Abstract
Endogenous protein phosphorylation patterns were compared in occluded and non-occluded cytosol fractions prepared from rat forebrain. The occluded fraction was taken as representative of synaptosomal cytosol. One- and two-dimensional autoradiographs revealed the presence in non-occluded cytosol of a substrate for cAMP- and Ca2+/calmodulin-dependent protein kinase activities of Mr 300kD, corresponding to phosphorylated microtubule-associated protein-2 (MAP-2); this protein was absent in occluded cytosol. In contrast, a major substrate for protein kinase C was observed exclusively in occluded cytosol after phosphorylation under basal conditions. However, after phosphorylation in the presence of exogenous lipids, approximately equal amounts of the 82kD substrate were detected in both fractions, suggesting that protein kinase C in the occluded fraction was present in a partially activated state. Other minor differences in phosphorylation patterns between the two fractions were observed.
Similar content being viewed by others
References
Nestler, E., andGreengard, P. 1984. Protein Phosphorylation in the Nervous System. Wiley Interscience: New York.
Rodnight, R. 1982. Aspects of protein phosphorylation in the nervous system with particular reference to synaptic transmission. Prog. Brain Res. 56:1–25.
Gower, H., andRodnight, R. 1982. Intrinsic protein phosphorylation in synaptic plasma membrane fragments from the rat. General characteristics and migration behaviour on polyacrylamide gels of the main phosphate acceptors. Biochem. Biophys. Acta 716:45–52.
Mahler, H. R., Kleine, L. P., Ratner, N., andSorensen, R. G. 1982. Identification and topography of synaptic phosphoproteins. Prog. Brain Res. 56:27–48.
Nestler, E., andGreengard, P. 1983. Protein phosphorylation in the brain. Nature (London) 305:583–588.
De Camilli, P., Cameron R., andGreengard, P. 1983. 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.
De Camilli, P., Harris, S. M., Huttner, W. B., andGreengard, P. 1983. 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.
O'Callaghan, J. P., Dunn, L. A., andLovenberg, W. 1980. Calcium-regulated phosphorylation in synaptosomal cytosol: dependence on calmodulin. Proc. Natn. Acad. Sci. (USA) 77:5812–5816.
Wu, W. C-S, Walaas, S. I., Nairn, A. C., andGreengard, P. 1982. Calcium/phospholipid regulates phosphorylation of a Mr “87K” substrate protein in brain synaptosomes. Proc. Natn. Acad. Sci. (USA) 79:5249–5253.
Wrenn, R. W., Katoh, N., Wise, B. D., andKuo, J. F. 1980. Stimulation by phosphatidylserine and calmodulin of calcium-dependent phosphorylation of endogenous proteins from cerebral cortex. J. Biol. Chem. 255:12042–12046.
Juskevich, J. C., Kuhn, D. M., andLovenberg, W 1983. Phosphorylation of brain cytosol proteins. Effects of phospholipids and calmodulin. J. Biol. Chem. 258:1950–1953.
Walaas, S. I., Nairn, A. C., andGreengard, P. 1983. Regional distribution of calcium and cyclic, adenosine 3′:5′-monophosphate protein phosphorylation systems in mammalian brain. II. Soluble systems. J. Neuroscience 3:302–311.
Holmes, H., andRodnight, R. 1981. Ontogeny of membrane-bound protein phosphorylating systems in the rat. Development Neuroscience 4:79–88.
Rodnight, R., Gower, H., andHughes, C. 1983. Stimulation and inhibition by magnesium ions of, intrinsic protein phosphorylating systems in synaptosomal membrane fragments from rat brain. Neurochem. Res. 8:1441–1449.
Rodnight, R., Trotta, E. E., andPerrett, C. 1985. A simple and economical method for studying protein phosphorylation in vivo in the rat brain. J. Neurosci. Methods 13:87–95.
Bligh, E. G., andDyer, W. J. 1959. A rapid method of total lipid extraction and purification. Canad. J. Biochem. 37:911–917.
Peterson, G. L. 1977. A simplification of the protein assay method of Lowry et al. which is more generelly applicable. Analyt. Biochem. 83:346–356.
Bennett, V., Davis, J., andFowler, W. L. 1982. Brain spectrin: a membrane-associated protein related in structure and formation to erythrocyte spectrin. Nature (London) 299:126–131.
Levine, J., andWillard, M. 1981. Fodrin: axonally transported polypeptides associated with the internal periphery of many cells. J. Cell. Biol. 90:631–643.
Kakiuchi, S. K., Sobue, K., Morimoto, K., andKanda, K. 1982. A spectrin-like calmodulin-binding protein (calspectin) of brain. Biochem. Int. 5:755–762.
Bennett, M. K., Erondu, N. E., andKennedy, M. B. 1983. Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain. J. Biol. Chem. 258:12735–12744.
Goldenring, J. R., McGuire, J. S., andDe Lorenzo, R. J. 1984. Identification of the major postsynaptic density protein as homologous with the major calmodulin-binding sub-unit of a calmodulin-dependent protein kinase. J. Neurochem. 42:1077–1084.
Sobue, K., Kanda, K., Yamagami, K., andKakiuchi, 5 1982. Ca2+-and calmodulin-dependent phosphorylation of calspectin (spectrin-like calmodulin-binding protein: fodrin) by protein kinase systems in synaptosomal cytosol and membranes. Biomed. Res. 3:561–570.
Burgoyne, R. D., andCumming, R. 1983. Characterization of microtubule-associated proteins at the synapse: absence of MAP2. Europ. J. Cell. Biol. 30:154–158.
Huber, C., andMatus, A. 1984. Differences in the cellular distributions of two microtubule-associated proteins MAPI AND MAP2 in rat brain. J. Neuroscience 4:151–160.
Caceres, A., Binder, L. I., Payne, M. R., Bender, P., Rebhun, L., andSteward, O. 1984. Differential subcellular localization of tubulin and and the microtubule-associated protein MAP2 in brain tissues as revealed by immunocytochemistry with monoclonal hybridoma antibodies. J.Neuroscience 4:394–410.
Lohmann, S. M., Walter, U., andGreengard, P. 1980. Identification of endogenous substrate proteins for cAMP-dependent protein kinase in bovine brain. J. Biol. Chem. 255:9985–9992.
Dosemeci, A. 1985. Protein phosphorylation systems in synaptic structures: localisation, nature of attachment and functional dependency on lipids. PhD Thesis. London University.
Nishizuka, Y., Takai, Y., Hashimoto, E., Kishimoto, A., Kuroda, Y., Sakai, K., andYamamura, H. 1979. Regulatory and functional compartment of three multifunctional protein kinase systems. Molec. and Cell. Biochem. 23:153–165.
Albert, K. A., Wu, W. C-S., Nairn, A. C., andGreengard, P. 1984. Inhibition by calmodulin of calcium/phospholipid dependent protein phosphorylation. Proc. Natl. Acad. Sci. 81:3622–3625.
Zagon, I. S., McLaughlin, P. J., andGoodman, S. R. 1984. Localization of spectrin in mammalian brain. J. Neuroscience 4:3089–3100.
Author information
Authors and Affiliations
Additional information
Special Issue dedicated to Prof. Eduardo De Robertis.
Rights and permissions
About this article
Cite this article
Rodnight, R., Perrett, C. & Dosemeci, A. Acceptors for cyclic AMP-dependent and calcium ion-dependent protein kinases in rat brain cytosol fractions: A comparison of occluded (synaptosomal) cytosol with non-occluded cytosol. Neurochem Res 11, 1049–1060 (1986). https://doi.org/10.1007/BF00965593
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00965593