The ultimate goal of the experiments proposed herein is to identify tyrosine kinase substrates and their function in developing and mature brain. Affinity purified polyclonal antibodies to phosphotyrosine will be used to determine the dynamics of tyrosine phosphorylation at cellular, subcellular and substrate levels. Previous immunohistochemical studies indicate that the majority of tyrosine phosphorylation detectable by these methods is in microglia and is associated with growing axons. The primary focus will be on establishing which proteins are tyrosine-phosphorylated in these two compartments, and on determining their subcellular location. Two tyrosine kinase substrates in cerebellar homogenates (135 and 95 kD) have a developmental pattern of phosphotyrosine-immunoreactivity coincident with that of axonal growth in the cerebellum. Consequently, antibodies will be generated to these two species for use in cellular and subcellular localization experiments to determine whether these may be axonal growth-associated proteins (GAPs). As an alternative approach, GAPs in developing optic nerve will be probed with anti-phosphotyrosine antibodies for the presence of these or other tyrosine kinase substrates. To investigate the second phosphotyrosine- immunoreactive compartment, experiments are proposed to determine the tyrosine kinase substrates of primary cultures enriched in microglia. Established in vitro protocols will enable a study of the dynamics of tyrosine phosphorylation during microglial proliferation, differentiation, activation and subsequent secretion of glial promoting factors (GFPs). The inability of CNS neurons to regenerate may be due to the formation of glial scars, which might discourage axon growth and are formed at least in part through the action of microglial GPFs. As the preliminary data suggests tyrosine phosphorylation occurs in growing axons and microglia, the information gathered in the proposed experiments has the potential to promote our understanding of and ability to influence repair mechanisms in the CNS.
