Neurochemical Research

, Volume 11, Issue 11, pp 1583–1595 | Cite as

Ca2+-dependent phosphorylation by endogenous kinases of Mr 95 K and 50 K-55 K proteins in PC12 pheochromocytoma cells

  • Kuniko Okumura-Noji
  • Taiji Kato
  • Ryo Tanaka
Original Articles
  • 9 Downloads

Abstract

Endogenous protein phosphorylation of PC12 cells was investigated with the homogenate as well as intact cells. In the case of the homogenate, the major proteins that were phosphorylated in the presence of Ca2+ were found to be of Mr 95 K and Mr 50 K-55 K. Ca2+/calmodulin-dependent protein kinase appeared to be responsible for phosphorylation of Mr 50 K-55 K proteins and partly of Mr 95 K protein. The apparentKm's for Ca2+ of Mr 95 K and 50 K-55 K protein phosphorylation were 2.2×10−7 M and around 1.5×10−6 M, respectively. Since several cell lines of neuroblastoma exhibited Mr 95 K protein phosphorylation of similar type, the protein phosphorylation may be a common process shared by neuronal cells. Depolarization of intact PC12 cells by high K+ concentrations induced Mr 95 K protein phosphorylation. The results suggest that a physiological increase by excitation in the intracellular Ca2+ concentration triggers phosphorylation of Mr 95 K protein in neuronal cells and this phosphorylation may play a role in the regulation of transmitter release.

Keywords

Protein Kinase PC12 Cell Neuronal Cell Neuroblastoma Intact Cell 

References

  1. 1.
    Krueger, B. K., Forn, J., andGreengard, P. 1977. Depolarization-induced phosphorylation of specific proteins, mediated by calcium ion influx, in rat brain synaptosomes. J. Biol. Chem. 252:2764–2773.Google Scholar
  2. 2.
    DeLorenzo, R. J. 1981. The calmodulin hypothesis of neurotransmission. Cell Calcium 2:365–385.Google Scholar
  3. 3.
    Wu, W. C.-S., Walaas, S. I., Nairn, A. G., andGreengard, P. 1982. Calcium/phospholipid regulates phosphorylation of a Mr “87 k” substrate protein in brain synaptosomes. Proc. Natl. Acad. Sci. USA 79:5249–5253.Google Scholar
  4. 4.
    Robinson, P. J., andDunkley, P. R. 1983. Depolarisation-dependent protein phosphorylation in rat cortical synaptosomes: Factors determining the magnitude of the response. J. Neurochem. 41:909–918.Google Scholar
  5. 5.
    Gagliardino, J. J., Harrioson, D. E., Christie, M. R., Gagliardino, E. E., andAshcroft, S. J. H. 1980. Evidence for the participation of calmodulin in stimulussecretion coupling in the pancreatic β-cell. Biochem. J. 192:919–927.Google Scholar
  6. 6.
    Schubart, U. K., Erlichman, J., andFleischer, N. 1980. The role of calmodulin in the regulation of protein phosphorylation and insulin release in hamster insulinoma cells. J. Biol. Chem. 255:4120–4124.Google Scholar
  7. 7.
    Maizels, E. T., andJungmann, R. A. 1982. Ca2+-dependent phosphorylation of rat ovary proteins. Biochem. Biophys. Res. Commun. 107:32–37.Google Scholar
  8. 8.
    Sieghart, W., Theoharides, T. C., Alper, S. L., Douglas, W. W., andGreengard, P. 1978. Calcium-dependent protein phosphorylation during secretion by exocytosis in the mast cell. Nature 275:329–331.Google Scholar
  9. 9.
    Sobel, A., andTashjian, A. H., Jr. 1983. Distinct patterns of cytoplasmic protein phosphorylation related to regulation of synthesis and release of prolactin by GH cells. J. Biol. Chem. 258:10312–10324.Google Scholar
  10. 10.
    Amy, C. M., andKirshner, N. 1981. Phosphorylation of adrenal medulla cell proteins in conjunction with stimulation of catecholamine secretion. J. Neurochem. 36:847–854.Google Scholar
  11. 11.
    Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., andNishizuka, Y. 1979. Calcium-dependent activation of a multifunctional protein kinase by membrane phospholipids. J. Biol. Chem. 254:3692–3695.Google Scholar
  12. 12.
    Castagna, M., Takai, Y., Kaibuchi, K., Sana, K., Kikkawa, U., andNishizuka, Y. 1982. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J. Biol. Chem. 257:7847–7851.Google Scholar
  13. 13.
    Pozzan, T., Gatti, G., Dozio, N., Vicentini, L. M., andMeldolesi, J. 1984. Ca2+-dependent and-independent release of neurotransmitters from PC12 cells: A role for protein kinase C activation? J. Cell Biol. 99:628–638.Google Scholar
  14. 14.
    Greene, L. A., andRein, G. 1977. Release, storage and uptake of catecholamines by a clonal cell line of nerve growth factor (NGF) responsive pheochromocytoma cells. Brain Res. 129:247–263.Google Scholar
  15. 15.
    Meldolesi, J., Huttner, W. B., Tsien, R. Y., andPozzan, T. 1984. Free cytoplasmic Ca2+ and neurotransmitter release: Studies on PC12 cells and synaptosomes exposed to α-latrotoxin. Proc. Natl. Acad. Sci. USA 81:620–624.Google Scholar
  16. 16.
    Kato, T., Chiu, T.-C., Lim, R., Troy, S. S., Turriff, D. E. 1979. Multiple molecular forms of glia maturation factor. Biochim. Biophys. Acta 579:216–227.Google Scholar
  17. 17.
    Felice, L. J., Felice, J. D., andKissinger, P. T. 1978. Determination of catecholamines in rat brain parts by reverse-phase ion-pair liquid chromatography. J. Neurochem. 31:1461–1465.Google Scholar
  18. 18.
    Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.Google Scholar
  19. 19.
    Kilpatrick, D. L., Slepetis, R. J., Corcoran, J. J., andKirshner, N. 1982. Calcium uptake and catecholamine secretion by cultured bovine adrenal medulla cells. J. Neurochem. 38:427–435.Google Scholar

Copyright information

© Plenum Publishing Corporation 1986

Authors and Affiliations

  • Kuniko Okumura-Noji
    • 1
  • Taiji Kato
    • 1
  • Ryo Tanaka
    • 1
  1. 1.Department of BiochemistryNagoya City University Medical SchoolNagoyaJapan

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