Surface protein phosphorylation by ecto-protein kinase is required for the maintenance of hippocampal long-term potentiation.

Abstract

During the induction of long-term potentiation (LTP) in hippocampal slices adenosine triphosphate (ATP) is secreted into the synaptic cleft, and a 48 kDa/50 kDa protein duplex becomes phosphorylated by extracellular ATP. All the criteria required as evidence that these two proteins serve as principal substrates of ecto-protein kinase activity on the surface of hippocampal pyramidal neurons have been fulfilled. This phosphorylation activity was detected on the surface of pyramidal neurons assayed after synaptogenesis, but not in immature neurons nor in glial cells. Addition to the extracellular medium of a monoclonal antibody termed mAb 1.9, directed to the catalytic domain of protein kinase C (PKC), inhibited selectively this surface protein phosphorylation activity and blocked the stabilization of LTP induced by high frequency stimulation (HFS) in hippocampal slices. This antibody did not interfere with routine synaptic transmission nor prevent the initial enhancement of synaptic responses observed during the 1-5 min period immediately after the application of HFS (the induction phase of LTP). However, the initial increase in the slope of excitatory postsynaptic potentials, as well as the elevated amplitude of the population spike induced by HFS, both declined gradually and returned to prestimulus values within 30-40 min after HFS was applied in the presence of mAb 1.9. A control antibody that binds to PKC but does not inhibit its activity had no effect on LTP. The selective inhibitory effects observed with mAb 1.9 provide the first direct evidence of a causal role for ecto-PK in the maintenance of stable LTP, an event implicated in the process of learning and the formation of memory in the brain.The long-term potentiation (LTP) of synaptic strength in hippocampal pyramidal neurons is a neurophysiological process iinplicated in the formation of memory traces in the brain (1, 2). While the initial chain of events that triggers this process is well-known, the mechanisms that determine the duration and stability of LTP have not yet been fully elucidated. Although it is generally accepted that the phosphorylation of proteins by several different kinases is involved in this stabilization, their exact localization and roles are still obscure (2, 3). Protein phosphorylation, a ubiquitous step in intracellular pathways that produce transient changes in neuronal activity, was found to serve also as a key mechanism of molecular adaptation in processes underlying the induction of longlasting alterations in synaptic function (for reviews, see ref. 4), including learning and memory formation (5-7). The first study that implicated protein phosphorylation in the process of learning (5) identified a synaptic phosphoprotein that was later shown to be a major neuronal substrate of protein kinase C (PKC), that plays a role in the maintenance-phase of LTP (8, 9). The specific PKC-isozyme involved in the maintenance of LTP was reported to be PKC C, a member of the atypical class (not stimulated by diacylglycerol or phorbol ...

A Novel Method for the Simultaneous Enrichment, Identification, and Quantification of Phosphopeptides and Sialylated Glycopeptides Applied to a Temporal Profile of Mouse Brain Development

“…Very little is known about the kinases/phosphatases involved in this process however, preliminary evidence suggests that extracellular phosphorylation plays a role in neuronal signal transduction (53,54) and long-term potentiation in hippocampal neurons (55). The combination of the presented method with specific analysis of extracellular extracts and/or the use of cell impermeable kinase inhibitors (56) may be a valuable approach to investigate this relatively unexplored phenomenon.…”

Phosphorylation of the β-Amyloid Precursor Protein at the Cell Surface by Ectocasein Kinases 1 and 2

“…Ectoprotein kinases phosphorylate membranebound proteins as well as soluble extracellular proteins. Phosphorylation of cell-surface proteins has been implicated in the regulation of certain cellular functions, including long-term potentiation in neurons (38) and synaptogenesis (39). However, the relevant protein substrates that are phosphorylated by ectoprotein kinases in these processes remain to be identified.…”

Ecto-Phosphorylation and Regeneration of the Adult Central Nervous System

“…A 48/50 kDa protein at the surface of hippocampal pyramidal neurons becomes phosphorylated during the amplification [45]. Addition of a neutralising antibody against PKC inhibits the extracellular 48/50 kDa protein phosphorylation, and it blocks the stabilisation of long-term potentiation in hippocampal slices [45]. Thus, PKC-mediated extracellular phosphorylation is required for maintenance of hippocampal long-term potentiation.…”

“…Extracellular phosphorylation of neuronal surface proteins is implicated in long-term potentiation in the hippocampus [44][45][46]. Using mouse hippocampal slices, addition of either ATP or ATPgammaS, but not AppNHp, was reported to amplify permanently the magnitude of the population spike [44].…”

“…Using mouse hippocampal slices, addition of either ATP or ATPgammaS, but not AppNHp, was reported to amplify permanently the magnitude of the population spike [44]. A 48/50 kDa protein at the surface of hippocampal pyramidal neurons becomes phosphorylated during the amplification [45]. Addition of a neutralising antibody against PKC inhibits the extracellular 48/50 kDa protein phosphorylation, and it blocks the stabilisation of long-term potentiation in hippocampal slices [45].…”