Phillip J. Robinson

Chapter 22 Depolarization-dependent protein phosphorylation in synaptosomes: mechanisms and significance

Publisher Summary This chapter reviews the methods used to study synaptosomal protein phosphorylatio to evaluate the limitations of the procedures and the likelihood of introducing in vitro artefacts. It describes the major synaptosomal phosphoproteins and the effect of depolarization on their labeling with 32Pi. The chapter focuses on phosphoproteins having molecular weights between 40 and 90 kDa because these proteins have been extensively investigated in disrupted synaptic tissue. The chapter describes mechanisms of protein kinase activation and control, which account for the observed phosphorylation of proteins before and after depolarization, and outlines the possible functions of the major synaptosomal phosphoproteins in relation to neurotransmitter release. A number of experimental systems have been used to investigate the role(s) of protein phosphorylation in neurotransmitter release and, out of these, synaptosome preparations constitute an excellent model. These subcellular organelles have the particular advantage of being an in vitro nerve terminal preparation that retains the capacity to release neurotransmitters in a physiologically relevant manner. They are relatively homogeneous compared to neuronal cell cultures containing axons, cell bodies, dendrites and nuclei, and brain slices, which contain nonneuronal cells.

Depolarisation-dependent protein phosphorylation and dephosphorylation in rat cortical synaptosomes is modulated by calcium.

Abstract: The effect of calcium on protein phosphorylation was investigated using intact synaptosomes isolated from rat cerebral cortex and prelabelled with 32Pi. For nondepolarised synaptosomes a group of calcium‐sensitive phosphoproteins were maximally labelled in the presence of 0.1 mM calcium. The phosphorylation of these proteins was slightly decreased in the presence of strontium and absent in the presence of barium, consistent with the decreased ability of these cations to activate calcium‐stimulated protein kinases. Addition of calcium alone to synaptosomes prelabelled in its absence increased phosphorylation of a number of proteins. On depolarisation in the presence of calcium certain of the calcium‐sensitive phosphoproteins were further increased in labelling above nondepolarised levels. These increases were maximal and most sustained after prelabelling at 0.1 mM calcium. On prolonged depolarisation at this calcium concentration a slow decrease in labelling was observed for most phosphoproteins, whereas a greater rate and extent of decrease occurred at higher calcium concentrations. At 2.5 mM calcium a rapid and then a subsequent slow dephosphorylation was observed, indicating two distinct phases of dephosphorylation. Of all the phosphoproteins normally stimulated by depolarisation, only phosphoprotein 59 did not exhibit the rapid phase of dephosphorylation at high calcium concentrations. Replacing calcium with strontium markedly decreased the extent of change observed on depolarisation whereas barium decreased phosphorylation changes even further. Taken together these data suggest that an influx of calcium into synaptosomes initially activates protein phosphorylation, but as the levels of intrasynaptosomal calcium rise protein dephosphorylation predominates. Other phosphoproteins were dephosphorylated immediately on depolarisation in the presence of calcium. The fine control of protein phosphorylation levels exerted by calcium supports the idea that the synaptosomal phosphoproteins could play a role in modulating events such as neurotransmitter release in the nerve terminal.

Dephosphorylation of synaptosomal proteins P96 and P139 is regulated by both depolarization and calcium, but not by a rise in cytosolic calcium alone

Abstract: Depolarization of intact synaptosomes activates calcium channels, leads to an influx of calcium, and increases the phosphorylation of several neuronal proteins. In contrast, there are two synaptosomal phosphoproteins labeled in intact synaptosomes with 32P1, termed P96 and P 139, which appear to be dephosphorylated following depolarization. Within intact synaptosomes P96 was found in the cytosol whereas P 139 was present largely in membrane fractions. Depolarization‐stimulated dephosphorylation was fully reversible and continued for up to five cycles of depolarization/repolarization, suggesting a physiological role for the phenomenon. The basal phosphorylation of these proteins was at least partly regulated by cyclic AMP, since dibutyryl cyclic AMP produced small but significant increases in P96 and P 139 labeling, even in the presence of fluphenazine at concentrations that inhibited calcium‐stimulated protein kinases. Depolarization‐dependent dephosphorylation was independent of a rise in intracellular calcium, since agents such as guanidine and low concentrations of A23187, which increase intracellular calcium without activating the calcium channel, did not initiate P96 or P 139 dephosphorylation. These agàents did sustain increases in the phosphorylation of a number of other proteins including synapsin I and protein III. The results suggest that the phosphorylation of these two synaptosomal proteins is intimately linked to the membrane potential and that their dephosphorylation is dependent on both the mechanism of calcium entry and calcium itself, rather than simply on a rise in intracellular free calcium.

Depolarization-dependent protein phosphorylation in rat cortical synaptosomes: the effects of calcium, strontium and barium.

Depolarization of synaptosomes increases the phosphorylation of a number of proteins in a calcium-dependent manner. The concentration of calcium required for optimum stimulation was 0.1 mM, with higher concentrations up to 2.5 mM being progressively less effective. Calcium was significantly better than strontium at increasing depolarization-dependent protein phosphorylation, while barium had no stimulating effect at concentrations above 0.1 mM. The order of potency of these ions is consistent with a calmodulin-stimulated protein kinase being activated on entry of calcium into synaptosomes, but is not consistent with the known efficacy of these ions in stimulating neurotransmitter release. The data show for the first time that phosphorylation of proteins may not be a prerequisite for neurotransmitter release.

Dopamine and serotonin in two populations of synaptosomes isolated by percoll gradient centrifugation.

A technique has recently been developed for the isolation of synaptosomes by centrifugation through percoll gradients. Utilizing this procedure, striatal synaptosomes were separated into two fractions, termed fractions 3 and 4, by their different sedimentation characteristics in percoll. The aim of this investigation was to determine whether there were any neurotransmitter differences between these fractions. The content of endogenous neurotransmitters dopamine (DA) and serotonin (5-HT) significantly differed between these fractions. Fraction 3 contained greater levels of 5-HT, while fraction 4 was enriched for DA. Both fractions were capable of releasing DA or 5-HT upon K(+) depolarization. The results raise the possibility that a relative enrichment of dopaminergic synaptosomes in fraction 4 and of serotonergic synaptosomes in fraction 3 has been achieved.

Calcium-stimulated protein kinases from rat cerebral cortex are inactivated by preincubation.

Summary Preincubation of a lysed P2 fraction from rat cerebral cortex caused a marked decrease in the phosphorylation of the major polypeptides labelled by calcium-stimulated protein kinases. The rate and extent of this decrease was essentially identical for these polypeptides and depended on the availability of calcium during the preincubation period. It is recommended that preincubation procedures should be carefully standardised in order to maintain reproducible phosphoprotein profiles and calcium-stimulated protein kinase activity.

Depolarisation‐Dependent Protein Phosphorylation in Rat Cortical Synaptosomes Is Inhibited by Fluphenazine at a Step After Calcium Entry

Abstract: The sequence of molecular events linking depolarisation‐dependent calcium influx to calcium‐stimulated protein phosphorylation is unknown. In this study the effect of the neuroleptic drug fluphenazine on depolarisation‐dependent protein phosphorylation was investigated using an intact postmitochondrial pellet isolated from rat cerebral cortex. Fluphenazine, in a dose‐dependent manner, completely inhibited the increases in protein phosphorylation observed previously. The concentration of fluphenazine required for 50% inhibition varied for different phosphoproteins but for synapsin I was 123 μM. Other neuroleptics produced effects similar to fluphenazine with their order of potency being thioridazine > haloperidol > trifluoperazine > fluphenazine > chlorpromazine. Fluphenazine also increased the phosphorylation of proteins in nondepolarised controls at concentrations of 20 and 60 μM. The inhibition of depolarisation‐dependent phosphorylation was apparently not due to a loss of synaptosomal integrity or viability, a decrease in calcium uptake, a change in substrate availability, or to a change in protein phosphatase activity. The data are most consistent with an inhibition of protein kinase activity by blockade of calmodulin or phospholipid activation.

The in vitro phosphorylation of actin from rat cerebral cortex

Actin was phosphorylated by a cyclic AMP-stimulated protein kinase in a lysed synaptosomal fraction incubated with [γ-32P]ATP, while calcium had no effect on endogenous labeling of the protein. Incubation of an intact synaptosomal fraction with32P-inorganic phosphate did not lead to any detectable phosphorylation of actin in the presence or absence of dibutryl-cyclic AMP, or chemical depolarization. It is suggested that actin is not phosphorylated in the physiologically relevant intact synaptosomes but gains access to protein kinases on lysis.

Phosphorylation of synaptosomal cytoplasmic proteins: inhibition of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) by Bay K 8644

The phosphorylation of specific substrates of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was examined in striatal synaptosomal cytoplasm. The phosphoprotein substrata were termed group C phosphoprotems and were divided into two subgroups: group C(1) phosphoproteins (P83, P45A, P21 and P18) were found in both cytoplasm and synaptosomal membranes and, although stimulated by phosphatidylserine, only required exogamous calcium for their labeling; group C(2) phosphoproteins (P120, P96, P21.5, P18.5 and P16) were found predominantly in the cytoplasm and were absolutely dependent upon exogenous calcium and phosphatidylserme for their labeling. Several criteria were used to identify these proteins as specific protein kinase C substrates: (a) their phosphorylation was stimulated to a greater extent by Ca(2+) /phosphatidylserine/diolein than by Ca(2+) alone or Cal(2+) /calmodulin (group C(1)) or was completely dependent upon Ca(2+) /phosphatdylserine/diolein (group C(2)); (b) supermaximal concentrations of the cAMP-dependent protein kinase inhibitor were without effect; (c) their phosphorylation was stimulated by oleic acid, which selectively activates protein kinase C in the absence of Ca(2+); (d) NaCl, which inhibited cAMP- and Ca(2+)/calmodulindependent phosphorylation, slightly increased phosphorylation of group C(1) and slightly decreased phosphorylation of group C(2) phosphoproteins. Maximal phosphorylation of P96 and other group C phosphoproteins occurred within 60 s and was followed by a slow decay rate while substrata of calmodulin-dependent protein kinase were maximally labeled within 20-30 s and rapidly dephosphorylated. The phosphorylation of all group C phosphoproteins was inhibited by the calcium channel agomst BAY K 8644, however, group C(2) phosphoproteins were considerably more sensitive. The IC(50) for inhibition of P96 labeling was 19 ?M. but for P83 was 190 ?M. Group B phosphoproteins were also slightly inhibited, and the IC(50) for P63 was 290 ?M. No inhibitory effects of another dihydropyridine, nifedipine, or of verapamil were detected in this concentration range. BAY K 8644 did not displace [(3)H]phorbol-12,13-dibutyrate binding, nor was the inhibition decreased by increasing phosphatidylserine concentrations. BAY K 8644 had no effect on the rate of dephosphorylation of any phosphoprotein, indicating that it is unlikely to inhibit a protein phosphatase. BAY K 8644 may, therefore, prove to be a valuable tool for discriminating protein kinase C activity from the activity of other protein kinases. We conclude that BAY K 8644 interacts either with a specific subgroup of protein kinase C substrata or with one of two putative forms of protein kinase C.