GROWTH CONE ENRICHMENT AND CYTOSKELETAL ASSOCIATION OF NONRECEPTOR TYROSINE KINASES

Fetal rat brain (E18) expresses at least three c-src-like, membrane-as sociated non-receptor tyrosine kinases: c-src, fyn, and lyn. c-src and fyn are the most abundant and are highly enriched in a subcellular fr action of nerve growth cones (GCPs). To study the cytoskeletal associa tion of these tyrosine kinases, Triton X-100-resistant fractions were prepared from GCPs. All three non-receptor tyrosine kinases are associ ated with the cytoskeleton to a significant degree with the relative a ffinities: fyn > c-src > lyn. The binding is sensitive to ionic streng th and to phosphotyrosine, but not to phosphoserine or phosphothreonin e. To investigate the regulation of this association we used phosphata se inhibitors to increase phosphotyrosine levels in GCPs. This resulte d in the release of c-src from the cytoskeleton. Under these condition s tyrosine phosphorylation was increased selectively in released c-src and primarily on tyrosine 527. Cytoskeletally bound c-src had a highe r specific kinase activity than Triton X-100-soluble c-src. These find ings indicate that src family members interact in a regulated manner w ith the cytoskeleton in non-transformed cells. This regulation is expl ained 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. (C) 1995 Wiley-Li ss, Inc.