Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1984 Aug 1;99(2):550–557. doi: 10.1083/jcb.99.2.550

Identification of calmodulin-binding proteins in chicken embryo fibroblasts

PMCID: PMC2113283  PMID: 6086671

Abstract

We recently reported the detection of multiple classes of calmodulin- binding proteins in subcellular fractions of chicken embryo fibroblasts by using a gel binding procedure (Van Eldik, L.J., and W.H. Burgess, 1983, J. Biol. Chem., 258:4539-4547). In this report we identify many of these calmodulin-binding proteins and provide further evidence for the existence of multiple classes of calmodulin-binding proteins based on the interaction of these proteins with calmodulin and other calcium- modulated proteins. The fact that, in some cases, the same calmodulin- binding protein can bind troponin C and S100 alpha suggests that similar functional domains may be present in these distinct calcium- modulated proteins. We also have used protocols based on purification steps for calmodulin-binding proteins and calmodulin-regulated activities from other systems, in conjunction with enzymatic assays and various immunological methods, to identify many of the calmodulin- binding proteins in chicken embryo fibroblasts. The identities of these proteins suggest in vivo roles for calmodulin in the regulation of cell shape and motility, cyclic nucleotide metabolism, and possibly nucleic acid and protein turnover in fibroblasts.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adelstein R. S., Klee C. B. Purification of smooth muscle myosin light-chain kinase. Methods Enzymol. 1982;85(Pt B):298–308. doi: 10.1016/0076-6879(82)85029-5. [DOI] [PubMed] [Google Scholar]
  2. Amphlett G. W., Vanaman T. C., Perry S. V. Effect of the troponin C-like protein from bovine brain (brain modulator protein) on the Mg2+-stimulated ATPase of skeletal muscle actinomyosin. FEBS Lett. 1976 Dec 15;72(1):163–168. doi: 10.1016/0014-5793(76)80836-8. [DOI] [PubMed] [Google Scholar]
  3. Andreasen T. J., Luetje C. W., Heideman W., Storm D. R. Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes. Biochemistry. 1983 Sep 27;22(20):4615–4618. doi: 10.1021/bi00289a001. [DOI] [PubMed] [Google Scholar]
  4. Blumenthal D. K., Stull J. T. Activation of skeletal muscle myosin light chain kinase by calcium(2+) and calmodulin. Biochemistry. 1980 Nov 25;19(24):5608–5614. doi: 10.1021/bi00565a023. [DOI] [PubMed] [Google Scholar]
  5. Bowen B., Steinberg J., Laemmli U. K., Weintraub H. The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res. 1980 Jan 11;8(1):1–20. doi: 10.1093/nar/8.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brostrom C. O., Bocckino S. B., Brostrom M. A. Identification of a Ca2+ requirement for protein synthesis in eukaryotic cells. J Biol Chem. 1983 Dec 10;258(23):14390–14399. [PubMed] [Google Scholar]
  7. Burgess W. H., Jemiolo D. K., Kretsinger R. H. Interaction of calcium and calmodulin in the presence of sodium dodecyl sulfate. Biochim Biophys Acta. 1980 Jun 26;623(2):257–270. doi: 10.1016/0005-2795(80)90254-8. [DOI] [PubMed] [Google Scholar]
  8. Burridge K. Changes in cellular glycoproteins after transformation: identification of specific glycoproteins and antigens in sodium dodecyl sulfate gels. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4457–4461. doi: 10.1073/pnas.73.12.4457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burridge K., Kelly T., Mangeat P. Nonerythrocyte spectrins: actin-membrane attachment proteins occurring in many cell types. J Cell Biol. 1982 Nov;95(2 Pt 1):478–486. doi: 10.1083/jcb.95.2.478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Carlin R. K., Bartelt D. C., Siekevitz P. Identification of fodrin as a major calmodulin-binding protein in postsynaptic density preparations. J Cell Biol. 1983 Feb;96(2):443–448. doi: 10.1083/jcb.96.2.443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Carlin R. K., Grab D. J., Siekevitz P. Function of a calmodulin in postsynaptic densities. III. Calmodulin-binding proteins of the postsynaptic density. J Cell Biol. 1981 Jun;89(3):449–455. doi: 10.1083/jcb.89.3.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Collatz E., Ulbrich N., Tsurugi K., Lightfoot H. N., MacKinlay W., Lin A., Wool I. G. Isolation of eukaryotic ribosomal proteins. Purification and characterization of the 40 S ribosomal subunit proteins Sa, Sc, S3a, S3b, S5', S9, S10, S11, S12, S14, S15, S15', S16, S17, S18, S19, S20, S21, S26, S27', and S29. J Biol Chem. 1977 Dec 25;252(24):9071–9080. [PubMed] [Google Scholar]
  13. Dabrowska R., Sherry J. M., Aromatorio D. K., Hartshorne D. J. Modulator protein as a component of the myosin light chain kinase from chicken gizzard. Biochemistry. 1978 Jan 24;17(2):253–258. doi: 10.1021/bi00595a010. [DOI] [PubMed] [Google Scholar]
  14. Frederiksen D. W. Physical properties of myosin from aortic smooth muscle. Biochemistry. 1979 May 1;18(9):1651–1656. doi: 10.1021/bi00576a003. [DOI] [PubMed] [Google Scholar]
  15. Glenney J. R., Jr, Glenney P., Weber K. Erythroid spectrin, brain fodrin, and intestinal brush border proteins (TW-260/240) are related molecules containing a common calmodulin-binding subunit bound to a variant cell type-specific subunit. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4002–4005. doi: 10.1073/pnas.79.13.4002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Irie S., Sezaki M. A quantitative determination of the relative amount of histones in polyacrylamide gel by silver stain. Anal Biochem. 1983 Oct 15;134(2):471–478. doi: 10.1016/0003-2697(83)90325-1. [DOI] [PubMed] [Google Scholar]
  17. Klee C. B., Krinks M. H. Purification of cyclic 3',5'-nucleotide phosphodiesterase inhibitory protein by affinity chromatography on activator protein coupled to Sepharose. Biochemistry. 1978 Jan 10;17(1):120–126. doi: 10.1021/bi00594a017. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. LaPorte D. C., Gidwitz S., Weber M. J., Storm D. R. Relationship between changes in the calcium dependent regulatory protein and adenylate cyclase during viral transformation. Biochem Biophys Res Commun. 1979 Feb 28;86(4):1169–1177. doi: 10.1016/0006-291x(79)90240-7. [DOI] [PubMed] [Google Scholar]
  20. LaPorte D. C., Toscano W. A., Jr, Storm D. R. Cross-linking of iodine-125-labeled, calcium-dependent regulatory protein to the Ca2+-sensitive phosphodiesterase purified from bovine heart. Biochemistry. 1979 Jun 26;18(13):2820–2825. doi: 10.1021/bi00580a021. [DOI] [PubMed] [Google Scholar]
  21. Lacks S. A., Springhorn S. S. Renaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. J Biol Chem. 1980 Aug 10;255(15):7467–7473. [PubMed] [Google Scholar]
  22. 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]
  23. Lahav M., Schoenfeld N., Epstein O., Atsmon A. A method for obtaining high recovery of purified subcellular fractions of rat liver homogenate. Anal Biochem. 1982 Mar 15;121(1):114–122. doi: 10.1016/0003-2697(82)90563-2. [DOI] [PubMed] [Google Scholar]
  24. Lin A., Wittmann-Liebold B., McNally J., Wool I. G. The primary structure of the acidic phosphoprotein P2 from rat liver 60 S ribosomal subunits. Comparison with ribosomal 'A' proteins from other species. J Biol Chem. 1982 Aug 10;257(15):9189–9197. [PubMed] [Google Scholar]
  25. Marshak D. R., Watterson D. M., Van Eldik L. J. Calcium-dependent interaction of S100b, troponin C, and calmodulin with an immobilized phenothiazine. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6793–6797. doi: 10.1073/pnas.78.11.6793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Martelly I., Molla A., Thomasset M., Le Moigne A. Planarian regeneration: in vivo and in vitro effects of calcium and calmodulin on DNA synthesis. Cell Differ. 1983 Sep;13(1):25–34. doi: 10.1016/0045-6039(83)90073-8. [DOI] [PubMed] [Google Scholar]
  27. Matsudaira P. T., Burgess D. R. SDS microslab linear gradient polyacrylamide gel electrophoresis. Anal Biochem. 1978 Jul 1;87(2):386–396. doi: 10.1016/0003-2697(78)90688-7. [DOI] [PubMed] [Google Scholar]
  28. Palfrey H. C., Schiebler W., Greengard P. A major calmodulin-binding protein common to various vertebrate tissues. Proc Natl Acad Sci U S A. 1982 Jun;79(12):3780–3784. doi: 10.1073/pnas.79.12.3780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pallen C. J., Wang J. H. Calmodulin-stimulated dephosphorylation of p-nitrophenyl phosphate and free phosphotyrosine by calcineurin. J Biol Chem. 1983 Jul 25;258(14):8550–8553. [PubMed] [Google Scholar]
  30. Palmiter R. D. Magnesium precipitation of ribonucleoprotein complexes. Expedient techniques for the isolation of undergraded polysomes and messenger ribonucleic acid. Biochemistry. 1974 Aug 13;13(17):3606–3615. doi: 10.1021/bi00714a032. [DOI] [PubMed] [Google Scholar]
  31. Perry S. V., Cole H. A. Phosphorylation of troponin and the effects of interactions between the components of the complex. Biochem J. 1974 Sep;141(3):733–743. doi: 10.1042/bj1410733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Picton C., Klee C. B., Cohen P. Phosphorylase kinase from rabbit skeletal muscle: identification of the calmodulin-binding subunits. Eur J Biochem. 1980 Oct;111(2):553–561. doi: 10.1111/j.1432-1033.1980.tb04971.x. [DOI] [PubMed] [Google Scholar]
  33. Van Eldik L. J., Burgess W. H. Analytical subcellular distribution of calmodulin and calmodulin-binding proteins in normal and virus-transformed fibroblasts. J Biol Chem. 1983 Apr 10;258(7):4539–4547. [PubMed] [Google Scholar]
  34. Van Eldik L. J., Watterson D. M. Characterization of a calcium-modulated protein from transformed chicken fibroblasts. J Biol Chem. 1979 Oct 25;254(20):10250–10255. [PubMed] [Google Scholar]
  35. Van Eldik L. J., Watterson D. M. Reproducible production of antiserum against vertebrate calmodulin and determination of the immunoreactive site. J Biol Chem. 1981 May 10;256(9):4205–4210. [PubMed] [Google Scholar]
  36. Van Eldik L. J., Zendegui J. G., Marshak D. R., Watterson D. M. Calcium-binding proteins and the molecular basis of calcium action. Int Rev Cytol. 1982;77:1–61. doi: 10.1016/s0074-7696(08)62463-8. [DOI] [PubMed] [Google Scholar]
  37. Veigl M. L., Sedwick W. D., Vanaman T. C. Calmodulin and Ca2+ in normal and transformed cells. Fed Proc. 1982 May;41(7):2283–2288. [PubMed] [Google Scholar]
  38. Villar-Palasi C., Oshiro D. L., Kretsinger R. H. Interaction of calmodulin and glycogen phosphorylase. Biochim Biophys Acta. 1983 May 4;757(1):40–46. doi: 10.1016/0304-4165(83)90150-2. [DOI] [PubMed] [Google Scholar]
  39. Watterson D. M., Harrelson W. G., Jr, Keller P. M., Sharief F., Vanaman T. C. Structural similarities between the Ca2+-dependent regulatory proteins of 3':5'-cyclic nucleotide phosphodiesterase and actomyosin ATPase. J Biol Chem. 1976 Aug 10;251(15):4501–4513. [PubMed] [Google Scholar]
  40. Watterson D. M., Iverson D. B., Van Eldik L. J. Rapid separation and quantitation of 3',5'-cyclic nucleotides and 5'-nucleotides in phosphodiesterase reaction mixtures using high-performance liquid chromatography. J Biochem Biophys Methods. 1980 Mar;2(3):139–146. doi: 10.1016/0165-022x(80)90021-4. [DOI] [PubMed] [Google Scholar]
  41. Watterson D. M., Sharief F., Vanaman T. C. The complete amino acid sequence of the Ca2+-dependent modulator protein (calmodulin) of bovine brain. J Biol Chem. 1980 Feb 10;255(3):962–975. [PubMed] [Google Scholar]
  42. Watterson D. M., Vanaman T. C. Affinity chromatography purification of a cyclic nucleotide phosphodiesterase using immobilized modulator protein, a troponin C-like protein from brain. Biochem Biophys Res Commun. 1976 Nov 8;73(1):40–46. doi: 10.1016/0006-291x(76)90494-0. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES