SRC BINDING TO THE CYTOSKELETON, TRIGGERED BY GROWTH CONE ATTACHMENT TO LAMININ, IS PROTEIN-TYROSINE PHOSPHATASE-DEPENDENT

The interaction of the non-receptor tyrosine kinase, Src, with the cyt oskeleton of adhesion sites was studied in nerve growth cones isolated from fetal rat brain. Of particular interest was the role of protein tyrosine phosphatases in the regulation of Src-cytoskeleton binding. G rowth cones were found to contain a high level of protein tryrosine ph osphatase activity, most of it membrane-associated and forming large, multimeric and wheat germ agglutinin-binding complexes. The receptor t yrosine phosphatase PTP alpha seems to be the most prevalent species a mong the membrane-associated enzymes. As seen by immunofluorescence, P TP alpha is present throughout the plasmalemma of the growth cone incl uding filopodia, and it forms a punctate pattern consistent with that of integrin pl For adhesion site analysis, isolated growth cones were either plated onto the neurite growth substratum, laminin, or kept in suspension. Plating growth cones on laminin triggered an g-fold increa se in Src binding to the adherent cytoskeleton, This effect was blocke d completely with the protein tyrosine phosphatase inhibitor, vanadate , Growth cone plating also increased the association with adhesion sit es of tyrosine phosphatase activity (14-fold) and of PTP alpha immunor eactivity (6-fold). Vanadate blocked the enzyme activity but not the r ecruitment of PTP alpha to the adhesion sites. In conjunction with our previous results on growth cones, these data suggest that integrin bi nding to laminin triggers the recruitment of PTP alpha (and perhaps ot her protein tyrosine phosphatases) to adhesion sites, resulting in de- phosphorylation of Src's tyr 527. As a result Src unfolds, becomes kin ase-active, and its SH2 domain can bind to an adhesion site protein, T his implies a critical role for protein tyrosine phosphatase activity in the earliest phases of adhesion site assembly.