Protein phosphorylation in isolated synaptic junctional structures: changes during development

Endogenous phosphorylation in subcellular fractions enriched in synaptic plasma membranes (SPM) and purified synaptic junctions (SJ) has been examined in vitro at various stages of postnatal development. Protein kinase activity was measured using both endogenous and exogenous (histones) proteins as substrates. Protein phosphorylation that is regulated by cyclic AMP also was investigated. SPM and SJ fractions displayed large increments in kinase activities and cyclic AMP-stimulated protein phosphorylation between postnatal days 5 and 15. SJ fractions exhibited a dramatic age-dependent change in the cyclic AMP-stimulated phosphorylation of endogenous proteins of molecular weights 73,000 (1a), 68,000 (1b), 65,000 (1c) and 55,000. Experiments in which isolated SJs were phosphorylated with soluble cyclic AMP-dependent kinase showed that the phosphorylation of proteins 1a, 1b and 1c resulted from their de novo appearance in newborn SJ fractions and not from a maturation-dependent coupling of kinases and substrates that were already present in newborn SJ fractions. The levels of regulatory (R) subunits of cyclic AMP-dependent kinases in synaptic fractions were measured by [3H]cyclic AMP binding and photoaffinity labeling with [32P]8-N3-cyclic AMP and were observed to change little during postnatal development. These findings show that there is a strong correlation between the in situ appearance of synapses and the enzymatic maturation of endogenous, cyclic AMP-stimulated protein phosphorylation in SJ fractions isolated at different stages of development.