The study of retroviral oncogenes has revealed unexpected probes for neuronal development. The normal cellular c-src proto-oncogene encodes a non receptor protein tyrosine kinase (PTK), termed pp60(c-src), that is preferentially expressed in nerve growth cones of developing and regenerating neurons. The fyn and c-yes proto-oncogenes, which encode closely related PTKs of unknown function, are also expressed in developing neurons. We have identified a functional association between pp60(c-src) and the neural cell adhesion molecules L1 and NCAM, which mediate neurite outgrowth and fasciculation. Triggering of L1 and NGAM in growth cone- enriched membranes from fetal rat brain with specific ligands or antibodies inhibits pp60(c-src)-dependent phosphorylation of tubulin and other endogenous substrates. Moreover, cerebellar neurons from homozygous mutant mice lacking a functional c-src gene display reduced neurite outgrowth on Ll, but not laminin. This proposal will investigate the hypothesis that nonreceptor PTKs encoded by the c-src, fyn, and yes proto- oncogenes function in neural cell adhesion signaling pathways, and may alter the growth cone cytoskeleton directly or indirectly to modulate neurite growth or pathfinding. In a molecular biological, biochemical, and cell biological approach we shall (i) analyze neurite outgrowth on adhesion molecules in cerebellar and sensory neuronal cultures from mice lacking functional src, fyn, or yes genes to reveal redundant or distinct signaling pathways; (2) determine by phosphopeptide mapping if L1 or NCAM regulates PTK activity in growth cone membranes and intact cells by altering the phosphorylation of specific residues in pp60(c-src), and test for direct physical association of pp60(c-src) and adhesion molecules by chemical crosslinking and coimmunoprecipitation. We will also (3) identify tyrosine kinase substrates and second messenger pathways regulated by pp60(c-src) in neuronal cultures; (4) measure effects of PTK activity on the growth conecytoskeleton and microtubule dynamics by video-enhanced- differential interference contrast microscopy; and finally (5) clone and characterize protein tyrosine phosphatase genes expressed in fetal rat brain as potential regulators of PTK activity in growth cones. This research will provide insight into the molecular mechanism of axon outgrowth essential for the development and regeneration of the mammalian nervous system.
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