Modulation of synaptosomal protein phosphorylation/dephosphorylation by calcium is antagonised by inhibition of protein phosphatases with okadaic acid.

Neuroscience Group, Faculty of Medicine, University of Newcastle, N.S.W., Australia.
Neuroscience Letters (Impact Factor: 2.06). 06/1991; 126(2):203-6. DOI: 10.1016/0304-3940(91)90554-7
Source: PubMed

ABSTRACT The protein phosphatase inhibitor okadaic acid was used to investigate the protein phosphatases involved in the endogenous dephosphorylation of proteins in intact synaptosomes. Despite the fact that the calcium-dependent protein phosphatase (calcineurin) is most concentrated in synaptosomes and accounts for approximately 0.3% of synaptoplasmic protein, the majority of the dephosphorylation activity under both basal and depolarisation conditions is due to protein phosphatase type 1 (PP1) and/or protein phosphatase type 2A (PP2A). Nevertheless our results do suggest that calcineurin is active in synaptosomes and has 2 effects: a rapid, direct dephosphorylation of a limited range of substrates and an indirect activation of PP1 presumably by dephosphorylation of protein phosphatase 1 inhibitor-1.

  • [Show abstract] [Hide abstract]
    ABSTRACT: The effect of external Ca2+ ([Ca2+]e) on the incorporation of [] into total protein, cytoskeletal proteins and the heat shock protein HSP27, was studied in primary cultures of astrocytes from the rat hippocampus. Zero [Ca2+]e increased total -incorporation into astrocyte protein and when this was normalized to 100%, incorporation was significantly increased into glial fibrillary acidic protein (GFAP), vimentin (VIM) and HSP27. The difference in total -incorporation between zero [Ca2+]e and 1 mM [Ca2+]e was reversed by incubation of the cells with the protein phosphatase inhibitor okadaic acid in the range 1–10 nM; higher concentrations of okadaic acid (50–100 nM) further increased total -incorporation. In zero [Ca2+]e the non-specific channel blocker Co2+ (1 mM) decreased total -incorporation by approximately 30%. The results were compared with a previous study [S.T. Wofchuk, R. Rodnight, Age-dependent changes in the regulation by external calcium ions of the phosphorylation of glial fibrillary acidic protein in slices of rat hippocampus, Dev. Brain Res. 85 (1995) 181–186] in which it was shown that in immature hippocampal slices zero [Ca2+]e compared with 1 mM [Ca2+]e increased -incorporation into GFAP without changing total incorporation. The difference between the results for total -incorporation obtained in cultured astrocytes and immature brain tissue was found to be related to the concentration of [Ca2+]e in the medium since in slices concentrations of [Ca2+]e higher than 1 mM progressively decreased total incorporation. The difference may reflect a higher Ca2+-permeability of the plasma membrane in cultured astrocytes and/or to the complex structure of the slice tissue. In two-dimensional electrophoresis HSP27, in contrast to GFAP and VIM, was separated into 3 immunodetectable isoforms only two of which were normally phosphorylated. After labelling in the presence of okadaic acid both immunodetectable and phosphorylated HSP27 focussed as a single polypeptide. Phorbol dibutyrate (1 μM) and zero [Ca2+]e stimulated the phosphorylation of both isoforms, but in the case of zero [Ca2+]e the effect on the more acidic isoform was proportionally greater.
    Brain Research 08/1999; 833(2-833):142-149. DOI:10.1016/S0006-8993(99)01503-6 · 2.83 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The protein B-50 is dephosphorylated in rat cortical synaptic plasma membranes (SPM) by protein phospha-tase type 1 and 2A (PP-1 and PP-2A)-like activities. The present studies further demonstrate that B-50 is dephosphorylated not only by a spontaneously active PP-1-like enzyme, but also by a latent form after pretreatment of SPM with 0.2 mM cobalt/20 μg of trypsin/ml. The activity revealed by cobalt/trypsin was inhibited by inhibitor-2 and by high concentrations (μM) of okadaic acid, identifying it as a latent form of PP-1. In the presence of inhibitor-2 to block PP-1, histone H1 (16–64 μg/ml) and spermine (2 mM) increased B-50 dephosphorylation. This sensitivity to poly-cations and the reversal of their effects on B-50 dephosphorylation by 2 nM okadaic acid are indicative of PP-2A-like activity. PP-1- and PP-2A-like activities from SPM were further displayed by using exogenous phosphorylase a and histone H1 as substrates. Both PP-1 and PP-2A in rat SPM were immunologically identified with monospecific antibodies against the C-termini of catalytic subunits of rabbit skeletal muscle PP-1 and PP-2A. Okadaic acid-induced alteration of B-50 phosphorylation, consistent with inhibition of protein phosphatase activity, was demonstrated in rat cortical synaptosomes after immunoprecipitation with affinity-purified anti-B-50 immunoglobulin G. These results provide further evidence that SPM-bound PP-1 and PP-2A-like enzymes that share considerable similarities with their cytosolic counterparts may act as physiologically important phosphatases for B-50.
    Journal of Neurochemistry 06/1992; 59(1):364 - 374. DOI:10.1111/j.1471-4159.1992.tb08913.x · 4.24 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Exocytosis from nerve terminals is triggered by depolarization-evoked Ca2+ entry, which also activates calmodulin and stimulates protein phosphorylation. Ba2+ is believed to replace Ca2+ in triggering exocytosis without activation of calmodulin and can therefore be used to unravel aspects of presynaptic function. We have analysed the cellular actions of Ba2+ in relation to its effect on transmitter release from isolated nerve terminals. Barium evoked specific release of amino acid transmitters, catecholamines and neuropeptides (EC50 0.2-0.5 mM), similar to K-/Ca(2+)-evoked release both in extent and kinetics. Ba(2+)-and Ca(2+)-evoked release were not additive. In contrast to Ca2+, Ba2+ triggered release which was insensitive to trifluoperizine and hardly stimulated protein phosphorylation. These observations are in accordance with the ability of Ba2+ to replace Ca2+ in exocytosis without activating calmodulin. Nevertheless, calmodulin appears to be essential for regular (Ca(2+)-triggered) exocytosis, given its sensitivity to trifluoperizine. Both Ba(2+)-and Ca(2+)-evoked release were blocked by okadaic acid. Furthermore, anti-calcineurin antibodies decreased Ba(2+)-evoked release. In conclusion, Ba2+ replaces Ca2+/calmodulin in the release of the same transmitter pool. Calmodulin-dependent phosphorylation appears not to be essential for transmitter release. Instead, our data implicate both Ca(2+)-dependent and -independent dephosphorylation in the events prior to neurotransmitter exocytosis.
    European Journal of Pharmacology 12/1995; 291(3). DOI:10.1016/0922-4106(95)90081-0 · 2.68 Impact Factor