Src family kinases are major regulators of various integrin-mediated biological processes, although their functional roles and substrates in cancer metastasis are unknown. We explored the roles of Src family tyrosine kinases in cell migration and the spread of K-1735 murine melanoma cell lines with low or high metastatic potential. Corresponding to elevated cell motility and spreading ability, Fyn was selectively activated among Src family kinases, and the cell motility was blocked by an inhibitor of Src family kinases. Significant tyrosine phosphorylation of cortactin, stable complex formation between activated Fyn and cortactin, and co-localization of cortactin with Fyn at cell membranes were all observed only in cells with high metastatic potential. Both integrin-mediated Fyn activation and hyperphosphorylation of cortactin were observed 2-5 h after stimulation in highly metastatic cells, and they required de novo protein synthesis. We demonstrate that cortactin is a specific substrate and cooperative effector of Fyn in integrin-mediated signaling processes regulating metastatic potential.Malignant tumors are thought to contain subpopulations of cells with differential metastatic capabilities (1). Processes of tumor metastasis consist of multiple steps linked together, including invasion, detachment, intravasation, circulation, adhesion, extravasation, and growth in distant organs (2). Cell locomotion and spreading are key functions of the cells required in most of these processes. Analysis of differences in expression and modification of signaling molecules associated with cell migration and spreading among tumor sublines with different metastatic potentials in a tumor is expected to provide precise information for understanding the molecular mechanisms underlying the development of cancer metastasis.There are studies demonstrating that Src family tyrosine kinases play essential roles in the signaling of integrin-mediated biological processes such as actin organization and cell migration (3-7). In addition, recent reports show that Src is highly activated in colon cancers, particularly in those metastatic to the liver (8). Involvement of Src kinase during metastatic spread of carcinoma cells in NBT-II rat carcinoma cell lines has also been suggested (5). Fyn has been suggested a factor governing the metastatic potential of tumors including murine methylcholanthrene-induced fibrosarcoma cells (9), although the precise mechanism is unknown.Increasing numbers of studies have shown that Src family kinases function through collaboration with their substrates, such as FAK, cortactin, p130Cas (a Crk-associated substrate), and paxillin in cytoskeleton organization and cell migration. FAK is activated upon cell binding to extracellular matrix proteins and forms transient signaling complexes with Src family kinases (10). Cortactin plays essential roles in cortical actin cytoskeleton organization, primarily affecting cell motility and invasion (11-13). A docking protein, p130Cas , plays essential roles in cell attachme...

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