p130(Cas) (crk associated substrate) has the structural characteristics of an adapter protein, containing multiple consensus SH2 binding sites, an SH3 domain, and a proline-rich domain. The structure of p130(Cas) suggests that it may act to provide a framework for protein-protein interactions; however, as yet, its functional role in cells is unknown. In this report we show that p130(Cas) is localized to focal adhesions. We demonstrate that p130(Cas) associates both in vitro and in vivo with pp125(FAK) (focal adhesion kinase), a kinase implicated in signaling by the integrin family of cell adhesion receptors. p130(Cas) also associates with pp41/43(FRNK) (pp125(FAK)-related, non-kinase), an autonomously expressed form of pp125(FAK) composed of only the C-terminal noncatalytic domain. We show that the association of p130(Cas) with pp125(Fak) and pp41/43(FRNK) is direct, and is mediated by the binding of the SH3 domain of p130(Cas) to a proline-rich sequence present in both the C terminus of pp125(FAK) and in pp41/43(FRNK). In agreement with recent studies we show that p130(Cas) is tyrosine-phosphorylated upon integrin mediated cell adhesion. The association of p130(Cas) with pp125(FAK), a kinase which is activated upon cell adhesion, is likely to be functionally important in integrin mediated signal transduction.
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...
The protein tyrosine phosphatase PTP-PEST displays remarkable substrate specificity, in vitro and in vivo for p130cas a signalling intermediate implicated in mitogenic signalling, cell-adhesion induced signalling, and in transformation by a variety of oncogenes. We have identified a high affinity interaction between the SH3 domain of p130cas and a proline-rich sequence (P335PPKPPR) within the C-terminal segment of PTP-PEST. Mutation of proline 337 within this sequence to alanine significantly impairs the ability of PTP-PEST to recognise tyrosine phosphorylated p130cas as a substrate, without qualitatively affecting the selectivity of the interaction. Thus the highly specific nature of the interaction between PTP-PEST and p130cas appears to result from a combination of two distinct substrate recognition mechanisms; the catalytic domain of PTP-PEST contributes specificity to the interaction with p130cas, whereas the SH3 domain-mediated association of p130cas and PTP-PEST dramatically increases the efficiency of the interaction. Furthermore, our results indicate that one important function of the p130cas SH3 domain is to associate with PTP-PEST and thereby facilitate the dephosphorylation of p130cas, resulting in the termination of tyrosine phosphorylation-dependent signalling events downstream of p130cas.
Recombinant secretory leukoprotease inhibitor (rSLPI), a recombinant form of a natural airway inhibitor of neutrophil elastase (NE), is a potential therapeutic agent for cystic fibrosis (CF), a condition characterized by airway derangement mediated in part by the large burden of NE on the CF respiratory epithelial surface. After in vitro studies that demonstrated that aerosolized rSLPI retains its form and function, rSLPI was administered via aerosol to normal individuals and individuals with CF to determine the pharmacokinetics of in vivo rSLPI augmentation of the anti-NE defenses of the respiratory epithelial surface. After rSLPI aerosolization to normal individuals (100 mg single dose or 100 mg twice daily for 1 wk) there was a marked increase in SLPI levels and anti-NE capacity in airway epithelial lining fluid (ELF) at 1 h, diminishing gradually over 4 to 12 h. Interestingly, the ELF SLPI levels and anti-NE capacity achieved 12 h after 1 wk of rSLPI aerosols were no different than those 12 h after a single dose of rSLPI, suggesting that rSLPI does not accumulate on the respiratory epithelial surface after aerosolization. The ability of rSLPI to suppress NE in vivo was evaluated by aerosolization of rSLPI to individuals with CF, first as an escalating dose to assess safety, and then at doses of 100 mg twice daily for 1 wk or 50 mg twice daily for 2 wk.(ABSTRACT TRUNCATED AT 250 WORDS)
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