SRC family kinases play essential roles in a variety of cellular functions, including proliferation, survival, differentiation, and apoptosis. The activities of these kinases are regulated by intramolecular interactions and by heterologous binding partners that modulate the transition between active and inactive structural conformations. p130 CAS (CAS) binds directly to both the SH2 and SH3 domains of c-SRC and therefore has the potential to structurally alter and activate this kinase. In this report, we demonstrate that overexpression of full-length CAS in COS-1 cells induces c-SRC-dependent tyrosine phosphorylation of multiple endogenous cellular proteins. A carboxy-terminal fragment of CAS (CAS-CT), which contains the c-SRC binding site, was sufficient to induce c-SRC-dependent protein tyrosine kinase activity, as measured by tyrosine phosphorylation of cortactin, paxillin, and, to a lesser extent, focal adhesion kinase. A single amino acid substitution located in the binding site for the SRC SH3 domain of CAS-CT disrupted CAS-CT's interaction with c-SRC and inhibited its ability to induce tyrosine phosphorylation of cortactin and paxillin. Murine C3H10T1/2 fibroblasts that expressed elevated levels of tyrosine phosphorylated CAS and c-SRC-CAS complexes exhibited an enhanced ability to form colonies in soft agar and to proliferate in the absence of serum or growth factors. CAS-CT fully substituted for CAS in mediating growth in soft agar but was less effective in promoting serum-independent growth. These data suggest that CAS plays an important role in regulating specific signaling pathways governing cell growth and/or survival, in part through its ability to interact with and modulate the activity of c-SRC.Homeostasis in multicellular organisms is maintained through the integration of diverse environmental signals for survival, proliferation, differentiation, and apoptosis. These signals are sensed by a variety of cell surface receptors that are coupled to complex networks of cytoplasmic regulatory proteins. SRC family nonreceptor protein tyrosine kinases (PTKs) are important components of many of these signaling networks, including those originating from integrin receptors, receptor PTKs, G-protein-coupled receptors, and cytokine receptors (for reviews, see references 1, 45, 54, 70 and 77). The activities of SRC kinases are tightly regulated and repressed under most circumstances. The importance of this negative regulation is highlighted by the fact that expression of constitutively activated forms of c-SRC results in cellular transformation, characterized by uncontrolled cell proliferation and deregulated cell survival (56, 61).The unique structure of SRC family kinases allows them to be regulated by substrate availability, as well as by the presence of other interacting proteins (31,45,54,55,70,72,74,77,86,87,91). Activity is down-modulated by a series of intramolecular interactions that impose conformational constraints on the catalytic domain, making it inaccessible to the substrate. This inactive confo...

Uptake of Yersinia pseudotuberculosis into mammalian cells involves engagement of β1 integrin receptors by the bacterial protein invasin. This triggers a host response that involves tyrosine phosphorylation of proteins and the induction of actin rearrangements that lead to cellular uptake of bacteria. In this report, we show that the focal adhesion protein CAS plays an important role in Yersinia uptake, and that its function is linked to the phosphorylation‐dependent interaction between CAS and Crk. These studies demonstrate that Yersinia binding to host cell receptors initiates a cascade of events involving tyrosine phosphorylation of CAS, subsequent formation of functional CAS–Crk complexes and the activity of the small GTP‐binding protein Rac1. The delineation of this pathway lends support for a model in which Yersinia uptake into human epithelial cells is dependent upon aspects of host signalling pathways that govern actin cytoskeleton remodelling and cell migration.

The adapter molecule CAS is localized primarily within focal adhesions in fibroblasts. Because many of the cellular functions attributed to CAS are likely to be dependent on its presence in focal adhesions, this study was undertaken to identify regions of the protein that are involved in its localization. The SH3 domain of CAS, when expressed in isolation from the rest of the protein, was able to target to focal adhesions, whereas a variant containing a point mutation that rendered the SH3 domain unable to associate with FAK remained cytoplasmic. However, in the context of full-length CAS, this mutation did not prevent CAS localization to focal adhesions. Two other variants of CAS that contained deletions of either the SH3 domain alone, or the SH3 domain together with an adjoining proline-rich region, also retained the capacity to localize to focal adhesions. A second focal adhesion targeting region was mapped to the extreme carboxy terminus of CAS. The identification of this second focal adhesion targeting domain in CAS ascribes a previously unknown function to the highly conserved C terminus of CAS. The regulated targeting of CAS to focal adhesions by two independent domains may reflect the important role of CAS within this subcellular compartment.

The interplay between pathogen-encoded virulence factors and host cell signaling networks is critical for both the establishment and clearance of microbial infections. Yersinia uptake into host cells serves as an in vitro model for exploring how host cells respond to Yersinia adherence. In this study, we provide insight into the molecular nature and regulation of signaling networks that contribute to the uptake process. Using a reconstitution approach in Fak-/- fibroblasts, we have been able to specifically address the interplay between Fak, Cas and Pyk2 in this process. We show that both Fak and Cas play roles in the Yersinia uptake process and that Cas can function in a novel pathway that is independent of Fak. Fak-dependent Yersinia uptake does not appear to involve Cas-Crk signaling. By contrast, Cas-mediated uptake in the absence of Fak requires Crk as well as the protein tyrosine kinases Pyk2 and Src. In spite of these differences, the requirement for Rac1 activity is a common feature of both pathways. Furthermore, blocking the function of either Fak or Cas induces similar morphological defects in Yersinia internalization, which are manifested by incomplete membrane protrusive activity that is consistent with an inhibition of Rac1 activity. Pyk2 also functions in Yersinia uptake by macrophages, which are physiologically important for clearing Yersinia infections. Taken together, these data provide new insight into the host cellular signaling networks that are initiated upon infection with Y. pseudotuberculosis. Importantly, these findings also contribute to a better understanding of other cellular processes that involve actin remodeling, including the host response to other microbial pathogens, cell adhesion and migration.