Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1995 Nov 15;312(Pt 1):197–204. doi: 10.1042/bj3120197

The activity of calmodulin is altered by phosphorylation: modulation of calmodulin function by the site of phosphate incorporation.

D B Sacks 1, B Mazus 1, J L Joyal 1
PMCID: PMC1136245  PMID: 7492313

Abstract

Calmodulin transduces Ca2+ signals by binding to and activating essential regulatory enzymes. The large number of intracellular targets for calmodulin raises the possibility that mechanisms in addition to Ca2+ may modulate calmodulin activity. Phosphocalmodulin is found in cells and tissues, and calmodulin phosphorylation is enhanced by several mitogens. Phosphorylation of calmodulin on serine/threonine residues by casein kinase II decreased its ability to activate Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II). The major effect was a 2.5-fold increase in the concentration at which half-maximal velocity (K0.5) was attained, with no apparent alteration in the Vmax, or the K0.5 for Ca2+. In contrast, calmodulin phosphorylated on tyrosine residues by the insulin receptor kinase produced an increase in the Vmax, with no alteration in the affinity for CaM-kinase II or the K0.5 for Ca2+. Direct determination by surface plasmon resonance of the dissociation constants with a synthetic peptide corresponding to the calmodulin-binding domain of CaM-kinase II revealed that phosphorylation on serine/threonine residues of calmodulin significantly decreased its affinity for the peptide, while tyrosine phosphorylation had no effect on binding. In contrast to CaM-kinase II, neither serine/threonine nor tyrosine phosphorylation of calmodulin altered its ability to activate calcineurin. These data indicate that phosphorylation of calmodulin differentially modifies its interaction with individual target enzymes. Moreover, the amino acid residues phosphorylated provide an additional level of control. These results demonstrate that phosphorylation is an in vitro regulatory mechanism in the targeting of calmodulin responses and, coupled with the stoichiometric phosphorylation of calmodulin in rat hepatocytes, suggest that it may be relevant in intact cells.

Full text

PDF
197

Images in this article

199Figure 1
on p.199

Selected References

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

  1. Ackerman P., Glover C. V., Osheroff N. Stimulation of casein kinase II by epidermal growth factor: relationship between the physiological activity of the kinase and the phosphorylation state of its beta subunit. Proc Natl Acad Sci U S A. 1990 Jan;87(2):821–825. doi: 10.1073/pnas.87.2.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Babu Y. S., Sack J. S., Greenhough T. J., Bugg C. E., Means A. R., Cook W. J. Three-dimensional structure of calmodulin. Nature. 1985 May 2;315(6014):37–40. doi: 10.1038/315037a0. [DOI] [PubMed] [Google Scholar]
  3. Benguría A., Hernández-Perera O., Martínez-Pastor M. T., Sacks D. B., Villalobo A. Phosphorylation of calmodulin by the epidermal-growth-factor-receptor tyrosine kinase. Eur J Biochem. 1994 Sep 15;224(3):909–916. doi: 10.1111/j.1432-1033.1994.00909.x. [DOI] [PubMed] [Google Scholar]
  4. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Brickey D. A., Colbran R. J., Fong Y. L., Soderling T. R. Expression and characterization of the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II using the baculovirus expression system. Biochem Biophys Res Commun. 1990 Dec 14;173(2):578–584. doi: 10.1016/s0006-291x(05)80074-9. [DOI] [PubMed] [Google Scholar]
  7. Chabbert M., Lukas T. J., Watterson D. M., Axelsen P. H., Prendergast F. G. Fluorescence analysis of calmodulin mutants containing tryptophan: conformational changes induced by calmodulin-binding peptides from myosin light chain kinase and protein kinase II. Biochemistry. 1991 Jul 30;30(30):7615–7630. doi: 10.1021/bi00244a034. [DOI] [PubMed] [Google Scholar]
  8. Cheung W. Y. Calmodulin plays a pivotal role in cellular regulation. Science. 1980 Jan 4;207(4426):19–27. doi: 10.1126/science.6243188. [DOI] [PubMed] [Google Scholar]
  9. Colca J. R., DeWald D. B., Pearson J. D., Palazuk B. J., Laurino J. P., McDonald J. M. Insulin stimulates the phosphorylation of calmodulin in intact adipocytes. J Biol Chem. 1987 Aug 25;262(24):11399–11402. [PubMed] [Google Scholar]
  10. Craig T. A., Watterson D. M., Prendergast F. G., Haiech J., Roberts D. M. Site-specific mutagenesis of the alpha-helices of calmodulin. Effects of altering a charge cluster in the helix that links the two halves of calmodulin. J Biol Chem. 1987 Mar 5;262(7):3278–3284. [PubMed] [Google Scholar]
  11. Fukami Y., Nakamura T., Nakayama A., Kanehisa T. Phosphorylation of tyrosine residues of calmodulin in Rous sarcoma virus-transformed cells. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4190–4193. doi: 10.1073/pnas.83.12.4190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Furukawa K., Tawada Y., Shigekawa M. Protein kinase C activation stimulates plasma membrane Ca2+ pump in cultured vascular smooth muscle cells. J Biol Chem. 1989 Mar 25;264(9):4844–4849. [PubMed] [Google Scholar]
  13. Gopalakrishna R., Anderson W. B. Ca2+-induced hydrophobic site on calmodulin: application for purification of calmodulin by phenyl-Sepharose affinity chromatography. Biochem Biophys Res Commun. 1982 Jan 29;104(2):830–836. doi: 10.1016/0006-291x(82)90712-4. [DOI] [PubMed] [Google Scholar]
  14. Graves C. B., Gale R. D., Laurino J. P., McDonald J. M. The insulin receptor and calmodulin. Calmodulin enhances insulin-mediated receptor kinase activity and insulin stimulates phosphorylation of calmodulin. J Biol Chem. 1986 Aug 5;261(22):10429–10438. [PubMed] [Google Scholar]
  15. Hagiwara T., Ohsako S., Yamauchi T. Studies on the regulatory domain of Ca2+/calmodulin-dependent protein kinase II by expression of mutated cDNAs in Escherichia coli. J Biol Chem. 1991 Sep 5;266(25):16401–16408. [PubMed] [Google Scholar]
  16. Hanley R. M., Means A. R., Kemp B. E., Shenolikar S. Mapping of calmodulin-binding domain of Ca2+/calmodulin-dependent protein kinase II from rat brain. Biochem Biophys Res Commun. 1988 Apr 15;152(1):122–128. doi: 10.1016/s0006-291x(88)80688-0. [DOI] [PubMed] [Google Scholar]
  17. Hashimoto Y., Soderling T. R. Regulation of calcineurin by phosphorylation. Identification of the regulatory site phosphorylated by Ca2+/calmodulin-dependent protein kinase II and protein kinase C. J Biol Chem. 1989 Oct 5;264(28):16524–16529. [PubMed] [Google Scholar]
  18. Haystead T. A., Campbell D. G., Hardie D. G. Analysis of sites phosphorylated on acetyl-CoA carboxylase in response to insulin in isolated adipocytes. Comparison with sites phosphorylated by casein kinase-2 and the calmodulin-dependent multiprotein kinase. Eur J Biochem. 1988 Aug 1;175(2):347–354. doi: 10.1111/j.1432-1033.1988.tb14203.x. [DOI] [PubMed] [Google Scholar]
  19. Heppel L. A., Newton D. L., Klee C. B., Draetta G. F. The phosphorylation of calmodulin and calmodulin fragments by kinase fractions from bovine brain. Biochim Biophys Acta. 1988 Oct 28;972(1):69–78. doi: 10.1016/0167-4889(88)90104-8. [DOI] [PubMed] [Google Scholar]
  20. Hurwitz M. Y., Putkey J. A., Klee C. B., Means A. R. Domain II of calmodulin is involved in activation of calcineurin. FEBS Lett. 1988 Sep 26;238(1):82–86. doi: 10.1016/0014-5793(88)80230-8. [DOI] [PubMed] [Google Scholar]
  21. Ikura M., Clore G. M., Gronenborn A. M., Zhu G., Klee C. B., Bax A. Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science. 1992 May 1;256(5057):632–638. doi: 10.1126/science.1585175. [DOI] [PubMed] [Google Scholar]
  22. Joyal J. L., Sacks D. B. Insulin-dependent phosphorylation of calmodulin in rat hepatocytes. J Biol Chem. 1994 Nov 25;269(47):30039–30048. [PubMed] [Google Scholar]
  23. Kallen R. G., Smith J. E., Sheng Z., Tung L. Expression, purification and characterization of a 41 kDa insulin receptor tyrosine kinase domain. Biochem Biophys Res Commun. 1990 Apr 30;168(2):616–624. doi: 10.1016/0006-291x(90)92365-7. [DOI] [PubMed] [Google Scholar]
  24. Karlsson R., Michaelsson A., Mattsson L. Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system. J Immunol Methods. 1991 Dec 15;145(1-2):229–240. doi: 10.1016/0022-1759(91)90331-9. [DOI] [PubMed] [Google Scholar]
  25. Klarlund J. K., Czech M. P. Insulin-like growth factor I and insulin rapidly increase casein kinase II activity in BALB/c 3T3 fibroblasts. J Biol Chem. 1988 Nov 5;263(31):15872–15875. [PubMed] [Google Scholar]
  26. Klee C. B., Draetta G. F., Hubbard M. J. Calcineurin. Adv Enzymol Relat Areas Mol Biol. 1988;61:149–200. doi: 10.1002/9780470123072.ch4. [DOI] [PubMed] [Google Scholar]
  27. Kretsinger R. H. Calmodulin and myosin light chain kinase: how helices are bent. Science. 1992 Oct 2;258(5079):50–51. doi: 10.1126/science.1439768. [DOI] [PubMed] [Google Scholar]
  28. Malmqvist M. Biospecific interaction analysis using biosensor technology. Nature. 1993 Jan 14;361(6408):186–187. doi: 10.1038/361186a0. [DOI] [PubMed] [Google Scholar]
  29. Meador W. E., Means A. R., Quiocho F. A. Modulation of calmodulin plasticity in molecular recognition on the basis of x-ray structures. Science. 1993 Dec 10;262(5140):1718–1721. doi: 10.1126/science.8259515. [DOI] [PubMed] [Google Scholar]
  30. Meggio F., Brunati A. M., Pinna L. A. Polycation-dependent, Ca2+-antagonized phosphorylation of calmodulin by casein kinase-2 and a spleen tyrosine protein kinase. FEBS Lett. 1987 May 11;215(2):241–246. doi: 10.1016/0014-5793(87)80154-0. [DOI] [PubMed] [Google Scholar]
  31. Nakajo S., Hayashi K., Daimatsu T., Tanaka M., Nakaya K., Nakamura Y. Phosphorylation of rat brain calmodulin in vivo and in vitro. Biochem Int. 1986 Oct;13(4):687–693. [PubMed] [Google Scholar]
  32. Payne M. E., Fong Y. L., Ono T., Colbran R. J., Kemp B. E., Soderling T. R., Means A. R. Calcium/calmodulin-dependent protein kinase II. Characterization of distinct calmodulin binding and inhibitory domains. J Biol Chem. 1988 May 25;263(15):7190–7195. [PubMed] [Google Scholar]
  33. Perrino B. A., Fong Y. L., Brickey D. A., Saitoh Y., Ushio Y., Fukunaga K., Miyamoto E., Soderling T. R. Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform. J Biol Chem. 1992 Aug 5;267(22):15965–15969. [PubMed] [Google Scholar]
  34. Persechini A., Blumenthal D. K., Jarrett H. W., Klee C. B., Hardy D. O., Kretsinger R. H. The effects of deletions in the central helix of calmodulin on enzyme activation and peptide binding. J Biol Chem. 1989 May 15;264(14):8052–8058. [PubMed] [Google Scholar]
  35. Persechini A., Jarrett H. W., Kosk-Kosicka D., Krinks M. H., Lee H. G. Activation of enzymes by calmodulins containing intramolecular cross-links. Biochim Biophys Acta. 1993 Jun 4;1163(3):309–314. doi: 10.1016/0167-4838(93)90167-p. [DOI] [PubMed] [Google Scholar]
  36. Plancke Y. D., Lazarides E. Evidence for a phosphorylated form of calmodulin in chicken brain and muscle. Mol Cell Biol. 1983 Aug;3(8):1412–1420. doi: 10.1128/mcb.3.8.1412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Putkey J. A., Ono T., VanBerkum M. F., Means A. R. Functional significance of the central helix in calmodulin. J Biol Chem. 1988 Aug 15;263(23):11242–11249. [PubMed] [Google Scholar]
  38. Quadroni M., James P., Carafoli E. Isolation of phosphorylated calmodulin from rat liver and identification of the in vivo phosphorylation sites. J Biol Chem. 1994 Jun 10;269(23):16116–16122. [PubMed] [Google Scholar]
  39. Roskoski R., Jr Assays of protein kinase. Methods Enzymol. 1983;99:3–6. doi: 10.1016/0076-6879(83)99034-1. [DOI] [PubMed] [Google Scholar]
  40. Sacks D. B., Davis H. W., Crimmins D. L., McDonald J. M. Insulin-stimulated phosphorylation of calmodulin. Biochem J. 1992 Aug 15;286(Pt 1):211–216. doi: 10.1042/bj2860211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sacks D. B., Davis H. W., Williams J. P., Sheehan E. L., Garcia J. G., McDonald J. M. Phosphorylation by casein kinase II alters the biological activity of calmodulin. Biochem J. 1992 Apr 1;283(Pt 1):21–24. doi: 10.1042/bj2830021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sacks D. B., McDonald J. M. Calmodulin as substrate for insulin-receptor kinase. Phosphorylation by receptors from rat skeletal muscle. Diabetes. 1989 Jan;38(1):84–90. doi: 10.2337/diab.38.1.84. [DOI] [PubMed] [Google Scholar]
  43. Sacks D. B., McDonald J. M. Effects of cationic polypeptides on the activity, substrate interaction, and autophosphorylation of casein kinase II: a study with calmodulin. Arch Biochem Biophys. 1992 Dec;299(2):275–280. doi: 10.1016/0003-9861(92)90275-2. [DOI] [PubMed] [Google Scholar]
  44. Sacks D. B., McDonald J. M. Insulin-stimulated phosphorylation of calmodulin by rat liver insulin receptor preparations. J Biol Chem. 1988 Feb 15;263(5):2377–2383. [PubMed] [Google Scholar]
  45. Saville M. K., Houslay M. D. Phosphorylation of calmodulin on Tyr99 selectively attenuates the action of calmodulin antagonists on type-I cyclic nucleotide phosphodiesterase activity. Biochem J. 1994 May 1;299(Pt 3):863–868. doi: 10.1042/bj2990863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Schworer C. M., Colbran R. J., Soderling T. R. Reversible generation of a Ca2+-independent form of Ca2+(calmodulin)-dependent protein kinase II by an autophosphorylation mechanism. J Biol Chem. 1986 Jul 5;261(19):8581–8584. [PubMed] [Google Scholar]
  47. Sommercorn J., Mulligan J. A., Lozeman F. J., Krebs E. G. Activation of casein kinase II in response to insulin and to epidermal growth factor. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8834–8838. doi: 10.1073/pnas.84.24.8834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tanasijevic M. J., Myers M. G., Jr, Thoma R. S., Crimmins D. L., White M. F., Sacks D. B. Phosphorylation of the insulin receptor substrate IRS-1 by casein kinase II. J Biol Chem. 1993 Aug 25;268(24):18157–18166. [PubMed] [Google Scholar]
  49. Tuazon P. T., Traugh J. A. Casein kinase I and II--multipotential serine protein kinases: structure, function, and regulation. Adv Second Messenger Phosphoprotein Res. 1991;23:123–164. [PubMed] [Google Scholar]
  50. Wang Q. M., Fiol C. J., DePaoli-Roach A. A., Roach P. J. Glycogen synthase kinase-3 beta is a dual specificity kinase differentially regulated by tyrosine and serine/threonine phosphorylation. J Biol Chem. 1994 May 20;269(20):14566–14574. [PubMed] [Google Scholar]
  51. Weber P. C., Lukas T. J., Craig T. A., Wilson E., King M. M., Kwiatkowski A. P., Watterson D. M. Computational and site-specific mutagenesis analyses of the asymmetric charge distribution on calmodulin. Proteins. 1989;6(1):70–85. doi: 10.1002/prot.340060107. [DOI] [PubMed] [Google Scholar]
  52. Williams J. P., Jo H., Sacks D. B., Crimmins D. L., Thoma R. S., Hunnicutt R. E., Radding W., Sharma R. K., McDonald J. M. Tyrosine-phosphorylated calmodulin has reduced biological activity. Arch Biochem Biophys. 1994 Nov 15;315(1):119–126. doi: 10.1006/abbi.1994.1479. [DOI] [PubMed] [Google Scholar]
  53. Wolff D. J., Poirier P. G., Brostrom C. O., Brostrom M. A. Divalent cation binding properties of bovine brain Ca2+-dependent regulator protein. J Biol Chem. 1977 Jun 25;252(12):4108–4117. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES