Synaptic vesicles isolated from 32P-prelabeled synaptosomes contain a phosphoprotein of apparent M(r) 65,000 (pp65), a possible substrate for PKC.
In the present study, we have investigated the subcellular localization of pp65, a synaptosomal phosphoprotein of apparent M(r) 65,000. The results obtained strongly support that pp65 is localized to synaptic vesicles. The solubility properties of pp65, especially its partitioning into the detergent phase of Triton X-114, indicated that it is tightly associated with the membrane of synaptic vesicles. pp65 is multiply phosphorylated exclusively on serine. By studying the decay of labeled phosphate following incubation of 32P-prelabeled synaptosomes in the presence of cold inorganic phosphate, we have found that pp65 shows an unusually high turnover of phosphate. Exposure of synaptosomes to 1 microM phorbol 12-myristate 13-acetate prior to prelabeling with 32P(i) led to a reduction in the steady state phosphorylation of pp65, and tryptic/chymotryptic mapping was shown to selectively affect phosphopeptide 4. Identical results were obtained following incubation of synaptosomes with the protein kinase C (PKC) inhibitor, GF 109203 X. These results indicated that one of the protein kinases involved in steady state phosphorylation of pp65 is PKC-dependent or is PKC itself. Several characteristics of pp65 reported in the present study suggest a regulatory role in nerve terminal function.
Gómez-Puertas
P
P
Departamento de Biología Molecular, Universidad Autonoma de Madrid, Spain.
Satrústegui
J
J
Bogónez
E
E
D000818:Animals; D010750:Phosphoproteins / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D012995:Solubility; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity; D013574:Synaptosomes / Q000378:metabolism*; D013696:Temperature
D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011493:Protein Kinase C