Activation Of Calmodulin-dependent Enzymes Research Articles

Phosphorylation within an Autoinhibitory Domain in Endothelial Nitric Oxide Synthase Reduces the Ca2+ Concentrations Required for Calmodulin To Bind and Activate the Enzyme

We have investigated the effects of phosphorylation at Ser-617 and Ser-635 within an autoinhibitory domain (residues 595-639) in bovine endothelial nitric oxide synthase on enzyme activity and the Ca (2+) dependencies for calmodulin binding and enzyme activation. A phosphomimetic S617D substitution doubles the maximum calmodulin-dependent enzyme activity and decreases the EC 50(Ca (2+)) values for calmodulin binding and enzyme activation from the wild-type values of 180 +/- 2 and 397 +/- 23 nM to values of 109 +/- 2 and 258 +/- 11 nM, respectively. Deletion of the autoinhibitory domain also doubles the maximum calmodulin-dependent enzyme activity and decreases the EC 50(Ca (2+)) values for calmodulin binding and calmodulin-dependent enzyme activation to 65 +/- 4 and 118 +/- 4 nM, respectively. An S635D substitution has little or no effect on enzyme activity or EC 50(Ca (2+)) values, either alone or when combined with the S617D substitution. These results suggest that phosphorylation at Ser-617 partially reverses suppression by the autoinhibitory domain. Associated effects on the EC 50(Ca (2+)) values and maximum calmodulin-dependent enzyme activity are predicted to contribute equally to phosphorylation-dependent enhancement of NO production during a typical agonist-evoked Ca (2+) transient, while the reduction in EC 50(Ca (2+)) values is predicted to be the major contributor to enhancement at resting free Ca (2+) concentrations.

Role of the Neurogranin Concentrated in Spines in the Induction of Long-Term Potentiation

Synaptic plasticity in CA1 hippocampal neurons depends on Ca2+ elevation and the resulting activation of calmodulin-dependent enzymes. Induction of long-term depression (LTD) depends on calcineurin, whereas long-term potentiation (LTP) depends on Ca2+/calmodulin-dependent protein kinase II (CaMKII). The concentration of calmodulin in neurons is considerably less than the total concentration of the apocalmodulin-binding proteins neurogranin and GAP-43, resulting in a low level of free calmodulin in the resting state. Neurogranin is highly concentrated in dendritic spines. To elucidate the role of neurogranin in synaptic plasticity, we constructed a computational model with emphasis on the interaction of calmodulin with neurogranin, calcineurin, and CaMKII. The model shows how the Ca2+ transients that occur during LTD or LTP induction affect calmodulin and how the resulting activation of calcineurin and CaMKII affects AMPA receptor-mediated transmission. In the model, knockout of neurogranin strongly diminishes the LTP induced by a single 100 Hz, 1 s tetanus and slightly enhances LTD, in accord with experimental data. Our simulations show that exchange of calmodulin between a spine and its parent dendrite is limited. Therefore, inducing LTP with a short tetanus requires calmodulin stored in spines in the form of rapidly dissociating calmodulin-neurogranin complexes.

Ni2+, a Double-Acting Inhibitor of Neuronal Nitric Oxide Synthase Interfering with [formula omitted]-Arginine Binding and Ca2+/Calmodulin-Dependent Enzyme Activation

Ni2+, a toxic and carcinogenic pollutant and one of the leading causes of contact dermatitis, is shown to inhibit neuronal nitric oxide synthase (nNOS) in a competitive, reversible manner with respect to the substrate l-arginine (Ki = 30 ± 4 μM). The IC50 values were dependent on calmodulin (CaM) concentration, but proved independent of Ca2+, tetrahydrobiopterin (BH4) and other essential cofactors. Ni2+ also inhibited CaM-dependent cytochrome c reduction, NADPH oxidation, and H2O2 production by nNOS. Overall, the action profile of Ni2+ was suggestive of an unusual, double-acting inhibitor of nNOS affecting l-arginine-binding and Ca2+/CaM-dependent enzyme activation.

Role of calcium-binding protein regucalcin in regenerating rat liver.

Regucalcin is a novel calcium-binding protein which does not contain EF-hand motif as a Ca2+ -binding domain. The organization of the rat regucalcin gene consists of seven exons and six introns. Its mRNA is mainly present in liver but slightly in kidney with a size of 1.8 kb. Hepatic regucalcin mRNA expression is stimulated by various factors including calcium, calcitonin, insulin, and oestrogen in rats. The mRNA is also expressed in hepatoma cells (Morris hepatoma and HepG2). Regucalcin plays a role in the maintenance of cytosolic Ca2+ homeostasis in liver cells. Moreover, regucalcin has an inhibitory effect on Ca2+ /calmodulin-dependent enzyme activation, protein kinase C activation, and many Ca2+ -activated enzymes, indicating a role in the regulation of the Ca2+ -signalling system. Recently, regucalcin has been demonstrated to regulate nuclear function in liver cells. Regucalcin can inhibit Ca2+ -activated nuclear DNA fragmentation in rat isolated liver nuclei. Furthermore, the liver nuclear DNA and RNA syntheses are inhibited by regucalcin. Such an effect of regucalcin is also seen in the nuclei of regenerating rat liver. The regucalcin mRNA level is increased in regenerating liver. These findings suggest that regucalcin plays a regulatory role in the suppression for overexpression of proliferative cells.

Target recognition by calmodulin: dissecting the kinetics and affinity of interaction using short peptide sequences.

The interaction between calmodulin (CaM) and peptide M13, its target binding sequence from skeletal muscle myosin light chain kinase, involves predominantly two sets of interactions, between the N-terminal target residues and the C-domain of calmodulin, and between the C-terminal target residues and the N-domain of calmodulin (Ikura M et al., 1992, Science 256:632-638). Using short synthetic peptides based on the two halves of the target sequence, the interactions with calmodulin and its separate C-domain have been studied by fluorescence and CD spectroscopy, calcium binding, and kinetic techniques. Peptide WF10 (residues 1-10 of M13) binds to CaM with Kd approximately 1 microM; peptide FW10 (residues 9-18 of M13, with Phe-17-->Trp substitution) binds to CaM with Kd approximately 100 microM. The effect of peptide WF10 on calcium binding to calmodulin produces a biphasic saturation curve, with marked enhancement of affinity for the binding of two calcium ions to the C-domain, forming a stable half-saturated complex, Ca2-CaM-peptide, and confirming the functional importance of the interaction of this sequence with the C-domain. Stopped-flow studies show that the EGTA-induced dissociation of WF10 from Ca4-CaM proceeds by a reversible relaxation mechanism from a kinetic intermediate state, also involving half-saturation of CaM, and the same mechanism is evident for the full target peptide. Interaction of the N-terminal target residues with the C-domain is energetically the most important component, but interaction of calmodulin with the whole target sequence is necessary to induce the full cooperative interaction of the two contiguous elements of the target sequence with both N- and C-domains of calmodulin. Thus, the interaction of calmodulin with the M13 sequence can be dissected on both a structural and kinetic basis into partial reactions involving intermediates comprising distinct regions of the target sequence. We propose a general mechanism for the calcium regulation of calmodulin-dependent enzyme activation, involving an intermediate complex formed by interaction of the calmodulin C-domain and the corresponding part of the target sequence. This intermediate species can function to regulate the overall calcium sensitivity of activation and to determine the affinity of the calmodulin target interaction.

Intramolecular binding contributes to the activation of CDPK, a protein kinase with a calmodulin-like domain.

The activity of calmodulin-like domain protein kinase (CDPK) is regulated by the direct binding of Ca2+. Unmodified soybean CDPK alpha and a chimeric enzyme in which the calmodulin-like domain (CLD) was replaced by VU-1 calmodulin had similar values of Vmax(app) (3.19, 3.46, and 3.60, 3.93 mumol/ min/mg, respectively), and each was activated 30-70-fold by Ca2+. To determine if activation results from the binding of the CLD to the autoinhibitory (junction) domain of CDPK alpha in a manner analogous to the activation of calmodulin-dependent enzymes by calmodulin, recombinant CLD and truncation mutants of CDPK alpha were expressed in bacteria and highly purified. In blot overlays, biotinylated CLD bound to mutants containing residues 312-328 of the junction domain. In an electrophoretic mobility shift assay CLD bound synthetic peptides containing residues 318-332 in a calcium-dependent manner, providing direct evidence for binding of CLD to a site in the junction domain. Mutants of CDPK alpha from which all or part of the CLD had been deleted were constitutively inactive. Addition of 20 microM CLD to these mutants in the presence, but not the absence, of calcium stimulated their activities, but to various degrees. His6-CDPK alpha (1-328), which contained none of the CLD, was activated only 5-fold, but the activity of His6-CDPK alpha (1-398), which retained nearly half of the CLD in its sequence, was stimulated 64-fold. The latter activity approached that of unmodified CDPK alpha and was half maximal at a CLD concentration of 7 microM. Our results suggest that binding of CLD to the junction domain contributes to, but is not sufficient for activation. Although calmodulin supported full activity of the chimeric enzyme, its addition to His6-CDPK alpha (1-398) resulted in activity that was only 6% of that of the unmodified enzyme and which was half-maximal at 20 microM Arabidopsis calmodulin. These results support the conclusion that simple binding of the calmodulin-like domain to the junction domain is not sufficient for activation.

Activation of calmodulin-dependent enzymes can be selectively inhibited by histone H1.

Calmodulin (CaM) and its target enzymes are important regulators of a variety of cellular processes including gene expression and cell cycle progression (Bading, H., Ginty, D. D., and Greenberg, M. E. (1993) Science 260, 181-186; Rasmussen, C. D., and Means, A. R. (1989) EMBO J. 8, 73-82). It has been previously accepted that regulation of CaM-dependent enzyme activity occurs via calcium/calmodulin-dependent activation. We have found that histone H1 is a potent inhibitor of the CaM-dependent activation of mouse calcium/calmodulin-dependent protein kinase II (CaMKII) and of the CaM-dependent protein phosphatase, calcineurin. Inhibition is mediated only by free histone H1; addition of DNA abolishes the inhibitory effect. The effect is not due to a simple interaction of basic (histone) and acidic (CaM) proteins since myelin basic protein and histone H2B, CaMKII substrates more basic than histone H1, did not affect autophosphorylation of CaMKII, and myelin basic protein had no effect on calcineurin activity. The effect is specific to CaM since addition of parvalbumin, a related Ca(2+)-binding protein, did not reverse the effect of histone H1, whereas addition of CaM recovered enzyme activity. These results indicate that free histone H1 levels may specifically affect the ability of CaM to activate its target enzymes and suggests a novel level of control of CaM-dependent enzymes in eukaryotic cells.

Phosphoinositide Metabolism and Hormone Action

Calcium-mobilizing hormones elicit diverse cellular responses by activating plasma membrane receptors coupled to guanine nucleotide regulatory proteins that promote activation of phospholipase C and hydrolysis of polyphosphoinositides. The cleavage products of phosphatidylinositol bisphosphate, inositol 1,4,5-trisphosphate and diacylglycerol, act as primary signals for calcium mobilization and activation of calmodulin-dependent enzymes and protein kinase C. The metabolism of inositol 1,4,5-trisphosphate to several higher inositol phosphates provides additional compounds with potential regulatory actions on calcium movement and cell responses, and the discovery of multiple isomers of protein kinase C reveals a mechanism for diversification and specificity of hormone action among target cells controlled by the same basic transduction system. In addition to its rapid activation by many hormones and neurotransmitters, the calcium-phospholipid signaling system is utilized by several growth factors and oncogenes, consistent with its pervasive role in the control of both immediate and long-term responses of cells to external messenger molecules.

Calcium in the regulation of aldosterone secretion and vascular smooth muscle contraction.

A model of angiotensin II action has been developed in which the flow of information from cell surface to cell interior proceeds by two temporally distinct branches: a calmodulin branch largely responsible for initiating the response; and a C-kinase branch for sustaining it. There are at least two initial events: a prompt and sustained increase in calcium influx rate, and prompt hydrolysis of phosphatidylinositol 4,5-bisphosphate. The latter leads to the generation of water-soluble inositol 1,4,5-trisphosphate and lipid soluble diacylglycerol. The rise in inositol 1,4,5-trisphosphate concentration causes the redistribution of intracellular calcium, a transient rise in the calcium concentration in the cytosol, and the activation of calmodulin-dependent enzymes, including protein kinase(s). As a result, several cellular proteins are rapidly phosphorylated and initiate the cellular response. The rise in calcium and these initial phosphorylation events are transient, however, so that an additional mechanism is necessary to sustain the response. The rise in diacylglycerol content, along with the transient rise in cytosolic calcium, leads to a shift of the C-kinase from a calcium-insensitive to a calcium-sensitive, plasma membrane-associated form. In this location, the activity of C-kinase is regulated by the rate of calcium flux across the plasma membrane. As a result of the activity of the C-kinase, a second set of cellular proteins becomes phosphorylated, and these control the sustained phase of the response.

Inhibition of human neutrophil activation by the allergic mediator release inhibitor, CI-922: Mechanism of inhibitory activity

3,7-Dimethoxy-4-phenyl-N-1H tetrazol-5-yl-4H-furo[3,2-b]-indole-2- carboxamide, L-arginate (CI-922) is a potent inhibitory of human neutrophil functions in response to a variety of stimuli. In this report, the effects of CI-922 on specific processes involved in stimulus-response coupling are evaluated. CI-922 does not inhibit human neutrophil phospholipase C or protein kinase C activities. CI-922 is shown to inhibit calmodulin-dependent enzyme activation. The calmodulin antagonist activity is confirmed by calmodulin-Sepharose affinity chromatography. These results suggest that CI-922 inhibits neutrophil activation by preventing the activation of calmodulin-dependent enzymes, implying a critical role for such enzymes in stimulus-response coupling.

Calmodulin inhibitors protect against cadmium-induced testicular damage in mice.

Recent reports showing that cadmium can interact with calmodulin and activate calmodulin-sensitive enzymes have lead us to examine the effects of calmodulin inhibitors on cadmium-induced testicular toxicity in mice. Male CF-1 mice were pretreated with the various calmodulin inhibitors or inactive analogs and then, one hour later, given CdCl2 X 2 1/2 H2O (32 mumoles/kg). After 24 hours, the mice were killed and the testes were removed and weighed. The extent of hemorrhaging was quantified by determining the absorbance of hemoglobin at 414 nm in the soluble fraction of testicular homogenates. Exposure to Cd2+ increased the mean testicular weight from 118 +/- 5 mg to 146 +/- 4 mg and the hemoglobin absorbance from 0.096 +/- 0.006 to 0.767 +/- 0.138. Pretreatment with the calmodulin inhibitors, trifluoperazine (40 mumoles/kg), chlorpromazine (40 mumoles/kg) or W-7 (140 mumoles/kg) greatly attenuated the CD2+-induced increase in both parameters, whereas pretreatment with chlorpromazine sulfoxide (140 mumoles/kg), pentobarbital (140 mumoles/kg), verapamil (80 mumoles/kg) or ethylenediaminetetraacetate (140 mumoles/kg) did not. These results indicate that calmodulin inhibitors can protect against certain toxic effects of cadmium and are consistent with the hypothesis that some of the effects of cadmium may result from the improper activation of calmodulin-dependent enzymes.

Protective effects of the calmodulin antagonist bepridil on ischaemia induced in the rat myocardium.

The effects of a block of the slow calcium channel by bepridil and its antagonising effects on calmodulin were determined in the ischaemic rat myocardium. When isolated rat hearts were treated with a cardioplegic solution containing bepridil or verapamil there was a significant diminution in ischaemic damage in the myocardium. Bepridil was more effective than verapamil in protecting against this ischaemic damage. Bepridil effectively inhibited the activation of calmodulin dependent enzymes, such as calcium calmodulin dependent cyclic nucleotide phosphodiesterase and myosin light chain kinase. The inhibitory effect of verapamil on these enzymes was weaker than that of bepridil. Since bepridil protects the myocardium and minimises ischaemia induced damage these events might be related to a calmodulin antagonistic action as well as a calcium channel blocking action.

Vitamin D-3 induces the de novo synthesis of calmodulin in Phaseolus vulgaris root segments growing in vitro

Vitamin D-3 affects growth and Ca 2+ transport in Phaseolus vulgaris roots by a mechanism dependent on de novo protein synthesis. The objective of this work was to identify the protein(s) induced by the sterol. Phaseolus vulgaris root segments cultured in vitro were used. Protein extracts of control cultures and cultures treated with 10 −9 M vitamin D-3 for 2 h labelled with [ 14C]leucine and [ 3H]leucine, respectively, were mixed and electrophoresed on sodium dodecyl sulfate polyacrylamide gels. Examination of 3H: 14C ratios in slices of gels revealed that vitamin D-3 stimulates production of a protein which exhibited Ca 2+-dependent electrophoretic mobility. The apparent molecular weight was 14500 in the presence of 1 mM Ca 2+ and 18 000 in the presence of 5 mM EGTA. This protein was heat- and acid-stable, bound 45Ca 2+ and had an isoelectric pH of 3.8-4.1. These data are consistent with the properties of calmodulin. In agreement with these observations, higher levels of calmodulin were detected in roots treated with vitamin D-3 than in control roots by means of assays based on the activation of calmodulin-dependent enzymes. Moreover, estimation of calmodulin relative synthesis and degradation rates in roots cultures in the absence and presence of vitamin D-3 indicated that the sterol increases calmodulin synthesis without affecting its degradation. In addition, the results suggest that the synthesis of other low-molecular-weight Ca 2+-binding proteins may be affected by vitamin D-3.

Structural Characterization of a Higher Plant Calmodulin

Calmodulin is a eukaryotic calcium binding protein which has several calcium-dependent in vitro activities. Presented in this report is a structural characterization of calmodulin from spinach leaves (Spinacia oleracea). Spinach calmodulin may be representative of higher plant calmodulins in general since calmodulin from the monocotyledon barley (Hordeum vulgare) is indistinguishable by a variety of physical, chemical, and functional criteria (Schleicher, Lukas, Watterson 1983 Plant Physiol 73: 666-670). Spinach calmodulin is homologous to bovine brain calmodulin with only 13 identified amino acid sequence differences, excluding a blocked NH(2)-terminal tripeptide whose sequence has not been elucidated. Two extended regions of sequence identity are in the NH(2)-terminal half of the molecule, while nine of the 13 identified differences are in the COOH-terminal half of the molecule. Two of the changes, a cysteine at residue 26 and a glutamine at residue 96, require a minimum of two base changes in the nucleotide codons. Both of these changes occur in the proposed calcium binding loops of the molecule. Five additional amino acid differences found in spinach calmodulin had not been observed previously in a calmodulin. As described in an accompanying report (Roberts, Burgess, Watterson 1984 Plant Physiol 75: 796-798), these limited number of amino acid sequence variations appear to result in differential effects on the activation of calmodulin-dependent enzymes by plant and vertebrate calmodulins.

In vitro studies on basal and stimulated prolactin release by rat anterior pituitary: a possible role for calmodulin.

The mechanism by which PRL is released from mammotrophs is a calcium-dependent process. Although calcium seems to function as a second messenger, its regulatory mechanism in PRL release has not been clarified. The binding of calcium to calmodulin and the activation of calmodulin-dependent enzymes have been suggested to be important steps during stimulus-secretion coupling in various cells. In the present work we investigated the in vitro effect of penfluridol, a potent neuroleptic that also possesses the ability to inhibit calmodulin's biological activity, on basal and stimulated PRL release. The effect of pimozide and haloperidol on basal PRL release was also investigated. Penfluridol, pimozide, and haloperidol inhibited basal PRL secretion in a dose-related manner, with the EC50 ranging from 0.5-1 microM for penfluridol to 1-2 microM for pimozide and more than 3 microM for haloperidol. These concentrations are similar to those necessary for the inactivation of calmodulin-dependent enzymes in vitro. Ionophore A-23187, a compound whose ability to mobilize extracellular calcium is not affected by neuroleptics, stimulated PRL secretion in vitro. This effect, however, was blocked by penfluridol pretreatment. The site of action of penfluridol may occur after calcium mobilization, with calmodulin a possible target for penfluridol's inhibitory action on PRL secretion. TRH, K+, (Bu)2cAMP, and theophylline, compounds that affect calcium mobilization, also significantly stimulated PRL release. The coincubation of varying concentrations of penfluridol with 70 nM TRH, 50 mM K+, 3 mM (Bu)2cAMP, or 5 mM theophylline resulted in a dose-related inhibition of secretagogue-stimulated PRL secretion. Perifusion of dispersed anterior pituitary cells with 1 microM penfluridol reduced the ability of 70 nM TRH to stimulate PRL release by approximately 50%, whereas removal of the penfluridol perifusion allowed the cells to again be fully responsive to TRH. These results are consistent with the hypothesis that calmodulin is involved in the stimulus-secretion coupling of PRL.

Ca 2+-dependent regulation of calmodulin binding and adenylate cyclase activation in bovine cerebellar membranes

The binding parameters of 125I-labeled calmodulin to bovine cerebellar membranes have been determined and correlted with the activation of adenylate cyclase by calmodulin. In the presence of saturating levels of free Ca 2+, calmodulin binds to a finite number of specific membrane sites with a dissociation constant ( K d) of 1.2 nM. Furthermore, Scatchard analysis reveals a second population of binding sites with a 100-fold lower affinity for calmodulin. The Ca 2+-dependence of calmodulin binding and of adenylate cyclase activation varies with the amount of calmodulin present, as can be infered from the model of sequential equilibrium reactions which describes the activation of calmodulin-dependent enzymes. On the basis of this model, a quantitative analysis of the effect of free Ca 2+ and of free calmodulin concentration on both binding and activation of adenylate cyclase was carried out. This analysis shows that both processes take place only when calmodulin is complexed with at least three Ca 2+ atoms. The concentration of the active calmodulin ·Ca 2+ species required for half-maximal activation of adenylate cyclase is very similar to the K d of the high affinity binding sites on brain membranes. A Hill coefficient of approx. 1 was found for both processes indicating an absence of cooperativity. Phenothiazines and thioxanthenes antipsychotic agents inhibit calmodulin binding to membranes and calmodulin-dependent activation of adenylate cyclase with a similar order of potency. These results suggest that the Ca 2+-dependent binding of calmodulin to specific high affinity sites on brain membranes regulates the activation of adenylate cyclase by calmodulin.

Methylxanthine effects on cyclic adenosine 3′:5′ monophosphate phosphodiesterase activity in preparations of neonatal rat cerebellum: Modification by trifluoperazine

Paradoxically, caffeine was found to stimulate the activity of 3′:5′-cyclic AMP phosphodiesterase at substrate concentrations of 14μM, in cerebellar tissue from 10-day-old rats. Pretreatment with trifluoperazine, an inhibitor of calmodulin-dependent enzyme activation, converted the stimulatory effect of caffeine to the expected inhibitory action. Trifluoperazine pretreatment also increased the inhibitory action of theophylline on the cerebellar phosphodiesterase, but had no effect on the inhibitory action of 3-isobutyl-1-methylxanthine. It is suggested that caffeine and to a lesser extent theophylline in addition to their intrinsic phosphodiesterase inhibitory activity can also cause calmodulin dependent effects on cerebellar phosphodiesterase due to calcium mobilisation.