Determination of Changes in the Phosphorylation State of the Neuron‐Specific Protein Kinase C Substrate B‐50 (GAP43) by Quantitative Immunoprecipitation

Abstract

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 ³²P_i or in synaptosomal plasma membranes with [γ-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 >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 4β-phorbol 12,13-dibutyrate stimulated B-50 phosphorylation, whereas 4α-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.