Focal adhesions are specialized regions of the cell surface where integrin receptors and associated proteins link the extracellular matrix to the actin cytoskeleton. To define the cellular role of the focal adhesion protein zyxin, we characterized the phenotype of fibroblasts in which the zyxin gene was deleted by homologous recombination. Zyxin-null fibroblasts display enhanced integrin-dependent adhesion and are more migratory than wild-type fibroblasts, displaying reduced dependence on extracellular matrix cues. We identified differences in the profiles of 75- and 80-kD tyrosine-phosphorylated proteins in the zyxin-null cells. Tandem array mass spectrometry identified both modified proteins as isoforms of the actomyosin regulator caldesmon, a protein known to influence contractility, stress fiber formation, and motility. Zyxin-null fibroblasts also show deficits in actin stress fiber remodeling and exhibit changes in the molecular composition of focal adhesions, most notably by severely reduced accumulation of Ena/VASP proteins. We postulate that zyxin cooperates with Ena/VASP proteins and caldesmon to influence integrin-dependent cell motility and actin stress fiber remodeling.

Transforming growth factor-␤1 (TGF-␤1Consistent with these biochemical findings, kindlerin is present at focal adhesions, sites of integrin-rich, membrane-substratum adhesion. Additionally, kindlerin is required for normal cell spreading. Taken together, these data suggest a role for kindlerin in mediating cell processes that depend on integrins.The survival of cancer patients with solid tumors decreases dramatically when tumors are invasive and have an increased likelihood of metastasizing to distal sites. The enhanced invasiveness of tumor cells is attributed to epithelial to mesenchymal transition (EMT), 1 (5-8) a normal biological process that is critical for wound healing and development. EMT is characterized by a change in cell shape from a polarized epithelial cell to a flattened fibroblast-like morphology, a decrease in cell-cell junctions concurrent with a decrease in E-cadherin expression, and an increase in cell motility (5, 6). Transforming growth factor-␤1 (TGF-␤) is a major contributor to tumor progression and metastasis (9 -11), and several studies have shown that TGF-␤ promotes tumor cell invasiveness by promoting EMT (9,(12)(13)(14)(15). Despite the key role established for TGF-␤ in stimulating EMT and tumor progression, the molecular mechanisms by which TGF-␤ promotes EMT have not been fully elucidated.Microarray analysis of a TGF-␤-responsive cell line, human mammary epithelial cells (HMEC), led us to identify kindlerin as a TGF-␤-inducible gene. Kindlerin is mutated in Kindler syndrome, a rare autosomal-recessive genodermatosis (1, 3). Early in life patients with Kindler syndrome endure blistering of the skin and photosensitivity, which progresses to diffuse poikiloderma followed by cutaneous atrophy (16, 17). The clinical presentation of this disease is similar to patients with junctional epidermolysis bullosa harboring mutations in ␣ 6 and ␤ 4 integrin genes (18,19).Kindlerin (also known as URP1 for UNC-112 related protein 1 or kindlin) is a member of a newly recognized protein family, which also includes Mig-2 and URP2 (2, 3). An apparent kindlerin orthologue, UNC-112, has been studied in Caenorhabditis elegans (20). The unc-112 gene is essential for embryogenesis, and it displays a genetic interaction with integrins (20). Because integrins play a critical role in mammalian cell adhesion and migration, we postulated that kindlerin may be involved in TGF-␤-stimulated EMT through interactions with integrins.Here we report that kindlerin expression is responsive to TGF-␤ levels, that kindlerin localizes in focal adhesions (integrin-rich signaling centers that integrate extracellular matrix attachment and cytoskeletal organization), and that kindlerin forms complexes with integrin ␤ subunit cytoplasmic domains. Furthermore, cell spreading is perturbed upon reduction of kindlerin protein. Taken together with the observation that kindlerin is overexpressed in colon and lung carcinomas (2), our data support a role for kindlerin in mammalian cell adhesion and suggest that kindlerin may mediate TGF...

Integrin binding to extracellular matrix proteins induces formation of signaling complexes at focal adhesions. Zyxin co-localizes with integrins at sites of cellsubstratum adhesion and is postulated to serve as a docking site for the assembly of multimeric protein complexes involved in regulating cell motility. Recently, we identified a new member of the zyxin family called TRIP6. TRIP6 is localized at focal adhesions and overexpression of TRIP6 slows cell migration. In an effort to define the molecular mechanism by which TRIP6 affects cell migration, the yeast two-hybrid assay was employed to identify proteins that directly bind to TRIP6. This assay revealed that both TRIP6 and zyxin interact with CasL/HEF1, a member of the Cas family. This association is mediated by the LIM region of the zyxin family members and the SH2 domain-binding region of CasL/ HEF1. Furthermore, the association between p130 Cas and the two zyxin family members was demonstrated to occur in vivo by co-immunoprecipitation. Zyxin and Cas family members may cooperate to regulate cell motility.Integrin-mediated cell adhesion to extracellular matrix (ECM) 1 components is crucial for many cell activities including cell survival, proliferation, migration, and differentiation (1-5). Upon binding to the substratum, integrins recruit many cytoskeletal components to the sites of cell adhesion and establish a structural link between the elements of the ECM and actin filaments. In addition to contributing to the physical link between the extracellular and intracellular environments, integrin engagement also regulates several signaling pathways (2, 6, 7). Although the cytoplasmic domains of integrins do not exhibit any enzymatic activity, they can activate intracellular signaling pathways by recruiting a number of signaling proteins to focal adhesions (8 -10).Recent studies have identified a number of proteins that participate in integrin-dependent signaling pathways (10 -13). These signaling molecules include non-receptor tyrosine kinases (14, 15), serine/threonine kinases (7, 16 -18), a lipid kinase (19), protein-tyrosine phosphatases (20 -24), and small GTPases in the Ras and Rho families (25-29). In addition to proteins that harbor catalytic domains, integrins recruit several adaptor proteins that facilitate the assembly of multicomponent signaling complexes (30 -32). For instance, upon substratum adhesion, the adaptor protein p130 Cas (p130 Crk-associated substrate ) is recruited to integrin-rich sites where it docks several regulatory molecules including Src, Crk, and FAK (focal adhesion kinase) (33-35).Members of the zyxin family have also been postulated to function in integrin-mediated signaling (36). Zyxin, the founding member of the family, is a phosphoprotein that is localized at focal adhesions and along actin filaments (37, 38). The protein displays an NH 2 -terminal proline-rich region, one or more leucine-rich nuclear export signals (depending on the species) and three copies of the LIM motif at its COOH terminus (Fig. 1A) (36, 39, 4...

Advances in understanding the role of transforming growth factor (TGF)-beta in tumorigenesis have led to the development of TGF-beta inhibitors for cancer treatment. Three platforms of TGF-beta inhibitors have evolved: antisense oligonucleotides, monoclonal antibodies and small molecules. In this review, the current stage of development of each known TGF-beta inhibitor will be discussed. As part of the risk/benefit assessment of TGF-beta inhibitors, the known effects of TGF-beta deficiency in mice, non-clinical toxicology studies with TGF-beta inhibitors in rats, and the clinical studies with monoclonal antibodies against TGF-beta will be summarised.

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