During periods of active growth, neurons increase synthesis of a class of proteins that function in the establishment of normal structure and synaptic relations. Among these is GAP(growth-associated)-43 or B-50. A role for this protein in determination of synaptic plasticity is suggested by the co-ordination between its synthesis and axonal growth, its continued expression throughout adult life in the associative areas of the brain and its relationship to long-term potentiation. Neurochemical functions of B-50, which include regulation of phosphoinositide metabolism and Ca2+-dependent processes, are related to its phosphorylation. Well- characterized as a substrate for protein kinase C (PKC), more recent work has demonstrated that B-50 is an in vitro substrate for casein kinase II (CKII) and phosphorylated in intact neurons by an unidentified kinase which recognizes serine or threonine in close proximity to proline residues (MAP kinase-like sites). This project will analyze in vitro phosphorylation of B-50 to identify the latter protein kinase and to examine interactions between the substrate sites in B-50 that allow phosphorylation by each protein kinase. The functional significance of each phosphorylation will be compared to that of PKC with regard to calmodulin binding. The site phosphorylated by each kinase will by characterized with purified substrate and in presynaptic membranes. Dephosphorylation of each substrate site by specific protein phosphatases will be characterized, since such enzymes may limit the extent of in vivo phosphorylation. Preliminary work with growth cones prepared from neonatal rat brains has demonstrated phosphorylation of B-50 on at least one other site in addition to serine 41, the PKC site. These site(s) will be fully characterized. Comparison of B-50 phosphorylation sites and protein kinase activities in growth cones will be compared to those seen in synaptosomes from adult rat brain to determine if the modification of these sites is developmentally-regulated. Analysis of the abilities of PKC, CKII and MAP kinases to phosphorylate B-50 will yield information on how growth stimuli are selectively and temporally coupled to neuronal second messenger systems.
