The relationship between postnatal age and protein tyrosine kinase activity in synaptosomes prepared from the rat forebrain was studied. Synaptosomal particulate and soluble fraction, as well as total homogenates, the cell soluble fraction, and P3, were prepared from rats ranging in postnatal age from 5 to 60 days and analyzed for (a) tyrosine kinase activity using polyglutamyltyrosine (4:1) as the substrate, (b) the presence of endogenous substrates for tyrosine phosphorylation using polyclonal antibodies specific for phosphotyrosine, and (c) levels of pp60src. Enzyme activity, expressed per milligram of protein, in the total homogenate, P3, and both the cell and synaptosomal soluble fractions was highest in the brains of young animals (postnatal days 5-10) and decreased thereafter to adult levels. In contrast, tyrosine kinase activity in the synaptosomal particulate fraction exhibited a unique biphasic developmental profile, increasing to maxima at postnatal days 10 and 20 before decreasing to adult values. Endogenous substrates for tyrosine phosphorylation were identified by incubating subcellular fractions with 2 mM ATP in the presence of sodium orthovanadate and probing nitrocellulose blots of proteins separated by gel electrophoresis with antiphosphotyrosine antibodies. Several phosphotyrosine-containing proteins were detected in the synaptosomal particulate and P3 fractions, including proteins of M(r) 180K, 145K, 120K, 100K, 77K, 68K, 62K, 54K, 52K, and 42K. In the cell soluble fraction a protein doublet of M(r) 54/52K and a 120K protein were the major phosphotyrosine-containing proteins. The 54/52K doublet was the major protein tyrosine kinase substrate in the synaptosomal soluble fraction. There was a general decrease in the immunostaining of phosphotyrosine-containing proteins in the P3 fraction after postnatal day 10. In contrast, the tyrosine phosphorylation of several proteins in the synaptosomal particulate fraction increased to maximal levels during the third and fourth week after birth before declining to adult levels. Western blotting using monoclonal antibody 327, specific for pp60src, showed that pp60src levels decreased throughout development in both homogenate and P3 fractions. The concentration of pp60src in the synaptosomal particulate fraction and postsynaptic densities, in contrast, increased 80-100% during the third and fourth weeks before decreasing slowly to adult levels. The results indicate that the expression of tyrosine kinase and the phosphorylation of synaptic proteins on tyrosine residues at the developing synapse are transiently increased during the latter stages of synaptogenesis and suggest that the phosphorylation of proteins on tyrosine residues may be involved in the regulation of synapse formation.
