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. 1984 Jul 1;99(1 Pt 1):11–19. doi: 10.1083/jcb.99.1.11

Phosphorylation of microtubule-associated proteins by a Ca2+/calmodulin- dependent protein kinase

PMCID: PMC2275617  PMID: 6736124

Abstract

In an earlier study I demonstrated that rat brain cytosol contains a Ca2+/calmodulin-dependent protein kinase activity that phosphorylates microtubule-associated protein 2 (MAP-2) but not MAP-1. Comparison of sites of phosphate incorporated in MAP-2 catalyzed by the Ca2+/calmodulin-dependent kinase activity and the cyclic AMP-dependent protein kinase activity in cytosolic extracts revealed distinct sites of phosphorylation (Schulman, H., 1984, Mol. Cell. Biol., 4:1175-1178; abstract by me and J.A. Kuret and K.H. Spitzer [1983, Fed. Proc., 42:2250]. I have now used MAP-2 as a substrate to purify the Ca2+/calmodulin-dependent protein kinase responsible for MAP-2 phosphorylation. The brain appears to contain a single predominant Ca2+/calmodulin-dependent protein kinase that phosphorylates MAP-2. The enzyme was purified to apparent homogeneity by column chromatography using DEAE-cellulose, phosphocellulose, hydroxylapatite, Sepharose 6B, and a calmodulin-Sepharose affinity column. The 580,000-dalton holoenzyme consists of 51,000- and 60,000-dalton subunits. The purified enzyme phosphorylates MAP-2 at the same "sites" that are phosphorylated in cytosolic extracts and thus has the same specificity as the activity present in cytosol. Analysis of phosphorylated MAP-2.1 and MAP-2.2, the two components of MAP-2, suggests considerable homology in their phosphorylated domains.

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Selected References

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  1. Asnes C. F., Wilson L. Isolation of bovine brain microtubule protein without glycerol: polymerization kinetics change during purification cycles. Anal Biochem. 1979 Sep 15;98(1):64–73. doi: 10.1016/0003-2697(79)90706-1. [DOI] [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. Bloom G. S., Vallee R. B. Association of microtubule-associated protein 2 (MAP 2) with microtubules and intermediate filaments in cultured brain cells. J Cell Biol. 1983 Jun;96(6):1523–1531. doi: 10.1083/jcb.96.6.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burns R. G., Islam K. Characterisation of the chick brain high molecular weight multiply phosphorylated microtubule associated protein. Eur J Biochem. 1982 Feb;122(1):25–29. doi: 10.1111/j.1432-1033.1982.tb05843.x. [DOI] [PubMed] [Google Scholar]
  5. Burns R. G., Pollard T. D. A dynein-like protein from brain. FEBS Lett. 1974 Apr 1;40(2):274–280. doi: 10.1016/0014-5793(74)80243-7. [DOI] [PubMed] [Google Scholar]
  6. Caceres A., Payne M. R., Binder L. I., Steward O. Immunocytochemical localization of actin and microtubule-associated protein MAP2 in dendritic spines. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1738–1742. doi: 10.1073/pnas.80.6.1738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carlin R. K., Grab D. J., Siekevitz P. Function of a calmodulin in postsynaptic densities. III. Calmodulin-binding proteins of the postsynaptic density. J Cell Biol. 1981 Jun;89(3):449–455. doi: 10.1083/jcb.89.3.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  9. Cleveland D. W., Hwo S. Y., Kirschner M. W. Purification of tau, a microtubule-associated protein that induces assembly of microtubules from purified tubulin. J Mol Biol. 1977 Oct 25;116(2):207–225. doi: 10.1016/0022-2836(77)90213-3. [DOI] [PubMed] [Google Scholar]
  10. Connolly J. A., Kalnins V. I., Cleveland D. W., Kirschner M. W. Intracellular localization of the high molecular weight microtubule accessory protein by indirect immunofluorescence. J Cell Biol. 1978 Mar;76(3):781–786. doi: 10.1083/jcb.76.3.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dedman J. R., Brinkley B. R., Means A. R. Regulation of microfilaments and microtubules by calcium and cyclic AMP. Adv Cyclic Nucleotide Res. 1979;11:131–174. [PubMed] [Google Scholar]
  12. Dentler W. L., Granett S., Witman G. B., Rosenbaum J. L. Directionality of brain microtubule assembly in vitro. Proc Natl Acad Sci U S A. 1974 May;71(5):1710–1714. doi: 10.1073/pnas.71.5.1710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fellous A., Francon J., Lennon A. M., Nunez J. Microtubule assembly in vitro. Purification of assembly-promoting factors. Eur J Biochem. 1977 Aug 15;78(1):167–174. doi: 10.1111/j.1432-1033.1977.tb11726.x. [DOI] [PubMed] [Google Scholar]
  14. Fifková E., Van Harreveld A. Long-lasting morphological changes in dendritic spines of dentate granular cells following stimulation of the entorhinal area. J Neurocytol. 1977 Apr;6(2):211–230. doi: 10.1007/BF01261506. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Gaskin F., Kramer S. B., Cantor C. R., Adelstein R., Shelanski M. L. A dynein-like protein associated with neurotubules. FEBS Lett. 1974 Apr 1;40(2):281–286. doi: 10.1016/0014-5793(74)80244-9. [DOI] [PubMed] [Google Scholar]
  17. Gill G. N., Holdy K. E., Walton G. M., Kanstein C. B. Purification and characterization of 3':5'-cyclic GMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3918–3922. doi: 10.1073/pnas.73.11.3918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goldenring J. R., Gonzalez B., McGuire J. S., Jr, DeLorenzo R. J. Purification and characterization of a calmodulin-dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubule-associated proteins. J Biol Chem. 1983 Oct 25;258(20):12632–12640. [PubMed] [Google Scholar]
  19. 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]
  20. Greene L. A., Liem R. K., Shelanski M. L. Regulation of a high molecular weight microtubule-associated protein in PC12 cells by nerve growth factor. J Cell Biol. 1983 Jan;96(1):76–83. doi: 10.1083/jcb.96.1.76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Griffith L. M., Pollard T. D. The interaction of actin filaments with microtubules and microtubule-associated proteins. J Biol Chem. 1982 Aug 10;257(15):9143–9151. [PubMed] [Google Scholar]
  22. Hathaway G. M., Traugh J. A. Casein kinases--multipotential protein kinases. Curr Top Cell Regul. 1982;21:101–127. [PubMed] [Google Scholar]
  23. Herzog W., Weber K. Fractionation of brain microtubule-associated proteins. Isolation of two different proteins which stimulate tubulin polymerization in vitro. Eur J Biochem. 1978 Dec 1;92(1):1–8. doi: 10.1111/j.1432-1033.1978.tb12716.x. [DOI] [PubMed] [Google Scholar]
  24. Islam K., Burns R. Multiple phosphorylation sites of microtubule-associated protein (MAP2) observed at high ATP concentrations. FEBS Lett. 1981 Jan 26;123(2):181–185. doi: 10.1016/0014-5793(81)80282-7. [DOI] [PubMed] [Google Scholar]
  25. Izant J. G., McIntosh J. R. Microtubule-associated proteins: a monoclonal antibody to MAP2 binds to differentiated neurons. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4741–4745. doi: 10.1073/pnas.77.8.4741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jameson L., Caplow M. Modification of microtubule steady-state dynamics by phosphorylation of the microtubule-associated proteins. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3413–3417. doi: 10.1073/pnas.78.6.3413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kelly P. T., Cotman C. W. Synaptic proteins. Characterization of tubulin and actin and identification of a distinct postsynaptic density polypeptide. J Cell Biol. 1978 Oct;79(1):173–183. doi: 10.1083/jcb.79.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kennedy M. B., Bennett M. K., Erondu N. E. Biochemical and immunochemical evidence that the "major postsynaptic density protein" is a subunit of a calmodulin-dependent protein kinase. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7357–7361. doi: 10.1073/pnas.80.23.7357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kennedy M. B., McGuinness T., Greengard P. A calcium/calmodulin-dependent protein kinase from mammalian brain that phosphorylates Synapsin I: partial purification and characterization. J Neurosci. 1983 Apr;3(4):818–831. doi: 10.1523/JNEUROSCI.03-04-00818.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kikkawa U., Takai Y., Minakuchi R., Inohara S., Nishizuka Y. Calcium-activated, phospholipid-dependent protein kinase from rat brain. Subcellular distribution, purification, and properties. J Biol Chem. 1982 Nov 25;257(22):13341–13348. [PubMed] [Google Scholar]
  31. Kim H., Binder L. I., Rosenbaum J. L. The periodic association of MAP2 with brain microtubules in vitro. J Cell Biol. 1979 Feb;80(2):266–276. doi: 10.1083/jcb.80.2.266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kishimoto A., Takai Y., Mori T., Kikkawa U., Nishizuka Y. Activation of calcium and phospholipid-dependent protein kinase by diacylglycerol, its possible relation to phosphatidylinositol turnover. J Biol Chem. 1980 Mar 25;255(6):2273–2276. [PubMed] [Google Scholar]
  33. Klee C. B., Krinks M. H. Purification of cyclic 3',5'-nucleotide phosphodiesterase inhibitory protein by affinity chromatography on activator protein coupled to Sepharose. Biochemistry. 1978 Jan 10;17(1):120–126. doi: 10.1021/bi00594a017. [DOI] [PubMed] [Google Scholar]
  34. Landis D. M., Reese T. S. Cytoplasmic organization in cerebellar dendritic spines. J Cell Biol. 1983 Oct;97(4):1169–1178. doi: 10.1083/jcb.97.4.1169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lee K. S., Schottler F., Oliver M., Lynch G. Brief bursts of high-frequency stimulation produce two types of structural change in rat hippocampus. J Neurophysiol. 1980 Aug;44(2):247–258. doi: 10.1152/jn.1980.44.2.247. [DOI] [PubMed] [Google Scholar]
  36. Leterrier J. F., Liem R. K., Shelanski M. L. Interactions between neurofilaments and microtubule-associated proteins: a possible mechanism for intraorganellar bridging. J Cell Biol. 1982 Dec;95(3):982–986. doi: 10.1083/jcb.95.3.982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Maeno H., Reyes P. L., Ueda T., Rudolph S. A., Greengard P. Autophosphorylation of adenosine 3',5'-monophosphate-dependent protein kinase from bovine brain. Arch Biochem Biophys. 1974 Oct;164(2):551–559. doi: 10.1016/0003-9861(74)90066-6. [DOI] [PubMed] [Google Scholar]
  38. Matus A., Ackermann M., Pehling G., Byers H. R., Fujiwara K. High actin concentrations in brain dendritic spines and postsynaptic densities. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7590–7594. doi: 10.1073/pnas.79.23.7590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Matus A., Bernhardt R., Hugh-Jones T. High molecular weight microtubule-associated proteins are preferentially associated with dendritic microtubules in brain. Proc Natl Acad Sci U S A. 1981 May;78(5):3010–3014. doi: 10.1073/pnas.78.5.3010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Miller P., Walter U., Theurkauf W. E., Vallee R. B., De Camilli P. Frozen tissue sections as an experimental system to reveal specific binding sites for the regulatory subunit of type II cAMP-dependent protein kinase in neurons. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5562–5566. doi: 10.1073/pnas.79.18.5562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Murphy D. B., Borisy G. G. Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2696–2700. doi: 10.1073/pnas.72.7.2696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Murphy D. B., Vallee R. B., Borisy G. G. Identity and polymerization-stimulatory activity of the nontubulin proteins associated with microtubules. Biochemistry. 1977 Jun 14;16(12):2598–2605. doi: 10.1021/bi00631a004. [DOI] [PubMed] [Google Scholar]
  43. Nosé P., Schulman H. Protein phosphorylation system in bovine brain cytosol dependent on calcium and calmodulin. Biochem Biophys Res Commun. 1982 Aug;107(3):1082–1090. doi: 10.1016/0006-291x(82)90632-5. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. PORTZEHL H., CALDWELL P. C., RUEEGG J. C. THE DEPENDENCE OF CONTRACTION AND RELAXATION OF MUSCLE FIBRES FROM THE CRAB MAIA SQUINADO ON THE INTERNAL CONCENTRATION OF FREE CALCIUM IONS. Biochim Biophys Acta. 1964 May 25;79:581–591. doi: 10.1016/0926-6577(64)90224-4. [DOI] [PubMed] [Google Scholar]
  46. Palfrey H. C., Schiebler W., Greengard P. A major calmodulin-binding protein common to various vertebrate tissues. Proc Natl Acad Sci U S A. 1982 Jun;79(12):3780–3784. doi: 10.1073/pnas.79.12.3780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rosen O. M., Erlichman J. Reversible autophosphorylation of a cyclic 3':5'-AMP-dependent protein kinase from bovine cardiac muscle. J Biol Chem. 1975 Oct 10;250(19):7788–7794. [PubMed] [Google Scholar]
  48. Runge M. S., el-Maghrabi M. R., Claus T. H., Pilkis S. J., Williams R. C., Jr A MAP-2-stimulated protein kinase activity associated with neurofilaments. Biochemistry. 1981 Jan 6;20(1):175–180. doi: 10.1021/bi00504a029. [DOI] [PubMed] [Google Scholar]
  49. Sattilaro R. F., Dentler W. L., LeCluyse E. L. Microtubule-associated proteins (MAPs) and the organization of actin filaments in vitro. J Cell Biol. 1981 Aug;90(2):467–473. doi: 10.1083/jcb.90.2.467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Schulman H. Differential phosphorylation of MAP-2 stimulated by calcium-calmodulin and cyclic AMP. Mol Cell Biol. 1984 Jun;4(6):1175–1178. doi: 10.1128/mcb.4.6.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schulman H., Greengard P. Ca2+-dependent protein phosphorylation system in membranes from various tissues, and its activation by "calcium-dependent regulator". Proc Natl Acad Sci U S A. 1978 Nov;75(11):5432–5436. doi: 10.1073/pnas.75.11.5432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Selden S. C., Pollard T. D. Phosphorylation of microtubule-associated proteins regulates their interaction with actin filaments. J Biol Chem. 1983 Jun 10;258(11):7064–7071. [PubMed] [Google Scholar]
  53. Sherline P., Schiavone K. Immunofluorescence localization of proteins of high molecular weight along intracellular microtubules. Science. 1977 Dec 9;198(4321):1038–1040. doi: 10.1126/science.337490. [DOI] [PubMed] [Google Scholar]
  54. Sloboda R. D., Dentler W. L., Rosenbaum J. L. Microtubule-associated proteins and the stimulation of tubulin assembly in vitro. Biochemistry. 1976 Oct 5;15(20):4497–4505. doi: 10.1021/bi00665a026. [DOI] [PubMed] [Google Scholar]
  55. Sloboda R. D., Rudolph S. A., Rosenbaum J. L., Greengard P. Cyclic AMP-dependent endogenous phosphorylation of a microtubule-associated protein. Proc Natl Acad Sci U S A. 1975 Jan;72(1):177–181. doi: 10.1073/pnas.72.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Solomon F., Magendantz M., Salzman A. Identification with cellular microtubules of one of the co-assemlbing microtubule-associated proteins. Cell. 1979 Oct;18(2):431–438. doi: 10.1016/0092-8674(79)90062-x. [DOI] [PubMed] [Google Scholar]
  57. Suprenant K. A., Dentler W. L. Association between endocrine pancreatic secretory granules and in-vitro-assembled microtubules is dependent upon microtubule-associated proteins. J Cell Biol. 1982 Apr;93(1):164–174. doi: 10.1083/jcb.93.1.164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Theurkauf W. E., Vallee R. B. Extensive cAMP-dependent and cAMP-independent phosphorylation of microtubule-associated protein 2. J Biol Chem. 1983 Jun 25;258(12):7883–7886. [PubMed] [Google Scholar]
  59. Theurkauf W. E., Vallee R. B. Molecular characterization of the cAMP-dependent protein kinase bound to microtubule-associated protein 2. J Biol Chem. 1982 Mar 25;257(6):3284–3290. [PubMed] [Google Scholar]
  60. 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]
  61. Valdivia M. M., Avila J., Coll J., Colaço C., Sandoval I. V. Quantitation and characterization of the microtubule associated MAP2 in porcine tissues and its isolation from porcine (PK15) and human (HeLa) cell lines. Biochem Biophys Res Commun. 1982 Apr 29;105(4):1241–1249. doi: 10.1016/0006-291x(82)90920-2. [DOI] [PubMed] [Google Scholar]
  62. Vallee R. B., Borisy G. G. Removal of the projections from cytoplasmic microtubules in vitro by digestion with trypsin. J Biol Chem. 1977 Jan 10;252(1):377–382. [PubMed] [Google Scholar]
  63. Vallee R. B., Davis S. E. Low molecular weight microtubule-associated proteins are light chains of microtubule-associated protein 1 (MAP 1). Proc Natl Acad Sci U S A. 1983 Mar;80(5):1342–1346. doi: 10.1073/pnas.80.5.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Vallee R. Structure and phosphorylation of microtubule-associated protein 2 (MAP 2). Proc Natl Acad Sci U S A. 1980 Jun;77(6):3206–3210. doi: 10.1073/pnas.77.6.3206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Voter W. A., Erickson H. P. Electron microscopy of MAP 2 (microtubule-associated protein 2). J Ultrastruct Res. 1982 Sep;80(3):374–382. doi: 10.1016/s0022-5320(82)80051-8. [DOI] [PubMed] [Google Scholar]
  66. Wang J. H., Waisman D. M. Calmodulin and its role in the second-messenger system. Curr Top Cell Regul. 1979;15:47–107. doi: 10.1016/b978-0-12-152815-7.50006-5. [DOI] [PubMed] [Google Scholar]
  67. Weingarten M. D., Lockwood A. H., Hwo S. Y., Kirschner M. W. A protein factor essential for microtubule assembly. Proc Natl Acad Sci U S A. 1975 May;72(5):1858–1862. doi: 10.1073/pnas.72.5.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Yamauchi T., Fujisawa H. Disassembly of microtubules by the action of calmodulin-dependent protein kinase (Kinase II) which occurs only in the brain tissues. Biochem Biophys Res Commun. 1983 Jan 14;110(1):287–291. doi: 10.1016/0006-291x(83)91293-7. [DOI] [PubMed] [Google Scholar]
  69. Yamauchi T., Fujisawa H. Purification and characterization of the brain calmodulin-dependent protein kinase (kinase II), which is involved in the activation of tryptophan 5-monooxygenase. Eur J Biochem. 1983 Apr 15;132(1):15–21. doi: 10.1111/j.1432-1033.1983.tb07319.x. [DOI] [PubMed] [Google Scholar]
  70. de Jonge H. R., Rosen O. M. Self-phosphorylation of cyclic guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung. Effect of cyclic adenosine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate and histone. J Biol Chem. 1977 Apr 25;252(8):2780–2783. [PubMed] [Google Scholar]

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