Tyrosine phosphorylation and immunodetection of vinculin in growth cone particle (GCP) fraction and in GCP-cytoskeletal subfractions.

The growth cone, the motile tip of developing neuronal processes, is considered responsible for the exact guidance of axons and synaptogenesis. High activity of tyrosine kinases in growth cones may contribute to the functions of growth cones. Our previous work revealed that vinculin is one of the endogenous substrates for intrinsic tyrosine kinases in the growth cone particle (GCP) fraction isolated from fetal rat brain. In the present study, we examined tyrosine phosphorylation and immunoblot analysis of vinculin in various fractions from fetal rat brains and adult synaptosomal fraction. Tyrosine phosphorylation of vinculin in the GCP fraction was more prominent than in any other fraction from fetal brain or synaptosomes from adult. Compared to other fractions, however, the enrichment of vinculin in the GCP fraction was not observed. Tyrosine phosphorylation of vinculin in the fraction was inhibited by genistein, a specific tyrosine kinase inhibitor. Although vinculin was also phosphorylated by protein kinase C in the GCP fraction, it incorporated a much smaller amount of 32P than MARCKS protein or GAP-43. The cytoskeletal subfraction from the GCP fraction contained a considerable amount of vinculin and it was one of the major substrates for tyrosine kinases in the GCP cytoskeleton. The membrane skeleton from the GCP fraction contained a low amount of vinculin but showed high kinase activity that phosphorylated vinculin. Taken together, our results suggest that tyrosine phosphorylation of vinculin contributes to the cytoskeletal organization of growth cones.