Determination of Changes in the Phosphorylation State of the Neuron‐Specific Protein Kinase C Substrate B‐50 (GAP43) by Quantitative Immunoprecipitation
Аннотация: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 32 P i or in synaptosomal plasma membranes with [γ‐ 32 P]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 32 P 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 32 P‐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 EC 50 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.
