Organs and tissues adapt to acute or chronic mechanical stress by remodeling their actin cytoskeletons. Cells that are stimulated by cyclic stretch or shear stress in vitro undergo bimodal cytoskeletal responses that include rapid reinforcement and gradual reorientation of actin stress fibers; however, the mechanism by which cells respond to mechanical cues has been obscure. We report that the application of either unidirectional cyclic stretch or shear stress to cells results in robust mobilization of zyxin from focal adhesions to actin filaments, whereas many other focal adhesion proteins and zyxin family members remain at focal adhesions. Mechanical stress also induces the rapid zyxin-dependent mobilization of vasodilator-stimulated phosphoprotein from focal adhesions to actin filaments. Thickening of actin stress fibers reflects a cellular adaptation to mechanical stress; this cytoskeletal reinforcement coincides with zyxin mobilization and is abrogated in zyxin-null cells. Our findings identify zyxin as a mechanosensitive protein and provide mechanistic insight into how cells respond to mechanical cues.
Full-length cDNAs for three human proteasome activator subunits, called REG␣, REG, and REG␥, have been expressed in Escherichia coli, and the purified recombinant proteins have been characterized. Recombinant ␣ or ␥ subunits form heptameric species; recombinant  subunits are found largely as monomers or small multimers. Each recombinant REG stimulates cleavage of fluorogenic peptides by human red cell proteasomes. The pattern of activated peptide hydrolysis is virtually identical for REG␣ and REG. These two subunits, alone or in combination, stimulate cleavage after basic, acidic, and most hydrophobic residues in many peptides. Recombinant ␣ and  subunits bind each other with high affinity, and the REG␣/ heteromeric complex activates hydrolysis of LLVY-methylcoumaryl-7-amide (LLVY-MCA) and LLE--nitroanilide (LLE-NA) more than REG␣ or REG alone. Using filter binding and gel filtration assays, recombinant REG␥ subunits were shown to bind themselves but not ␣ or  subunits. REG␥ differs from REG␣ and REG in that it markedly stimulates hydrolysis of peptides with basic residues in the P1 position but only modestly activates cleavage of LLVY-MCA or LLE-NA by the proteasome. REG␥ binds the proteasome with higher affinity than REG␣ or REG yet with lower affinity than complexes containing both REG␣ and REG. In summary, each of the three REG homologs is a proteasome activator with unique biochemical properties.
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.
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...
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