We previously showed that activation of the small GTPase Cdc42 promotes breast cell migration on a collagen matrix. Here we further define the signaling pathways that drive this response and show that Cdc42-mediated migration relies on the adaptor molecule p130Cas . Activated Cdc42 enhanced p130Cas phosphorylation and its binding to Crk. Cdc42-driven migration and p130 Cas phosphorylation were dependent on the Cdc42 effector Ack1 (activated Cdc42-associated kinase). Ack1 formed a signaling complex that also included Cdc42, p130Cas , and Crk, formation of which was regulated by collagen stimulation. The interaction between Ack1 and p130Cas occurred through their respective SH3 domains, while the substrate domain of p130 Cas was the major site of Ack1-dependent phosphorylation. Signaling through this complex is functionally relevant, because treatment with either p130Cas or Ack1 siRNA blocked Cdc42-induced migration. These results suggest that Cdc42 exerts its effects on cell migration in part through its effector Ack1, which regulates p130Cas signaling.Integrin-mediated cell interactions with components of the extracellular matrix regulate several aspects of both epithelial polarization and migration. Integrins are involved in signaling pathways that include activation of small GTPases, scaffolding molecules, and protein kinases (1-3). These signaling pathways regulate various aspects of cell migration on integrin ligands, such as collagen.Members of the Rho family of GTPases, Rho, Rac, and Cdc42, are important regulators of the actin cytoskeleton and have been shown to mediate cell migration (4 -10, for review see Refs. 3 and 11). Although much work has focused on understanding the mechanism by which Rho GTPases regulate the actin cytoskeleton, relatively less is known about the effects they have on integrin signaling pathways.Ack1 and Ack2 are homologous tyrosine kinases that bind exclusively to activated Cdc42-GTP, but not Rac or Rho (12, 13). The structure of Ack includes a kinase domain, an SH3 2 domain, a Cdc42/Rac-interactive binding (CRIB) domain, and a proline-rich C terminus, which is the key determinant between Ack1, a 120-kDa protein (12,14) and Ack2 an 83-kDa isoform (13). Multiple interactions have been identified for Ack1. In vitro binding to the proline-rich region has been demonstrated for Nck (15), Grb2 (16), Src (15), and Hck (17), whereas the SH3 domain binds HSH2, an adaptor in hematopoietic cells (18). Ack1 can also be co-immunoprecipitated with clathrin (16) and sorting nexin 9 (19), components of vesicle dynamics. Ack kinases have been implicated in a variety of signaling pathways. Tyrosine phosphorylation of Ack1 and Ack2 has been demonstrated downstream of growth factors (13, 16), cell adhesion (14, 20), and muscarinic receptors (21). Ack1 phosphorylates and activates Dbl, an exchange factor for Rho (22), as well as Ras/GRF1, an exchange factor for Ras (23). Ack1 also mediates p130Cas phosphorylation downstream of melanoma chondroitin sulfate proteoglycan (24), whereas Ack2 is implicated...