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.

Caldesmon is an actin-, tropomyosin-, myosin-, and calmodulin-binding protein that is alternatively spliced to yield either a 93-kDa protein (high molecular mass caldesmon (h-CaD)

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The C-terminal region of smooth muscle caldesmon (CaD) interacts with calmodulin (CaM) and reverses CaD's inhibitory effect on the actomyosin ATPase activity. We have previously shown that the major CaM-binding site (site A) in this region is within the segment from Met-658 to Ser-666 (Zhan, Q., Wong, S. S., and Wang, C.-L. A. (1991) J. Biol. Chem. 266, 21810-21814). Recently, another segment (site B), Asn-675 to Lys-695, was reported to bind CaM (Mezgueldi, M., Derancourt, J., Calas, B., Kassab, R., and Fattoum, A. (1994) J. Biol. Chem. 269, 12824-12832). To assess the functional relevance of these two putative CaM-binding sites, we have examined three synthetic peptides regarding their effects on CaM's ability to reverse CaD-induced inhibition of actomyosin ATPase activity: GS17C (Gly-651 to Ser-667), VG29C (Val-685 to Gly-713), each containing one CaM-binding site, and MG56C (Met-658 to Gly-713), which contains both sites. We found that although VG29C did bind CaM, its affinity was weakened by GS17C, and it failed to compete with CaD for CaM under the conditions where GS17C effectively displaced CaD from CaM. MG56C had an effect similar to that of GS17C. These experiments demonstrated that site A for CaM binding is involved in regulating the inhibitory property of CaD.

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Actin polymerization and depolymerization plays a central role in controlling a wide spectrum of cellular processes. There are many actin-binding proteins in eukaryotic cells. Their roles in the remodeling of the actin architecture and whether they work cooperatively await further study. Caldesmon (CaD) is an actin-binding protein present in nearly all mammalian cells. Cortactin is another actin-binding protein found mainly in the cell cortex. There have been no reports suggesting that CaD and cortactin interact with each other or work as partners. Here we present evidence that CaD binds cortactin directly by overlay, pull-down assays, ELISA and by column chromatography. The interaction involves the N-terminal region of cortactin and the C-terminal region of CaD, and appears to be enhanced by divalent metal ions. Cortactin competes with both full-length CaD and its C-terminal fragment for actin binding. Binding of cortactin partially alleviates the inhibitory effect of CaD on the actomyosin ATPase activity. Not only can binding be demonstrated in vitro, the two proteins also co-localize in activated cells at the cortex. Whether such interactions bear any functional significance awaits further investigation.Actin polymerization and depolymerization plays a central role in controlling a wide spectrum of cellular phenomena and intracellular processes. This is especially true in cells like smooth muscle cells (SMCs), which undergo cell shape changes during contraction and relaxation. The ability to recover cell shape after either transient or sustained deformation, depends on the dynamic and constant rearrangement of actin cytoskeleton (Bursac et al., 2005;Seow, 2005). Cytoskeletal disassembly is known to contribute to the hyporesponsiveness of SMC and mesangial cells to vasopressors both in vitro and in vivo (Zhou et al., 1995;Cortes et al., 2000). It was also found that under pathological conditions that mimic cytochalasin D treatment, mesangial cells respond with an up-regulation of a group of actin cytoskeleton regulatory proteins in an attempt to restore the cytoskeletal integrity (Clarkson et al., 2002).The canonical mechanism of actin regulation involves cofilin (an actin disassembly factor), profilin (a G-actin sequester), Arp2/3 (a branching factor), formin (a barbed end capping protein that promotes actin assembly) and capping proteins (that controls the disassembly at the pointed end). However, there are many more other actin-binding proteins in the eukaryotic cells. These include gelsolin (a severing protein) and tropomyosin (a filament stabilizer). Individual actin filaments are further organized into higher order structures by α -actinin (a bundling protein), filamin and spectrin (crosslinking proteins), as well as myosins (motors). The actin filaments are anchored to the cell membrane to provide a framework that allows force-bearing; this is achieved by a group of membrane-associated proteins, such as dystrophin, utrophin, talin, vinculin, paxillin and integrin. While the players are identifie...

Caldesmon (CaD) is thought to regulate smooth muscle contraction, because it binds actin and inhibits actomyosin interactions. A synthetic actin-binding peptide (GS17C) corresponding to Gly666-Ser682 of chicken gizzard CaD has been shown to induce force development in permeabilized smooth muscle cells. The mechanism of GS17C's action remains unclear, although a structural effect was postulated. By photo-crosslinking and fluorescence quenching experiments with a gizzard CaD fragment (H32K; Met563-Pro771) and its mutants, we showed that GS17C indeed dissociated the C-terminal region of H32K from actin, in a manner similar to extracellular signal-regulated kinase-mediated phosphorylation, thereby reversing the CaD-imposed inhibition and enabling the actomyosin interaction.