Abstract. Carcinoma cells selected for their ability to migrate in vitro showed enhanced invasive properties in vivo. Associated with this induction of migration was the anchorage-dependent phosphorylation of p130CAS (Crk-associated substrate), leading to its coupling to the adaptor protein c-CrkII (Crk). In fact, expression of CAS or its adaptor protein partner Crk was sufficient to promote cell migration, and this depended on CAS tyrosine phosphorylation facilitating an SH2-mediated complex with Crk. Cytokine-stimulated cell migration was blocked by CAS lacking the Crk binding site or Crk containing a mutant SH2 domain. This migration response was characterized by CAS/Crk localization to membrane ruffles and blocked by the dominant-negative GTPase, Rac, but not Ras. Thus, CAS/Crk assembly serves as a “molecular switch” for the induction of cell migration and appears to contribute to the invasive property of tumors.

Src kinase activity was found to protect endothelial cells from apoptosis during vascular endothelial growth factor (VEGF)-, but not basic fibroblast growth factor (bFGF)-, mediated angiogenesis in chick embryos and mice. In fact, retroviral targeting of kinase-deleted Src to tumor-associated blood vessels suppressed angiogenesis and the growth of a VEGF-producing tumor. Although mice lacking individual Src family kinases (SFKs) showed normal angiogenesis, mice deficient in pp60c-src or pp62c-yes showed no VEGF-induced vascular permeability (VP), yet fyn-/- mice displayed normal VP. In contrast, inflammation-mediated VP appeared normal in Src-deficient mice. Therefore, VEGF-, but not bFGF-, mediated angiogenesis requires SFK activity in general, whereas the VP activity of VEGF specifically depends on the SFKs, Src, or Yes.

Adhesion to ECM is required for many cell functions including cytoskeletal organization, migration, and proliferation. We observed that when cells first adhere to extracellular matrix, they spread rapidly by extending filopodia-like projections and lamellipodia. These structures are similar to the Rac-and Cdc42-dependent structures observed in growth factor-stimulated cells. We therefore investigated the involvement of Rac and Cdc42 in adhesion and spreading on the ECM protein fibronectin. We found that integrin-dependent adhesion led to the rapid activation of p21-activated kinase, a downstream effector of Cdc42 and Rac, suggesting that integrins activate at least one of these GTPases. Dominant negative mutants of Rac and Cdc42 inhibit cell spreading in such a way as to suggest that integrins activate Cdc42, which leads to the subsequent activation of Rac; both GTPases then contribute to cell spreading. These results demonstrate that initial integrin-dependent activation of Rac and Cdc42 mediates cell spreading. INTRODUCTIONMost cell types respond to surfaces coated with ECM proteins by adhering and then spreading out to acquire a flattened morphology. This process of cell adhesion and spreading is mediated by integrins and involves complex dynamic rearrangements of the actin cytoskeleton. These dynamics appear to be coordinated in space and time by intracellular signaling pathways involving tyrosine kinases, protein kinase C, arachidonic acid metabolism, and, in some cases, intracellular calcium Jacobson, 1992, 1993;Pelletier et al., 1992;Auer et al., 1993;Vuori and Ruoslahti, 1993;Romer et al., 1994). How specific signaling pathways regulate the cytoskeleton is, however, poorly understood.Cells spread by putting out extensions that contact the surface, form adhesions, and then exert tension to induce outward movement. This process is reminiscent of the extensions and adhesions induced by the small GTP-binding proteins Rac and Cdc42. These proteins are closely related members of the Ras superfamily of GTPases, which, like other Ras family members, act as guanine nucleotide-regulated switches. Cdc42 mediates formation of long, thin, actin-dependent extensions called filopodia, whereas Rac mediates formation of curtain-like extensions called lamellipodia and ruffles Nobes and Hall, 1995). Both can induce formation of small substrate adhesions called focal complexes.Rac and Cdc42 interact with a number of effector proteins. The best characterized effectors are the p21-activated kinases (PAKs). Both Rac and Cdc42 in the GTP-bound state interact specifically with PAKs and strongly stimulate PAK kinase activity (Manser et al., 1994;Knaus et al., 1995;Martin et al., 1995). Mutants of Rac and Cdc42 that do not bind and activate PAK1 can still induce lamellipodia and filopodia, respectively (Joneson et al., 1996;Lamarche et al., 1996), however, activated PAK1 mutants themselves induce lamellipodia and cytoskeletal rearrangements (Sells et al., 1997). Thus, the role of PAK1 in mediating effects of small GTPases on the ...