Advertisement

Journal of Molecular Neuroscience

, Volume 5, Issue 3, pp 193–206 | Cite as

Purification and characterization of the Ca2+/calmodulin-dependent protein kinase II from chicken forebrain

  • Nian Liu
  • Nigel G. F. Cooper
Article

Abstract

CaM kinase II is known to be enriched in mammalian and avian brains. To determine the holoenzymic composition and functional characteristics of this kinase, a new approach for isolation was applied to isolate it from the chicken forebrain. Forebrains of hatched 45-d chicken were dissected, homogenized, and centrifuged. The supernatant was loaded onto a CaM-agarose affinity column and the calmodulin-binding proteins were eluted with EGTA. Selected eluates were loaded onto the antibody-agarose affinity column, which was prepared with monoclonal antibody (MAb) (6G9) to the CaM kinase II α subunit. Samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and either silver-stained or blotted onto a nitrocellulose membrane. The protein composition and the immunoreactivity of the antibody-agarose affinity eluate fractions were analyzed with a densitometric scanner. Silver staining of gels showed that the β subunit doublet, the β′ subunit, and a putative substrate were coeluted with the α subunit from the antibody affinity column although only the α subunit bound the 6G9 antibody. Scintillation counting showed that the autophosphorylation of the kinase was significantly reduced in the eluate from the antibody affinity column. Whereas silver staining indicated an increase in the relative amount of α subunit had occurred during purification, phosphorylation assays indicated an increase in the relative amount of the α subunit after the last purification step. A possible reason for this is discussed. The presence of β/β′ subunits in the antibody-agarose affinity eluate indicated the existence of an αβ/β′ heteropolymer. The phosphorylation assay was not a good indication of the amount of purification because of the loss of enzyme activity following purification. In contrast, the immunoassay indicated a 97-fold purification from the cytosolic fraction was achieved using the method. In conclusion, the data indicate the existence of the CaM kinase II αβ/β′ heteropolymer in the chicken forebrain.

Index Entries

Multifunctional CaM kinase heteropolymer autophosphorylation antibody affinity chromatography 6G9 monoclonal antibody 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Baudier J. and Cole R. D. (1987) Phosphorylation of tau proteins to a state like that in Alzheimer’s brain is catalyzed by a calcium/calmodulin-dependent kinase and modulated by phospholipids.J. Biol. Chem. 262, 17,577–17,583. (1983) Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain.J. Biol. Chem. 258, 12,735−12,744.Google Scholar
  2. Bronstein J. M., Wasterlain C. G., and Farber D. B. (1988a) A retinal calmodulin-dependent kinase: calcium/calmodulin-stimulated and -inhibited states.J. Neurochem. 50, 1438–1446.PubMedCrossRefGoogle Scholar
  3. Bronstein J. M., Wasterlain C. G., Bok D., Lasher R., and Farber D. B. (1988b) Localization of retinal calmodulin kinase.Exp. Eye Res. 47, 391–402.PubMedCrossRefGoogle Scholar
  4. Bronstein J., Wasterlain C. G., Lasher R., and Farber D. B. (1989) Dark-induced changes in activity and compartmentalization of retinal calmodulin kinase in the rat.Brain Res. 495, 83–88.PubMedCrossRefGoogle Scholar
  5. DeLorenzo R. J., Freedman S. D., Yohe W. B., and Maurer S. C. (1979) Stimulation of Ca2+-dependent neurotransmitter release and presynaptic nerve terminal protein phosphorylation by calmodulin and a calmodulin-like protein isolated from synaptic vesicles.Proc. Natl. Acad. Sci. USA 76, 1838–1842.PubMedCrossRefGoogle Scholar
  6. Erondu N. E. and Kennedy M. B. (1985) Regional distribution of type II Ca2+/calmodulin-dependent protein kinase in rat brain.J. Neurosci. 5, 3270–3277.PubMedGoogle Scholar
  7. Goldenring J. R., Gonzalez B., McGuire J. S. Jr., and DeLorenzo R. J. (1983) Purification and characterization of a calmodulin-dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubule-associated proteins.J. Biol. Chem. 258, 12,632–12,640.Google Scholar
  8. Gupta R. P., Lapadula D. M., and Abou-Donia M. B. (1992) Ca2+/calmodulin-dependent protein kinase II from hen brain. Purification and characterization.Biochem. Pharmacol. 43, 1975–1988.PubMedCrossRefGoogle Scholar
  9. Hendry S. H. C. and Kennedy M. B. (1985) Immunoreactivity for a calmodulin-dependent protein kinase is selectively increased in macaque striate cortex after monocular deprivation.Proc. Natl. Acad. Sci. USA 83, 1536–1541.CrossRefGoogle Scholar
  10. Kanaseki T., Ikeuchi Y., Sugiura H., and Yamauchi T. (1991) Structural features of Ca2+/calmodulin-dependent protein kinase II revealed by electron microscopy.J. Cell Biol. 115, 1049–1060.PubMedCrossRefGoogle Scholar
  11. Kapiloff M. S., Mathis J. M., Nelson C. A., Lin C. R., and Rosenfeld M. G. (1991) Calcium/calmodulin-dependent protein kinase mediates a pathway for transcriptional regulation.Proc. Natl. Acad. Sci. USA 88, 3710–3714.PubMedCrossRefGoogle Scholar
  12. Kato M., Hagiwara M., and Hidaka H. (1992) Identification of a 80 kDa calmodulin-binding protein as a new Ca2+/calmodulin-dependent kinase by renaturation blotting assay (RBA).Biochem. J. 281, 339–342.PubMedGoogle Scholar
  13. Kelly P. T. and Vernon P. (1985) Changes in the subcellular distribution of calmodulin-kinase II during brain development.Dev. Brain Res. 18, 211–224.CrossRefGoogle Scholar
  14. Kennedy M. B., Bennett M. K., and Erondu N. E. (1983) Biochemical and immunochemical evidence that the “major postsynaptic density protein” is a subunit of a calmodulin-dependent protein kinase.Proc. Natl. Acad. Sci. USA 80, 7357–7361.PubMedCrossRefGoogle Scholar
  15. Kuret J. and Schulman H. (1984) Purification and characterization of a Ca2+/calmodulin-dependent protein kinase from rat brain.Biochemistry 23, 5495–5504.PubMedCrossRefGoogle Scholar
  16. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–685.PubMedCrossRefGoogle Scholar
  17. Liu N., Xin W., and Cooper N. G. F. (1995) Ca2+/calmodulin-dependent kinase in developing chick retina.Invest. Ophthal. Vis. Sci. ARVOSuppl. 36, S 59a.Google Scholar
  18. Llinas R., Gruner J. A., Sugimori M., McGuinness T. L., and Greengard P. (1991) Regulation by synapsin I and Ca2+-calmodulin-dependent protein kinase II of transmitter release in squid giant synapse.J. Physiol. 436, 257–282.PubMedGoogle Scholar
  19. McGuinness T. L., Lai Y., and Greengard P. (1985) Ca2+/calmodulin-dependent protein kinase II. Isoenzymic forms from rat forebrain and cerebellum.J. Biol. Chem. 260, 1696–1704.PubMedGoogle Scholar
  20. Miller S. G. and Kennedy M. B. (1985) Distinct forebrain and cerebellar isozymes of type II Ca2+/calmodulin-dependent protein kinase associate differently with the postsynaptic density fraction.J. Biol. Chem. 260, 9039–9046.PubMedGoogle Scholar
  21. Miller S. G. and Kennedy M. B. (1986) Regulation of brain type II Ca2+/calmodulin-dependent protein kinase by autophosphorylation: a Ca2+-triggered molecular switch.Cell 44, 861–870.PubMedCrossRefGoogle Scholar
  22. Ochiishi T., Terashima T., Sugiura H., and Yamauchi T. (1994) Immunohistochemical localization of Ca2+/calmodulin-dependent protein kinase II in the rat retina.Brain Res. 634, 257–265.PubMedCrossRefGoogle Scholar
  23. Onodera H., Hara H., Kogure K., Fukunaga K., Ohta Y., and Miyamoto E. (1990) Ca2+/calmodulin-dependent protein kinase II immunoreactivity in the rat hippocampus after forebrain ischemia.Neurosci. Lett. 113, 134–138.PubMedCrossRefGoogle Scholar
  24. Rostas J. A. P., Seccombe M., and Weinberger R. P. (1988) Two developmentally regulated isozymes of calmodulin-stimulated protein kinase II in rat forebrain.J. Neurochem. 50, 945–953.PubMedCrossRefGoogle Scholar
  25. Rostas J. A. P., Brent V. A., Seccombe M., Weinberger R. P., and Dunkley P. R. (1989) Purification and characterization of calmodulin-stimulated protein kinase II from two-day and adult chicken forebrain.J. Mol. Neurosci. 1, 93–104.PubMedGoogle Scholar
  26. Saitoh T. and Schwartz J. H. (1985) Phosphorylation-dependent subcellular translocation of a Ca2+/calmodulin-dependent protein kinase produces an autonomous enzyme inAplysia neurons.J. Cell Biol. 100, 835–842.PubMedCrossRefGoogle Scholar
  27. Schulman H. (1988) The multifunctional Ca2+/calmodulin-dependent protein kinase, inAdvances in Second Messenger and Phosphoprotein Research, vol. 22 (Greengard P. and Robinson R., eds.) Raven, New York, pp. 39–112.Google Scholar
  28. Sheng M., Thompson M. A., and Greenberg M. E. (1991) CREB: a Ca2+-regulated transcription factor phosphorylated by calmodulin-dependent kinases.Science 252, 1427–1430.PubMedCrossRefGoogle Scholar
  29. Sugiura H. and Yamauchi T. (1992) Developmental changes in the levels of Ca2+/calmodulin-dependent protein kinase II α and β proteins in soluble and particulate fractions of the rat brain.Brain Res. 593, 97–104.PubMedCrossRefGoogle Scholar
  30. 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.PubMedCrossRefGoogle Scholar
  31. Vallano R. L. (1988) Identification and regional distribution of a type II calcium/calmodulin-dependent kinase in mouse brain.Biochem. Pharmacol. 37, 2381–2388.PubMedCrossRefGoogle Scholar
  32. Weinberger R. P. and Rostas J. A. P. (1986) Subcellular distribution of a calmodulin-dependent protein kinase activity in rat cerebral cortex during development.Dev. Brain Res. 29, 37–50.CrossRefGoogle Scholar
  33. Willmund R., Mitschulat H., and Schneider K. (1986) Long-term modulation of Ca2+-stimulated autophosphorylation and subcellular distribution of the Ca2+/calmodulin-dependent protein kinase in the brain ofDrosophila.Proc. Natl. Acad. Sci. USA 83, 9789–9793.PubMedCrossRefGoogle Scholar
  34. Yamamoto H., Fukunaga K., Lee K., and Soderling T. (1992) Ischemia-induced loss of brain calcium/calmodulin-dependent protein kinase II.J. Neurochem. 58, 1110–1117.PubMedCrossRefGoogle Scholar
  35. Yamauchi T., Nakata H., and Fujisawa H. (1981) A new activator protein that activates tryptophan 5-monooxygenase and tyrosine 3-monooxygenase in the presence of Ca2+-, calmodulin-dependent protein kinase. Purification and characterization.J. Biol. Chem. 256, 5404–5409.PubMedGoogle Scholar
  36. Yamauchi T. and Hitoshi F. (1982) Phosphorylation of microtubule-associated protein 2 by calmodulin-dependent protein kinase (kinase II) which occurs only in the brain tissues.Biochem. Biophys. Res. Commun. 109, 975–981.PubMedCrossRefGoogle Scholar
  37. Yamauchi T., Ohsako S., and Deguchi T. (1989) Expression and characterization of calmodulin-dependent protein kinase II from cloned cDNAs in Chinese hamster ovary cells.J. Biol. Chem. 264, 19,108–19,116.Google Scholar

Copyright information

© Humana Press Inc 1995

Authors and Affiliations

  • Nian Liu
    • 1
  • Nigel G. F. Cooper
    • 1
  1. 1.Department of Anatomical Sciences and NeurobiologyUniversity of Louisville School of MedicineLouisville

Personalised recommendations