Pharmacological evidence for the implication of both cyclic GMP-dependent and -independent transduction pathways within auxin-induced stomatal opening in Commelina communis (L.)

It has been previously suggested that auxin-induced stomatal opening results from at least two transduction pathways, one of which involves cyclic GMP (cGMP) as the mediator within a Ca²⁺ signalling cascade. This hypothesis was investigated further in epidermal peels of Commelina communis by comparing the effects of potential inhibitors of plant Ca²⁺-dependent enzymes on the stomatal opening responses to the auxin indolyl-3-butyric acid (IBA) and to the cGMP membrane-permeable derivative 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP). In the 30–50 μM range, the potential plant calmodulin (CaM) antagonist N-(aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7) positively interacted with IBA but not with 8-Br-cGMP to open the stomata. The CaM antagonists W-7 (in the 10–20 μM range) and N-(aminohexyl)-1-naphthalenesulphonamide (40 μM), the potential inhibitors of plant protein kinases 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (20 and 200 μM) and K-252a (0.6 μM), and cyclosporine A and FK506, potential inhibitors of plant homologs of Ca²⁺–CaM complex (Ca²⁺/CaM)-dependent protein phosphatase 2B, prevented the IBA and 8-Br-cGMP responses by about 70% and 100%, respectively. Together, these results provide indirect pharmacological evidence that, in addition to the cGMP-dependent pathway, the auxin signal is transduced through at least one cGMP-independent pathway.     

Involvement of Calmodulin and Calmodulin-Dependent Myosin Light Chain Kinase in Blue Light-Dependent H Pumping by Guard Cell Protoplasts from Vicia faba L

Signal transduction processes involved in blue light-dependent proton pumping were investigated using guard cell protoplasts from Vicia faba. N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide, an inhibitor of cyclic AMP- and cyclic GMP-dependent protein kinases, had no effect. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) and calphostin C, inhibitors of protein kinase C, produced slight inhibition of the blue light-dependent proton pumping. 1-[N, O-Bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl] -4-phenylpiperazine, a specific inhibitor of Ca²⁺/calmodulin (CaM)-dependent protein kinase II, did not inhibit the proton pumping, but 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine and 1-(5-chloro-naphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), inhibitors of Ca²⁺/CaM-dependent myosin light chain kinase, strongly suppressed the proton pumping. A CaM antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), inhibited blue light-dependent proton pumping, whereas its less active structural analog, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), had little effect on the response. Other CaM antagonists, trifluoperazine, compound 48/80, prenylamine, and 3-(2-benzothiazolyl)-4,5-dimethoxy-N-[3-(4-phenyl-piperidinyl)- propylbenzenesulfonamide inhibited the proton pumping. In accord with these results, light-induced stomatal opening in the epidermis of Commelina benghalensis ssp. was inhibited by ML-9 and W-7, but not by H-7 and W-5. Thus, it is concluded that CaM and Ca²⁺/CaM-dependent myosin light chain kinase are the components of the signal transduction process in blue light-dependent proton pumping in guard cells.     

NMR analysis of free and lipid nanodisc anchored CEACAM1 membrane proximal peptides with Ca2+/CaM

CEACAM1, a homotypic transmembrane receptor with 12 or 72 amino acid cytosolic domain isoforms, is converted from inactive cis-dimers to active trans-dimers by calcium-calmodulin (Ca²⁺/CaM). Previously, the weak binding of Ca²⁺/CaM to the human 12 AA cytosolic domain was studied using C-terminal anchored peptides. We now show the binding of ¹⁵N labeled Phe-454 cytosolic domain peptides in solution or membrane anchored using NMR demonstrates a significant role for the lipid bilayer. Although binding is increased by the mutation Phe454Ala, this mutation was previously shown to abrogate actin binding. On the other hand, Ca²⁺/CaM binding is abrogated by phosphorylation of nearby Thr-457, a post-translation modification required for actin binding and subsequent in vitro lumen formation. Binding of Ca²⁺/CaM to a membrane proximal peptide from the long 72 AA cytosolic domain anchored to lipid nanodiscs was very weak compared to lipid free conditions, suggesting membrane specific effects between the two isoforms. NMR analysis of ¹⁵N labeled Ca²⁺/CaM with unlabeled peptides showed the C-lobe of Ca²⁺/CaM is involved in peptide interactions, and hydrophobic residues such as Met-109, Val-142 and Met-144 play important roles in binding peptide. This information was incorporated into transmembrane models of CEACAM1 binding to Ca²⁺/CaM. The lack of Ca²⁺/CaM binding to phosphorylated Thr-457, a residue we have previously shown to be phosphorylated by CaMK2D, also dependent on Ca²⁺/CaM, suggests stepwise binding of the cytosolic domain first to Ca²⁺/CaM and then to actin.     

Unique Structural Changes in Calcium-Bound Calmodulin Upon Interaction with Protein 4.1R FERM Domain: Novel Insights into the Calcium-dependent Regulation of 4.1R FERM Domain Binding to Membrane Proteins by Calmodulin

Calmodulin (CaM) binds to the FERM domain of 80 kDa erythrocyte protein 4.1R (R30) independently of Ca²⁺ but, paradoxically, regulates R30 binding to transmembrane proteins in a Ca²⁺-dependent manner. We have previously mapped a Ca²⁺-independent CaM-binding site, pep11 (A²⁶⁴KKLWKVCVEHHTFFR), in 4.1R FERM domain and demonstrated that CaM, when saturated by Ca²⁺ (Ca²⁺/CaM), interacts simultaneously with pep11 and with Ser¹⁸⁵ in A¹⁸¹KKLSMYGVDLHKAKD (pep9), the binding affinity of Ca²⁺/CaM for pep9 increasing dramatically in the presence of pep11. Based on these findings, we hypothesized that pep11 induced key conformational changes in the Ca²⁺/CaM complex. By differential scanning calorimetry analysis, we established that the C-lobe of CaM was more stable when bound to pep11 either in the presence or absence of Ca²⁺. Using nuclear magnetic resonance spectroscopy, we identified 8 residues in the N-lobe and 14 residues in the C-lobe of pep11 involved in interaction with CaM in both of presence and absence of Ca²⁺. Lastly, Kratky plots, generated by small-angle X-ray scattering analysis, indicated that the pep11/Ca²⁺/CaM complex adopted a relaxed globular shape. We propose that these unique properties may account in part for the previously described Ca²⁺/CaM-dependent regulation of R30 binding to membrane proteins.     

Physiologically Relevant Free Ca2+ Ion Concentrations Regulate STRA6-Calmodulin Complex Formation via the BP2 Region of STRA6

The interaction of calmodulin (CaM) with the receptor for retinol uptake, STRA6, involves an α-helix termed BP2 that is located on the intracellular side of this homodimeric transporter (Chen et al., 2016 [1]). In the absence of Ca²⁺, NMR data showed that a peptide derived from BP2 bound to the C-terminal lobe (C-lobe) of Mg²⁺-bound CaM (^MgCaM). Upon titration of Ca²⁺ into ^MgCaM-BP2, NMR chemical shift perturbations (CSPs) were observed for residues in the C-lobe, including those in the EF-hand Ca²⁺-binding domains, EF3 and EF4 (^CaK_D = 60 ± 7 nM). As higher concentrations of free Ca²⁺ were achieved, CSPs occurred for residues in the N-terminal lobe (N-lobe) including those in EF1 and EF2 (^CaK_D = 1000 ± 160 nM). Thermodynamic and kinetic Ca²⁺ binding studies showed that BP2 addition increased the Ca²⁺-binding affinity of CaM and slowed its Ca²⁺ dissociation rates (k_off) in both the C- and N-lobe EF-hand domains, respectively. These data are consistent with BP2 binding to the C-lobe of CaM at low free Ca²⁺ concentrations (<100 nM) like those found at resting intracellular levels. As free Ca²⁺ levels approach 1000 nM, which is typical inside a cell upon an intracellular Ca²⁺-signaling event, BP2 is shown here to interact with both the N- and C-lobes of Ca²⁺-loaded CaM (^CaCaM-BP2). Because this structural rearrangement observed for the ^CaCaM-BP2 complex occurs as intracellular free Ca²⁺ concentrations approach those typical of a Ca²⁺-signaling event (^CaK_D = 1000 ± 160 nM), this conformational change could be relevant to vitamin A transport by full-length ^CaCaM-STRA6.     

Calcium/Calmodulin Inhibits the Binding of Specific [125I]Omega-Conotoxin GVIA to Chick Brain Membranes

The effect of Ca²⁺/calmodulin (CaM) on the specific binding of [¹²⁵I]omega-conotoxin GVIA (¹²⁵I--CTX) to crude membranes from chick brain was investigated. When we examined the effects of the activation of various endogenous protein kinases on specific [¹²⁵I]-CTX binding to crude membranes, we observed that Ca²⁺/CaM had an inhibitory effect regardless of whether or not the standard medium contained ATP (0.5 mM). Ca²⁺/CaM also had an inhibitory effect in a simple binding-assay medium containing HEPES-HCl buffer, BSA, Ca²⁺ and CaM, and this effect was dependent on the concentration of Ca²⁺. The effect of Ca²⁺/CaM was attenuated by the CaM antagonists W-7 and CaM-kinase II fragment (290–309). An experiment with modified ELISA using purified anti -CTX antibody indicated that Ca²⁺/CaM did not affect the direct binding of [¹²⁵I]-CTX and CaM. These results suggest that Ca²⁺/CaM either directly or indirectly affects specific [¹²⁵I]-CTX binding sites, probably N-type Ca²⁺ channels in crude membranes from chick whole brain.     

Conformation of <Emphasis Type="Italic">Thermus thermophilus</Emphasis> ribosomal protein S1 in solution at different ionic strengths

Small-angle x-ray and neutron scattering were used to study the structure of the ribosomal protein S1 (61 kDa) from Thermus thermophilus in solution at low and moderate ionic strength (0 and 100 mM NaCl). The protein was found to be globular in both cases. Modeling of the S1 structure comprising six homologous domains on the basis of the NMR data for one domain showed that the best fit to scattering data was provided by compact domain packing. The calculated gyration radius was 28–29 Å, as typical of globular proteins about 60 kDa. The protein was prone to self-association, forming mainly dimers and trimers at moderate ionic strength and higher compact associates at low ionic strength. Neutron scattering assays in heavy water at 100 mM NaCl revealed markedly elongated associates. The translational diffusion coefficient calculated for S1 at 100 mM NaCl from dynamic light scattering was markedly lower than the one expected for its globular monomer (D _20,w = (2.7 ± 0.1)·10^?7 versus (5.8–6.0)·10^?7 cm² s^?1), confirming protein association under equilibrium conditions.     

The involvement of calmodulin in motile activities of fish chromatophores

1. Effects of W-7 and W-5, calmodulin antagonists, on the pigment aggregation within melanophores and coloring response of iridophores were examined in the blue damselfish, Chrysiptera cyanea.2. W-7 was found to antagonize norepinephrine-induced responses of the chromatophores, whereas W-5 had only a slight effect on inhibition of the responses.3. H-7, a specific antagonist of protein kinase C, did not arrest the responses of melanophores and iridophores at all.4. The chromatophores responded normally to norepinephrine in Ca²⁺, Mg²⁺-free saline solution.5. These results indicate that it is a Ca²⁺/calmodulin-regulated enzyme and not protein kinase C that is involved in motile activities of fish chromatophores. Ca²⁺ may be supplied from an intracellular store.     

ATP-dependent Ca2+ transport and Ca2+-ATPase activities in an enriched plasma membrane fraction from gypsy moth larval midgut tissue

A subcellular fraction enriched in plasma membranes was obtained from gypsy moth (Lymantria dispar) larval midgut tissue. Using [⁴⁵Ca]²⁺ as a tracer, Ca²⁺ transport activity by membrane vesicles in the enriched fraction was measured and shown to be ATP-dependent, with a very high affinity for Ca²⁺ (apparent K_m for [Ca²⁺ _free]

1 Abbreviations used: [Ca²⁺free] = concentration of free (unbound) calcium ion;CaM = calmodulin; F = fraction; IOV = inside-out membrane vesicles; W-5 = N-(6-aminohexyl)-1-naphthalenesulfonamide; W-7 = N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide.

= 22 nM). Ca²⁺ transport was abolished upon addition of the calcium ionophore, A23187. Ca²⁺-stimulated, Mg²⁺-dependent ATPase activity peaked between 100 and 200 nM Ca²⁺_free. Ca²⁺-Mg²⁺-ATPase activity was inhibited by vanadate, 2 phenothiazine drugs (trifluoperazine and chlorpromazine), and the naphthalene sulfonamide, W-7; the related compound, W-5, and ouabain had a negligible effect. These results suggest the presence of a high affinity plasma membrane Ca²⁺ pump in gypsy moth larval midgut cells and are discussed in light of earlier work involving calcium transport in isolated midguts of larval Hyalophora cecropia. Ionic and other conditions that characterize the midgut physiology of larval Lepidoptera (e.g., luminal pH; electrochemical gradient for Ca²⁺; effect of certain ions and inhibitors on Ca²⁺ transport) contrast significantly with those found in adult Diptera. The implications that these differences may have for calcium regulation are discussed. © 1992 Wiley-Liss, Inc.     

Ca-Calmodulin Increases RyR2 Open Probability yet Reduces Ryanoid Association with RyR2

We have shown that physiological levels of Ca²⁺-calmodulin (Ca²⁺CaM; 50-100 nM) activate cardiac ryanodine receptors (RyR2) incorporated into bilayers and increase the frequency of Ca²⁺ sparks and waves in cardiac cells. In contrast, it is well known that Ca²⁺CaM inhibits [³H]ryanodine binding to cardiac sarcoplasmic reticulum. Since the [³H]ryanodine binding technique does not reflect the effects of Ca²⁺CaM on RyR2 open probability (Po), we have investigated, using the reversible ryanoid, ryanodol, whether Ca²⁺CaM can directly influence the binding of ryanoids to single RyR2 channels independently of Po. We demonstrate that Ca²⁺CaM reduces the rate of ryanodol association to RyR2 without affecting the rate of dissociation. We also find that ryanodol-bound channels fluctuate between at least two distinct subconductance states, M₁ and M₂, in a voltage-dependent manner. Ca²⁺CaM significantly alters the equilibrium between these two states. The results suggest that Ca²⁺CaM binding to RyR2 causes a conformation change to regions of the channel that include the ryanoid binding site, thereby leading to a decrease in ryanoid association rate and modulation of gating within the ryanoid/RyR2 bound state. Our data provide a possible explanation for why the effects of Ca²⁺CaM at the single-channel level are not mirrored by [³H]ryanodine binding studies.     

Rapid Desensitization of Serotonin 5-HT2C Receptor-Stimulated Intracellular Calcium Mobilization in CHO Cells Transfected with Cloned Human 5-HT2C Receptors

Abstract: Serotonin 5-HT_2C receptor-mediated intracellular Ca²⁺ mobilization was investigated in Chinese hamster ovary (CHO) cells transfected with 5-HT_2C receptors. Fura-2 acetoxymethyl ester was used to investigate the regulation of 5-HT_2C receptor function. CHO cells, transfected with a cDNA clone for the 5-HT_2C receptor, expressed 287 fmol/mg of the receptor protein as determined by mianserin-sensitive [³H]mesulergine binding (K_D = 0.49 nM). The addition of 5-HT mobilized intracellular Ca²⁺ in a dose-dependent fashion, ranging from a basal level of 99 ± 1.8 up to 379 ± 18 nM, with an EC₅₀ value for 5-HT of 0.029 µM. Exposure to 5-HT, 1-(3-chlorophenyl)piperazine dihydrochloride (a 5-HT_2C agonist), and 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (a 5-HT_2C and 5-HT_2A agonist) resulted in increased intracellular Ca²⁺ levels. Mianserin, mesulergine, ritanserin, and ketanserin each blocked 5-HT-mediated intracellular Ca²⁺ mobilization more effectively than spiperone. The receptor was rapidly desensitized by preexposure to 5-HT in a time- and concentration-dependent manner. Mezerein and phorbol 12-myristate 13-acetate, protein kinase C activators, weakly inhibited the intracellular Ca²⁺ mobilization induced by 10 µM 5-HT. Furthermore, the protein kinase C inhibitor H-7 partially prevented the protein kinase C activator-induced inhibition of the 5-HT-mediated increase in intracellular Ca²⁺ concentration. The desensitization induced by pretreatment with 5-HT was blocked by W-7, added in conjunction with 5-HT, and partially inhibited by W-5, a nonselective inhibitor of protein kinases and weak analogue of W-7. Therefore, the 5-HT_2C receptor may be connected with protein kinase C and calcium/calmodulin turnover. These results suggest that 5-HT_2C receptor activation mobilizes Ca²⁺ in CHO cells and that the acute desensitization of the receptor may be due to calmodulin kinase-mediated feedback.     

Mechanisms of Calmodulin Regulation of Different Isoforms of Kv7.4 K+ Channels

Calmodulin (CaM), a Ca²⁺-sensing protein, is constitutively bound to IQ domains of the C termini of human Kv7 (hKv7, KCNQ) channels to mediate Ca²⁺-dependent reduction of Kv7 currents. However, the mechanism remains unclear. We report that CaM binds to two isoforms of the hKv7.4 channel in a Ca²⁺-independent manner but that only the long isoform (hKv7.4a) is regulated by Ca²⁺/CaM. Ca²⁺/CaM mediate reduction of the hKv7.4a channel by decreasing the channel open probability and altering activation kinetics. We took advantage of a known missense mutation (G321S) that has been linked to progressive hearing loss to further examine the inhibitory effects of Ca²⁺/CaM on the Kv7.4 channel. Using multidisciplinary techniques, we demonstrate that the G321S mutation may destabilize CaM binding, leading to a decrease in the inhibitory effects of Ca²⁺ on the channels. Our study utilizes an expression system to dissect the biophysical properties of the WT and mutant Kv7.4 channels. This report provides mechanistic insights into the critical roles of Ca²⁺/CaM regulation of the Kv7.4 channel under physiological and pathological conditions.     

Calcium-dependent stoichiometries of the KCa2.2 (SK) intracellular domain/calmodulin complex in solution

Ca²⁺ activates SK Ca²⁺-activated K⁺ channels through the protein Ca²⁺ sensor, calmodulin (CaM). To understand how SK channels operate, it is necessary to determine how Ca²⁺ regulates CaM binding to its target on SK. Tagless, recombinant SK peptide (SKp), was purified for binding studies with CaM at low and high Ca²⁺ concentrations. Composition gradient multi-angle light scattering accurately measures the molar mass, stoichiometry, and affinity of protein complexes. In 2 mM Ca²⁺, SKp and CaM bind with three different stoichiometries that depend on the molar ratio of SKp:CaM in solution. These complexes include 28 kD 1SKp/1CaM, 39 kD 2SKp/1CaM, and 44 kD 1SKp/2CaM. A 2SKp/2CaM complex, observed in prior crystallographic studies, is absent. At <5 nM Ca²⁺, 1SKp/1CaM and 2SKp/1CaM were observed; however, 1SKp/2CaM was absent. Analytical ultracentrifugation was used to characterize the physical properties of the three SKp/CaM stoichiometries. In high Ca²⁺, the sedimentation coefficient is smaller for a 1SKp:1CaM solution than it is for either 2SKp:1CaM or 1SKp:2CaM. At low Ca²⁺ and at >100 µM protein concentrations, a molar excess of SKp over CaM causes aggregation. Aggregation is not observed in Ca²⁺ or with CaM in molar excess. In low Ca²⁺ both 1SKp:1CaM and 1SKp:2CaM solutions have similar sedimentation coefficients, which is consistent with the absence of a 1SKp/2CaM complex in low Ca²⁺. These results suggest that complexes with stoichiometries other than 2SKp/2CaM are important in gating.     

Cytosolic Calmodulin Is Increased in SK-N-SH Human Neuroblastoma Cells Due to Release of Calcium from Intracellular Stores

Abstract: Muscarinic receptor stimulation elicits a redistribution of calmodulin (CaM) from the membrane fraction to cytosol in the human neuroblastoma cell line SK-N-SH. Increasing the intracellular Ca²⁺ concentration with ionomycin also elevates cytosolic CaM. The aim of this study was to investigate the roles of extracellular and intracellular Ca²⁺ pools in the muscarinic receptor-mediated increases in cytosolic CaM in SK-N-SH cells. Stimulus-mediated changes in intracellular Ca²⁺ were monitored in fura-2-loaded cells, and CaM was measured by radioimmunoassay in the 100,000-g cytosol and membrane fractions. The influx of extracellular Ca²⁺ normally seen with carbachol treatment in SK-N-SH cells was eliminated by pretreatment with the nonspecific Ca²⁺ channel blocker Ni²⁺. Blocking the influx of extracellular Ca²⁺ had no effect on carbachol-mediated increases in cytosolic CaM (168 ± 18% of control values for carbachol treatment alone vs. 163 ± 28% for Ni²⁺ and carbachol) or decreases in membrane CaM. Similarly, removal of extracellular Ca²⁺ from the medium did not affect carbachol-mediated increases in cytosolic CaM (168 ± 26% of control). On the other hand, prevention of the carbachol-mediated increase of intracellular free Ca²⁺ by pretreatment with the cell-permeant Ca²⁺ chelator BAPTA/AM did attenuate the carbachol-mediated increase in cytosolic CaM (221 ± 37% of control without BAPTA/AM vs. 136 ± 13% with BAPTA/AM). The effect of direct entry of extracellular Ca²⁺ into the cell by K⁺ depolarization was assessed. Incubation of SK-N-SH cells with 60 mM K⁺ elicited an immediate and persistent increase in intracellular free Ca²⁺ concentration, but there was no corresponding alteration in CaM localization. On the contrary, in cells where intracellular Ca²⁺ was directly elevated by thapsigargin treatment, cytosolic CaM was elevated for at least 30 min while particulate CaM was decreased. In addition, treatment with ionomycin in the absence of extracellular Ca²⁺, which releases Ca²⁺ from intracellular stores, induced an increase in cytosolic CaM (203 ± 30% of control). The mechanism for the CaM release may involve activation of the α isozyme of protein kinase C, which was translocated from cytosol to membranes much more profoundly by thapsigargin than by K⁺ depolarization. These data demonstrate that release of Ca²⁺ from the intracellular store is important for the carbachol-mediated redistribution of CaM in human neuroblastoma SK-N-SH cells.     

Neutron scattering study of the 13 S fragment of 16 S RNA and its complex with ribosomal protein S4

A fragment with a molecular weight of 170,000 and a sedimentation coefficient of 13 S which is capable of specifically binding ribosomal protein S4 has been obtained by digestion of Escherichia coli 16 S RNA with ribonuclease A. The 13 S fragment of 16 S RNA and its complex with protein S4 have been studied by different physical methods; in the first place, by neutron scattering. It has been shown that this fragment is very compact in solution. The radii of gyration of this fragment (50 ± 3 Å) and of protein S4 within the complex (17 ± 3 Å) coincide, within the limits of experimental error, with the radii of gyration for the free RNA fragment (47 ± 2 Å) and the free ribosomal protein S4 in solution (18 ± 2 Å). Hence the conclusion is drawn that the compactness of the RNA fragment and the ribosomal protein does not change on complex formation. The compact 13 S fragment of 16 S RNA is shown to be contrast-matched in solvent containing 70% ²H₂O which corresponds to a value for the partial specific volume of RNA of 0.537 cm³/g.     

Calcium/calmodulin-dependent inhibition of microtubule assembly by brain synaptic junction

The effect of synaptic junction (SJ) on microtubule assembly was examined. After preincubation with ATP at 37°C, rat SJ decreased the initial velocity and the extent of the porcine brain microtubule assembly (initiated by the addition of GTP) in a Ca²⁺/calmodulin (CaM)-dependent manner. The degree of the inhibition reached 35% of the control assembly (0-min preincubation) after 20-min preincubation with ATP. There was no inhibition either with heat-treated SJ, at 0°C, or in the presence of EGTA or W-7 (CaM antagonist). The inhibition was due neither to protease(s) nor CaM contaminating the preparations. Free Ca²⁺ concentration level required for the inhibition of microtubule assembly was 10^–6 M. Phosphorylation of microtubule proteins was inhibited by SJ in a Ca²⁺/CaM-dependent manner, and the inhibition occurred in a physiological increase range of intracellular Ca²⁺ concentration (10^–6M) The heat-treated SJ caused no inhibition. The result suggested that the microtubule assembly in the postsynaptic region was regulated by a Ca²⁺/CaM-dependent protein kinase associated with SJ; i. e., major postsynaptic density protein.Abbreviations used CaM calmodulin - DTT dithiothreitol - MAPs microtubule-associated proteins - MES 2-(N-morphorino)ethanesulfonic acid - mPSDp major postsynaptic density protein - PSD postsynaptic density - SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride     

Ca2+/Calmodulin and Apo-Calmodulin Both Bind to and Enhance the Tyrosine Kinase Activity of c-Src

Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca²⁺ signal generated in cells by different effectors, where the Ca²⁺-receptor protein calmodulin (CaM) plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca²⁺-dependent and Ca²⁺-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR), in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca²⁺/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca²⁺-free CaM (apo-CaM) exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca²⁺/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca²⁺ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred.     

Small-angle X-ray scattering studies of Escherichia colil-asparaginase

Small angle X-ray scattering studies on Escherichia colil-asparaginase solutions show that the enzyme has a radius of gyration of 34.0 Å ± 0.5 Å at pH 7. The radius of gyration of the dissociated monomer is 16.0 Å ± 1.0 Å; it has the general shape of a prolate ellipsoid with an axial ratio of 1.4. A tetramer of four such ellipsoids arranged with 222 symmetry gives good agreement between measured and calculated radii of gyration if the distance between subunit centers is 43 Å. The tetramer dissociates on dilution below 1% and at pH values below 3.0. Acid-induced denaturation at pH 2.0 is irreversible in contrast to the reversible guanidine-HCl-induced denaturation.     

A rapid separation of bovine brain S-100a and S-100b proteins and related conformation studies

S-100 protein absorbs to the calmodulin antagonist W-7 coupled to epoxy-activated Sepharose 6B in the presence of Ca²⁺ and is eluted by ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid buffer. S-100a and S-100b were separated and isolated by Ca²⁺-dependent affinity chromatography on W-7 Sepharose. The Ca²⁺-induced conformational changes of S-100a and S-100b were examined using circular dichroism, ultraviolet difference spectra, and a fluorescence probe. Differences in Ca²⁺-dependent conformational changes between S-100a and S-100b became apparent. Circular dichroism studies revealed that both S-100a and S-100b undergo a conformational change upon binding of Ca²⁺ in the aromatic and far-uv range. In the presence or absence of Ca²⁺, the aromatic CD spectrum of S-100a differed completely from that of S-100b, possibly due to the single tryptophan residue of S-100a. Far-uv studies indicate that α-helical contents of both S-100a and S-100b decreased with addition of Ca²⁺. Ca²⁺-induced conformational changes of S-100a and S-100b were also detected by uv difference spectra. The spectrum of S-100a also differed from that of S-100b. Fluorescence studies using 2-p-toluidinylnaphthalene-6-sulfonate (TNS), a hydrophobic probe for protein, revealed a slight difference in conformational changes of these two components. The interaction of TNS and S-100b was observed with concentrations above 3 μm Ca²⁺; on the other hand, S-100a required concentrations above 8 μm. This finding was supported by the difference in the binding affinities of S-100a and S-100b to the W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide)-Sepharose column; both S-100a and S-100b bound the column in the presence of Ca²⁺ but S-100a was eluted prior to S-100b. These results suggest that S-100a and S-100b differ in their dependence on Ca²⁺ and that the affinity-chromatographic separation of S-100a from S-100b on the W-7-Sepharose column makes feasible a rapid purification of these two components.     

Mutational tests of the NMR-docked structure of the staphylococcal nuclease–metal–3′,5′-pdTp complex

In the X-ray structure of the staphylococcal nuclease–Ca²⁺ ?3′,5′-pdTp complex, the conformation of the inhibitor 3′,5′-pdTp is distroteed Lys-70* and Lys-71* from an adjacent molecule of staphylococcal nuclease (Loll, P.J., Lattman, E.E. Proteins 5 : 183-201, 1989). In order to correct this crystal packing problem, the solution conformation of enzyme-bound 3′,5′-pdTp in the staphylococcal nuclease–metal–pdTp Complex determined by NMR methods was docked into the X-ray structure of the enzyme [Weber, D. J., Serpersu, E. H., Gittis, A. G., Lattman, E. E., Mildvan, A. S. (preceding paper)]. In the NMR-docked structure, the 5′-phophate of 3′,5′-pdTp overlaps with that in the X-ray Structure. However the 3′-phosphate accepts a hydrogen bond from Lys-49 (2.89Å) rather than from Lys-84 (8.63 Å), and N3 of thymine donates a hydrogen bond to the OH of Tyr-115 (3.16 Å) which does not occur in the X-ray structure (5.28 Å). These interactions have been tested by binding studies of 3′,5′-pdTp, Ca²⁺, and Mn²⁺ to the K49A, K84A, and Y115A mutants of staphylococcal nuclease using water proton relaxation rate and EPR methods. Each mutant was fully active and structurally intact, as found by CD and two-dimensional NMR spectroscopy, but bound Ca²⁺ 9.1- to 9.9-fold more weakly than the wild-type enzyme. While thye K84A mutation did not significantly weaken 3′,5′-pdTp binding to the enzyme (1.5 ± 0.7 fold), the K49A mutation weakened 3′,5′-pdTp binding to the enzyme by the factor of 4.4 ± 1.8-fold. Similarly, the Y115A mutation weakened 3′,5′-pdTp binding to the enzyme 3.6 ± 1.6-fold. Comparable weakening effects of these mutations were found on the binding of Ca²⁺-3′,5′-pdTp. These results are more readily explained by the NMR-docked structure of staphylococcal nuclease-metal-3′,5′-pdTp than by the X-ray structure. © 1993 Wiley-Liss, Inc.