Modulation of synaptosomal protein phosphorylation/dephosphorylation by calcium is antagonised by inhibition of protein phosphatases with okadaic acid.
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.
- SourceAvailable from: Margaret E Gnegy[Show abstract] [Hide abstract]
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 01/1997; 26(3):281-91. DOI:10.1002/(SICI)1098-2396(199707)26:3<281::AID-SYN9>3.0.CO;2-3 · 2.43 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 · 5.29 Impact Factor
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ABSTRACT: A unique feature of neuronal calcium/calmodulin-stimulated protein kinase II (CaM-PK II) is its autophosphorylation. A number of sites are involved and, depending on the in vitro conditions used, three serine and six threonine residues have been tentatively identified as autophosphorylation sites in the alpha subunit. These sites fall into three categories. Primary sites are phosphorylated in the presence of calcium and calmodulin, but under limiting conditions of temperature, ATP, Mg2+, or time. Secondary sites are phosphorylated in the presence of calcium and calmodulin under nonlimiting conditions. Autonomous sites are phosphorylated in the absence of calcium and calmodulin after initial phosphorylation of Thr-286. Mechanisms that lead to a decrease in CaM-PK II autophosphorylation include the thermolability of the enzyme and the activity of protein phosphatases. A range of in vitro inhibitors of CaM-PK II autophosphorylation have recently been identified. Autophosphorylation of CaM-PK II leads to a number of consequences in vitro, including generation of autonomous activity and subcellular redistribution, as well as alterations in conformation, activity, calmodulin binding, substrate specificity, and susceptibility to proteolysis. It is established that CaM-PK II is autophos-phorylated in neuronal cells under basal conditions. Depolarization and/or activation of receptors that lead to an increase in intracellular calcium induces a marked rise in the autophosphorylation of CaM-PK II in situ. The incorporation of phosphate is mainly found on Thr-286, but other sites are also phosphorylated at a slower rate. One consequence of the increase in CaM-PK II autophosphorylation in situ is an increase in the level of autonomous kinase activity. It is proposed that the formation of an autonomous enzyme is only one of the consequences of CaM-PK II autophosphorylation in situ and that some of the other consequences observed in vitro will also be seen. CaM-PK II is involved in the control of neuronal plasticity, including neurotransmitter release and long-term modulation of postreceptor events. In order to understand the function of CaM-PK II, it will be essential to ascertain more fully the mechanisms of its autophosphorylation in situ, including especially the sites involved, the consequences of this autophosphorylation for the kinase activity, and the relationships between the state of CaM-PK II autophosphorylation and the physiological events within neurons.Molecular Neurobiology 02/1991; 5(2-4):179-202. DOI:10.1007/BF02935545 · 5.29 Impact Factor