Skip to main content
SpringerLink
Log in

Protein phosphorylation systems in postmortem human brain

  • Published:
Journal of Molecular Neuroscience Aims and scope Submit manuscript

Abstract

Protein phosphorylation systems regulated by cyclic adenosine 3′, 5′-monophosphate (cyclic AMP), or calcium in conjunction with calmodulin or phospholipid/ diacylglycerol, have been studied by phosphorylation in vitro of particulate and soluble fractions from human postmorten brain samples. One-dimensional or two-dimensional gel electrophoretic protein separations were used for analysis. Protein phosphorylation catalyzed by cyclic AMP-dependent protein kinase was found to be highly active in both particulate and soluble preparations throughout the human CNS, with groups of both widely distributed and region-specific substrates being observed in different brain nuclei. Dopamine-innervated parts of the basal ganglia and cerebral cortex contained the phosphoproteins previously observed in rodent basal ganglia. In contrast, calcium/phospholipid-dependent and calcium/calmodulin-dependent protein phosphorylation systems were less prominent in human postmortem brain than in rodent brain, and only a few widely distributed substrates for these protein kinases were found. Protein staining indicated that postmortem proteolysis, particularly of high-molecular-mass proteins, was prominent in deeply located, subcortical regions in the human brain. Our results indicate that it is feasible to use human postmortem brain samples, when obtained under carefully controlled conditions, for qualitative studies on brain protein phosphorylation. Such studies should be of value in studies on human neurological and/or psychiatric disorders.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Agrawal, H.C., O'Connell, K., Randle, C.L., Agrawal, D. (1982). Phosphorylation in vivo of four basic proteins of rat brain myelin. Biochem J. 201:39–47.

    PubMed  CAS  Google Scholar 

  • Albert, K.A., Wu, W.C.-S., Nairn, A.C., Greengard, P. (1984). Inhibition by calmodulin of calcium/phospholipid-dependent protein phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 81:3622–3625.

    Article  PubMed  CAS  Google Scholar 

  • Albert, K.A., Walaas, S.I., Wang, J.K.-T., Greengard, P., (1986). Widespread occurrence of “87 kDa,” a major specific substrate for protein kinase C. Proc. Natl. Acad. Sci. U.S.A. 83:2822–2826.

    Article  PubMed  CAS  Google Scholar 

  • Cash, R., Raisman, R., Ploska, A., Agid, Y. (1987). Dopamine D-1 receptor and cyclic AMP-dependent phosphorylation in Parkinson's disease. J. Neurochem. 49:1075–1083.

    Article  PubMed  CAS  Google Scholar 

  • Cleveland, D.W., Fischer, S.G., Kirschner, M.W., Laemmli, U.K. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sulfate, and analysis by gel electrophoresis. J. Biol. Chem. 252:1102–1106

    PubMed  CAS  Google Scholar 

  • DeCamilli, P., Harris, S.M., Huttner, W.B., Greengard, P. (1983a). Synapsin I (protein I), a nerve terminal-specific phosphoprotein: II. Its specific association with synaptic vesicles demonstrated by immunocytochemistry in agarose-embedded synaptosomes. J. Cell Biol. 96:1355–1373.

    Article  CAS  Google Scholar 

  • DeCamilli, P., Cameron, R., Greengard, P. (1983b). Synapsin I (protein I), a nerve terminal-specific phosphoprotein: I. Its general distribution in synapses of the central and peripheral nervous system demonstrated by immunofluorescence in frozen and plastic sections. J. Cell Biol. 96:1337–1354.

    Article  CAS  Google Scholar 

  • DeCamilli, P., Miller, P.E., Navone, F., Theurkauf, W.F., Vallee, R.B. (1984). Distribution of microtubule associated protein 2 in the nervous system of the rat studied by immunofluorescence. Neuroscience 11:817–846.

    CAS  Google Scholar 

  • Freifelder, D. (1982). Physical Biochemistry. Applications to Biochemistry and Molecular Biology. 2nd ed. W.H. Freeman and Co., San Francisco.

    Google Scholar 

  • Helmerhorst, E. and Stokes, G.B. (1980). Microcentrifuge desalting: A rapid, quantitative method for desalting small amounts of protein. Anal. Biochem. 104:130–135.

    Article  PubMed  CAS  Google Scholar 

  • Hemmings, H.C., Jr., Walaas, S.I., Ouimet, C.C., Greengard, P. (1987). Dopamine receptors: Regulation of protein phosphorylation. Receptor Biochemistry and Methodology, Vol. 9: Structure and Function of Dopamine Receptors. I. Creese, C.M. Fraser, (eds). Alan R. Liss, New York, pp 115–151.

    Google Scholar 

  • Huttner, W.B., Greengard, P. (1979). Multiple phosphorylation sites in protein I, and their differential regulation by cyclic AMP and calcium. Proc. Natl. Acad. Sci. U.S.A. 76:5402–5406.

    Article  PubMed  CAS  Google Scholar 

  • Inoue, M., Kishimoto, A., Takai, Y., Nishizuka, Y. (1977): Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. J. Biol. Chem. 252:7610–7616.

    PubMed  CAS  Google Scholar 

  • Jäckle, H. (1979). Visualization of proteins after isoelectric focusing during two-dimensional gel electrophoresis. Anal. Biochem. 98:81–84.

    Article  PubMed  Google Scholar 

  • Kelly, P.T., Luttges, M.W. (1975). Electrophoretic separation of nervous system proteins on exponential gradient polyacrylamide gels. J. Neurochem. 24:1077–1079.

    Article  PubMed  CAS  Google Scholar 

  • Kennedy, M.B., McGuinness, T.L., Greengard, P. (1983). A calcium/calmodulin-dependent protein kinase from mammalian brain that phosphorylates synapsin I: Partial purification and characterization. J. Neurosci. 3:818–831.

    PubMed  CAS  Google Scholar 

  • Kikkawa, U., Takai, Y., Minakuchi, R., Inohara, S., Nishizuka, Y. (1982). Calcium-activated, phospholipiddependent protein kinase from rat brain. Subcellular distribution, purification and properties. J. Biol. Chem. 257:13341–13348.

    PubMed  CAS  Google Scholar 

  • Lohmann, S.M., Walter, U., Greengard, P. (1980). Identification of endogenous substrate proteins for cAMP-dependent protein kinase in bovine brain. J. Biol. Chem. 250:9985–9992.

    Google Scholar 

  • Martinez-Millan, L., Rodnight, R. (1982). Intrinsic protein phosphorylation in synaptosomal plasma membrane fragments: A comparison of cerebral cortex tissue from several species, including human biopsy specimens. J. Neurochem. 39:1302–1307.

    Article  PubMed  CAS  Google Scholar 

  • McGuinness, T.L., Lai, Y., Ouimet, C.C., Greengard, P. (1985). Calcium/calmodulin-dependent protein phosphorylation in the nervous system. Calcium in Biological Systems. R.P. Rubin, G.B. Weiss, J.W. Putney, Jr., (eds). Plenum Press, New York, pp 291–305

    Google Scholar 

  • Nairn, A.C., Hemings, H.C., Jr., Greengard. P. (1985). Protein kinases in the brain. Annu. Rev Biochem. 54:931–976.

    Article  PubMed  CAS  Google Scholar 

  • Nestler, E.J., Greengard, P., (1984). Protein Phosphorylation in the Nervous System. John Wiley, New York.

    Google Scholar 

  • O'Farrell, P.H. (1975). High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007–4021.

    PubMed  Google Scholar 

  • O'Farrell, P.Z., Goodman, H.W., O'Farrell, P.H. (1977). High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12:1133–1142.

    Article  PubMed  Google Scholar 

  • Perdahl, E., Wu, W.C.-S., Browning, M.D., Winblad, B., Greengard, P. (1984). Protein III, a neuron-specific phosphoprotein: Variant forms found in human brain. Neurobehav. Toxicol. Teratol. 6:425–431.

    PubMed  CAS  Google Scholar 

  • Peterson, G. (1977). A simplification of the protein assay of Lowry et al. which is more generally applicable. Anal. Biochem. 83:346–356.

    Article  PubMed  CAS  Google Scholar 

  • Routtenberg, A., Morgan, D.G., Conway, R.G., Schmidt, M.J., Ghetti, B. (1981). Human brain protein phosphorylation in vitro: Cyclic AMP stimulation of electrophoretically-separated substrates. Brain Res. 222:323–333.

    Article  PubMed  CAS  Google Scholar 

  • Turner, R.S., Chou, C.-H.J., Kibler, R.F., Kuo, J.F. (1982): Basic protein in brain myelin is phosphorylated by endogenous phospholipid-sensitive Ca2+-dependent protein kinase. J. Neurochem. 39:1397–1404.

    Article  PubMed  CAS  Google Scholar 

  • Walaas, S.I., Greengard, P. (1984). DARPP-32, a dopamine-and adenosine 3′:5′-monophosphate-regulated phosphoprotein enriched in dopamine-innervated brain regions. I. Regional and cellular distribution in rat brain. J. Neurosci. 4:84–98.

    PubMed  CAS  Google Scholar 

  • Walaas, S.I., Greengard, P. (1987). Phosphorylation of brain proteins. The Enzymes, Vol. XVIII, Control by Phosphorylation, Part B. P.D. Boyer, E.G. Krebs (eds). Academic Press, Orlando, FL, pp. 285–317.

    Google Scholar 

  • Wallas, S.I., Nairn, A.C., Greengard, P. (1983a). Regional distribution of calcium- and adenosine 3′:5′-monophosphate-regulated protein phosphorylation systems in mammalian brain. I. Particulate systems. J. Neurosci. 3:291–301.

    Google Scholar 

  • Walaas, S.I., Nairn, A.C., Greengard, P. (1983b). Regional distribution of calcium- and adenosine 3′:5′-monophosphate-regulated protein phosphorylation systems in mammalian brain. II. Soluble systems. J. Neurosci. 3:302–311.

    PubMed  CAS  Google Scholar 

  • Walaas, S.I., Nairn, A.C., Greengard, P. (1986). PCPP-260, a Purkinje cell-specific cyclic AMP-regulated membrane phosphoprotein of M r 260,000. J. Neurosci. 6:954–961.

    PubMed  CAS  Google Scholar 

  • Wu W.C.-S., Walaas, S.I., Nairn, A.C., Greengard, P. (1982). Calcium/phospholipid regulates phosphorylation of a Mr “87k” substrate protein in brain synaptosomes. Proc. Natl. Acad. Sci. U.S.A. 79:5249–5253.

    Article  PubMed  CAS  Google Scholar 

  • Yahr, M.D. (1976). The Basal Ganglia. Raven Press. New York.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, 1230 York Avenue, 10021, New York, NY, USA

    S. Ivar Walaas, Eva Perdahl-Wallace & Paul Greengard

  2. Department of Geriatric Medicine, Karolinska Institute, Huddinge Hospital, S 141 86, Huddinge, Sweden

    Bengt Winblad

Authors
  1. S. Ivar Walaas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eva Perdahl-Wallace
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bengt Winblad
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Paul Greengard
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

This study was supported by United States Public Health Services grants MH 40899 and Environmental Protection Agency Award CR-813826-01-0, and grants from the Swedisn Medical Research Council (no. 5664), the Swedish Society of Medical Sciences, and the Norwegian Council for Science and the Humanities.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Walaas, S.I., Perdahl-Wallace, E., Winblad, B. et al. Protein phosphorylation systems in postmortem human brain. J Mol Neurosci 1, 105–116 (1989). https://doi.org/10.1007/BF02918896

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02918896

Keywords

Search

Navigation