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. 1988 Jun 1;106(6):1973–1983. doi: 10.1083/jcb.106.6.1973

Characterization of 83-kilodalton nonmuscle caldesmon from cultured rat cells: stimulation of actin binding of nonmuscle tropomyosin and periodic localization along microfilaments like tropomyosin

PMCID: PMC2115152  PMID: 3384851

Abstract

Nonmuscle caldesmon purified from cultured rat cells shows a molecular weight of 83,000 on SDS gels, Stokes radius of 60.5 A, and sedimentation coefficient (S20,w) of 3.5 in the presence of reducing agents. These values give a native molecular weight of 87,000 and a frictional ratio of 2.04, suggesting that the molecule is a monomeric, asymmetric protein. In the absence of reducing agents, the protein is self-associated, through disulfide bonds, into oligomers with a molecular weight of 230,000 on SDS gels. These S-S oligomers appear to be responsible for the actin-bundling activity of nonmuscle caldesmon in the absence of reducing agents. Actin binding is saturated at a molar ratio of one 83-kD protein to six actins with an apparent binding constant of 5 X 10(6) M-1. Because of 83-kD nonmuscle caldesmon and tropomyosin are colocalized in stress fibers of cultured cells, we have examined effects of 83-kD protein on the actin binding of cultured cell tropomyosin. Of five isoforms of cultured rat cell tropomyosin, tropomyosin isoforms with high molecular weight values (40,000 and 36,500) show higher affinity to actin than do tropomyosin isoforms with low molecular weight values (32,400 and 32,000) (Matsumura, F., and S. Yamashiro-Matsumura. 1986. J. Biol. Chem. 260:13851-13859). At physiological concentration of KCl (100 mM), 83-kD nonmuscle caldesmon stimulates binding of low molecular weight tropomyosins to actin and increases the apparent binding constant (Ka from 4.4 X 10(5) to 1.5 X 10(6) M-1. In contrast, 83-kD protein has slight stimulation of actin binding of high molecular weight tropomyosins because high molecular weight tropomyosins bind to actin strongly in this condition. As the binding of 83-kD protein to actin is regulated by calcium/calmodulin, 83-kD protein regulates the binding of low molecular weight tropomyosins to actin in a calcium/calmodulin-dependent way. Using monoclonal antibodies to visualize nonmuscle caldesmon along microfilaments or actin filaments reconstituted with purified 83-kD protein, we demonstrate that 83-kD nonmuscle caldesmon is localized periodically along microfilaments or actin filaments with similar periodicity (36 +/- 4 nm) as tropomyosin. These results suggest that 83- kD protein plays an important role in the organization of microfilaments, as well as the control of the motility, through the regulation of the binding of tropomyosin to actin.

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Selected References

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  1. Bretscher A., Lynch W. Identification and localization of immunoreactive forms of caldesmon in smooth and nonmuscle cells: a comparison with the distributions of tropomyosin and alpha-actinin. J Cell Biol. 1985 May;100(5):1656–1663. doi: 10.1083/jcb.100.5.1656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bretscher A. Smooth muscle caldesmon. Rapid purification and F-actin cross-linking properties. J Biol Chem. 1984 Oct 25;259(20):12873–12880. [PubMed] [Google Scholar]
  3. Bretscher A. Thin filament regulatory proteins of smooth- and non-muscle cells. Nature. 1986 Jun 19;321(6072):726–727. doi: 10.1038/321726b0. [DOI] [PubMed] [Google Scholar]
  4. Cooper H. L., Feuerstein N., Noda M., Bassin R. H. Suppression of tropomyosin synthesis, a common biochemical feature of oncogenesis by structurally diverse retroviral oncogenes. Mol Cell Biol. 1985 May;5(5):972–983. doi: 10.1128/mcb.5.5.972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Criss W. E., Kakiuchi S. Calcium: calmodulin and cancer. Fed Proc. 1982 May;41(7):2289–2291. [PubMed] [Google Scholar]
  6. Dabrowska R., Goch A., Gałazkiewicz B., Osińska H. The influence of caldesmon on ATPase activity of the skeletal muscle actomyosin and bundling of actin filaments. Biochim Biophys Acta. 1985 Sep 27;842(1):70–75. doi: 10.1016/0304-4165(85)90295-8. [DOI] [PubMed] [Google Scholar]
  7. Dingus J., Hwo S., Bryan J. Identification by monoclonal antibodies and characterization of human platelet caldesmon. J Cell Biol. 1986 May;102(5):1748–1757. doi: 10.1083/jcb.102.5.1748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eaton B. L., Kominz D. R., Eisenberg E. Correlation between the inhibition of the acto-heavy meromyosin ATPase and the binding of tropomyosin to F-actin: effects of Mg2+, KCl, troponin I, and troponin C. Biochemistry. 1975 Jun 17;14(12):2718–2725. doi: 10.1021/bi00683a025. [DOI] [PubMed] [Google Scholar]
  9. Eaton B. L. Tropomyosin binding to F-actin induced by myosin heads. Science. 1976 Jun 25;192(4246):1337–1339. doi: 10.1126/science.131972. [DOI] [PubMed] [Google Scholar]
  10. Fujii T., Imai M., Rosenfeld G. C., Bryan J. Domain mapping of chicken gizzard caldesmon. J Biol Chem. 1987 Feb 25;262(6):2757–2763. [PubMed] [Google Scholar]
  11. Fürst D. O., Cross R. A., De Mey J., Small J. V. Caldesmon is an elongated, flexible molecule localized in the actomyosin domains of smooth muscle. EMBO J. 1986 Feb;5(2):251–257. doi: 10.1002/j.1460-2075.1986.tb04206.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hendricks M., Weintraub H. Multiple tropomyosin polypeptides in chicken embryo fibroblasts: differential repression of transcription by Rous sarcoma virus transformation. Mol Cell Biol. 1984 Sep;4(9):1823–1833. doi: 10.1128/mcb.4.9.1823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hendricks M., Weintraub H. Tropomyosin is decreased in transformed cells. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5633–5637. doi: 10.1073/pnas.78.9.5633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Horiuchi K. Y., Miyata H., Chacko S. Modulation of smooth muscle actomyosin ATPase by thin filament associated proteins. Biochem Biophys Res Commun. 1986 May 14;136(3):962–968. doi: 10.1016/0006-291x(86)90426-2. [DOI] [PubMed] [Google Scholar]
  15. Ishiwata S., Fujime S. Effect of calcium ions on the flexibility of reconstituted thin filaments of muscle studied by quasielastic scattering of laser light. J Mol Biol. 1972 Jul 28;68(3):511–522. doi: 10.1016/0022-2836(72)90103-9. [DOI] [PubMed] [Google Scholar]
  16. Kennett R. H. Cell fusion. Methods Enzymol. 1979;58:345–359. doi: 10.1016/s0076-6879(79)58149-x. [DOI] [PubMed] [Google Scholar]
  17. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Leonardi C. L., Warren R. H., Rubin R. W. Lack of tropomyosin correlates with the absence of stress fibers in transformed rat kidney cells. Biochim Biophys Acta. 1982 Apr 29;720(2):154–162. doi: 10.1016/0167-4889(82)90007-6. [DOI] [PubMed] [Google Scholar]
  20. Lin J. J., Helfman D. M., Hughes S. H., Chou C. S. Tropomyosin isoforms in chicken embryo fibroblasts: purification, characterization, and changes in Rous sarcoma virus-transformed cells. J Cell Biol. 1985 Mar;100(3):692–703. doi: 10.1083/jcb.100.3.692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lynch W. P., Riseman V. M., Bretscher A. Smooth muscle caldesmon is an extended flexible monomeric protein in solution that can readily undergo reversible intra- and intermolecular sulfhydryl cross-linking. A mechanism for caldesmon's F-actin bundling activity. J Biol Chem. 1987 May 25;262(15):7429–7437. [PubMed] [Google Scholar]
  22. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  23. Marston S. B., Smith C. W. The thin filaments of smooth muscles. J Muscle Res Cell Motil. 1985 Dec;6(6):669–708. doi: 10.1007/BF00712237. [DOI] [PubMed] [Google Scholar]
  24. Matsumura F., Lin J. J. Visualization of monoclonal antibody binding to tropomyosin on native smooth muscle thin filaments by electron microscopy. J Mol Biol. 1982 May 5;157(1):163–171. doi: 10.1016/0022-2836(82)90520-4. [DOI] [PubMed] [Google Scholar]
  25. Matsumura F., Lin J. J., Yamashiro-Matsumura S., Thomas G. P., Topp W. C. Differential expression of tropomyosin forms in the microfilaments isolated from normal and transformed rat cultured cells. J Biol Chem. 1983 Nov 25;258(22):13954–13964. [PubMed] [Google Scholar]
  26. Matsumura F., Yamashiro-Matsumura S., Lin J. J. Isolation and characterization of tropomyosin-containing microfilaments from cultured cells. J Biol Chem. 1983 May 25;258(10):6636–6644. [PubMed] [Google Scholar]
  27. Matsumura F., Yamashiro-Matsumura S. Modulation of actin-bundling activity of 55-kDa protein by multiple isoforms of tropomyosin. J Biol Chem. 1986 Apr 5;261(10):4655–4659. [PubMed] [Google Scholar]
  28. Matsumura F., Yamashiro-Matsumura S. Purification and characterization of multiple isoforms of tropomyosin from rat cultured cells. J Biol Chem. 1985 Nov 5;260(25):13851–13859. [PubMed] [Google Scholar]
  29. Maupin-Szamier P., Pollard T. D. Actin filament destruction by osmium tetroxide. J Cell Biol. 1978 Jun;77(3):837–852. doi: 10.1083/jcb.77.3.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ngai P. K., Walsh M. P. Inhibition of smooth muscle actin-activated myosin Mg2+-ATPase activity by caldesmon. J Biol Chem. 1984 Nov 25;259(22):13656–13659. [PubMed] [Google Scholar]
  31. Owada M. K., Hakura A., Iida K., Yahara I., Sobue K., Kakiuchi S. Occurrence of caldesmon (a calmodulin-binding protein) in cultured cells: comparison of normal and transformed cells. Proc Natl Acad Sci U S A. 1984 May;81(10):3133–3137. doi: 10.1073/pnas.81.10.3133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sato K., Slesinski R. S., Littlefield J. W. Chemical mutagenesis at the phosphoribosyltransferase locus in cultured human lymphoblasts. Proc Natl Acad Sci U S A. 1972 May;69(5):1244–1248. doi: 10.1073/pnas.69.5.1244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Siegel L. M., Monty K. J. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta. 1966 Feb 7;112(2):346–362. doi: 10.1016/0926-6585(66)90333-5. [DOI] [PubMed] [Google Scholar]
  34. Smith C. W., Pritchard K., Marston S. B. The mechanism of Ca2+ regulation of vascular smooth muscle thin filaments by caldesmon and calmodulin. J Biol Chem. 1987 Jan 5;262(1):116–122. [PubMed] [Google Scholar]
  35. Sobue K., Muramoto Y., Fujita M., Kakiuchi S. Purification of a calmodulin-binding protein from chicken gizzard that interacts with F-actin. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5652–5655. doi: 10.1073/pnas.78.9.5652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sobue K., Takahashi K., Tanaka T., Kanda K., Ashino N., Kakiuchi S., Maruyama K. Crosslinking of actin filaments is caused by caldesmon aggregates, but not by its dimers. FEBS Lett. 1985 Mar 11;182(1):201–204. doi: 10.1016/0014-5793(85)81184-4. [DOI] [PubMed] [Google Scholar]
  37. Sobue K., Takahashi K., Wakabayashi I. Caldesmon150 regulates the tropomyosin-enhanced actin-myosin interaction in gizzard smooth muscle. Biochem Biophys Res Commun. 1985 Oct 30;132(2):645–651. doi: 10.1016/0006-291x(85)91181-7. [DOI] [PubMed] [Google Scholar]
  38. Sobue K., Tanaka T., Kanda K., Ashino N., Kakiuchi S. Purification and characterization of caldesmon77: a calmodulin-binding protein that interacts with actin filaments from bovine adrenal medulla. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5025–5029. doi: 10.1073/pnas.82.15.5025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Szpacenko A., Dabrowska R. Functional domain of caldesmon. FEBS Lett. 1986 Jul 7;202(2):182–186. doi: 10.1016/0014-5793(86)80683-4. [DOI] [PubMed] [Google Scholar]
  40. Takebayashi T., Morita Y., Oosawa F. Electronmicroscopic investigation of the flexibility of F-actin. Biochim Biophys Acta. 1977 Jun 24;492(2):357–363. doi: 10.1016/0005-2795(77)90086-1. [DOI] [PubMed] [Google Scholar]
  41. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wegner A. Kinetic analysis of actin assembly suggests that tropomyosin inhibits spontaneous fragmentation of actin filaments. J Mol Biol. 1982 Oct 25;161(2):217–227. doi: 10.1016/0022-2836(82)90149-8. [DOI] [PubMed] [Google Scholar]
  43. Yamashiro-Matsumura S., Matsumura F. Intracellular localization of the 55-kD actin-bundling protein in cultured cells: spatial relationships with actin, alpha-actinin, tropomyosin, and fimbrin. J Cell Biol. 1986 Aug;103(2):631–640. doi: 10.1083/jcb.103.2.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yamashiro-Matsumura S., Matsumura F. Purification and characterization of an F-actin-bundling 55-kilodalton protein from HeLa cells. J Biol Chem. 1985 Apr 25;260(8):5087–5097. [PubMed] [Google Scholar]

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