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 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.

Determination of the endogenous phosphorylation state of B‐50/GAP‐43 and neurogranin in different brain regions by electrospray mass spectrometry

“…The degree of phosphorylation in vitro is therefore thought to represent the number of non-phosphorylated sites in vivo. An alternative to this post-hoc phosphorylation assay, which more closely measures the degree of phosphorylation in vivo, involves in situ labelling of the proteins by incubating intact tissues with 32p and subsequent immunoprecipitation of the protein of interest (for instance, see [2]). …”

Long-term potentiation and synaptic protein phosphorylation

“…Intracellular injection of PKC (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31), a peptide fragment representing the pseudosubstrate region of the regulatory domain of PKC, prevented the maintenance of LTP in the injected cell whereas the neighbouring cells showed normal LTP [48]. These results indicate that postsynaptic PKC is involved in the maintenance of LTP.…”

“…This post-hoc phosphorylation assay has some interpretation problems and therefore a new method of quantitative immunoprecipitation was developed to determine the in situ phosphorylation of B-50 in the hippocampal slice [22]. Using this assay we have performed a series of experiments with the following experimental design: three hippocampal slices (450 #m) were placed in a recording chamber, in two slices stimulation and recording electrodes were placed, hereafter slices were labelled with 100/~Ci/ml [32p]orthophosphate for 90 rain.…”

“…One of the slices received a high frequency train of pulses (100 Hz, 1 s, test intensity), whereafter experiments were continued for 10, 30, 60, 90 or 120 min, the electrodes were removed, and the slices were homogenized. B-50 phosphorylation in these slices was then determined after quantitative immunoprecipitation [22].…”

Methods and approaches for the comprehensive characterization and quantification of cellular proteomes using mass spectrometry

“…Immunoprecipitation of proteins from complex mixtures with antibodies against phosphorylated amino acid residues is a common approach in phosphoproteomics (25,32,47,48). Most analyses have successfully used pTyr antibodies (84,126), and recently attempts have been made to precipitate proteins with pSer and pThr antibodies (35).…”