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. 1988 May;85(10):3279–3283. doi: 10.1073/pnas.85.10.3279

Calmodulin-dependent multifunctional protein kinase in Aspergillus nidulans.

D C Bartelt 1, S Fidel 1, L H Farber 1, D J Wolff 1, R L Hammell 1
PMCID: PMC280192  PMID: 2835766

Abstract

A Ca2+/calmodulin (CaM)-dependent multifunctional protein kinase has been isolated from Aspergillus nidulans and purified to homogeneity. Unlike any CaM-dependent multifunctional protein kinase described previously, the native enzyme from Aspergillus behaves as a monomer. The calculated molecular weight is 41,200. NaDodSO4/PAGE reveals a single protein band with an apparent Mr of 51,000. Two-dimensional isoelectric focusing/NaDodSO4/PAGE of the purified enzyme showed one major and one minor more acidic Coomassie blue-stained spot, both of which bind 125I-labeled calmodulin in a calcium-dependent manner. The kinase is autophosphorylated in a calcium- and CaM-dependent manner, yielding an increase in the amount and number of more acidic forms of the enzyme. The Aspergillus kinase catalyzes the Ca2+/CaM-dependent phosphorylation of known substrates of type II Ca2+/CaM-dependent protein kinases, including glycogen synthase, microtubule-associated protein 2, synapsin, tubulin, gizzard myosin light chain, and casein. Cross-reactivity between antiserum raised against native rat brain protein kinase II and 125I-labeled Aspergillus kinase has been detected. Two forms of CaM have been isolated from Aspergillus nidulans, both of which activate the Aspergillus kinase at lower concentrations than that required for activation by bovine brain CaM.

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Selected References

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  1. Bartelt D. C., Moroney S., Wolff D. J. Purification, characterization and substrate specificity of calmodulin-dependent myosin light-chain kinase from bovine brain. Biochem J. 1987 Nov 1;247(3):747–756. doi: 10.1042/bj2470747. [DOI] [PMC free article] [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. Berridge M. J. The interaction of cyclic nucleotides and calcium in the control of cellular activity. Adv Cyclic Nucleotide Res. 1975;6:1–98. [PubMed] [Google Scholar]
  5. Chou Y. H., Rebhun L. I. Purification and characterization of a sea urchin egg Ca2+-calmodulin-dependent kinase with myosin light chain phosphorylating activity. J Biol Chem. 1986 Apr 25;261(12):5389–5395. [PubMed] [Google Scholar]
  6. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  7. DePaoli-Roach A. A., Roach P. J., Larner J. Rabbit skeletal muscle phosphorylase kinase. Comparison of glycogen synthase and phosphorylase as substrates. J Biol Chem. 1979 May 25;254(10):4212–4219. [PubMed] [Google Scholar]
  8. DeRiemer S. A., Kaczmarek L. K., Lai Y., McGuinness T. L., Greengard P. Calcium/calmodulin-dependent protein phosphorylation in the nervous system of Aplysia. J Neurosci. 1984 Jun;4(6):1618–1625. doi: 10.1523/JNEUROSCI.04-06-01618.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Edelman A. M., Blumenthal D. K., Krebs E. G. Protein serine/threonine kinases. Annu Rev Biochem. 1987;56:567–613. doi: 10.1146/annurev.bi.56.070187.003031. [DOI] [PubMed] [Google Scholar]
  10. Farber L. H., Wilson F. J., Wolff D. J. Calmodulin-dependent phosphatases of PC12, GH3, and C6 cells: physical, kinetic, and immunochemical properties. J Neurochem. 1987 Aug;49(2):404–414. doi: 10.1111/j.1471-4159.1987.tb02880.x. [DOI] [PubMed] [Google Scholar]
  11. Ferrari F. A., Ferrari E., Hoch J. A. Chromosomal location of a Bacillus subtilis DNA fragment uniquely transcribed by sigma-28-containing RNA polymerase. J Bacteriol. 1982 Nov;152(2):780–785. doi: 10.1128/jb.152.2.780-785.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Flanagan S. D., Yost B. Calmodulin-binding proteins: visualization by 125I-calmodulin overlay on blots quenched with Tween 20 or bovine serum albumin and poly(ethylene oxide). Anal Biochem. 1984 Aug 1;140(2):510–519. doi: 10.1016/0003-2697(84)90202-1. [DOI] [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. Gorelick F. S., Cohn J. A., Freedman S. D., Delahunt N. G., Gershoni J. M., Jamieson J. D. Calmodulin-stimulated protein kinase activity from rat pancreas. J Cell Biol. 1983 Oct;97(4):1294–1298. doi: 10.1083/jcb.97.4.1294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grab D. J., Carlin R. K., Siekevitz P. Function of a calmodulin in postsynaptic densities. II. Presence of a calmodulin-activatable protein kinase activity. J Cell Biol. 1981 Jun;89(3):440–448. doi: 10.1083/jcb.89.3.440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Krueger B. K., Forn J., Greengard P. Depolarization-induced phosphorylation of specific proteins, mediated by calcium ion influx, in rat brain synaptosomes. J Biol Chem. 1977 Apr 25;252(8):2764–2773. [PubMed] [Google Scholar]
  17. Kuret J., Schulman H. Purification and characterization of a Ca2+/calmodulin-dependent protein kinase from rat brain. Biochemistry. 1984 Nov 6;23(23):5495–5504. doi: 10.1021/bi00318a018. [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. 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]
  20. Lin C. R., Kapiloff M. S., Durgerian S., Tatemoto K., Russo A. F., Hanson P., Schulman H., Rosenfeld M. G. Molecular cloning of a brain-specific calcium/calmodulin-dependent protein kinase. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5962–5966. doi: 10.1073/pnas.84.16.5962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. MacSween J. M., Eastwood S. L. Recovery of antigen from staphylococcal protein A--antibody adsorbents. Methods Enzymol. 1981;73(Pt B):459–471. [PubMed] [Google Scholar]
  22. May G. S., Gambino J., Weatherbee J. A., Morris N. R. Identification and functional analysis of beta-tubulin genes by site specific integrative transformation in Aspergillus nidulans. J Cell Biol. 1985 Sep;101(3):712–719. doi: 10.1083/jcb.101.3.712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Nairn A. C., Bhagat B., Palfrey H. C. Identification of calmodulin-dependent protein kinase III and its major Mr 100,000 substrate in mammalian tissues. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7939–7943. doi: 10.1073/pnas.82.23.7939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  27. Palfrey H. C., Rothlein J. E., Greengard P. Calmodulin-dependent protein kinase and associated substrates in Torpedo electric organ. J Biol Chem. 1983 Aug 10;258(15):9496–9503. [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Rasmussen H., Kojima I., Kojima K., Zawalich W., Apfeldorf W. Calcium as intracellular messenger: sensitivity modulation, C-kinase pathway, and sustained cellular response. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1984;18:159–193. [PubMed] [Google Scholar]
  31. Ryazanov A. G. Ca2+/calmodulin-dependent phosphorylation of elongation factor 2. FEBS Lett. 1987 Apr 20;214(2):331–334. doi: 10.1016/0014-5793(87)80081-9. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  34. Schulman H., Kuret J., Jefferson A. B., Nose P. S., Spitzer K. H. Ca2+/calmodulin-dependent microtubule-associated protein 2 kinase: broad substrate specificity and multifunctional potential in diverse tissues. Biochemistry. 1985 Sep 24;24(20):5320–5327. doi: 10.1021/bi00341a008. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. 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]
  38. Shortle D., Haber J. E., Botstein D. Lethal disruption of the yeast actin gene by integrative DNA transformation. Science. 1982 Jul 23;217(4557):371–373. doi: 10.1126/science.7046050. [DOI] [PubMed] [Google Scholar]
  39. Siegel L. M., Monty K. J. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta. 1966 Feb 7;112(2):346–362. doi: 10.1016/0926-6585(66)90333-5. [DOI] [PubMed] [Google Scholar]
  40. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]

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