Determination of changes in the phosphorylation state of the neuron-specific protein kinase C substrate B-50 (GAP43) by quantitative immunoprecipitation.
To determine changes in the degree of phosphorylation of the protein kinase C substrate B-50 in vivo, a quantitative immunoprecipitation assay for B-50 (GAP43, F1, pp46) was developed. B-50 was phosphorylated in intact hippocampal slices with 32Pi or in synaptosomal plasma membranes with [gamma-32P]ATP. Phosphorylated B-50 was immunoprecipitated from slice homogenates or synaptosomal plasma membranes using polyclonal anti-B-50 antiserum. Proteins in the immunoprecipitate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the incorporation of 32P into B-50 was quantified by densitometric scanning of the autoradiogram. Only a single 48-kilodalton phosphoband was detectable in the immunoprecipitate, but this band was absent when preimmune serum was used. The B-50 immunoprecipitation assay was quantitative under the following condition chosen, as (1) recovery of purified 32P-labelled B-50 added to slice homogenates or synaptosomal plasma membranes was greater than 95%; and (2) modulation of B-50 phosphorylation in synaptosomal plasma membranes with adrenocorticotrophic hormone, polymyxin B, or purified protein kinase C in the presence of phorbol diester resulted in EC50 values identical to those obtained without immunoprecipitation. With this immunoprecipitation assay we found that treatment of hippocampal slices with 4 beta-phorbol 12,13-dibutyrate stimulated B-50 phosphorylation, whereas 4 alpha-phorbol 12,13-didecanoate was inactive. Thus, we conclude that the B-50 immunoprecipitation assay is suitable to monitor changes in B-50 phosphorylation in intact neuronal tissue.
De Graan
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Division of Molecular Neurobiology, Rudolf Magnus Institute for Pharmacology, Utrecht, The Netherlands.
Dekker
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Oestreicher
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Van der Voorn
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D000818:Animals; D003366:Cosyntropin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D019922:GAP-43 Protein; D007163:Immunosorbent Techniques; D008297:Male; D008970:Molecular Weight; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011112:Polymyxin B / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D019922:GAP-43 Protein; D010703:Phorbol Esters; D010750:Phosphoproteins; D003366:Cosyntropin; C008876:phorbol-12,13-didecanoate; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D011112:Polymyxin B