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: 2.03).
06/1991; 126(2):203-6. DOI: 10.1016/0304-3940(91)90554-7
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.
Available from: Margaret E Gnegy
- "To examine this, we determined whether incubation with AMPH would increase the level of site 3-phospho- synapsin I in the presence of okadaic acid. Okadaic acid is an inhibitor of phosphatases 1 and 2A, which can dephosphorylate synapsin I (Sim et al., 1991). At higher concentrations, okadaic acid also inhibits phosphatase 2B. "
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ABSTRACT: Amphetamine is taken up through the dopamine transporter in nerve terminals and enhances the release of dopamine. We previously found that incubation of rat striatal synaptosomes increases phosphorylation of the presynaptic neural-specific protein, neuromodulin (Gnegy et al., Mol. Brain Res. 20:289-293, 1993). Using a state-specific antibody, we now demonstrate that incubation of rat striatal synaptosomes with amphetamine increases levels of neuromodulin phosphorylated at ser41, the protein kinase C substrate site. Phosphorylation was maximal at 5 min at 37 degrees C at concentrations from 100 nM to 10 microM amphetamine. The effect of amphetamine on the phosphorylation of synapsin I at a site specifically phosphorylated by Ca2+/calmodulin-dependent protein kinase II (site 3), was examined using a state-specific antibody for site 3-phosphosynapsin I. Incubation with concentrations of amphetamine from 1 to 100 nM increased the level of site 3-phospho-synapsin I at times from 30 sec to 2 min. The effect of amphetamine on synapsin I phosphorylation was blocked by nomifensine. The presence of calcium in the incubating buffer was required for amphetamine to increase the level of site 3-phospho-synapsin I. The amphetamine-mediated increase in the content of phosphoser41-neuromodulin was less sensitive to extrasynaptosomal calcium. The amphetamine-mediated increase in the content of site 3-phospho-synapsin I persisted in the presence of 10 microM okadaic acid and was not significantly altered by D1 or D2 dopamine receptor antagonists. Preincubation of striatal synaptosomes with 10 microM of the protein kinase C inhibitor, Ro-31-8220, blocked the amphetamine-mediated increases in the levels of both phosphoser41-neuromodulin and site 3-phospho-synapsin I. Our results demonstrate that amphetamine can alter phosphorylation-related second messenger activities in the synaptosome.
Synapse 07/1997; 26(3):281-91. DOI:10.1002/(SICI)1098-2396(199707)26:3<281::AID-SYN9>3.0.CO;2-3 · 2.13 Impact Factor
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ABSTRACT: Data emerging from a number of different systems indicate that protein phosphatases are highly regulated and potentially responsive to changes in the levels of intracellular second messengers produced by extracellular stimulation. They may therefore be involved in the regulation of many cell functions. The protein phosphatases in the nervous system have not been well studied. However, a number of neuronal-specific regulators (such as DARPP-32 and G-substrate) exist, and brain protein phosphatases appear to have particularly low specific activity, suggesting that neuronal protein phosphatases possess considerable and unique potential for regulation. Several early events following depolarization or receptor activation appear to involve specific dephosphorylations, indicating that regulation of protein phosphatase activity is important for the control of many neuronal functions. This article reviews the current literature concerning the identification, regulation, and function of serine/threonine protein phosphatases in the brain, with particular emphasis on the regulation of the major protein phosphatases, PP1 and PP2A, and their potential roles in modulating neurotransmitter release and postsynaptic responses.
Molecular Neurobiology 02/1991; 5(2-4):229-46. DOI:10.1007/BF02935548 · 5.14 Impact Factor
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ABSTRACT: This article focuses on the role of protein phosphorylation, especially that mediated by protein kinase C (PKC), in neurotransmitter release. In the first part of the article, the evidence linking PKC activation to neurotransmitter release is evaluated. Neurotransmitter release can be elicited in at least two manners that may involve distinct mechanisms: Evoked release is stimulated by calcium influx following chemical or electrical depolarization, whereas enhanced release is stimulated by direct application of phorbol ester or fatty acid activators of PKC. A markedly distinct sensitivity of the two pathways to PKC inhibitors or to PKC downregulation suggests that only enhanced release is directly PKC-mediated. In the second part of the article, a framework is provided for understanding the complex and apparently contrasting effects of PKC inhibitors. A model is proposed whereby the site of interaction of a PKC inhibitor with the enzyme dictates the apparent potency of the inhibitor, since the multiple activators also interact with these distinct sites on the enzyme. Appropriate PKC inhibitors can now be selected on the basis of both the PKC activator used and the site of inhibitor interaction with PKC. In the third part of the article, the known nerve terminal substrates of PKC are examined. Only four have been identified, tyrosine hydroxylase, MARCKS, B-50, and dephosphin, and the latter two may be associated with neurotransmitter release. Phosphorylation of the first three of these proteins by PKC accompanies release. B-50 may be associated with evoked release since antibodies delivered into permeabilized synaptosomes block evoked, but not enhanced release. Dephosphin and its PKC phosphorylation may also be associated with evoked release, but in a unique manner. Dephosphin is a phosphoprotein concentrated in nerve terminals, which, upon stimulation of release, is rapidly dephosphorylated by a calcium-stimulated phosphatase (possibly calcineurin [CN]). Upon termination of the rise in intracellular calcium, dephosphin is phosphorylated by PKC. A priming model of neurotransmitter release is proposed where PKC-mediated phosphorylation of such a protein is an obligatory step that primes the release apparatus, in preparation for a calcium influx signal. Protein dephosphorylation may therefore be as important as protein phosphorylation in neurotransmitter release.
Molecular Neurobiology 02/1991; 5(2-4):87-130. DOI:10.1007/BF02935541 · 5.14 Impact Factor
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