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Calcium Signaling Protocols pp 325–341Cite as

Assay and Purification of Calmodulin-Dependent Protein Kinase

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 114))

Abstract

Posttranslation covalent modification of proteins by phosphorylation represents an important mechanism in the control of many cellular functions (1,2). Protein kinases catalyze the transfer of the γ-phosphoryl group of ATP to an acceptor protein substrate. The activity of the enzyme is determined by the transfer of 32P from [γ-32P] ATP to protein substrate

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References

  1. Cohen, P. (1992) Signal integration at the level of protein kinases, protein phosphatases and their substrates. Trends Biochem. Sci. 17, 408–413.

    Article  PubMed  CAS  Google Scholar 

  2. Hunter, T. (1995) Protein kinases and phosphatases: The yin and yang of protein phosphorylation and signalling. Cell 80, 225–236.

    Article  PubMed  CAS  Google Scholar 

  3. Berridge, M. J. (1975) The interaction of cyclic nucleotides and calcium in the control of cellular activity. Adv. Cyclic Nucleotide Res. 6, 1–98.

    PubMed  CAS  Google Scholar 

  4. Clapham, D. E. (1995) Calcium signalling. Cell 80, 259–268.

    Article  PubMed  CAS  Google Scholar 

  5. Sharma, R. K. and Kalra, J. (1994) Molecular interaction between cAMP and calcium in calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme system. Clin. Invest. Med. 17, 374–382.

    PubMed  CAS  Google Scholar 

  6. Klee, C. B. (1988) Interaction of calmodulin with Ca2+ and target proteins, in Molecular Aspects of Cell Regulation, vol. 5, (Cohen, P. and Klee, C., eds.), Elsevier Science, New York, pp. 35–56.

    Google Scholar 

  7. Nairn, A. C. and Picciotto, M. R. (1994) Calcium/calmodulin dependent protein kinase. Semin. Cancer Biol. 5, 295–303.

    PubMed  CAS  Google Scholar 

  8. Schulman, H. (1988) The multifunctional Ca2+/calmodulin-dependent protein kinase. Advances in Second Messenger and Phosphoprotein Research 22, 39–112.

    PubMed  CAS  Google Scholar 

  9. Kemp, B. E. and Pearson, R. B. (1990) Protein kinase recognition sequence motifs. Trends Biochem. Sci. 15, 342–346.

    Article  PubMed  CAS  Google Scholar 

  10. Soderling, T. R. (1995) Calcium-dependent protein kinases in learning and memory. Advances in Second Messenger and Phosphoprotein Research 30, 175–189.

    PubMed  CAS  Google Scholar 

  11. Hashimoto, Y., Schworer, C. M., Colbran, R. J., and Soderling, T. R. (1987) Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II: Effects on total and Ca2+-independent activities and kinetic parameters. J. Biol. Chem. 262, 8051–8055.

    PubMed  CAS  Google Scholar 

  12. Zhang, G. Y., Wang, J. H., and Sharma, R. K. (1993) Bovine brain calmodulindependent protein kinase II: Molecular mechanisms of autophosphorylation. Biochem. Biophys. Res. Commun. 191, 669–674.

    Article  PubMed  CAS  Google Scholar 

  13. Zhang, G. Y., Wang, J. H., and Sharma, R. K. (1993) Purification and characterization of bovine brain calmodulin-dependent protein kinase II. The significance of autophosphorylation in the regulation of 63 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme. Mol. Cell. Biochem. 122, 159–169.

    Article  PubMed  CAS  Google Scholar 

  14. Kakkar, R., Raju, R. V. S., and Sharma, R. K. (1996) Calmodulindependent protein kinase II from bovine cardiac muscle: purification and differential activation by calcium. Cell Calcium 20, 347–353.

    Article  PubMed  CAS  Google Scholar 

  15. Bennett, M. K., Erondu, N. E., and Kennedy, M. B. (1983) Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain. J. Biol. Chem. 258, 12,735–12,744.

    PubMed  CAS  Google Scholar 

  16. Iwasa, T., Inoue, N., Fukunaga, K., Isobe, T., Okuyama, T., and Miyamoto, E. (1986) Purification and characterization of a multifunctional calmodulin-dependent protein kinase from canine myocardial cytosol. Arch. Biochem. Biophys. 248, 21–29.

    Article  PubMed  CAS  Google Scholar 

  17. Yang, S. D., Chang, S. Y., and Soderling, T. R. (1987) Characterization of an autophosphorylation-dependent multifunctional protein kinase from liver. J. Biol. Chem. 262, 9421–9427.

    PubMed  CAS  Google Scholar 

  18. Gorelick, F.S., Cohn, J. A., Freedman, S. D., Delahunt, N. G., Gershoni, J. M., and Jamisen, J. D. (1983) Calmodulin-stimulated protein kinase activity from rat pancreas. J. Cell. Biol. 89, 440–448.

    Google Scholar 

  19. Bartelt, D. C., Fidel, S., Wolff, D. J., and Hammell, R. L. (1988) Calmodulin-dependent multifunctional protein kinase in Aspergillus nidulans. Proc. Natl. Acad. Sci. USA 85, 3279–3283.

    Article  PubMed  CAS  Google Scholar 

  20. Sharma, R. K. (1990) Purification and characterization of novel calmodulin-binding protein from cardiac muscle. J. Biol. Chem. 265, 1152–1157.

    PubMed  CAS  Google Scholar 

  21. Reimann, E. M., Walsh, D. A., and Krebs, E. G. (1971) Purification and properties of rabbit skeletal muscle adenosine 3′, 5′-monophosphate-dependent protein kinases. J. Biol. Chem. 246, 1986–1995.

    PubMed  CAS  Google Scholar 

  22. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

    Article  PubMed  CAS  Google Scholar 

  23. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.

    Article  PubMed  CAS  Google Scholar 

  24. Schworer, C. M., Colbran, R. J., and Soderling, T. R. (1986) Reversible generation of a Ca2+ independent form of Ca2+/calmodulin-dependent protein kinase II by an autophosphorylation mechanism. J. Biol. Chem. 261, 8581–8584.

    PubMed  CAS  Google Scholar 

  25. Lai, Y., Nairn, A. C., and Greengard, P. (1986) Autophosphorylation reversibly regulates the Ca2+/calmodulin-dependence of Ca2+/calmodulin-dependent protein kinase II. Proc. Natl. Acad. Sci. USA 83, 4253–4257.

    Article  PubMed  CAS  Google Scholar 

  26. Miller, S. G. and Kennedy, M. B. (1986) Regulation of brain type II Ca2+/calmodulin-dependent protein kinase by autophosphorylation: A calcium triggered molecular switch. Cell 44, 861–870.

    Article  PubMed  CAS  Google Scholar 

  27. Saitoh, Y., Yamamato, H., Fukunaga, K., Matsukado, Y., and Miyamoto, E. (1987) Inactivation and reactivation of the multifunctional calmodulin-dependent protein kinase from brain by autophosphorylation and dephosphorylation: Involvement of protein phosphatases from brain. J. Neurochem. 99, 1286–1292.

    Article  Google Scholar 

  28. Bronstein, J. M., Farber, D. B., and Wasterlain, C. G. (1993) Regulation of type II calmodulin kinase: Functional implications. Brain Res. Rev. 18, 135–147.

    Article  PubMed  CAS  Google Scholar 

  29. Sharma, R. K. and Wang, J. H. (1985) Differential regulation of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme by cAMP-dependent protein kinase and calmodulin dependent phosphatase. Proc. Natl. Acad. Sci. USA 82, 2603–2607.

    Article  PubMed  CAS  Google Scholar 

  30. Sharma, R. K. and Kalra, J. (1994) Characterization of calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes. Biochem. J. 299, 97–100.

    PubMed  CAS  Google Scholar 

  31. Huang, C. Y., Chau, V., Chock, P. B., Wang, J. H., and Sharma, R. K. (1981) Mechanism of activation of cyclic nucleotide phosphodiesterase: Requirement of the binding of four Ca2+ to calmodulin for activation. Proc. Natl. Acad. Sci. USA 78, 871–874.

    Article  PubMed  CAS  Google Scholar 

  32. Crouch, T. H. and Klee, C. B. (1980) Positive cooperative binding of calcium to bovine brain calmodulin. Biochemistry 19, 3692–3698.

    Article  PubMed  CAS  Google Scholar 

  33. Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354.

    Article  PubMed  CAS  Google Scholar 

  34. Means, A. R., Cruzalegui, F., Le Magueresse, B., Needleman, D. S., Slaughter, G. R., and Ono, T. (1991) A novel Ca2+/calmodulin dependent protein kinase and a male germ cell specific calmodulin binding protein are derived from the same gene. Mol. Cell. Biol. 11, 3960–3971.

    PubMed  CAS  Google Scholar 

  35. Tobimatsu, T. and Fujisawa, H. (1989) Tissue-specific expression of four types of rat calmodulin-dependent protein kinase II mRNAs. J. Biol. Chem. 264, 17,907–17,912.

    PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Pathology, Saskatoon Cancer Centre, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

    Rakesh Kakkar & Rajendra K. Sharma

Authors
  1. Rakesh Kakkar
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  2. Rajendra K. Sharma
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Editor information

Editors and Affiliations

  1. University of Leicester, UK

    David G. Lambert

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© 1999 Humana Press Inc., Totowa, NJ

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Kakkar, R., Sharma, R.K. (1999). Assay and Purification of Calmodulin-Dependent Protein Kinase. In: Lambert, D.G. (eds) Calcium Signaling Protocols. Methods in Molecular Biology™, vol 114. Humana Press. https://doi.org/10.1385/1-59259-250-3:325

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  • DOI: https://doi.org/10.1385/1-59259-250-3:325

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-597-3

  • Online ISBN: 978-1-59259-250-0

  • eBook Packages: Springer Protocols

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