Open in a separate window Figure 10 Requirement of Rac, but not Ras, for CAS/Crk-mediated cell migration. the induction of cell migration, little is known about the actual signal transduction events responsible for this process or how they impact the cells’ migration machinery. Ultimately, the biochemical signals responsible for controlling cell migration must impact adhesion and deadhesion of integrin receptors as well as organization of the actin and myosin cytoskeleton since these events are critical for movement (Huttenlocher et al., 1996). However, the complexity of cell motility suggests that multiple signaling mechanisms exist to regulate this process. For example, ABL Ras/MAP kinase (ERK1 and 2) signaling has been shown to promote phosphorylation of myosin light chain kinase leading to activation Nelonicline of the actin/myosin motor and cell migration (Klemke et al., 1997), whereas the Rho family of small GTPases (i.e., Rac, Rho, Cdc42) control actin business associated with cell motility (Ridley et al., 1992; Takaishi et al., 1993; Hotchin and Hall, 1995; Michiels et al., 1995). Furthermore, the adhesion-dependent signaling molecules focal adhesion kinase (FAK) and c-src may be involved in focal contact disassembly during cell migration (Ilic et al., Nelonicline 1995; Rodier et al., 1995; Cary et al., 1996; Gilmore and Romer, 1996; Parsons, 1996; Hanks and Polte, 1997). Phosphatidylinositol 3-kinase (Rodriguez- Viciana et al., 1997) and PLC (Chen et al., 1994) are also thought to regulate cell migration. Therefore, it is likely that cell migration depends on the coordinate regulation of a number of these signaling events. Ligation of integrin or cytokine Nelonicline receptors promote a cascade of biochemical signals, including the activation of tyrosine kinases leading to phosphorylation of multiple cellular substrates. A family of adaptor proteins (Nck, Crk, Grb2), which consist primarily of src homology 2 (SH2) and 3 (SH3) domains, coordinate these biochemical events by assembling signal-generating complexes. For example, the SH2 domains of these adaptor proteins bind specifically to phosphotyrosine-binding sites in many signaling molecules, while the SH3 domains are critical for coupling to effector molecules and targeting of signaling complexes to discrete sites within the cell (Bar-Sagi et al., 1993). The adaptor protein p130CAS (Crk-associated substrate) is usually a member of a family of structurally related proteins (i.e., Efs/Sin and Hef 1) that was originally identified as a prominent tyrosine-phosphorylated protein in v-crkC and v-srcCtransformed cells (Sakai et al., 1994; Greulich and Hanafusa, 1996; Matsuda and Kurata, 1996). CAS contains an SH3 domain name, two proline-rich regions, and a substrate domain name consisting of 15 potential SH2-binding motifs (Sakai et al., 1994). C-Crk II (Crk) is an adaptor protein Nelonicline that contains an SH2 and two SH3 domains capable of interacting with numerous effector molecules, including tyrosine-phosphorylated CAS (for reviews see Feller et al., 1994(Santa Cruz, CA). Anti-myc antibody 9E10 and anti-Ras (259) was from cocktail tablet; Axiovert microscope (Thornwood, NY) focused at the cellCsubstratum interface. Results Selection of FG Pancreatic Carcinoma Cells with Increased Migration In Vitro and Metastatic Properties In Vivo While cell adhesion to the extracellular matrix is required for cell migration, it is not sufficient. For example, FG pancreatic carcinoma cells attach to vitronectin but fail to migrate on this adhesive ligand, whereas, they readily migrate on collagen (Klemke et al., 1994). This may be explained by differences in adhesion-dependent signaling events that ultimately facilitate cell movement. To identify putative signaling molecules that are associated with the migratory phenotype, FG cells were allowed to attach to either collagen or vitronectin and examined for differences in their phosphoprotein profile. As shown in Fig. ?Fig.1,1, cell attachment to collagen, a migration-competent ligand, resulted in significantly enhanced tyrosine phosphorylation of a 70- and 125C130-kD protein(s), whereas, cell adhesion to vitronectin, which does not support FG cell migration, showed only little tyrosine phosphorylation of these proteins. Open in a separate window.