Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1987 Aug;84(16):5710–5714. doi: 10.1073/pnas.84.16.5710

Ca2+/calmodulin-dependent protein kinase II: identification of autophosphorylation sites responsible for generation of Ca2+/calmodulin-independence.

Y Lai, A C Nairn, F Gorelick, P Greengard
PMCID: PMC298932  PMID: 3475699

Abstract

Ca2+/calmodulin-dependent protein kinase II contains two types of subunit, alpha (Mr 50,000) and beta (Mr 60,000/58,000), both of which undergo Ca2+/calmodulin-dependent autophosphorylation. Autophosphorylation is known to convert the enzyme to a Ca2+/calmodulin-independent form. In the present study, we have characterized the autophosphorylation sites on rat forebrain Ca2+/calmodulin-dependent protein kinase II that are most likely to be responsible for the generation of Ca2+/calmodulin-independence. Under conditions (0 degree C, low concentrations of ATP) sufficient to generate close to maximal Ca2+/calmodulin-independence, only a few of the phosphorylatable sites on the enzyme became phosphorylated. These autophosphorylation sites were examined by phospho amino acid analysis, two-dimensional thermolytic phosphopeptide mapping, and high-performance liquid chromatography. The time course of phosphorylation of threonine in both alpha and beta subunits was similar to the time course of the generation of Ca2+/calmodulin-independence. Moreover, the time course of phosphorylation of one set of peptides, referred to as peptide 1/1', present in both alpha and beta subunits was similar to the time course of the generation of Ca2+/calmodulin-independence. Threonine was the only amino acid phosphorylated in peptide 1/1'. An additional peptide, referred to as peptide 2, was phosphorylated in the beta subunit. The time course of phosphorylation of peptide 2, which also contained only phosphothreonine, did not parallel the time course of the generation of Ca2+/calmodulin-independence. It is likely that the phosphorylation of a threonine residue on peptide 1/1' is responsible for the generation of Ca2+/calmodulin-independence of Ca2+/calmodulin-dependent protein kinase II.

Full text

PDF
5710

Images in this article

5712Image
on p.5712
5712Image
on p.5712

Selected References

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

  1. Ahmad Z., DePaoli-Roach A. A., Roach P. J. Purification and characterization of a rabbit liver calmodulin-dependent protein kinase able to phosphorylate glycogen synthase. J Biol Chem. 1982 Jul 25;257(14):8348–8355. [PubMed] [Google Scholar]
  2. Bennett M. K., Erondu N. E., Kennedy M. B. Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain. J Biol Chem. 1983 Oct 25;258(20):12735–12744. [PubMed] [Google Scholar]
  3. Bennett M. K., Kennedy M. B. Deduced primary structure of the beta subunit of brain type II Ca2+/calmodulin-dependent protein kinase determined by molecular cloning. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1794–1798. doi: 10.1073/pnas.84.7.1794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Detre J. A., Nairn A. C., Aswad D. W., Greengard P. Localization in mammalian brain of G-substrate, a specific substrate for guanosine 3',5'-cyclic monophosphate-dependent protein kinase. J Neurosci. 1984 Nov;4(11):2843–2849. doi: 10.1523/JNEUROSCI.04-11-02843.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fukunaga K., Yamamoto H., Matsui K., Higashi K., Miyamoto E. Purification and characterization of a Ca2+- and calmodulin-dependent protein kinase from rat brain. J Neurochem. 1982 Dec;39(6):1607–1617. doi: 10.1111/j.1471-4159.1982.tb07994.x. [DOI] [PubMed] [Google Scholar]
  6. Huttner W. B., DeGennaro L. J., Greengard P. Differential phosphorylation of multiple sites in purified protein I by cyclic AMP-dependent and calcium-dependent protein kinases. J Biol Chem. 1981 Feb 10;256(3):1482–1488. [PubMed] [Google Scholar]
  7. Kemp B. E., Pearson R. B., Guerriero V., Jr, Bagchi I. C., Means A. R. The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence. J Biol Chem. 1987 Feb 25;262(6):2542–2548. [PubMed] [Google Scholar]
  8. Kuret J., Schulman H. Mechanism of autophosphorylation of the multifunctional Ca2+/calmodulin-dependent protein kinase. J Biol Chem. 1985 May 25;260(10):6427–6433. [PubMed] [Google Scholar]
  9. Lai Y., Nairn A. C., Greengard P. Autophosphorylation reversibly regulates the Ca2+/calmodulin-dependence of Ca2+/calmodulin-dependent protein kinase II. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4253–4257. doi: 10.1073/pnas.83.12.4253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lou L. L., Lloyd S. J., Schulman H. Activation of the multifunctional Ca2+/calmodulin-dependent protein kinase by autophosphorylation: ATP modulates production of an autonomous enzyme. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9497–9501. doi: 10.1073/pnas.83.24.9497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. McGuinness T. L., Lai Y., Greengard P. Ca2+/calmodulin-dependent protein kinase II. Isozymic forms from rat forebrain and cerebellum. J Biol Chem. 1985 Feb 10;260(3):1696–1704. [PubMed] [Google Scholar]
  12. McGuinness T. L., Lai Y., Greengard P., Woodgett J. R., Cohen P. A multifunctional calmodulin-dependent protein kinase. Similarities between skeletal muscle glycogen synthase kinase and a brain synapsin I kinase. FEBS Lett. 1983 Nov 14;163(2):329–334. doi: 10.1016/0014-5793(83)80846-1. [DOI] [PubMed] [Google Scholar]
  13. Miller S. G., Kennedy M. B. Regulation of brain type II Ca2+/calmodulin-dependent protein kinase by autophosphorylation: a Ca2+-triggered molecular switch. Cell. 1986 Mar 28;44(6):861–870. doi: 10.1016/0092-8674(86)90008-5. [DOI] [PubMed] [Google Scholar]
  14. Nairn A. C., Greengard P. Purification and characterization of Ca2+/calmodulin-dependent protein kinase I from bovine brain. J Biol Chem. 1987 May 25;262(15):7273–7281. [PubMed] [Google Scholar]
  15. Nairn A. C., Hemmings H. C., Jr, Greengard P. Protein kinases in the brain. Annu Rev Biochem. 1985;54:931–976. doi: 10.1146/annurev.bi.54.070185.004435. [DOI] [PubMed] [Google Scholar]
  16. Payne M. E., Schworer C. M., Soderling T. R. Purification and characterization of rabbit liver calmodulin-dependent glycogen synthase kinase. J Biol Chem. 1983 Feb 25;258(4):2376–2382. [PubMed] [Google Scholar]
  17. Pearson R. B., Woodgett J. R., Cohen P., Kemp B. E. Substrate specificity of a multifunctional calmodulin-dependent protein kinase. J Biol Chem. 1985 Nov 25;260(27):14471–14476. [PubMed] [Google Scholar]
  18. Saitoh T., Schwartz J. H. Phosphorylation-dependent subcellular translocation of a Ca2+/calmodulin-dependent protein kinase produces an autonomous enzyme in Aplysia neurons. J Cell Biol. 1985 Mar;100(3):835–842. doi: 10.1083/jcb.100.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Schulman H. Phosphorylation of microtubule-associated proteins by a Ca2+/calmodulin-dependent protein kinase. J Cell Biol. 1984 Jul;99(1 Pt 1):11–19. doi: 10.1083/jcb.99.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Shenolikar S., Lickteig R., Hardie D. G., Soderling T. R., Hanley R. M., Kelly P. T. Calmodulin-dependent multifunctional protein kinase. Evidence for isoenzyme forms in mammalian tissues. Eur J Biochem. 1986 Dec 15;161(3):739–747. doi: 10.1111/j.1432-1033.1986.tb10502.x. [DOI] [PubMed] [Google Scholar]
  22. Shields S. M., Vernon P. J., Kelly P. T. Autophosphorylation of calmodulin-kinase II in synaptic junctions modulates endogenous kinase activity. J Neurochem. 1984 Dec;43(6):1599–1609. doi: 10.1111/j.1471-4159.1984.tb06084.x. [DOI] [PubMed] [Google Scholar]
  23. Stevens C. F. Neurotransmission. Are there two functional classes of glutamate receptors? Nature. 1986 Jul 17;322(6076):210–211. doi: 10.1038/322210a0. [DOI] [PubMed] [Google Scholar]
  24. Ueda T., Greengard P. Adenosine 3':5'-monophosphate-regulated phosphoprotein system of neuronal membranes. I. Solubilization, purification, and some properties of an endogenous phosphoprotein. J Biol Chem. 1977 Jul 25;252(14):5155–5163. [PubMed] [Google Scholar]
  25. Vulliet P. R., Woodgett J. R., Cohen P. Phosphorylation of tyrosine hydroxylase by calmodulin-dependent multiprotein kinase. J Biol Chem. 1984 Nov 25;259(22):13680–13683. [PubMed] [Google Scholar]
  26. Woodgett J. R., Davison M. T., Cohen P. The calmodulin-dependent glycogen synthase kinase from rabbit skeletal muscle. Purification, subunit structure and substrate specificity. Eur J Biochem. 1983 Nov 15;136(3):481–487. doi: 10.1111/j.1432-1033.1983.tb07766.x. [DOI] [PubMed] [Google Scholar]
  27. Yamauchi T., Fujisawa H. Tyrosine 3-monoxygenase is phosphorylated by Ca2+-, calmodulin-dependent protein kinase, followed by activation by activator protein. Biochem Biophys Res Commun. 1981 May 29;100(2):807–813. doi: 10.1016/s0006-291x(81)80246-x. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES