Characterization of Calponin Binding to Actin

Lu, Frank W. M.; Freedman, Michael V.; Chalovich, Joseph M.

doi:10.1021/bi00037a026

Cited by 41 publications

(53 citation statements)

References 59 publications

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“…Also, under the conditions used, the actin was saturated by a -actinin when the molar ratio for a -actinin/G-actin in the incubation mixture was 0.25-0.30. These results agree with the data reported in the literature (15,16).…”

Section: Resultssupporting

confidence: 93%

Simultaneous Interaction of Actin With a‐Actinin and Calponin

Panasenko

Gusev

2000

IUBMB Life

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SummaryInteraction of calponin and ®-actinin with actin was analyzed by means of cosedimentation and electron microscopy. G-actin was polymerized in the presence of calponin, ®-actinin, or both of these actin-binding proteins (ABPs). The single and bundled actin laments were separated, and the stoichiometry of ABPs and actin in both types of laments was determined. Binding of calponin to the single or bundled actin laments was not dependent on the presence of ®-actinin and did not displace ®-actinin from actin. In the presence of calponin, however, less ®-actinin was bound to the bundled actin laments, and the binding of ®-actinin was accompanied by a partial decrease in the calponin/actin stoichiometry in the bundles of actin laments. Calponin had no in uence on the binding of ®-actinin to the single actin laments. The structure of actin bundles formed in the presence of the two ABPs differed from that formed in the presence of either one singly. We conclude that calponin and ®-actinin can coexist on actin and that nearly each actin monomer can bind one of these ABPs. IUBMB Life, 49: 277 -282, 2000

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Section: Resultssupporting

confidence: 93%

Simultaneous Interaction of Actin With a‐Actinin and Calponin

Panasenko

Gusev

2000

IUBMB Life

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“…Similar results were obtained in the Chalovich laboratory (8). This suggested that calponin and caldesmon do not form a mutual complex on actin and reside on different populations of thin filaments in vivo.…”

supporting

confidence: 84%

Does Calponin Interact with Caldesmon?

Czuryło

Kulikova

Da̧browska

1997

Journal of Biological Chemistry

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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, and N-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.The primary mechanism for smooth muscle regulation, necessary to initiate contraction, involves phosphorylation of the 20-kDa myosin light chains by a specific Ca 2ϩ /calmodulin-dependent myosin light chain kinase (for reviews see Refs. 1-3). It is postulated that the specific thin filament-associated proteins caldesmon and calponin take part in the secondary mechanism of regulation, probably during the relaxation phase. This view is supported by in vitro experiments showing that both of these proteins inhibit actin-activated ATPase activity of myosin, mobility of actin filaments over immobilized myosin in motility assay, and skinned muscle contraction. This inhibition can be reversed by Ca 2ϩ /calmodulin, or other Ca 2ϩ -binding proteins (like S100 or caltropin), and by phosphorylation with protein kinase C or casein kinase II (Refs. 3-6 and references therein).Studies by Makuch et al. (7) revealed that calponin and caldesmon compete for the binding sites on actin filament, calponin being more effective at displacing caldesmon than vice versa. Similar results were obtained in the Chalovich laboratory (8). This suggested that calponin and caldesmon do not form a mutual complex on actin and reside on different populations of thin filaments in vivo. Immunofluorescence and immunogold electron microscopy confirmed this suggestion showing that, whereas caldesmon is present exclusively in the contractile domain, calponin is primarily, although not exclusively, located in the cytoskeletal domain of...

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“…The higher affinity between calponin and immobilized actin (8 nM, Figure 4B) in comparison with that previously reported (6 µM, [42]) may be due to the nature of the solid-phase assay conditions, which may facilitate the formation of the F-actin-calponin complex. Although detectable by the ELISA experiments, the binding between calponin and immobilized tropomyosin is significantly weaker in contrast to the binding to actin ( Figure 4A).…”

Section: Phosphorylation Of Serine-175 Reduces Calponin's Actin-bindicontrasting

confidence: 43%

A role for serine-175 in modulating the molecular conformation of calponin

Jin

Walsh

Sutherland

et al. 2000

Biochemical Journal

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Calponin is an actin filament-associated protein found in smooth muscle and non-muscle cells. Calponin inhibits actin-myosin interaction in a manner that is prevented by protein kinase C (PKC)-catalysed phosphorylation of serine-175. To investigate the molecular basis of serine-175-mediated regulation, we examined the effect of phosphorylation on the conformation of calponin using monoclonal antibody (mAb) epitope analysis. Eight mAbs against different epitopes on chicken gizzard calponin were developed to monitor the conformational changes in calponin induced by PKC-mediated phosphorylation or serine-175 alanine (S175A) substitution. The relative affinities of the mAbs for calponins immobilized on microtitre plates or bound to actin-tropomyosin thin filaments were determined, and epitope competitions between free and immobilized calponins were carried out. The changes in binding affinity between mAb paratopes and calponin epitopes demonstrate several serine-175 modification-induced conformational effects : (a) structures of calponin are reconfigured by serine-175 modification, supporting

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Characterization of Calponin Binding to Actin

Cited by 41 publications

References 59 publications

Simultaneous Interaction of Actin With a‐Actinin and Calponin

Simultaneous Interaction of Actin With a‐Actinin and Calponin

Does Calponin Interact with Caldesmon?

A role for serine-175 in modulating the molecular conformation of calponin

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