Growth cone enrichment and cytoskeletal association of non-receptor tyrosine kinases.

Fetal rat brain (E18) expresses at least three c-src-like, membrane-associated non-receptor tyrosine kinases: c-src, fyn, and lyn. c-src and fyn are the most abundant and are highly enriched in a subcellular fraction of nerve growth cones (GCPs). To study the cytoskeletal association of these tyrosine kinases, Triton X-100-resistant fractions were prepared from GCPs. All three non-receptor tyrosine kinases are associated with the cytoskeleton to a significant degree with the relative affinities: fyn > c-src > lyn. The binding is sensitive to ionic strength and to phosphotyrosine, but not to phosphoserine or phosphothreonine. To investigate the regulation of this association we used phosphatase inhibitors to increase phosphotyrosine levels in GCPs. This resulted in the release of c-src from the cytoskeleton. Under these conditions tyrosine phosphorylation was increased selectively in released c-src and primarily on tyrosine 527. Cytoskeletally bound c-src had a higher specific kinase activity than Triton X-100-soluble c-src. These findings indicate that src family members interact in a regulated manner with the cytoskeleton in non-transformed cells. This regulation is explained by a model in which c-src binds to the cytoskeleton via its SH2 domain and is released when phosphorylated tyrosine-527 binds to this domain intramolecularly, inhibiting kinase activity.