title: p80/85 cortactin associates with the Src SH2 domain and colocalizes with v-Src in transformed cells.
abstract: Expression of oncogenic variants of pp60src leads to dramatic changes in cytoskeletal organization characteristic of transformation. Activated Src associates with the cytoskeletal matrix, resulting in tyrosine phosphorylation of specific cytoskeletal substrates. We have previously shown that stable association of Src with the cytoskeletal matrix is mediated by the Src SH2 domain in a phosphotyrosine-dependent interaction. In this report, we demonstrate that one of the cytoskeletal binding partners of Src is p80/85 cortactin. The association was observed in lysates of transformed cells but was not seen in normal fibroblasts. The interaction could be reconstituted in vitro using transformed cell extracts and a glutathione S-transferase (GST) fusion protein containing the Src SH2 domain but not with GST-Src SH3 or with GST-Src SH2 containing a point mutation in the FLVRES sequence. Confocal microscopy revealed that cortactin redistributed and colocalized with v-Src and a Src SH3 deletion mutant in transformed cells. However, in cells expressing a Src SH2 deletion mutant, the redistribution of cortactin and colocalization with Src did not occur. Furthermore, biochemical fractionation 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 protein. Cortactin fractionated from cells expressing kinase-defective or myristylation-defective Src mutants did not exhibit this shift. These data suggest a molecular mechanism by which tyrosine phosphorylation of cortactin and association with the Src SH2 domain influence the cytoskeletal reorganization induced in Src-transformed cells.
authors.affiliation: Cell Biology and Genetics Program, Sloan-Kettering Institute, New York, New York 10021, USA.
authors.forename: H
authors.initials: H
authors.lastname: Okamura
authors.forename: M D
authors.initials: MD
authors.lastname: Resh
mesh_terms: D000818:Animals; D001665:Binding Sites; D002461:Cell Line, Transformed; D002642:Chick Embryo; D051356:Cortactin; D004591:Electrophoresis, Polyacrylamide Gel; D016391:Genes, src; D005982:Glutathione Transferase / Q000096:biosynthesis; D015151:Immunoblotting; D008840:Microfilament Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D015688:Oncogene Protein pp60(v-src) / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure; D018909:src Homology Domains
chemical_list: D051356:Cortactin; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D015688:Oncogene Protein pp60(v-src)