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 tyrosinephosphorylated 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.

Cortactin: coupling membrane dynamics to cortical actin assembly

“…In support of this, cortactin directly interacts with the recombinant SH2 domain of Src and other SH2 domain-containing proteins, an interaction that is prevented in the presence of a phosphopeptide containing the optimal Src SH2 binding sequence (-pYEEI-;Songyang et al, 1993;Okamura and Resh, 1995). These results indicate that tyrosine phosphorylation of cortactin creates phospho-dependent binding site(s) for Src and perhaps other SH2 domain-containing proteins.…”

“…Coimmunoprecipitation of Src with cortactin suggests that Src directly binds cortactin (Zhan et al, 1994;Okamura and Resh, 1995;van Damme et al, 1997). In support of this, cortactin directly interacts with the recombinant SH2 domain of Src and other SH2 domain-containing proteins, an interaction that is prevented in the presence of a phosphopeptide containing the optimal Src SH2 binding sequence (-pYEEI-;Songyang et al, 1993;Okamura and Resh, 1995).…”

An invasion-related complex of cortactin, paxillin and PKCμ associates with invadopodia at sites of extracellular matrix degradation

“…Cortactin binds to F-actin in vitro, colocalizes with cortical actin at ru ing membranes, and possesses actin-bundling activity that is modulated by c-Src, suggesting a role in membrane motility (Wu and Parsons, 1993;Huang et al, 1997). In v-Srcactivated ®broblasts transformed by Rous sarcoma virus, cell motility and invasiveness are increased, cortactin becomes phosphorylated on tyrosine residues enabling an association with v-Src in podosomes (Okamura and Resh, 1995). Overexpression of cortactin in NIH3T3 ®broblasts or endothelial cells increases cell motility in vitro (Patel et al, 1998;Huang et al, 1998).…”

A Drosophila Model of Multiple Endocrine Neoplasia Type 2

“…c Rab11 encodes a small GTPase that regulates endosomal trafficking of transmembrane receptors (Cullis et al 2002). d cortactin encodes a known substrate for Src kinases (Wu et al 1991;Okamura and Resh 1995). e RhoGap93B is an ortholog of RhoGAP, which itself is a regulator of Ret MEN2 activity (Chiariello et al 1998).…”