Advertisement

Neurochemical Research

, Volume 19, Issue 10, pp 1257–1264 | Cite as

Glutamate-induced protein phosphorylation in cerebellar granule cells: Role of protein kinase C

  • Maria Luisa Eboli
  • Delio Mercanti
  • Maria Teresa Ciotti
  • Angelo Aquino
  • Loriana Castellani
Original Articles

Abstract

Protein phosphorylation in response to toxic doses of glutamate has been investigated in cerebellar granule cells.32P-labelled cells have been stimulated with 100 μM glutamate for up to 20 min and analysed by one and two dimensional gel electrophoresis. A progressive incorporation of label is observed in two molecular species of about 80 and 43 kDa (PP80 and PP43) and acidic isoelectric point. Glutamate-stimulated phosphorylation is greatly reduced by antagonists of NMDA and non-NMDA glutamate receptors. The effect of glutamate is mimicked by phorbol esters and is markedly reduced by inhibitors of protein kinase C (PKC) such as staurosporine and calphostin C. PP80 has been identified by Western blot analysis as the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate), while antibody to GAP-43 (growth associated protein-43), the nervous tissue-specific substrate of PKC, failed to recognize PP43. Our results suggest that PKC is responsible for the early phosphorylative events induced by toxic doses of glutamate in cerebellar granule cells.

Key Words

Cerebellar granules glutamate excitotoxicity protein kinase C phorbol esters phosphoproteins 

Abbreviations

(NMDA)

N-methyl-D-aspartate

(PKC)

protein kinase C

(EAA)

excitatory aminoacids

(GAMSA)

γ-D-glutamylaminomethylsulfonate

(MK801)

(+)-10,11-dihydro-5-methyl-5-H-dibenzo-(a,d)-cyclohepten-5,10imine

(TPA)

phorbol 12-myristate 13-acetate

(MARCKS)

myristoylated alanine-rich C kinase substrate

(GAP-43)

growth-associated protein-43

(SDS)

sodium dodecyl sulfate

(PAGE)

polyacrylamide gel electrophoresis

(H7)

1-(5-isoquinolinesulfonyl)-2-methylpiperazine

(DIV)

days in vitro

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Mayer, M. L., and Westbrook, G. L. 1988. The physiology of excitatory amino acids in the vertebrate central nervous system. Progr. Neurobiol. 28:197–276.Google Scholar
  2. 2.
    Watkins, J. C., and Olverman, H. J. 1987. Agonist and antagonist for excitatory amino acid receptors. Trends Neurosci. 10:265–272.Google Scholar
  3. 3.
    Choi, D. W. 1990. Cerebral hypoxia: some new approaches and unanswered questions. J. Neurosci. 10:2493–2501.Google Scholar
  4. 4.
    McDonald, J. W., and Johnston, M. V. 1990. Physiological and pathophysiological roles of excitatory amino acids during central nervous system development. Brain Res. Rev. 15:41–70.Google Scholar
  5. 5.
    Beal, M. F. 1992. Role of excitotoxicity in human neurological disease. Current opinions in Neurobiology 2:657–662.Google Scholar
  6. 6.
    Nishizuka, Y. 1988. The molecular heterogeneity of protein kinase C and its implication for cellular regulation. Nature 334:661–665.Google Scholar
  7. 7.
    Olah, Z., Ikeda, J., Anderson, W. B., and Joo, F. 1990. Altered protein kinase C activity in different subfields of hippocampus following cerebral ischemia. Neurochem. Res. 15:515–518.Google Scholar
  8. 8.
    Wieloch, T., Cardell, M., Bingren, H., Zivin, J., and Saitoh, T. 1991. Changes in the activity of protein kinase C and the differential subcellular redistribution of its isozymes in the rat striatrum during and following transient forebrain ischemia. J. Neurochem. 56:1227–1235.Google Scholar
  9. 9.
    Domanaska-Janik, K., and Zalewska, T. 1992. Effect of brain ischemia on protein kinase C. J. Neurochem. 58:1432–1439.Google Scholar
  10. 10.
    Cardell, M., and Wieloch, T. 1993. Time course of the translocation and inhibition of protein kinase C during complete cerebral ischemia in the rat. J. Neurochem. 61:1308–1314.Google Scholar
  11. 11.
    Vaccarino, F., Guidotti, A., and Costa, E. 1987. Ganglioside inhibition of glutamate-mediated protein kinase C translocation in primary cultures of cerebellar neurons. Proc. Natl. Acad. Sci. USA 84:8707–8711.Google Scholar
  12. 12.
    Vaccarino, F. M., Liljequist S., and Tallman, J. F. 1991. Modulation of protein kinase C translocation by excitatory and inhibitory amino acids in primary cultures of neurons. J. Neurochem. 57:391–396.Google Scholar
  13. 13.
    Eboli, M. L., Ciotti, M. T., Mercanti, D., and Calissano, P. 1993. Differential involvement of protein kinase C in transmitter release and response to excitatory aminoacids in cultured cerebellar neurons. Neurochem. Res. 18:133–138.Google Scholar
  14. 14.
    Scholz, W. K., and Palfrey, H. C. 1991. Glutamate-stimulated protein phosphorylation in cultured hippocampal pyramidal neurons. J. Neurosci. 11:2422–2432.Google Scholar
  15. 15.
    Stumpo, D. J., Graff, J. M., Albert, K. A., Greengard, P. and Blackshear, P. J. 1989. Molecular cloning, characterization and expression of cDNA encoding the “80- to 87-kDa” myristoylated alanine-rich C kinase substrate: a major cellular substrate for protein kinase C. Proc. Natl. Acad. Sci. USA 86:4012–4016.Google Scholar
  16. 16.
    Andreasen, T. J., Luetje, C. W., Heidman, W., and Storm, D. R. 1983. Purification of a novel calmodulin binding protein from bovine cerebral cortex. Biochemistry 22:4615–4618.Google Scholar
  17. 17.
    Coggins, P. J., and Zwiers 1991. B-50 (GAP-43): Biochemistry and functional neurochemistry of a neuron-specific phosphoprotein. J. Neurochem. 56:1095–1106.Google Scholar
  18. 18.
    Levi, G., Aloisi F., Ciotti, M. T., and Gallo, V. 1984. Autoradiographic localization and depolarization-induced release of acidic amino acids in differentiating cerebellar granule cell cultures. Brain Res. 290:77–86.Google Scholar
  19. 19.
    Manev, H., Favaron, M., Siman, R., Guidotti, A., and Costa E. 1991. Glutamate neurotoxicity is independent of calpain I inhibition in primary cultures of cerebellar granule cells. J. Neurochem. 57:1288–1295.Google Scholar
  20. 20.
    Bruns, R. F., Miller, F. D., Merriman, R. L., Howbert, J. J., Heath, W. F., Kobayashi, I., Tamaoki, T., and Nakano, H. 1991. Inhibition of protein kinase C by calphostin C is light-dependent. Biochem. Biophys. Res. Commun. 176:288–293.Google Scholar
  21. 21.
    Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacter ophage T4. Nature (London) 227: 680–685.Google Scholar
  22. 22.
    Hochstrasser, D. F., Harrington, M. G., Hochstrasser, A.-C., Miller, M. J., and Merril, C. R. 1988. Methods for increasing the resolution of two-dimensional protein electrophoresis. Analytical Biochemistry 173:434–435.Google Scholar
  23. 23.
    Cupo, J. F., Lidgard, G. P., and Lichtman, W. F. 1990. A high resolution two-dimensional gel electrophoresis and silver staining protocol demonstrated with nuclear matrix proteins. Electrophoresis 11:500–504.Google Scholar
  24. 24.
    Towbin, H., Staehelin, T., and Gordon, J. 1979. Electroforetic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350–4354.Google Scholar
  25. 25.
    Aderem, A. A., Albert, K. A., Keum, M. M., Wang, J. K. T., Greengard, P., and Cohn, Z. A. 1988. Stimulus-dependent myristoylation of a major substrate for protein kinase C. Nature 332: 362–364.Google Scholar
  26. 26.
    Choi, D. W., Maulucci-Gedde, M., and Kriegstein, A. R. 1987. Glutamate neurotoxicity in cortical cell culture. J. Neurosci. 7: 357–368.Google Scholar
  27. 27.
    Favaron, M., Manev, H., Alho, H., Bertolino, M., Ferret, B., Guidotti, A., and Costa, E. 1988. Ganglioside prevent glutamate and kainate neurotoxicity in primary neuronal cultures of neonatal rat cerebellum and cortex. Proc. Natl. Acad. Sci. USA 85:7351–7355.Google Scholar
  28. 28.
    Wong, E. H. F., Kemp, J. A., Priestly, T., Knight, A. R., and Woodruff, G. N. 1986. The anti-convulsant MK-801 is a potent N-methyl-D-aspartate antagonist. Proc. Natl. Acad. Sci. USA 83: 7104–7108.Google Scholar
  29. 29.
    Davies, J., Evans, R. H., Jones, A. W., Smith, D. A. S., and Watkins, J. C. 1982. Differential activation and blockade of excitatory aminoacid receptors in the mammalian and amphibian central nervous systems. Comp. Biochem. Physiol. 72c:211–224.Google Scholar
  30. 30.
    Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. 1982. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J. Biol. Chem. 257:7847–7851.Google Scholar
  31. 31.
    Tamaoki, T. 1991. Use and specificity of staurosporine, UCN-01, and calphostin C as protein kinase C inhibitors. Meth. Enzymol. 201:340–347.Google Scholar
  32. 32.
    Favaron, M., Manev, H., Siman, R., Bertolino, M., Szekely, A. M., DeErausquin, G., Guidotti, A., and Costa, E. 1990. Down-regulation of protein kinase C protects cerebellar granule neurons in primary culture from glutamate-induced neuronal death. Proc. Natl. Acad. Sci. USA 87:1983–1987.Google Scholar
  33. 33.
    Felipo, V., Minana, M. D., and Grisolia, S. 1993. Inhibitors of protein kinase C prevent the toxicity of glutamate in primary neuronal cultures. Brain Res. 604:192–196.Google Scholar
  34. 34.
    Halpain, S., and Greengard, P. 1990. Activation of NMDA receptors induces rapid dephosphorylation of the cytoskeletal protein MAP2. Neuron. 5:237–246.Google Scholar
  35. 35.
    Wang, S., Hamberger, A., Ding, M., and Haglid, K. G. 1992. In vivo activation of kainate receptors induces dephosphorylation of the heavy neurofilament subunit. J. Neurochem. 59:1975–1978.Google Scholar
  36. 36.
    Cohen, P., Holmes, C. F. B., and Tsukitani, Y. 1990. Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem. Sci. 15:98–108.Google Scholar
  37. 37.
    Candeo, P., Favaron, M., Lengyel, I., Manev, R. M., Rimland, J. M., and Manev, H. 1992. Pathological phosphorylation causes neuronal death: Effect of okadaic acid in primary culture of cerebellar granule cells. J. Neurochem. 59:1558–1561.Google Scholar
  38. 38.
    Aderem, A. 1992. MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin. Nature 356:618–622.Google Scholar
  39. 39.
    Graham, M. E., and Burgoyne, R. D. 1993. Phosphoproteins of cultured cerebellar granule cells and response to the differentiation-promoting stimuli NMDA, high K+ and ionomycin. Eur. J. Neurosci. 5:575–583.Google Scholar
  40. 40.
    Thelen, M., Rosen, A., Nairn, A. C., and Aderem, A. 1991. Regulation by phosphorylation of reversible association of a myristoylated protein kinase C substrate with the plasma membrane. Nature 351, 320–322.Google Scholar
  41. 41.
    Hartwig, J. H., Thelen, M., Rosen, A., Janmey, P. A., Nairn, A. C., and Aderem, A. 1992. MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin. Nature 356:618–622.Google Scholar

Copyright information

© Plenum Publishing Corporation 1994

Authors and Affiliations

  • Maria Luisa Eboli
    • 1
  • Delio Mercanti
    • 2
  • Maria Teresa Ciotti
    • 2
  • Angelo Aquino
    • 3
  • Loriana Castellani
    • 3
    • 2
  1. 1.Institute of General PathologyCatholic UniversityRomeItaly
  2. 2.Institute of NeurobiologyC. N. R.RomeItaly
  3. 3.Department of Experimental Medicine and Biochemical SciencesUniversity of Rome “Tor Vergata”RomeItaly

Personalised recommendations