The protein kinase C phosphosite(s) in B-50 (GAP-43) are confined to 15K phosphofragments produced by Staphylococcus aureus V8 protease

Abstract

The neuron-specific phosphoprotein B-50 (= GAP-43/F1/pp46/P57) is an endogenous substrate of protein kinase C (PKC) in rat brain. To examine the location of the PKC phosphosites, phosphorylated B-50 was digested by Staphylococcus aureus V8 protease (SAP). The products migrated in SDS-polyacrylamide gel electrophoresis as two phosphoprotein bands of apparent molecular weight of 15 and 28 kDa (indicated as 15 and 28 K). This study reports further characterization of the 15 and 28 K phosphobands. ACTH 1–24, a characteristic inhibitor, inhibited equally effective the [ 32P]phosphate-incorporation into the 15 and 28 K phosphobands formed by SAP from B-50 endogenously phosphorylated in synaptosomal plasma membrane (SPM). Tests using immunoprecipitation or immunoblotting showed that all polyclonal rabbit B-50 antisera recognized the 28 K phosphoband, but only a minor population B-50 antibodies of a recently developed antiserum 8613 reacted with the 15 K phosphoband. The time course of the SAP digestion indicated that B-50 is degraded first to the 28 K band and then to the 15 K band. [ 32P]phosphate incorporated in B-50 was totally recovered in these phosphobands. Isoelectric focusing resolved the SAP products into one 28 K phosphopeptide of isoelectric point (IEP) 4.8 and the 15 K phosphofragment in at least 4 phosphopeptides, with IEP of 6.1, 6.6, 6.9 and 7.0, respectively. SAP digests of extensively phosphorylated B-50 analysed by isoelectric focusing in narrow pH gradients, showed microheterogeneity in the undigested B-50, the 28 and 15 K phosphofragments. The time course of SAP digestion of B-50 in endogenously phosphorylated SPM and in phosphorylated nerve growth cone membranes, demonstrated that in both types of membranes, 28 and 15 K phosphofragments are consecutively formed, with IEP identical to the phosphofragments derived from isolated B-50. Our findings suggest that the PKC phosphosite(s) in the B-50 protein are restricted to neutral 15 K peptides of the B-50 molecule.