P80 85 CORTACTIN ASSOCIATES WITH THE SRC SH2 DOMAIN AND COLOCALIZES WITH V-SRC IN TRANSFORMED-CELLS/

Expression of oncogenic variants of pp60(src) leads to dramatic change s in cytoskeletal organization characteristic of transformation. Activ ated Src associates with the cytoskeletal matrix, resulting in tyrosin e phosphorylation of specific cytoskeletal substrates. We have previou sly shown that stable association of Src with the cytoskeletal matrix is mediated by the Src SH2 domain in a phosphotyrosine-dependent inter action. In this report, we demonstrate that one of the cytoskeletal bi nding partners of Src is p80/85 cortactin. The association was observe d in lysates of transformed cells but was not seen in normal fibroblas ts. The interaction could be reconstituted in vitro using transformed cell extracts and a glutathione S transferase (GST) fusion protein con taining the Src SH2 domain but not with GST-Src SH3 or with GST-Src SH 2 containing a point mutation in the FLVRES sequence, Confocal microsc opy revealed that cortactin redistributed and colocalized with v-Src a nd a Src SH3 deletion mutant in transformed cells. However, in cells e xpressing a Src SH2 deletion mutant, the redistribution of cortactin a nd colocalization with Src did not occur, Furthermore, biochemical fra ctionation of transformed cells indicated that a significant increase in cortactin distribution to the cytoskeletal fraction occurred, which correlated with a shift in the tyrosine-phosphorylated form of the pr otein, Cortactin fractionated from cells expressing kinase-defective o r myristylation-defective Src mutants did not exhibit this shift. Thes e data suggest a molecular mechanism by which tyrosine phosphorylation of cortactin and association with the Src SH2 domain influence the cy toskeletal reorganization induced in Src transformed cells.