The Experts below are selected from a list of 135 Experts worldwide ranked by ideXlab platform
David B. Sacks - One of the best experts on this subject based on the ideXlab platform.
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Calmodulin Enhances the Stability of the Estrogen Receptor
Journal of Biological Chemistry, 2001Co-Authors: Zhigang Li, John L. Joyal, David B. SacksAbstract:Abstract The estrogen receptor mediates breast cell proliferation and is the principal target for chemotherapy of breast carcinoma. Previous studies have demonstrated that the estrogen receptor binds to Calmodulin-Sepharose in vitro. However, the association of endogenous Calmodulin with endogenous estrogen receptors in intact cells has not been reported, and the function of the interaction is obscure. Here we demonstrate by co-immunoprecipitation from MCF-7 human breast epithelial cells that endogenous estrogen receptors bind to endogenous Calmodulin. Estradiol treatment of the cells had no significant effect on the interaction. However, incubation of the cells with tamoxifen enhanced by 5–10-fold the association of Calmodulin with the estrogen receptor and increased the total cellular content of estrogen receptors by 1.5–2-fold. In contrast, the structurally distinct Calmodulin antagonists trifluoperazine and CGS9343B attenuated the interaction between Calmodulin and the estrogen receptor and dramatically reduced the number of estrogen receptors in the cell. Neither of these agents altered the amount of estrogen receptor mRNA, suggesting that Calmodulin stabilizes the protein. This hypothesis is supported by the observation that, in the presence of Ca2+, Calmodulin protected estrogen receptors from in vitro proteolysis by trypsin. Furthermore, overexpression of wild type Calmodulin, but not a mutant Calmodulin incapable of binding Ca2+, increased the concentration of estrogen receptors in MCF-7 cells, whereas transient expression of a Calmodulin inhibitor peptide reduced the estrogen receptor concentration. These data demonstrate that Calmodulin binds to the estrogen receptor in intact cells in a Ca2+-dependent, but estradiol-independent, manner, thereby modulating the stability and the steady state level of estrogen receptors.
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The activity of Calmodulin is altered by phosphorylation: modulation of Calmodulin function by the site of phosphate incorporation
Biochemical Journal, 1995Co-Authors: David B. Sacks, B Mazus, John L. JoyalAbstract:Calmodulin transduces Ca2+ signals by binding to and activating essential regulatory enzymes. The large number of intracellular targets for Calmodulin raises the possibility that mechanisms in addition to Ca2+ may modulate Calmodulin activity. PhosphoCalmodulin is found in cells and tissues, and Calmodulin phosphorylation is enhanced by several mitogens. Phosphorylation of Calmodulin on serine/threonine residues by casein kinase II decreased its ability to activate Ca2+/Calmodulin-dependent protein kinase II (CaM-kinase II). The major effect was a 2.5-fold increase in the concentration at which half-maximal velocity (K0.5) was attained, with no apparent alteration in the Vmax, or the K0.5 for Ca2+. In contrast, Calmodulin phosphorylated on tyrosine residues by the insulin receptor kinase produced an increase in the Vmax, with no alteration in the affinity for CaM-kinase II or the K0.5 for Ca2+. Direct determination by surface plasmon resonance of the dissociation constants with a synthetic peptide corresponding to the Calmodulin-binding domain of CaM-kinase II revealed that phosphorylation on serine/threonine residues of Calmodulin significantly decreased its affinity for the peptide, while tyrosine phosphorylation had no effect on binding. In contrast to CaM-kinase II, neither serine/threonine nor tyrosine phosphorylation of Calmodulin altered its ability to activate calcineurin. These data indicate that phosphorylation of Calmodulin differentially modifies its interaction with individual target enzymes. Moreover, the amino acid residues phosphorylated provide an additional level of control. These results demonstrate that phosphorylation is an in vitro regulatory mechanism in the targeting of Calmodulin responses and, coupled with the stoichiometric phosphorylation of Calmodulin in rat hepatocytes, suggest that it may be relevant in intact cells.
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Alteration of Calmodulin-protein interactions by a monoclonal antibody to Calmodulin
Biochimica et biophysica acta, 1994Co-Authors: David B. SacksAbstract:Abstract The effects of specific anti-Calmodulin monoclonal antibodies on the conformation and interaction of Calmodulin with two enzymes, the insulin receptor tyrosine kinase and casein kinase II, are examined. Addition of the anti-Calmodulin antibody 2D1 in vitro augments phosphorylation of Calmodulin by rat hepatocyte insulin receptors 4.9 ± 0.5-fold ( n = 7). Nonimmune immunoglobulin has no effect. Maximal phosphorylation is observed at a molar ratio of Calmodulin: antibody of approx. 2: 1, with higher concentrations of antibody producing lesser enhancement. Increasing Ca 2+ concentrations in the physiological range progressively inhibit phosphorylation both in the absence and presence of antibody 2D1. Phosphate is incorporated predominantly on Tyr-99, which is distant from the antibody binding site. Enhancement of casein kinase II-catalyzed Calmodulin phosphorylation is also produced by the antibody 2D1, implying that antibody binding induces a change in Calmodulin conformation. In contrast, two other anti-Calmodulin monoclonal antibodies, 4F4 and 4G2, decrease phosphorylation of Calmodulin by both the insulin receptor kinase and casein kinase II. These data indicate that secondary and tertiary structures are important in enzyme-substrate interactions and suggest that the antibodies may be useful in investigating the mechanism of Calmodulin function.
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Monoclonal antibody to Calmodulin: development, characterization, and comparison with polyclonal anti-Calmodulin antibodies
Analytical biochemistry, 1991Co-Authors: David B. Sacks, Sharon E. Porter, Jack H. Ladenson, Jay M. McdonaldAbstract:Specific anti-Calmodulin rabbit polyclonal and murine monoclonal antibodies have been produced with a thyroglobulin-linked peptide corresponding to amino acids 128-148 of bovine brain Calmodulin. The monoclonal antibody is IgG-1 with kappa light chains. Both sets of antibodies recognize native vertebrate Calmodulin, with the polyclonal antibody exhibiting an approximately fourfold higher sensitivity than the monoclonal antibody in a radioimmunoassay. The affinity of both polyclonal and monoclonal antibodies is approximately 2.5-fold higher for Ca(2+)-free Calmodulin than for Ca(2+)-Calmodulin. Other selected members of the Calmodulin family (S100, troponin, and parvalbumin) do not exhibit significant cross-reactivity with the monoclonal antibody. Troponin and S100 beta displace some 125I-Calmodulin from the polyclonal antibody, but require at least 900-fold excess concentration. The monoclonal antibody recognizes intact vertebrate Calmodulin in solution and also on solid-phase. In addition, plant Calmodulin and some forms of post-translationally modified Calmodulin (phosphorylated or glycated) bind the monoclonal antibody. The affinity of the monoclonal antibody is approximately 5 x 10(8) liters/mol determined by displacement of 125I-Calmodulin. On dot blotting the sensitivity for vertebrate Calmodulin is 50 pg. The epitope for the monoclonal antibody is in the carboxyl terminal region (residues 107-148) of Calmodulin. This highly specific anti-Calmodulin monoclonal antibody should be a useful reagent in elucidating the mechanism by which Calmodulin regulates intracellular metabolism.
Charles F. Louis - One of the best experts on this subject based on the ideXlab platform.
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Localization and functional role of the Calmodulin-binding domain of phospholamban in cardiac sarcoplasmic reticulum vesicles
Biochimica et biophysica acta, 1993Co-Authors: Gale M. Strasburg, Timothy P. Hanson, Helena X. Ouyang, Charles F. LouisAbstract:Limited proteolysis and affinity-labeling techniques have been used to localize the Calmodulin-binding domain of phospholamban, the major substrate for both cAMP- and Calmodulin-dependent protein kinases in cardiac sarcoplasmic reticulum (SR). SR vesicles, treated with increasing concentrations of trypsin (likely hydrolyzing at Arg-25 in the cytoplasmic region of phospholamban), exhibited a subsequent loss of both cAMP- and Calmodulin-dependent phosphorylation, as well as Calmodulin affinity-labeling of phospholamban. When SR vesicles were treated with increasing concentrations of chymotrypsin (which likely cleaves at Tyr-6 of phospholamban) there was no effect on the cAMP-dependent phosphorylation of phospholamban. However, similar concentrations of chymotrypsin resulted in a loss of both Calmodulin affinity-labeling and Calmodulin-dependent phosphorylation of phospholamban (at Thr-17). When SR vesicles were treated with increasing concentrations of Endoproteinase Lys-C (which hydrolyzes phospholamban at Lys-3) both the Calmodulin affinity-labeling and the Calmodulin-dependent, but not the cAMP-dependent, phosphorylation of phospholamban were inhibited. These data were complemented by 1H-NMR studies on the complex formed by Calmodulin and a phospholamban peptide. These data suggest that binding of Calmodulin to phospholamban may be an essential intermediate step in the Calmodulin-dependent phosphorylation of phospholamban.
Koichi Yagi - One of the best experts on this subject based on the ideXlab platform.
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A strange Calmodulin of yeast.
Muscle Physiology and Biochemistry, 1999Co-Authors: Michio Yazawa, Ken-ichi Nakashima, Koichi YagiAbstract:Calmodulin of Saccharomyces cerevisiae has different Ca2+ binding properties from other Calmodulins. We previously reported that the maximum number of Ca2+ binding was 3 mol/mol and the fourth binding site was defective, which was different from 4 mol/mol for others. Their macroscopic dissociation constants suggested the cooperative three Ca2+ bindings rather than a pair of cooperative two Ca2+ bindings of ordinary Calmodulin. Here we present evidence for yeast Calmodulin showing the intramolecular close interaction between the N-terminal half domain and the C-terminal half domain, while the two domains of ordinary Calmodulin are independent of each other. We will discuss the relationship of the shape and the shape change caused by the Ca2+ binding to the enzyme activation in yeast. The functional feature of Calmodulin in yeast will also be considered, which might be different from the one of vertebrate Calmodulin.
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Binding of calcium by Calmodulin: influence of the Calmodulin binding domain of the plasma membrane calcium pump
Biochemistry, 1992Co-Authors: Michio Yazawa, Thomas Vorherr, Peter James, Ernesto Carafoli, Koichi YagiAbstract: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.
Gale M. Strasburg - One of the best experts on this subject based on the ideXlab platform.
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Localization and functional role of the Calmodulin-binding domain of phospholamban in cardiac sarcoplasmic reticulum vesicles
Biochimica et biophysica acta, 1993Co-Authors: Gale M. Strasburg, Timothy P. Hanson, Helena X. Ouyang, Charles F. LouisAbstract:Limited proteolysis and affinity-labeling techniques have been used to localize the Calmodulin-binding domain of phospholamban, the major substrate for both cAMP- and Calmodulin-dependent protein kinases in cardiac sarcoplasmic reticulum (SR). SR vesicles, treated with increasing concentrations of trypsin (likely hydrolyzing at Arg-25 in the cytoplasmic region of phospholamban), exhibited a subsequent loss of both cAMP- and Calmodulin-dependent phosphorylation, as well as Calmodulin affinity-labeling of phospholamban. When SR vesicles were treated with increasing concentrations of chymotrypsin (which likely cleaves at Tyr-6 of phospholamban) there was no effect on the cAMP-dependent phosphorylation of phospholamban. However, similar concentrations of chymotrypsin resulted in a loss of both Calmodulin affinity-labeling and Calmodulin-dependent phosphorylation of phospholamban (at Thr-17). When SR vesicles were treated with increasing concentrations of Endoproteinase Lys-C (which hydrolyzes phospholamban at Lys-3) both the Calmodulin affinity-labeling and the Calmodulin-dependent, but not the cAMP-dependent, phosphorylation of phospholamban were inhibited. These data were complemented by 1H-NMR studies on the complex formed by Calmodulin and a phospholamban peptide. These data suggest that binding of Calmodulin to phospholamban may be an essential intermediate step in the Calmodulin-dependent phosphorylation of phospholamban.
Howard Schulman - One of the best experts on this subject based on the ideXlab platform.
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Substrate-directed Function of Calmodulin in Autophosphorylation of Ca2+/Calmodulin-dependent Protein Kinase II
The Journal of biological chemistry, 1998Co-Authors: Roger C. Rich, Howard SchulmanAbstract:Autophosphorylation of Thr286 in Ca2+/Calmodulin-dependent protein kinase II occurs within each holoenzyme by an intersubunit reaction and is essential for kinase function in vivo. In addition to a kinase-directed function of Calmodulin to activate the kinase, a second Calmodulin is required for the autophosphorylation of each Thr286 (Hanson, P. I., Meyer, T., Stryer, L., and Schulman, H. (1994) Neuron 12, 943-956). We have engineered heteromeric holoenzymes comprising distinct "kinase" and "substrate" subunits to test for kinase- and substrate-directed functions of Calmodulin. The obligate kinase subunits have aspartate residues substituted for threonine at positions 286, 305, and 306 (the autophosphorylation and Calmodulin-binding sites), making it constitutively active but unable to bind Calmodulin. Obligate substrate subunits are catalytically inactive (K42M mutation) but are able to bind Calmodulin. Phosphorylation of substrate subunits occurs specifically at Thr286 and is completely dependent upon the presence of Calmodulin. Blocking the ability of the substrate subunit to bind Calmodulin, either with inhibitor KN-93 or by mutagenesis of the Calmodulin-binding domain of the substrate subunit, prevents its phosphorylation, consistent with a substrate-directed function of Calmodulin that requires its direct binding to the subunit being phosphorylated.
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Selective Activation and Inhibition of Calmodulin‐Dependent Enzymes by a Calmodulin‐Like Protein Found in Human Epithelial Cells
European journal of biochemistry, 1994Co-Authors: Carl F. Edman, Samuel E. George, Anthony R. Means, Howard Schulman, Paul YaswenAbstract:A Calmodulin-like protein, which is identical in size and 85% identical to vertebrate Calmodulin, was recently identified by 'subtractive hybridization' comparison of transcripts expressed in normal versus transformed human mammary epithelial cells. Unlike the ubiquitous distribution of Calmodulin, Calmodulin-like protein expression is restricted to certain epithelial cells, and appears to be modulated during differentiation. In addition, Calmodulin-like protein levels are often significantly reduced in malignant tumor cells as compared to corresponding normal epithelial cells. The current studies compare Calmodulin-like protein functions with those of Calmodulin. We find that Calmodulin-like protein activation of multifunctional Ca2+/Calmodulin-dependent protein kinase II (Calmodulin kinase II) is equivalent to activation by Calmodulin, but that four other Calmodulin-dependent enzymes, cGMP phosphodiesterase, calcineurin, nitric-oxide synthase, and myosin-light-chain kinase, display much weaker activation by Calmodulin-like protein than by Calmodulin. In the case of myosin-light-chain kinase, Calmodulin-like protein competitively inhibits Calmodulin activation of the enzyme with a Ki value of 170 nM. Thus, Calmodulin-like protein may have evolved to function as a specific agonist of certain Calmodulin-dependent enzymes, and/or as a specific competitive antagonist of other Calmodulin-dependent enzymes.
