺ signals were visualized in dissociated DRG neurons using confocal scanning laser microspcopy and the Ca²⁺ indicator dye fluo-3. The depolarization-induced Ca²⁺ signals were highest in neurons during the first few days in culture, prior to neurite extension; during this time nuclear signals exceeded those of the cytoplasm severalfold. After several days in culture, neurons began to arborize, depolarization-induced Ca²⁺ signals became attenuated, and nuclear signals no longer exceeded those of the cytoplasm. Elevated Ca²⁺ signals were dependent upon both Ca²⁺ influx and intact intracellular Ca²⁺ stores, indicating that the signals are generated by calcuim-induced calcium release (CICR). Thapsigargin, an endoplasmic reticulum Ca²⁺ ATPase inhibitor, depleted intracellular Ca²⁺ stores and blocked the induction of the large nuclear Ca²⁺ signals. Treating DRG neurons briefly with thapsigargin (200 nM for 20 min) shortly after plating reduced subsequent neuritogenesis, impyling that intact Ca²⁺ stores are necessery for initiating neurite outgrowth. Immunostaining of DRG neurons with antibodies to Ca²⁺ /calmodulin-dependent kinase II (CaM kinase II) demonstrated that this enzyme is present in the nucleus at early times in culture. These observations are consistent with the idea that CICR triggered by Ca²⁺ entry subsequent to depolarization may elicit neurite outgrowth by activating nuclear enzymes appropriate for such outgrowth. © 1994 John Wile & Sons, Inc.     

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.     

The dynamics of PKC‐induced phosphorylation triggered by Ca2+ oscillations in mouse eggs

Fertilization of mammalian eggs is characterized by a series of Ca²⁺ oscillations triggered by a phospholipase C activity. These Ca²⁺ increases and the parallel generation of diacylglycerol (DAG) stimulate protein kinase C (PKC). However, the dynamics of PKC activity have not been directly measured in living eggs. Here, we have monitored the dynamics of PKC‐induced phosphorylation in mouse eggs, alongside Ca²⁺ oscillations, using fluorescent C‐kinase activity reporter (CKAR) probes. Ca²⁺ oscillations triggered either by sperm, phospholipase C zeta (PLCζ) or Sr²⁺ all caused repetitive increases in PKC‐induced phosphorylation, as detected by CKAR in the cytoplasm or plasma membrane. The CKAR responses lasted for several minutes in both the cytoplasm and plasma membrane then returned to baseline values before subsequent Ca²⁺ transients. High frequency oscillations caused by PLCζ led to an integration of PKC‐induced phosphorylation. The conventional PKC inhibitor, Gö6976, could inhibit CKAR increases in response to thapsigargin or ionomycin, but not the repetitive responses seen at fertilization. Repetitive increases in PKCδ activity were also detected during Ca²⁺ oscillations using an isoform‐specific δCKAR. However, PKCδ may already be mostly active in unfertilized eggs, since phorbol esters were effective at stimulating δCKAR only after fertilization, and the PKCδ‐specific inhibitor, rottlerin, decreased the CKAR signals in unfertilized eggs. These data show that PKC‐induced phosphorylation outlasts each Ca²⁺ increase in mouse eggs but that signal integration only occurs at a non‐physiological, high Ca²⁺ oscillation frequency. The results also suggest that Ca²⁺‐induced DAG formation on intracellular membranes may stimulate PKC activity oscillations at fertilization. J. Cell. Physiol. 228: 110–119, 2013. © 2012 Wiley Periodicals, Inc.     

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.     

Comparison of Ca2+/calmodulin-dependent protein kinase II purified from control and diisopropyl phosphorofluoridate (DFP)-treated hens

Diisopropyl phosphorofluoridate (DFP) produces type I organophosphorus ester-induced delayed neurotoxicity in humans and sensitive animal species. This is accompanied by enhanced Ca²⁺/CaM-dependent protein kinase II (CaM-kinase II) activity, and [¹²⁵I]calmodulin binding to CaM-kinase II in DFP-treated hen brain supernatant without increase in the enzyme quantity. We have purified CaM-kinase II from control and DFP-treated hen whole brains and compared various physical and biochemical properties. The two enzymes exhibited similar properties in many respects. However, there was a decrease in calcium-independent protein kinase II activity after autophosphorylation, and an increase in K_0.5 for free calcium and calmodulin of enzyme purified from DFP-treated hen brains. This change in kinetic parameters may result in greater percentage of total CaM-kinase II present in unphosphorylated form, which is consistent with the increased autophosphorylation of CaM-kinase II and [¹²⁵I]calmodulin binding in the brain supernatant of DFP-treated hens.Abbreviations used CaM calmodulin - CaM-kinase II Ca²⁺/calmodulin-dependent protein kinase II - MAP-2 microtubule associated protein-2 - DFP diisopropylphosphorofluoridate - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - NEPHGE nonequilibrium pH gradient electrophoresis - OPIDN organophosphorus ester-induced delayed neurotoxicity - PIPES 1,4-piperazinediethanesulfonic acid - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dedecyl sulfate-polyacrylamide gel electrophoresis - St. aureus V₈ protease Staphylococus aureus V₈ protease - TOCP tri-O-cresyl phosphate - TPCK N-tosyl-I-phenylalanine chloromethyl ketone