Characterization of the Regulatory Domain of Gizzard Calponin

Mezgueldi, Mohamed; Mendre, Christiane; Calas, Bernard; Kassab, Ridha; Fattoum, Abdellatif

doi:10.1074/jbc.270.15.8867

Cited by 73 publications

(107 citation statements)

References 38 publications

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“…Thus, it was first demonstrated that the major calmodulin-binding sites reside in the chymotryptic fragments corresponding to the COOH-terminal region of caldesmon (Wang et al, 1991b). Synthetic peptides corresponding to residues 658-666 (Zhan et al, 1991) and 675-695 (Mezgueldi et al, 1994) in chicken gizzard h-caldesmon were later shown to bind calmodulin in a Ca 2+ -dependent manner. Using recombinant fragments, Marston et al (1994) proposed that Ca 2+ -calmodulin interacts with three distinct caldesmon sites: residues 658-666 (site A), residues 687-695 (site B), and residues 717-726 (site B′).…”

Section: Discussionmentioning

confidence: 99%

“…It has been proposed that Ca 2+ -calmodulin interacts with three sites in chicken gizzard caldesmon: (Zhan et al, 1991;Marston et al, 1994;Mezgueldi et al, 1994). Each of the three sites contains a Trp residue, which is believed to be involved in calmodulin binding as has been shown by fluorescence measurements (Shirinsky et al, 1988).…”

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confidence: 99%

“…Smooth muscle h-caldesmon has a M r of 87 000 and contains three structurally and functionally distinct domains: an NH 2 -terminal domain (residues 1-250), a middle domain (residues 251-400), and a COOH-terminal domain (residues 401-756). While all three domains have been shown to bind tropomyosin in Vitro (Redwood & Marston, 1993;Graceffa, 1987;Watson et al, 1990), the NH 2 -terminal domain also binds myosin (Velaz et al, 1990;Ikebe & Reardon, 1988), and the COOHterminal domain interacts with actin (Bartegi et al, 1990;Mezgueldi et al, 1994) and the Ca 2+ -binding proteins calmodulin (Zhan et al, 1991;Zhuang et al, 1995;Marston et al, 1994) and caltropin (Mani et al, 1992). Both NH 2 -and COOH-terminal domains of smooth muscle caldesmon are conserved in nonmuscle caldesmon with similar primary structures and functions (Hayashi et al, 1991;Bryan & Lee, 1991).…”

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confidence: 99%

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Tryptophan Residues in Caldesmon Are Major Determinants for Calmodulin Binding

et al. 1997

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Calmodulin has been shown to interact with the COOH-terminal domain of gizzard h-caldesmon at three sites, A (residues 658-666), B (residues 687-695), and B′ (residues 717-725), each of which contains a Trp residue [Zhan et al. (1991) J. Biol. Chem. 266, 21810-21814; Marston et al. (1994) J. Biol. Chem. 269, 8134-8139; Mezgueldi et al. (1994) J. Biol. Chem. 269, 12824-12832]. To determine the contribution of each of the three Trp residues in the calmodulin-caldesmon interaction, we have mutated the Trp residues to Ala in the COOH-terminal domain of fibroblast caldesmon (CaD39) and studied the effects on calmodulin binding by fluorescence measurements and using immobilized calmodulin. Wild-type CaD39 binds with a K d of 0.13 × 10 -6 M and a stoichiometry of 1 mol of calmodulin per mol of caldesmon. Replacing Trp 659 at site A or Trp 692 at site B to Ala reduces binding by 22-and 31-fold (K d ) 2.9 × 10 -6 and 4.0 × 10 -6 M), respectively, and destabilizes the CaD39-calmodulin complex by 1.75 and 1.94 kcal mol -1 , respectively. Mutation of both Trp 659 and Trp 692 to Ala further reduces binding with a K d of 6.1 × 10 -6 M and destabilizes the complex by 2.17 kcal mol -1 . On the other hand, mutation of Trp 722 at site B′ to Ala causes a much smaller decrease in affinity (K d ) 0.6 × 10 -6 M) and results in a destabilization energy of 0.87 kcal mol -1 . To investigate the relative importance of the amino acid residues near each Trp residue in the caldesmon-calmodulin interaction, deletion mutants were constructed lacking site A, site B, and site A+B. Although deletion of site A decreases binding of CaD39 to calmodulin by 13-fold (K d ) 1.7 × 10 -6 M), it results in tighter binding than mutation of Trp 659 to Ala at this site, suggesting that the residues neighboring Trp 659 may contribute negatively to the interaction. Deletion of site B causes a similar reduction in binding (K d ) 4.1 × 10 -6 M) as observed for replacing Trp 692 to Ala at this site, indicating that Trp 692 is the major, if not the only, binding determinant at site B. Deletion of both site A and site B drastically reduces binding by 62-fold. Taken together, these results suggest that Trp 659 and Trp 692 are the major determinants in the caldesmoncalmodulin interaction and that Trp 722 in site B′ plays a minor role.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Tryptophan Residues in Caldesmon Are Major Determinants for Calmodulin Binding

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“…In all these cases calponin was eluted from the affinity matrix at about 70-100 mM of the salt and interaction of calponin with desmin is comparable with two other thin-filament-associated proteins. Limited chymotrypsinolysis of calponin results in production of long 22 kDa N-terminal and short 13 kDa C-terminal fragments [15] (Fig. 4).…”

Section: 4 6 8 112 14 16mentioning

confidence: 99%

Interaction of smooth muscle calponin and desmin

Wang

Gusev

1996

FEBS Letters

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Interaction of smooth-muscle calponin and desmin was analyzed by means of ultracentrifugation, fluorescent spectroscopy and affinity chromatography. At low and intermediate ionic strength (30-50 mM NaCI) eaiponin is cosedimented with desmin with an apparent dissociation constant 3-15 IxM and stoichiometry of 1 calponin/4-6 desmin. Calmodulin decreases the quantity of calponin bound to desmin. Increase of ionic strength up to 150 mM weakens calponin-desmin interaction, but even at this ionic strength part of calponin remains bound to desmin. Calponin increases the rate and extent of fluorescence quenching induced by polymerization of 5-iodoacetamidofluorescein-labeled desmin. Affmity chromatography data indicate that desmin-binding sites are located in the N-terminal 22 kDa fragment of calponin. Since calponin interacts with desmin with an affinity comparable with that of, e.g., tropomyosin and myosin we suppose that calponin-desmin interaction may be important for cytoskeleton organization.

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“…Calponin Proteolysis-Calponin was digested with ␣-chymotrypsin (Sigma) (19) with the Staphylococcus aureus V8 protease (Roche Molecular Biochemicals). V8 fragments were purified by reverse phase HPLC using a Vydac C18 column developed with a 0 -100% water acetonitrile gradient containing 0.1% trifluoroacetic acid.…”

Section: Methodsmentioning

confidence: 99%

Regulation of Protein Kinase C by the Cytoskeletal Protein Calponin

Leinweber

Parissenti

Gallant

et al. 2000

Journal of Biological Chemistry

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Previous studies from this laboratory have shown that, upon agonist activation, calponin co-immunoprecipitates and co-localizes with protein kinase C⑀ (PKC⑀) in vascular smooth muscle cells. In the present study we demonstrate that calponin binds directly to the regulatory domain of PKC both in overlay assays and, under native conditions, by sedimentation with lipid vesicles. Calponin was found to bind to the C2 region of both PKC⑀ and PKC␣ with possible involvement of C1B. The C2 region of PKC⑀ binds to the calponin repeats with a requirement for the region between amino acids 160 and 182. We have also found that calponin can directly activate PKC autophosphorylation. By using anti-phosphoantibodies to residue Ser-660 of PKC␤II, we found that calponin, in a lipid-independent manner, increased auto-phosphorylation of PKC␣, -⑀, and -␤II severalfold compared with control conditions. Similarly, calponin was found to increase the amount of 32 P-labeled phosphate incorporated into PKC from [␥-32 P]ATP. We also observed that calponin addition strongly increased the incorporation of radiolabeled phosphate into an exogenous PKC peptide substrate, suggesting an activation of enzyme activity. Thus, these results raise the possibility that calponin may function in smooth muscle to regulate PKC activity by facilitating the phosphorylation of PKC.

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Characterization of the Regulatory Domain of Gizzard Calponin

Cited by 73 publications

References 38 publications

Tryptophan Residues in Caldesmon Are Major Determinants for Calmodulin Binding

Tryptophan Residues in Caldesmon Are Major Determinants for Calmodulin Binding

Interaction of smooth muscle calponin and desmin

Regulation of Protein Kinase C by the Cytoskeletal Protein Calponin

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