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At least 19 recordsLinked to original sources
PubMed1992

Mechanism for ganglioside-mediated modulation of a calmodulin-dependent enzyme. Modulation of calmodulin-dependent cyclic nucleotide phosphodiesterase activity through binding of gangliosides to calmodulin and the enzyme.

Gangliosides were recently shown to bind to calmodulin (Higashi, H., Omori, A., and Yamagata, T. (1992) J. Biol. Chem. 267, 9831-9838). This prompted us to investigate the effects of gangliosides on the calmodulin-dependent enzyme, cyclic nucleotide phosphodiesterase. Several species of gangliosides competitively inhibited calmodulin-stimulated phosphodiesterase activity, with GD1b, GT1b, and GD1a being noted to do so particularly (group 1). GM1, GQ1b, and GM2 (group 2) were less inhibitory, and GM3, GM3(NeuGc), GalCer, sulfatide, GgOse4Cer, and oligosaccharide portions of inhibitory gangliosides showed no inhibition in accordance with the binding specificity of calmodulin to gangliosides. Trypsin-activated phosphodiesterase was inhibited by gangliosides with similar specificity, indicating interactions of gangliosides with the enzyme. Inhibition, however, was less than that of calmodulin-dependent activity by these compounds and, in both cases, was eliminated by excess calmodulin. In the absence of calmodulin, group 1 gangliosides at lower concentrations activated the intact enzyme but inhibited it over a certain range of increase in concentration. Ganglioside-dependent modulation of calmodulin-dependent phosphodiesterase activity is thus shown to be due to interactions of gangliosides with both calmodulin and the enzyme, and consequently, ganglioside-calmodulin binding is likely the mechanism for regulation of the enzyme.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1992

Binding of calcium by calmodulin: influence of the calmodulin binding domain of the plasma membrane calcium pump.

The interaction between calmodulin and synthetic peptides corresponding to the calmodulin binding domain of the plasma membrane Ca2+ pump has been studied by measuring Ca2+ binding to calmodulin. The largest peptide (C28W) corresponding to the complete 28 amino acid calmodulin binding domain enhanced the Ca2+ affinity of calmodulin by more than 100 times, implying that the binding of Ca2+ increased the affinity of calmodulin for the peptide by more than 10(8) times. Deletion of the 8 C-terminal residues from peptide C28W did not decrease the affinity of Ca2+ for the high-affinity sites of calmodulin, but it decreased that for the low-affinity sites. A larger deletion (13 residues) decreased the affinity of Ca2+ for the high-affinity sites as well. The data suggest that the middle portion of peptide C28W interacts with the C-terminal half of calmodulin. Addition of the peptides to a mixture of tryptic fragments corresponding to the N- and C-terminal halves of calmodulin produced a biphasic Ca2+ binding curve, and the effect of peptides was different from that on calmodulin. The result shows that one molecule of peptide C28W binds both calmodulin fragments. Interaction of the two domains of calmodulin through the central helix is necessary for the high-affinity binding of four Ca2+ molecules.

Amino Acid Sequence↗
PubMed1992

The lipid-binding peptide from the plasma membrane Ca2+ pump binds calmodulin, and the primary calmodulin-binding domain interacts with lipid.

Peptide G25 (KKAVKVPKKEKSVLQGKLTRLAVQI) representing the putative lipid-binding region (G region) of the erythrocyte Ca2+ pump was synthesized. This peptide interacted with acidic lipids, as shown by the increase in size of phosphatidylserine liposomes in its presence. This lipid interaction is consistent with the previous evidence suggesting that the portion of the pump from which this peptide was taken is responsible for the activation of the pump by acidic lipid. G25 also bound to calmodulin, as was shown by its cause of a shift in the fluorescence of 5-dimethylamino naphthalene-1-sulfonyl- (dansyl)-calmodulin, and by its competition with Ca2+ pump for calmodulin. Its Kd for dansyl-calmodulin was much higher (0.8 microM) than that of the peptides representing the primary calmodulin-binding region (C region) of the Ca2+ pump. Although the presence of the G region provided the possibility of a second calmodulin-binding site, activation of the pump by calmodulin always could be fitted by simple saturation kinetics. The calmodulin-binding peptide from the C region of the pump, C28R2, also interacted with lipid with even greater effectiveness than G25. When the C region of the pump was saturated with calmodulin, acidic lipid activation of the pump followed simple saturation kinetics. However, when calmodulin was omitted, a higher concentration of lipid was needed for saturation and the kinetics became complex. The data are consistent with the idea that calmodulin activates the pump only by interaction at the C region, but that acidic lipid activates by interaction at both of the C and G regions.

Animals↗
PubMed1992

Calmodulin, a ganglioside-binding protein. Binding of gangliosides to calmodulin in the presence of calcium.

Ca(2+)-dependent ganglioside-binding protein was isolated from a soluble cytosol fraction of mouse brains using a ganglioside affinity column prepared with a mixture of bovine brain gangliosides. It was identified as calmodulin based on the following features identical with those of calmodulin: molecular weight, pI, chromatographic profile and amino acid sequences of lysyl-endopeptidase digests, and ability to activate cyclic nucleotide phosphodiesterase. Bovine brain calmodulin derivatized with 5-dimethylaminonaphthalene-1-sulfonyl (dansyl-calmodulin), tetramethylrhodamine isothiocyanate, or biotin was also shown to bind to the ganglioside affinity column Ca2+ dependently and elute with gangliosides GD1a, GD1b, GT1b, GQ1b, GM1, and GM2, melittin, and trifluoperazine but not with GgOse4Cer and oligosaccharides of GM1, GD1a, and GT1b. Modification of the Lys94 residue of calmodulin by biotinylation drastically reduced the capacity for ganglioside binding. Ganglioside GD1b caused a blue shift and increase in intensity of the fluorescence emission spectrum of dansyl-calmodulin in the presence of Ca2+. The increment in fluorescence was proportional to the amount of GD1b added and was maximal at the molar ratio of GD1b to calmodulin, approximately 7.8. Gangliosides are thus shown to specifically bind to calmodulin, and this binding may be a general mechanism for regulating calmodulin-dependent enzymes with consequent cellular response, such as cell differentiation.

Amino Acid Sequence↗
PubMed1992

Protein A-calmodulin fusions: a novel approach for investigating calmodulin function in yeast.

A novel gene fusion approach which may be of more general use has been developed for investigating the function of calmodulin in the budding yeast Saccharomyces cerevisiae. By fusing a portion of the Staphylococcus aureus spa gene (encoding protein A) to CMD1, the S. cerevisiae gene encoding calmodulin, we have generated a yeast calmodulin with an affinity tag able to bind immunoglobulins. The chimaeric protein A-calmodulin (ProtA-CaM) polypeptide functions in vivo and shows Ca(2+)-dependent binding to calmodulin target proteins. The spa-CMD1 fusion has been used (i) to prepare (by affinity chromatography) a fraction of yeast proteins which interact with calmodulin, (ii) to isolate genes encoding calmodulin target proteins by direct screening of an expression library, and (iii) to visualize calmodulin-binding proteins in crude extracts by Western blot analysis.

Amino Acid Sequence↗
PubMed1992

Novel bovine heart calmodulin-dependent protein kinase which phosphorylates a high molecular weight calmodulin-binding protein.

A novel calmodulin-dependent protein kinase has been isolated from bovine cardiac muscle by successive chromatography on DEAE-Sepharose 6B, Calmodulin-Sepharose 4B affinity and Sepharose 6B chromatography columns. The protein kinase was shown by gel filtration chromatography to have a molecular mass of 36,000 daltons. The highly purified protein kinase stoichiometrically phosphorylated the high molecular weight calmodulin-binding protein from cardiac muscle [Sharma RK (1990) J Biol Chem 265, 1152-1157] in a Ca2+/calmodulin-dependent manner. The phosphorylation resulted in the maximal incorporation of 1 mol of phosphate/mol of the high molecular weight calmodulin-binding protein. Other Ca2+/calmodulin-dependent protein kinases failed to phosphorylate the high molecular weight calmodulin-binding protein. The distinct substrate specificity of this protein kinase indicates that it is not related to the known calmodulin-dependent protein kinases and therefore constitutes a novel protein kinase.

Animals↗
PubMed1992

Biochemical and immunological characterization of p190-calmodulin complex from vertebrate brain: a novel calmodulin-binding myosin.

We have recently identified a novel 190-kD calmodulin-binding protein (p190) associated with the actin-based cytoskeleton from mammalian brain (Larson, R. E., D. E. Pitta, and J. A. Ferro. 1988. Braz. J. Med. Biol. Res. 21:213-217; Larson, R. E., F. S. Espindola, and E. M. Espreafico. 1990. J. Neurochem. 54:1288-1294). These studies indicated that p190 is a phosphoprotein substrate for calmodulin-dependent kinase II and has calcium- and calmodulin-stimulated MgATPase activity. We now have biochemical and immunological evidence that this protein is a novel calmodulin-binding myosin whose properties include (a) Ca2+ dependent action activation of its Mg-ATPase activity, which seems to be mediated by Ca2+ binding directly to calmodulin(s) associated with p190 (maximal activation by actin requires the presence of Ca2+ and is further augmented by addition of exogenous calmodulin); (b) ATP-sensitive cross-linking of skeletal muscle F-actin, as demonstrated by the low-speed actin sedimentation assay; and (c) cross-reactivity with mAbs specific for epitopes in the head of brush border myosin I. We also show that p190 has properties distinct from conventional brain myosin II and brush border myosin I, including (a) separation of p190 from brain myosin II by gel filtration on a Sephacryl S-500 column; (b) lack by p190 of K(+)-stimulated EDTA ATPase activity characteristic of most myosins; (c) lack of immunological cross-reactivity of polyclonal antibodies which recognize p190 and brain myosin II, respectively; (d) lack of immunological recognition of p190 by mAbs against an epitope in the tail region of brush border myosin I; and (e) distinctive proteolytic susceptibility to calpain. A survey of rat tissues by immunoblotting indicated that p190 is expressed predominantly in the adult forebrain and cerebellum, and could be detected in embryos 11 d post coitus. Immunocytochemical studies showed p190 to be present in the perikarya and dendritic extensions of Purkinje cells of the cerebellum.

Adenosine Triphosphatases↗
PubMed1992

Calmodulin trapping by calcium-calmodulin-dependent protein kinase.

Multifunctional calcium-calmodulin-dependent protein kinase (CaM kinase) transduces transient elevations in intracellular calcium into changes in the phosphorylation state and activity of target proteins. By fluorescence emission anisotropy, the affinity of CaM kinase for dansylated calmodulin was measured and found to increase 1000 times after autophosphorylation of the threonine at position 286 of the protein. Autophosphorylation markedly slowed the release of bound calcium-calmodulin; the release time increased from less than a second to several hundred seconds. In essence, calmodulin is trapped by autophosphorylation. The shift in affinity does not occur in a site-directed mutant in which threonine at position 286 has been replaced by a non-phosphorylatable amino acid. These experiments demonstrate the existence of a new state in which calmodulin is bound to CaM kinase even though the concentration of calcium is basal. Calmodulin trapping provides for molecular potentiation of calcium transients and may enable detection of their frequency.

Amino Acid Sequence↗
PubMed1979

Stimulation of Ca2+-dependent neurotransmitter release and presynaptic nerve terminal protein phosphorylation by calmodulin and a calmodulin-like protein isolated from synaptic vesicles.

Synaptic vesicles have a Ca(2+)-dependent protein kinase system that may play a role in mediating Ca(2+)-stimulated neurotransmitter release and vesicle function. Calcium's ability to initiate norepinephrine release and protein phosphorylation in synaptic vesicle preparations was shown to be stimulated by the presence of an endogenous heat-stable vesicle protein fraction. The heat stability and characteristics of this endogenous vesicle fraction were similar to those of calmodulin (Ca(2+)-dependent regular protein) isolated from rat and bovine brain. Calmodulin, like endogenous heat-stable vesicle factor, restored calcium's ability to stimulate vesicle neurotransmitter release and protein kinase activity. Calmodulin-like vesicle protein and purified calmodulin were also equally effective in stimulating cyclic nucleotide-dependent phosphodiesterase, further indicating that these two proteins are functionally equivalent. Depolarization-dependent Ca(2+) uptake in intact synaptosomes simultaneously stimulated release of neurotransmitter and phosphorylation of particular synaptic vesicle proteins that were shown in the isolated vesicle preparation to be dependent on Ca(2+) and calmodulin. The results suggest that calcium's effects on neurotransmitter release and presynaptic nerve terminal protein phosphorylation may be mediated by endogenous calmodulin-like proteins.

Animals↗
PubMed1992

Casein kinase II-catalysed phosphorylation of calmodulin is altered by amino acid deletions in the central helix of calmodulin.

Calmodulin is phosphorylated by casein kinase II on Thr-79, Ser-81, Ser-101 and Thr-117. To determine the consensus sequences for casein kinase II in intact calmodulin, we examined casein kinase II-mediated phosphorylation of engineered calmodulins with 1-4 deletions in the central helical region (positions 81-84). Total casein kinase II-catalyzed phosphate incorporation into all deleted calmodulins was similar to control calmodulin. Neither CaM delta 84 (Glu-84 deleted) nor CaM delta 81-84 (Ser-81 to Glu-84 deleted) has phosphate incorporated into Thr-79 or Ser-81, but both exhibit increased phosphorylation of residues Ser-101 and Thr-117. These data suggest that phosphoserine in the +2 position may be a specificity determinant for casein kinase II in intact proteins and/or secondary structures are important in substrate recognition by casein kinase II.

Amino Acid Sequence↗
PubMed1992

Full activation without calmodulin of calmodulin-dependent cyclic nucleotide phosphodiesterase by acidic glycosphingolipids: GM3, sialosylneolactotetraosylceramide and sulfatide.

Among calmodulin-non-binding glycosphingolipids, GM3, sialosylneolactotetraosylceramide (LM1), and sulfatide potently activated calmodulin-dependent cyclic nucleotide phosphodiesterase with or without Ca2+ showing ED50 1-5 microM. In contrast to calmodulin-binding gangliosides, these glycosphingolipids activated the enzyme up to the maximum level achieved by Ca2+/calmodulin and did not inhibit the activity at higher concentrations. Competition studies with GD1b that bind both to calmodulin and the enzyme suggest that the calmodulin-non-binding glycosphingolipids activate the enzyme through interaction with the same site of the enzyme as GD1b interacts.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1992

Calmodulin and protein kinase C cross-talk: the MARCKS protein is an actin filament and plasma membrane cross-linking protein regulated by protein kinase C phosphorylation and by calmodulin.

The myristoylated, alanine-rich C kinase (PKC) substrate (MARCKS) is a major, specific substrate of PKC that is phosphorylated during macrophage and neutrophil activation, growth factor-dependent mitogenesis and neurosecretion. MARCKS is also a calmodulin-binding protein and binding of calmodulin inhibits phosphorylation of the protein by PKC. Several recent observations from our laboratories suggest a role for MARCKS in cellular morphology and motility. First, in macrophages MARCKS is located at points of cellular adherence where actin filaments insert at the plasma membrane and is released to the cytoplasm upon activation of PKC. Second, during neutrophil chemotaxis MARCKS undergoes a cycle of release from, and reassociation with, the plasma membrane. Third, in vitro, MARCKS is an F-actin cross-linking protein whose activity is inhibited by PKC-mediated phosphorylation and by binding to calmodulin. MARCKS therefore appears to be a regulated cross-bridge between actin and the plasma membrane. Regulation of the plasma membrane-binding and actin-binding properties of MARCKS represents a convergence of the PKC and calmodulin signal transduction pathways in the control of actin cytoskeleton-plasma membrane interactions.

Actins↗
PubMed1992

The effects of calcium site occupancy and reagent length on reactivity of calmodulin lysyl residues with heterobifunctional aryl azides. Mapping interaction domains with specific calmodulin photoprobe derivatives.

The relationship of structural and functional moieties on calmodulin is important in all venues of cell activity. In this study, we investigate the effect of lysine modification on calmodulin function. Azidosalicylate reagents containing different "linker arm" lengths, between the photoactive terminus and an amine-reactive N-hydroxysuccinimidyl ester moiety were used to modify calmodulin lysines at three different positions in a calcium-dependent manner. The short cross-linker, (ASNE-2 (where ASNE represents azidosalicylate N-hydroxysuccinimidyl ester), modifies Lys-75, whereas the longer reagent, ASNE-6, modifies lysines 21, 75, and 94. The modification of these different lysines is shown to be calcium-dependent. At 1-100 microM levels of calcium, only Lys-94 is modified, suggesting that modification of this residue is directed by both the binding of calcium to calcium-binding loops III and IV and the hydrophobic pocket exposed between these two loops as a result of calcium binding. At higher calcium concentrations (> 200 microM), where sites I and II become filled, modification of Lys-21 or Lys-75 also was observed. All the modified calmodulins were able to stimulate 3',5'-cyclic-nucleotide phosphodiesterase fully although the Kact for the Lys-75 and Lys-21 derivatives increased 10- and 50-fold, respectively. None of the modifications affected the activation of erythrocyte plasma membrane Ca(2+)-ATPase. Only the ASNE-6 Lys-75 derivative showed efficient (40%) photocross-linking to the Ca(2+)-ATPase. The ASNE-2 Lys-75 derivative as well as the ASNE-6 Lys-21 and Lys-94 derivatives did not show efficient calcium-dependent photocross-linking to this enzyme.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMedDate not supplied

Regulation of expression of calmodulin and calmodulin-related genes by environmental stimuli in plants.

Plants are very sensitive to environmental stimuli and have evolved the ability to adapt to many environmental stresses by altering development. In particular, mechanical stimuli such as touch or wind, result in growth changes that result in plants with greater resistance to such mechanical stimuli. We have initiated a molecular dissection of the pathways that enable perception of and responses to these environmental stimuli in plants. We have discovered five genes--termed the TCH genes--whose expression levels are strongly and rapidly increased in response to stimuli such as touch, wind, rain, wounding and darkness. Three of the TCH genes encode proteins related to calmodulin thereby implicating roles for calcium ions and calmodulin in the transduction of signals from the environment.

Calcium↗
PubMed1992

Carbachol stimulates binding of a photoreactive calmodulin derivative to calmodulin-binding proteins in intact SK-N-SH human neuroblastoma cells.

Calmodulin (CaM) mediates the Ca(2+)-dependent activation of many enzyme systems in accordance with its cellular localization. We have described previously a muscarinic receptor-mediated translocation of CaM from membranes into the cytosol of SK-N-SH human neuroblastoma cells. To explore the potential targets (CaM-binding proteins, CaMBP) for CaM upon translocation, a photoreactive CaM derivative was introduced into living SK-N-SH cells using a scrape-loading technique. Scrape-loading incorporated rhodamine isothiocyanate-labeled CaM with an efficiency of 38%. CaM-diazopyruvamide (CaM-DAP), a Ca(2+)-dependent and CaM-specific probe, was also introduced into the cells. The muscarinic agonist carbachol stimulated a translocation of CaM from membranes into cytosol in CaM-DAP-loaded SK-N-SH cells. Upon photochemical cross-linking, cross-linked adducts of CaM-CaMBP were detected by immunoblotting with anti-CaM antibody. Carbachol stimulated increased photoaffinity labeling of three proteins with relative adduct molecular masses of 70, 120, and 180 kDa. The time course of labeling for the 70- and 120-kDa adducts showed maximal increased by 15-30 min. The 180-kDa adduct displayed a slower time course of maximal labeling, with increases maintained for 2-4 h. Subtracting the molecular mass of CaM, carbachol stimulated binding to CaMBPs of 55, 105, and 163 kDa. Predominant cellular CaMBP were identified using a biotinylated CaM overlay procedure. Western blot analysis indicated the expression of specific CaM-dependent enzymes such as calcineurin, phosphodiesterase, the beta-isoform (rat brain) of CaM kinase II, and Ca(2+)-ATPase. Numerous cytoskeletal CaMBP were expressed such as microtubule-associated protein-2, spectrin, tubulin, caldesmon, adducin, and neuromodulin. Of the CaMBP expressed, phosphodiesterase, calcineurin, caldesmon, and adducin cross-linked with CaM-DAP in the loaded SK-N-SH cells. Carbachol stimulated the time-dependent CaM-DAP labeling of calcineurin and adducin. This study demonstrates the novel incorporation of a photoreactive CaM derivative into living cells, as well as muscarinic receptor-activated CaM-DAP interaction with several cellular CaMBP. We postulate that carbachol-stimulated CaM translocation in SK-N-SH cells may affect the activity of CaM-dependent enzymes and may alter aspects of cytoskeletal function.

Affinity Labels↗
PubMed1992

Calcium transport by sarcoplasmic reticulum of vascular smooth muscle: II. Effects of calmodulin and calmodulin inhibitors.

The role of calmodulin (CaM) in modulating calcium (Ca) uptake by sarcoplasmic reticulum (SR) of vascular smooth muscle was studied in saponin skinned strips of rat caudal artery. Exogenous CaM concentrations ranging from 0.3-1.8 microM did not statistically change the steady state MgATP-dependent Ca content, the MgATP-independent Ca content, or the oxalate-stimulated Ca influx. Calmidazolium (CDZ), W-7, and trifluoperazine (TFP) were used to examine the potential effect of an endogenous CaM pool on inward Ca transport. The IC50 of these antagonists for inhibition of Ca-CaM-stimulated phosphodiesterase activity and Ca-activated superprecipitation of canine aortic actomyosin was measured and found to be in the low micromolar range with a rank order of potency for inhibition of CDZ greater than TFP greater than W-7. In skinned tissues, micromolar concentrations of antagonists that inhibited CaM-mediated reactions in isolated enzyme systems did not reduce Ca content or oxalate-stimulated Ca influx. At higher concentrations of 100-200 microM, the MgATP-dependent Ca content was significantly reduced by TFP and W-7 but not by CDZ. The order of potency for inhibition of Ca uptake was TFP greater than W-7 greater than CDZ. The MgATP-independent Ca content was significantly decreased only by 200 microM TFP. Although none of these inhibitors significantly altered Ca efflux at concentrations up to 100 microM, Ca release was significantly stimulated by all three at 200 microM. The TFP-stimulated Ca release was partially inhibited by ruthenium red. The results indicate that neither exogenous CaM nor an endogenous CaM pool directly modulates inward Ca transport by the SR of saponin skinned caudal artery. The inhibition of Ca uptake produced by hundred micromolar concentrations of CaM antagonists fails to correlate with the order of and with the potency of inhibition measured in isolated enzyme systems. This suggests that the inhibition of Ca uptake produced by high concentrations of these antagonists may be independent of a specific interaction with CaM. The activation of Ca release by high concentrations of CaM antagonists may involve a nonspecific increase in membrane permeability as well as modulation of a membrane Ca channel.

Actomyosin↗
PubMed1992

Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex.

The crystal structure of calcium-bound calmodulin (Ca(2+)-CaM) bound to a peptide analog of the CaM-binding region of chicken smooth muscle myosin light chain kinase has been determined and refined to a resolution of 2.4 angstroms (A). The structure is compact and has the shape of an ellipsoid (axial ratio approximately 2:1). The bound CaM forms a tunnel diagonal to its long axis that engulfs the helical peptide, with the hydrophobic regions of CaM melded into a single area that closely covers the hydrophobic side of the peptide. There is a remarkably high pseudo-twofold symmetry between the closely associated domains. The central helix of the native CaM is unwound and expanded into a bend between residues 73 and 77. About 185 contacts (less than 4 A) are formed between CaM and the peptide, with van der Waals contacts comprising approximately 80% of this total.

Amino Acid Sequence↗