Protein Kinase C Activation Potentiates the Rapid Secretion of the Amyloid Precursor Protein from Rat Cortical Synaptosomes

Authors


  • Abbreviations used : AβPP, amyloid precursor protein ; AβPPs, soluble secreted N-terminal fragment of amyloid precursor protein ; AD, Alzheimer's disease ; ECL, enhanced chemiluminescent ; GAP43, growth-associated protein 43 ; GFAP, glial fibrillary acidic protein ; HBM, HEPES-buffered medium ; MARCKS, myristoylated alaninerich C kinase substrate ; PAGE, polyacrylamide gel electrophoresis ; PDBu, phorbol 12,13-dibutyrate ; PKC, protein kinase C ; SDS, sodium dodecyl sulphate ; T-TBS, 150 mM NaCl and 10 mM Tris-HCl (pH 7.4) containing 0.05% Tween 20.

Address correspondence and reprint requests to Dr. M. McLaughlin at Department of Pharmacology and Neuroscience, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland, U.K.

Abstract

Abstract : In this study we have used the presynaptic-rich rat cerebrocortical synaptosomal preparation to investigate the proteolytic cleavage of the amyloid precursor protein (AβPP) by the α-secretase pathway within the βA4 domain to generate a soluble secreted N-terminal fragment (AβPPs). AβPP was detected in crude cortical synaptosomal membranes, although at a lower density than that observed in whole-tissue homogenates. Protein kinase C (PKC) activation induced a translocation of the conventional PKC isoform β1 and novel PKCε from cytosol to membrane fractions, but there was no alteration in the proportion of AβPP associated with the Tritonsoluble and -insoluble fractions. AβPPs was constitutively secreted from cortical synaptosomes, with this secretion being enhanced significantly by the direct activation of PKC with phorbol ester. The PKC-induced secretion of AβPPs was only partially blocked by the PKC inhibitor GF109203X (2.5 μM), whereas the phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein was significantly inhibited by GF109203X. The differential sensitivities of the MARCKS phosphorylation and AβPPs secretion to GF109203X may imply that different PKC isoforms are involved in these two events in the synaptosomal system. These findings strongly suggest that the α-secretase activity leading to the secretion of AβPPs can occur at the level of the presynaptic terminal.

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