The Experts below are selected from a list of 252 Experts worldwide ranked by ideXlab platform
Steven B. Marston - One of the best experts on this subject based on the ideXlab platform.
-
role of Caldesmon in the ca2 regulation of smooth muscle thin filaments evidence for a cooperative switching mechanism
Journal of Biological Chemistry, 2008Co-Authors: Saira Ansari, Steven B. Marston, Mustapha Alahyan, Mohammed ElmezgueldiAbstract:Abstract Smooth muscle thin filaments are made up of actin, tropomyosin, Caldesmon, and a Ca2+-binding protein and their interaction with myosin is Ca2+-regulated. We suggested that Ca2+ regulation by Caldesmon and Ca2+-calmodulin is achieved by controlling the state of thin filament through a cooperative-allosteric mechanism homologous to troponin-tropomyosin in striated muscles. In the present work, we have tested this hypothesis. We monitored directly the thin filament transition between the ON and OFF state using the excimer fluorescence of pyrene iodoacetamide (PIA)-labeled smooth muscle αα-tropomyosin homodimers. In steady state fluorescence measurements, myosin subfragment 1 (S1) cooperatively switches the thin filaments to the ON state, and this is exhibited as an increase in the excimer fluorescence. In contrast, Caldesmon decreases the excimer fluorescence, indicating a switch of the thin filament to the OFF state. Addition of Ca2+-calmodulin increases the excimer fluorescence, indicating a switch of the thin filament to the ON state. The excimer fluorescence was also used to monitor the kinetics of the ON-OFF transition in a stopped-flow apparatus. When ATP induces S1 dissociation from actin-PIA-tropomyosin, the transition to the OFF state is delayed until all S1 molecules are dissociated actin. In contrast, Caldesmon switches the thin filament to the OFF state in a cooperative way, and no lag is displayed in the time course of the Caldesmon-induced fluorescence decrease. We have also studied Caldesmon and Ca2+-calmodulin-Caldesmon binding to actin-tropomyosin in the ON and OFF states. The results are used to discuss both Caldesmon inhibition and Ca2+-calmodulin-Caldesmon activation of actin-tropomyosin.
-
Actomyosin cross-linking by Caldesmon in non-muscle cells
FEBS Letters, 2001Co-Authors: E A Goncharova, Vladimir P. Shirinsky, Steven B. Marston, A. Shevelev, Alexander V. VorotnikovAbstract:The role of myosin-binding in cytoskeletal arrange- ment of non-muscle low molecular weight Caldesmon (l- Caldesmon) was studied. The N-terminal myosin-binding domain of Caldesmon (N152) colocalized with myosin in transiently transfected chicken fibroblasts. When added exogenously to the Triton-insoluble cytoskeleton, N152 enhanced l-Caldesmon dis- placement by exogenous C-terminal actin-binding fragment (H1). Thus, a significant fraction of l-Caldesmon cross-links actin and myosin. In contrast, in epithelioid HeLa cells most of l-Caldesmon was only actin-bound as H1 alone was enough for its displacement. Phosphorylation by mitogen-activated protein kinase reduced the capability of H1 to displace endogenous l-Caldesmon, suggesting it may represent a regulatory mechanism for actin^Caldesmon interaction in vivo. fl 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
-
Interaction of isoforms of S100 protein with smooth muscle Caldesmon
FEBS letters, 1998Co-Authors: Alexei A. Polyakov, Pia A. J. Huber, Steven B. Marston, Nikolai B. GusevAbstract:Interaction of S100a and S100b with duck gizzard Caldesmon was investigated by means of native gel electrophoresis, fluorescent spectroscopy and disulfide crosslinking. Both isoforms of S100 interact with intact Caldesmon and its C-terminal deletion mutant 606C (residues 606-756) with apparent Kd of 0.2-0.6 microM thus indicating that the S100-binding site is located in the C-terminal domain of Caldesmon. The single SH group of duck gizzard Caldesmon can be crosslinked to Cys-84 of the beta-chain or to Cys-85 of the alpha-chain of S100. Crosslinking of S100 reduces the inhibitory action of Caldesmon on actomyosin ATPase activity. S100 reverses the inhibitory action of intact Caldesmon and its deletion mutants 606C (residues 606-756) and H9 (residues 669-737) as effectively as calmodulin. S100a has higher affinity to Caldesmon and is more effective than S100b in reversing Caldesmon-induced inhibition of actomyosin ATPase activity. Although monomeric (calmodulin, troponin C) and dimeric (S100) Ca-binding proteins have different sizes and structures they interact with Caldesmon in a very similar fashion.
-
Affinity and structure of complexes of tropomyosin and Caldesmon domains
Biophysical journal, 1996Co-Authors: E.j. Hnath, Steven B. Marston, C.l. Wang, Pia A. J. Huber, George N. PhillipsAbstract:The interaction of Caldesmon domains with tropomyosin has been studied using x-ray crystallography and an optical biosensor. Only whole Caldesmon and the carboxyl-terminal domain of Caldesmon (CaD-4, chicken gizzard residues 597–756) bound to tropomyosin with greater than millimolar affinity at 100 and 150 microM salt. Under these conditions the affinities of whole Caldesmon and CaD-4 were both in the micromolar range. Data from the x-ray studies showed that whole Caldesmon bound to tropomyosin in several places, with the region of tightest interaction being at tropomyosin residues 70–100 and/or 230–260. Studies with CaD-4 revealed that this region corresponded to the strong binding site seen with whole Caldesmon. Weaker association of other regions of Caldesmon to tropomyosin residues 180–210 and 5–50 was also observed. The results suggest that the carboxyl-terminus of Caldesmon binds tightly to tropomyosin and that other regions of Caldesmon may interact with tropomyosin tightly only when they are held close to tropomyosin by the carboxyl-terminal domain. Four models are presented to show the possible interactions of Caldesmon with tropomyosin.
-
The Caldesmon content of vertebrate smooth muscle
Biochimica et biophysica acta, 1993Co-Authors: William Lehman, Deanna Denault, Steven B. MarstonAbstract:Abstract Caldesmon and tropomyosin can be selectively and quantitatively extracted from vascular and visceral smooth muscle following heat treatment; all other smooth muscle proteins are precipitated by this procedure. Estimates of the Caldesmon/tropomyosin molar ratio in heat-extracts determined by SDS-PAGE densitometry are 1 Caldesmon:5.1–5.3 tropomyosin for rabbit and sheep aorta, and 1 Caldesmon:5.9 tropomyosin for rabbit stomach and chicken gizzard. If the assumption is made that tropomyosin serves as a true reference of thin-filament content in intact muscle, it follows that the relative Caldesmon contents in the above tissues are similar to each other. Caldesmon in heat extracts was identified by Western blotting, by its anomalous migration on several different SDS-PAGE systems and by its position on two-dimensional PAGE. Values of Caldesmon contents in unfractionated total tissue homogenates were found to be similar to those cited above. Smooth muscles contain different thin-filament classes and only one type appears to possess Caldesmon. By comparing values for the molar composition of Caldesmon-specific filaments (1 Caldesmon:2 tropomyosin:14 actin) with the values above determined for intact tissue, we conclude that the Caldesmon filaments account for approx. 34–45% of he total thin-filament pool in arterial smooth muscle and slightly less in visceral muscles.
Nikolai B. Gusev - One of the best experts on this subject based on the ideXlab platform.
-
some properties of Caldesmon and calponin and the participation of these proteins in regulation of smooth muscle contraction and cytoskeleton formation
Biochemistry, 2001Co-Authors: Nikolai B. GusevAbstract:The interaction of Caldesmon with different Ca2+-binding proteins has been analyzed, and it is supposed that one of the conformers of calmodulin might be an endogenous regulator of Caldesmon. The arrangement of Caldesmon and Ca2+-binding proteins within their complexes has been analyzed by different methods. The central helix of calmodulin is supposed to be located near the single Cys residue in the C-terminal domain of Caldesmon. The N-terminal globular domain of calmodulin interacts with sites A and B" of Caldesmon, whereas the C-terminal globular domain of calmodulin binds to site B of Caldesmon. The complex of calmodulin and Caldesmon is very flexible; therefore, both parallel and antiparallel orientation of polypeptide chains of the two proteins is possible in experiments with short fragments of Caldesmon and calmodulin. The length, flexibility, and charge of the central helix of calmodulin play an important role in its interaction with Caldesmon. Phosphorylation of Caldesmon by different protein kinases in vitro has been analyzed. It was shown that phosphorylation catalyzed by casein kinase II of sites located in the N-terminal domain decreases the interaction of Caldesmon with myosin and tropomyosin. Caldesmon and calponin may interact with phospholipids. The sites involved in the interaction of these actinbinding proteins with phospholipids have been mapped. It is supposed that the interaction of calponin and Caldesmon with phospholipids may play a role in the formation of cytoskeleton. Calponin interacts with 90-kD heat shock protein (hsp90) that may be involved in transportation of calponin and its proper interaction with different elements of cytoskeleton. Calponin, filamin, and a-actinin can simultaneously interact with actin filaments. Simultaneous binding of two actin-binding proteins affects the structure of actin bundles and their mechanical properties and may be of great importance in formation of different elements of cytoskeleton.
-
Interaction of isoforms of S100 protein with smooth muscle Caldesmon
FEBS letters, 1998Co-Authors: Alexei A. Polyakov, Pia A. J. Huber, Steven B. Marston, Nikolai B. GusevAbstract:Interaction of S100a and S100b with duck gizzard Caldesmon was investigated by means of native gel electrophoresis, fluorescent spectroscopy and disulfide crosslinking. Both isoforms of S100 interact with intact Caldesmon and its C-terminal deletion mutant 606C (residues 606-756) with apparent Kd of 0.2-0.6 microM thus indicating that the S100-binding site is located in the C-terminal domain of Caldesmon. The single SH group of duck gizzard Caldesmon can be crosslinked to Cys-84 of the beta-chain or to Cys-85 of the alpha-chain of S100. Crosslinking of S100 reduces the inhibitory action of Caldesmon on actomyosin ATPase activity. S100 reverses the inhibitory action of intact Caldesmon and its deletion mutants 606C (residues 606-756) and H9 (residues 669-737) as effectively as calmodulin. S100a has higher affinity to Caldesmon and is more effective than S100b in reversing Caldesmon-induced inhibition of actomyosin ATPase activity. Although monomeric (calmodulin, troponin C) and dimeric (S100) Ca-binding proteins have different sizes and structures they interact with Caldesmon in a very similar fashion.
-
Interaction of smooth muscle Caldesmon with calmodulin mutants
FEBS letters, 1995Co-Authors: M. V. Medvedeva, Tatyana L. Bushueva, V.p. Shirinsky, Thomas J. Lukas, D. Martin Watterson, Nikolai B. GusevAbstract:The interaction of avian smooth muscle Caldesmon with calmodulin (CaM) was investigated by studying the ability of selected mutant calmodulins to induce fluorescence changes in Caldesmon. Different types of CaM mutants were used including point charge mutants, cluster mutations, and mutations which alter the calcium binding of CaM. The Caldesmon binding properties were only slightly affected by E84K-CaM or by the double mutation E84Q/E120Q-CaM. Affinity of calmodulin to Caldesmon was decreased 2–4 times by point mutation G33V-CaM, double mutation E84K/E120K-CaM, deletion of residues 82–84, and by cluster mutations DEE118-120 → KKK or EEE8284 → KKK. Mutations of the first (E31A-CaM) and the second (E67A-CaM) calcium binding sites reduced the affinity of calmodulin to Caldesmon by at least 5-fold; in addition these calmodulin mutants exhibited smaller changes in the fluorescence spectra of Caldesmon. Simultaneous mutation of the two negatively charged clusters of calmodulin EEE82-84 → KKK and DEE118-120 → KKK resulted in a more than 15-fold decrease in the affinity of calmodulin for Caldesmon. The data indicate that charged and uncharged amino acids in both halves of CaM play an important role in the binding of calmodulin to Caldesmon, and that Ca2+ binding must be maintained in the amino-terminal sites for maximal interaction with Caldesmon.
-
Isolation and study of certain properties of Caldesmon from cattle aorta
Biokhimiia (Moscow Russia), 1995Co-Authors: L.k. Skolysheva, E.a. Smirnova, M. V. Medvedeva, Nikolai B. GusevAbstract:A modified method for bovine aorta Caldesmon isolation has been developed. When isolated from large vessels, Caldesmon is copurified with connective tissue proteins whose molecular weight is similar to that of Caldesmon. Separation of these proteins can be achieved by stepwise anion- (Q-Sepharose) and cation (phosphocellulose)-exchange chromatography. Bovine aorta and duck gizzard Caldesmons have similar apparent molecular weights, absorption at 280 nm and one-dimensional peptide maps. Casein kinase II transfers about one mol of phosphate per mol of bovine aorta or duck gizzard Caldesmon. In both cases, the sites of phosphorylation are located in the N-terminal peptides of apparent molecular weights of 26-28 kDa. It is concluded that there are no substantial differences between the structures and properties of avian gizzard and mammalian vessel Caldesmons.
-
Phosphorylation of aorta Caldesmon by endogenous proteolytic fragments of protein kinase C
Journal of muscle research and cell motility, 1994Co-Authors: Alexander V. Vorotnikov, Nikolai B. Gusev, C Redwood, S. Hua, John H. Collins, Steve MarstonAbstract:Endogenous Caldesmon kinase activity in sheep aorta smooth muscle was purified and characterized. The enzyme was identified as a proteolytic fragment of protein kinase C by cross-reactivity with anti-protein kinase C antibodies, autophosphorylation, substrate specificity and the primary structure of the sites of phosphorylation on Caldesmon. The enzyme phosphorylated aorta Caldesmon both in native thin filaments and in the isolated state. Up to 2.9 mols of phosphate per mol of Caldesmon were transferred. Prolonged incubation of Caldesmon with the kinase resulted in phosphorylation of Ser-127, Ser-587, Ser-600, Ser-657, Ser-686, and Ser-726 (numbering corresponds to chicken gizzard Caldesmon sequence). Ser-600 and Ser-587 were the major sites of phosphorylation containing more than 30% of phosphate transferred. Phosphorylation did not significantly affect the interaction of Caldesmon with Ca2+-calmodulin. However, phosphorylation of both intact Caldesmon and of its C-terminal fragment (658C), containing residues 658–756, significantly decreased their ability to inhibit acto-heavy meromyosin ATPase. This seems to be partially due to a decrease in the binding of Caldesmon and 658C to actin-tropomyosin and partly due to an uncoupling of the binding-inhibition relationship.
Joseph M Chalovich - One of the best experts on this subject based on the ideXlab platform.
-
Additional Sites are Involved in the Regulation of Caldesmon by PAK Phosphorylation
Biophysical Journal, 2010Co-Authors: Svetlana S. Hamden, Mechthild M. Schroeter, Joseph M ChalovichAbstract:Caldesmon is an actin- and myosin-binding protein that is rich in smooth muscle. Caldesmon inhibits the actin activation of myosin catalyzed ATPase activity and may have additional functions in smooth muscle. The activity of Caldesmon is controlled by phosphorylation and by binding to other factors such as Ca++-calmodulin. Caldesmon is a substrate for p21-activated kinase, PAK, which is reported to phosphorylate chicken gizzard Caldesmon at two sites, Ser672 and Ser702. We investigated PAK phosphorylation of Caldesmon using a 22kDa C-terminal Caldesmon fragment. We also substituted Ser672 and Ser702 with either alanine or aspartic acid residues to mimic non-phosphorylated and constitutively phosphorylated states of Caldesmon, respectively. We found that the aspartic acid mutation of Caldesmon weakened calmodulin binding but had no effect on the inhibitory activity of Caldesmon. Phosphorylation of the aspartic acid double mutant with recombinant PAK resulted in additional phosphorylation at Thr627, Ser631, Ser635 and Ser642. Phosphorylation at these sites by PAK was slow, but produced further weakening of calmodulin binding and reduced the inhibitory activity of Caldesmon in the absence of calmodulin. Phosphorylation at the additional sites was without effect on Ca++-Calmodulin binding if Ser672 and Ser702 were not phosphorylated, but was sufficient to release inhibition of actomyosin ATPase activity. This work raises the possibility that phosphorylation in the region of residues 627-642 significantly alters the activity of Caldesmon.
-
Influence of ionic strength, actin state, and Caldesmon construct size on the number of actin monomers in a Caldesmon binding site.
Biochemistry, 2003Co-Authors: Scott Fredricksen, Anmei Cai, Boris S. Gafurov, And Andrea Resetar, Joseph M ChalovichAbstract:There is no consensus on the mechanism of inhibition of actin-myosin ATPase activity by Caldesmon. Various models are based on different assumptions for the number of actin monomers that constitute a Caldesmon binding site. Differences in binding behavior may be due to variations in the assay, the range of Caldesmon concentrations, the type of Caldesmon, and the method of data analysis used. We have evaluated these factors by measuring binding in the presence and absence of tropomyosin with both intact Caldesmon and a recombinant 35 kDa actin binding fragment and with actin initially in the polymerized state or monomeric state. In all cases Caldesmon binding could be simulated with a model having one class of binding sites. However, the number of actin monomers constituting a site was variable. Binding to F-actin at 165 mM ionic strength was best described with 7 actin monomers per site. When Caldesmon bound to actin during the polymerization of G-actin, the size of the binding site was 3. Binding of the expressed truncated fragment, Cad35, could be described with 3 monomers per site. A simple interpretation of the data is that Caldesmon binds tightly to 2-3 actin monomers. Additional parts of Caldesmon bind less tightly to actin, causing Caldesmon to cover approximately 7 actin monomers. The appendix contains an analysis of several binding curves with multiple binding site models. There is no compelling evidence for two classes of binding sites.
-
theoretical studies on competitive binding of Caldesmon and myosin s1 to actin prediction of apparent cooperativity in equilibrium and slow down in kinetics of s1 binding by Caldesmon
Biochemistry, 2003Co-Authors: Joseph M Chalovich, Yider ChenAbstract:Several laboratories have reported cooperative binding of S1 to actin in the presence of Caldesmon. This cooperative binding has been interpreted with a model similar to that proposed for the binding of S 1 to regulated actins in which the binding affinity of S 1 is controlled by the position of the tropomyosin filaments. In a recent paper [Sen, A., Chen, Y., Yan, B., and Chalovich, J. M. (2001) Biochemistry 40, 5757-64], we showed qualitatively that Sl binding resulted in rapid dissociation of Caldesmon from actin or actin-tropomyosin. This suggests that the cooperativity observed in the case of Caldesmon is not due to a conformational change in actin-Caldesmon but to the displacement of Caldesmon. We show in this paper that the pure competitive binding model, in which both Sl and Caldesmon are competing for the same binding sites on actin, can simulate quantitatively the effect of Caldesmon on both the equilibrium and the kinetics of Sl binding to actin. This model successfully predicts an apparent cooperativity for the binding of S 1 to actin-Caldesmon without the need to assume multiple actin-Caldesmon structures and produces a decreased rate of S 1 binding to actin in the presence of Caldesmon. This suggests that the inhibitory action of Caldesmon on the actin-activated ATPase activity of myosin in solution and on the generation of active force in a contracting muscle may be simply due to the blocking of myosin binding sites on actin by Caldesmon.
-
Caldesmon reduces the apparent rate of binding of myosin S1 to actin-tropomyosin.
Biochemistry, 2001Co-Authors: Anindita Sen, Yider Chen, Bo Yan, Joseph M ChalovichAbstract:Equilibrium measurements of the rate of binding of Caldesmon and myosin S1 to actin- tropomyosin from different laboratories have yielded different results and have led to different models of Caldesmon function. An alternate approach to answering these questions is to study the kinetics of binding of both Caldesmon and S1 to actin. We observed that Caldesmon decreased the rate of binding of S1 to actin in a concentration-dependent manner. The inhibition of the rate of S1 binding was enhanced by tropomyosin, but the effect of tropomyosin on the binding was small. Premixing actin with S1 reduced the amplitude (extent) of Caldesmon binding in proportion to the fraction of actin that contained bound S1, but the rate of binding of Caldesmon to free sites was not greatly altered. No evidence for a stable Caldesmon-actin-tropomyosin-S1 complex was observed, although S1 did apparently bind to gaps between Caldesmon molecules. These results indicate that experiments involving Caldesmon, actin, tropomyosin, and myosin are inherently complex. When the concentration of either S1 or Caldesmon is varied, the amount of the other component bound to actin-tropomyosin cannot be assumed to remain fixed. The results are not readily explained by a mechanism in which Caldesmon acts only by stabilizing an inactive state of actin-tropomyosin. The results support regulatory mechanisms that involve changes in the actin-S1 interaction.
-
Flexation of Caldesmon: effect of conformation on the properties of Caldesmon
Journal of Muscle Research and Cell Motility, 1995Co-Authors: R. E. Crosbie, Joseph M Chalovich, Emil ReislerAbstract:The contribution of the extended and bent forms of Caldesmon to its function was investigated by examining chemically modified forms of this protein. The bent 'hairpin' form of Caldesmon was enhanced between pH 6.0 and 8.0 and at low ionic strengths, as reported by an increase in excimer fluorescence of pyrene-labelled Caldesmon under these conditions. The presence of nucleotides also produced significant conformational changes in Caldesmon, as detected by fluorescence measurements and protease digestions. Titrations of pyrene Caldesmon with actin, heavy meromyosin, and calmodulin resulted in a decrease in excimer fluorescence. The function of the bent form of Caldesmon was investigated by using intramolecular 1-ethyl-3-(3-dimethylamino propyl) carbodiimide-crosslinked Caldesmon. The inhibition of acto-S-1 ATPase activity by crosslinked Caldesmon was less efficient compared with that by pyrene modified and control Caldesmons. Caldesmon's ability to switch from an activator to an inhibitor of actin-activated ATPase of myosin was also affected by the folding. Cosedimentation experiments revealed normal binding of crosslinked Caldesmon to smooth muscle myosin. These results indicate the importance of Caldesmon's transition from extended to folded forms and suggest possible functional roles for these different forms of Caldesmon
Alexander V. Vorotnikov - One of the best experts on this subject based on the ideXlab platform.
-
Smooth muscle myosin filament assembly under control of a kinase-related protein (KRP) and Caldesmon
Journal of Muscle Research & Cell Motility, 2002Co-Authors: Dmitry S. Kudryashov, Alexander V. Vorotnikov, Thomas J. Lukas, D. Martin Watterson, Tatyana V. Dudnakova, Olga V. Stepanova, James R. Sellers, Vladimir P. ShirinskyAbstract:Kinase-related protein (KRP) and Caldesmon are abundant myosin-binding proteins of smooth muscle. KRP induces the assembly of unphosphorylated smooth muscle myosin filaments in the presence of ATP by promoting the unfolded state of myosin. Based upon electron microscopy data, it was suggested that Caldesmon also possessed a KRP-like activity (Katayama et al. , 1995, J Biol Chem 270: 3919–3925). However, the nature of its activity remains obscure since Caldesmon does not affect the equilibrium between the folded and unfolded state of myosin. Therefore, to gain some insight into this problem we compared the effects of KRP and Caldesmon, separately, and together on myosin filaments using turbidity measurements, protein sedimentation and electron microscopy. Turbidity assays demonstrated that KRP reduced myosin filament aggregation, while Caldesmon had no effect. Additionally, neither Caldesmon nor its N-terminal myosin binding domain (N152) induced myosin polymerization at subthreshold Mg^2+ concentrations in the presence of ATP, whereas the filament promoting action of KRP was enhanced by Mg^2+. Moreover, the amino-terminal myosin binding fragment of Caldesmon, like the whole protein, antagonizes Mg^2+-induced myosin filament formation. In electron microscopy experiments, Caldesmon shortened myosin filaments in the presence of Mg^2+ and KRP, but N152 failed to change their appearance from control. Therefore, the primary distinction between Caldesmon and KRP appears to be that Caldesmon interacts with myosin to limit filament extension, while KRP induces filament propagation into defined polymers. Transfection of tagged-KRP into fibroblasts and overlay of fibroblast cytoskeletons with Cy3KRP demonstrated that KRP colocalizes with myosin structures in vivo . We propose a new model that through their independent binding to myosin and differential effects on myosin dynamics, Caldesmon and KRP can, in concert, control the length and polymerization state of myosin filaments.
-
Actomyosin cross-linking by Caldesmon in non-muscle cells.
FEBS letters, 2001Co-Authors: E A Goncharova, Vladimir P. Shirinsky, A Y Shevelev, S B Marston, Alexander V. VorotnikovAbstract:The role of myosin-binding in cytoskeletal arrangement of non-muscle low molecular weight Caldesmon (l-Caldesmon) was studied. The N-terminal myosin-binding domain of Caldesmon N152 colocalized with myosin in transiently transfected chicken fibroblasts. When added exogenously to the Triton-insoluble cytoskeleton, N152 enhanced l-Caldesmon displacement by exogenous C-terminal actin-binding fragment (H1). Thus, a significant fraction of l-Caldesmon cross-links actin and myosin. In contrast, in epithelioid HeLa cells most of l-Caldesmon was only actin-bound as H1 alone was enough for its displacement. Phosphorylation by mitogen-activated protein kinase reduced the capability of H1 to displace endogenous l-Caldesmon, suggesting it may represent a regulatory mechanism for actin-Caldesmon interaction in vivo.
-
Actomyosin cross-linking by Caldesmon in non-muscle cells
FEBS Letters, 2001Co-Authors: E A Goncharova, Vladimir P. Shirinsky, Steven B. Marston, A. Shevelev, Alexander V. VorotnikovAbstract:The role of myosin-binding in cytoskeletal arrange- ment of non-muscle low molecular weight Caldesmon (l- Caldesmon) was studied. The N-terminal myosin-binding domain of Caldesmon (N152) colocalized with myosin in transiently transfected chicken fibroblasts. When added exogenously to the Triton-insoluble cytoskeleton, N152 enhanced l-Caldesmon dis- placement by exogenous C-terminal actin-binding fragment (H1). Thus, a significant fraction of l-Caldesmon cross-links actin and myosin. In contrast, in epithelioid HeLa cells most of l-Caldesmon was only actin-bound as H1 alone was enough for its displacement. Phosphorylation by mitogen-activated protein kinase reduced the capability of H1 to displace endogenous l-Caldesmon, suggesting it may represent a regulatory mechanism for actin^Caldesmon interaction in vivo. fl 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
-
Phosphorylation of aorta Caldesmon by endogenous proteolytic fragments of protein kinase C
Journal of muscle research and cell motility, 1994Co-Authors: Alexander V. Vorotnikov, Nikolai B. Gusev, C Redwood, S. Hua, John H. Collins, Steve MarstonAbstract:Endogenous Caldesmon kinase activity in sheep aorta smooth muscle was purified and characterized. The enzyme was identified as a proteolytic fragment of protein kinase C by cross-reactivity with anti-protein kinase C antibodies, autophosphorylation, substrate specificity and the primary structure of the sites of phosphorylation on Caldesmon. The enzyme phosphorylated aorta Caldesmon both in native thin filaments and in the isolated state. Up to 2.9 mols of phosphate per mol of Caldesmon were transferred. Prolonged incubation of Caldesmon with the kinase resulted in phosphorylation of Ser-127, Ser-587, Ser-600, Ser-657, Ser-686, and Ser-726 (numbering corresponds to chicken gizzard Caldesmon sequence). Ser-600 and Ser-587 were the major sites of phosphorylation containing more than 30% of phosphate transferred. Phosphorylation did not significantly affect the interaction of Caldesmon with Ca2+-calmodulin. However, phosphorylation of both intact Caldesmon and of its C-terminal fragment (658C), containing residues 658–756, significantly decreased their ability to inhibit acto-heavy meromyosin ATPase. This seems to be partially due to a decrease in the binding of Caldesmon and 658C to actin-tropomyosin and partly due to an uncoupling of the binding-inhibition relationship.
-
Effect of 67 kDa calcimedin on Caldesmon functioning
FEBS letters, 1993Co-Authors: Natalia V. Bogatcheva, Alexander V. Vorotnikov, Michail P. Panaiotov, Nikolai B. GusevAbstract:Interaction of smooth muscle Caldesmon with calmodulin, troponin C, S-100 protein and 67 kDa calcimedin was analyzed. Native gel electrophoresis and crosslinking revealed the complex formation between Caldesmon and three EF-hand Ca-binding proteins, whereas calcimedin did not interact with Caldesmon. In the presence of Ca2+, calcimedin binds to actin-tropomyosin without affecting the interaction of Caldesmon with this complex. Although calcimedin reversed the inhibitory action of Caldesmon on the actomyosin ATPase activity at a lower concentration than three other Ca-binding proteins, this effect only slightly depends on Ca2+ and was observed at the concentration of calcimedin comparable to that of actin. It is concluded that calcimedin itself cannot be responsible for Ca-dependent regulation of Caldesmon functioning, but actin bundling induced by calcimedin (or by other actin binding proteins) decreases the inhibitory action of Caldesmon on the actomyosin ATPase activity.
Renata Dabrowska - One of the best experts on this subject based on the ideXlab platform.
-
Visualization of Caldesmon binding to synthetic filaments of smooth muscle myosin.
Journal of muscle research and cell motility, 2003Co-Authors: Natalia Kulikova, Robert Makuch, Zoya A. Podlubnaya, Renata DabrowskaAbstract:We have used synthetic filaments of unphosphorylated chicken gizzard myosin with a compact, highly ordered structure under relaxing conditions (in the absence of Ca2+ and in the presence of ATP) to visualize the mode of Caldesmon binding to myosin filaments by negative staining and immunogold electron microscopy. We demonstrate that the addition of Caldesmon to preformed myosin filaments leads to the appearance of numerous smooth projections curving out from the filament surface. The addition of Caldesmon or its N-terminal fragment resulted in the partial masking of myosin filament periodicity. However, it did not change the inner structure of the filaments. It is demonstrated that most Caldesmon molecules bind to myosin filaments through the N-terminal part, while the C-terminal parts protrude from the filament surface, as confirmed by immunoelectron microscopy visualization. Together with the available biochemical data on Caldesmon binding to both actin and myosin and electron microscopic observations on the mode of Caldesmon attachment to actin filaments with the C-termini of the molecules curving out from the filaments, the visualization of Caldesmon attachment to myosin filaments completes the scenario of actin to myosin tethering by Caldesmon.
-
The First Caldesmon-like Protein in Higher Plants
Biochemical and biophysical research communications, 1998Co-Authors: Katarzyna Krauze, Robert Makuch, Małgorzata Stępka, Renata DabrowskaAbstract:Abstract Using anti-Caldesmon polyclonal and monoclonal (raised against the N-terminal fragment of chicken gizzard Caldesmon) antibodies, a plant Caldesmon-like protein, 107 kDa as determined by SDS-gel electrophoresis, has been identified based on Western blotting of total extracts ofOrnithogalum virenspollen tubes. Biochemical investigations showed common properties of this protein with animal Caldesmon—it binds to actin and, in a Ca2+-dependent manner, to calmodulin. In contrast to animal Caldesmon, this plant cell counterpart is relatively resistant to proteolysis by endogenous proteases and sensitive to heat treatment. Our results show the presence of a Caldesmon-like protein in higher plants for the first time.
-
DOES CALPONIN INTERACT WITH Caldesmon
The Journal of biological chemistry, 1997Co-Authors: Edward A. Czuryło, Natalia Kulikova, Renata DabrowskaAbstract:Abstract The roles of calponin and Caldesmon and their interaction in regulation of smooth muscle contraction are controversial. Recently, strong binding between these two proteins has been reported (Graceffa, P., Adam, L. P., and Morgan, K. G. (1996) J. Biol. Chem. 271, 30336–30339). Results in this paper fail to confirm their data and are consistent with the concept of independent functions for calponin and Caldesmon. To examine the ability of duck gizzard Caldesmon to interact with calponin, three Caldesmon derivatives, each containing a different sulfhydryl-specific reporter probe (6-acryloyl-2-dimethylaminonaphtalene,N-(1-pyrenyl)iodoacetamide, andN-iodoacetyl-N′-(5-sulfo-1-naphtylo)ethylenediamine) attached to a single cysteine located in the C-terminal domain, were synthesized. Addition of calponin to labeled Caldesmon at both low and physiological salt concentrations did not induce any changes in fluorescence intensity or maximum shift. Under the same conditions, calmodulin and tropomyosin (known to bind to the C terminus of Caldesmon) produced substantial changes in these spectral parameters. Gel filtration of an equimolar Caldesmon-calponin mixture on a fast protein liquid chromatography Superose-12 column revealed two base-line-separated peaks, the first containing only Caldesmon and the second only calponin, thus confirming the lack of any interaction between these two proteins. Also, the addition of calponin did not change the fluorescence parameters of labeled Caldesmon in complexes with F-actin and F-actin-tropomyosin.
-
Functional interrelationship between calponin and Caldesmon.
Biochemical Journal, 1991Co-Authors: R Makuch, Konstantin G. Birukov, V.p. Shirinsky, Renata DabrowskaAbstract:Calponin and Caldesmon, constituents of smooth-muscle thin filaments, are considered to be potential modulators of smooth-muscle contraction. Both of them interact with actin and inhibit ATPase activity of smooth- and skeletal-muscle actomyosin. Here we show that calponin and Caldesmon could bind simultaneously to F-actin when used in subsaturating amounts, whereas each one used in excess caused displacement of the other from the complex with F-actin. Calponin was more effective than Caldesmon in this competition: when F-actin was saturated with calponin the binding of Caldesmon was eliminated almost completely, whereas even at high molar excess of Caldesmon one-third of calponin (relative to the saturation level) always remained bound to actin. The inhibitory effects of low concentrations of calponin and Caldesmon on skeletal-muscle actomyosin ATPase were additive, whereas the maximum inhibition of the ATPase attained at high concentration of each of them was practically unaffected by the other one. These data suggest that calponin and Caldesmon cannot operate on the same thin filaments. CA(2+)-calmodulin competed with actin for calponin binding, and at high molar excess dissociated the calponin-actin complex and reversed the calponin-induced inhibition of actomyosin ATPase activity.
-
Caldesmon-induced polymerization of actin from profilactin.
European journal of biochemistry, 1991Co-Authors: Barbara Gała̧zkiewicz, Folma Buss, Brigitte M. Jockusch, Renata DabrowskaAbstract:We have investigated the effect of Caldesmon, a Ca2+/calmodulin-regulated actin-binding protein, on the complex between profilin and G-actin (profilactin). We found that smooth muscle Caldesmon dissociates this complex rapidly and induces the polymerization of the released actin. Native profilactin (e. g. the complex isolated from calf thymus) proved more resistant to the attack of Caldesmon than a heterologous complex reconstituted from calf thymus profilin and skeletal muscle actin. The mode of Caldesmon-induced profilactin dissociation was similar to that described for Mg2+, and 2 mM MgCl2 potentiated the Caldesmon effect. Since both Caldesmon and profilin have been found enriched in ruffling membranes of animal cells, our in vitro findings may be relevant to the regulation of actin filaments in living cells.
