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...

Mechanical stimulation induces zyxin-dependent actin cytoskeletal reinforcement. Stretch induces MAPK activation, zyxin phosphorylation, and recruitment to actin stress fibers, independent of p130Cas. Zyxin's C-terminal LIM domains are required for stretch-induced targeting to stress fibers, and zyxin's N-terminus is necessary for actin remodeling.

Contractile actomyosin stress fibers are critical for maintaining the force balance between the interior of the cell and its environment. Consequently, the actin cytoskeleton undergoes dynamic mechanical loading. This results in spontaneous, stochastic, highly localized strain events, characterized by thinning and elongation within a discrete region of stress fiber. Previous work showed the LIM-domain adaptor protein, zyxin, is essential for repair and stabilization of these sites. Using live imaging, we show paxillin, another LIM-domain adaptor protein, is also recruited to stress fiber strain sites. Paxillin recruitment to stress fiber strain sites precedes zyxin recruitment. Zyxin and paxillin are each recruited independently of the other. In cells lacking paxillin, actin recovery is abrogated, resulting in slowed actin recovery and increased incidence of catastrophic stress fiber breaks. For both paxillin and zyxin, the LIM domains are necessary and sufficient for recruitment. This work provides further evidence of the critical role of LIM-domain proteins in responding to mechanical stress in the actin cytoskeleton.

Proteasomes can be markedly activated by associating with 19S regulatory complexes to form the 26S protease or by binding 11S protein complexes known as REG or PA28. Three REG subunits, ␣, ␤, and ␥, have been expressed in Escherichia coli, and each recombinant protein can activate human proteasomes. Combining PCR mutagenesis with an in vitro activity assay, we have isolated and characterized 36 inactive, single-site mutants of recombinant REG␣. Most are monomers that produce functional proteasome activators when mixed with REG␤ subunits. Five REG␣ mutants that remain inactive in the mixing assay contain amino acid substitutions clustered between Arg-141 and Gly-149. The crystal structure of the REG␣ heptamer shows that this region forms a loop at the base of each REG␣ subunit. One mutation in this loop (N146Y) yields a REG␣ heptamer that binds the proteasome as tightly as wild-type REG␣ but does not activate peptide hydrolysis. Corresponding amino acid substitutions in REG␤ (N135Y) and REG␥ (N151Y) produce inactive proteins that also bind the proteasome and inhibit proteasome activation by their normal counterparts. Our studies clearly demonstrate that REG binding to the proteasome can be separated from activation of the enzyme. Moreover, the dominant negative REGs identified here should prove valuable for elucidating the role(s) of these proteins in antigen presentation.A major mechanism for controlling viral infections involves cytotoxic T lymphocytes that recognize viral peptides presented on the cell surface by major histocompatability complex class I molecules and lyse the infected cells (1, 2). There is considerable evidence that some presented peptides, at least, are produced by the proteasome (3, 4). Crystal structures of Thermoplasma and yeast proteasomes reveal that they are cylindrical protein complexes, composed of four stacked rings (5, 6). The two end rings consist of catalytically inactive ␣-type subunits, whereas the two inner rings are composed of ␤-type subunits, some of which are catalytically active (7). The protease active sites are located within an inner chamber that is virtually sealed from the particles surface (5, 6). Thus, it seems clear that substrate entry to the sites of peptide bond hydrolysis must be tightly regulated.Two proteasome activators have been identified so far. The proteasome can either associate with a 19S regulatory complex to form the 26S protease, which is capable of degrading intact proteins (8-11), or the proteasome can bind an 11S activator called REG or PA28. This association greatly enhances fluorogenic peptide hydrolysis by the proteasome (12, 13). As isolated from human red blood cells, REG is a hexameric or heptameric ring formed from two homologous subunits, REG␣ and REG␤; these two subunits are, in turn, homologous to KI antigen or REG␥. cDNAs for all three proteins have been expressed in Escherichia coli, and each recombinant protein is capable of activating the proteasome in vitro (14).A variety of evidence suggests that REG is involved in antige...

Zyxin is an evolutionarily conserved protein that is concentrated at sites of cell adhesion, where it associates with members of the Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) family of cytoskeletal regulators and is postulated to play a role in cytoskeletal dynamics and signaling. Zyxin transcripts are detected throughout murine embryonic development, and the protein is widely expressed in adults. Here we used a reverse genetic approach to examine the consequences of loss of zyxin function in the mouse. Mice that lack zyxin function are viable and fertile and display no obvious histological abnormalities in any of the organs examined. Because zyxin contributes to the localization of Ena/VASP family members at certain subcellular locations, we carefully examined the zyxin ؊/؊ mice for evidence of defects that have been observed when Ena/VASP proteins are compromised in the mouse. Specifically, we evaluated blood platelet function, nervous system development, and skin architecture but did not detect any defects in these systems. Zyxin is the founding member of a family of proteins that also includes the lipoma preferred partner (LPP) and thyroid receptor-interacting protein 6 (TRIP6). These zyxin family members display patterns of expression that significantly overlap that of zyxin. Western blot analysis indicates that there is no detectable upregulation of either LPP or TRIP6 expression in tissues derived from zyxin-null mice. Because zyxin family members may have overlapping functions, a comprehensive understanding of the role of these proteins in the mouse will require the generation of compound mutations in which multiple zyxin family members are simultaneously compromised.