A growing number of actin-associated membrane proteins have been implicated in motile processes, adhesive interactions, and signal transduction to the cell nucleus. We report here that supervillin, an F-actin binding protein originally isolated from bovine neutrophil plasma membranes, contains functional nuclear targeting signals and localizes at or near vinculin-containing focal adhesion plaques in COS7-2 and CV1 cells. Overexpression of full-length supervillin in these cells disrupts the integrity of focal adhesion plaques and results in increased levels of F-actin and vinculin. Localization studies of chimeric proteins containing supervillin sequences fused with the enhanced green fluorescent protein indicate that: (1) the amino terminus promotes F-actin binding, targeting to focal adhesions, and limited nuclear localization; (2) the dominant nuclear targeting signal is in the center of the protein; and (3) the carboxy-terminal villin/gelsolin homology domain of supervillin does not, by itself, bind tightly to the actin cytoskeleton in vivo. Overexpression of chimeras containing both the amino-terminal F-actin binding site(s) and the dominant nuclear targeting signal results in the formation of large nuclear bundles containing F-actin, supervillin, and lamin. These results suggest that supervillin may contribute to cytoarchitecture in the nucleus, as well as at the plasma membrane.

Supervillin is a 205-kDa F-actin binding protein originally isolated from bovine neutrophils. This protein is tightly associated with both actin filaments and plasma membranes, suggesting that it forms a high-affinity link between the actin cytoskeleton and the membrane. Human supervillin cDNAs cloned from normal human kidney and from the cervical carcinoma HeLa S3 predict a bipartite structure with three potential nuclear localization signals in the NH2-terminus and three potential actin-binding sequences in the COOH-terminus. In fact, throughout its length, the COOH-terminal half of supervillin is similar to segments 2-6 plus the COOH-terminal "headpiece" of villin, an actin-binding protein in intestinal microvilli. A comparison of the bovine and human sequences indicates that supervillin is highly conserved at the amino acid level, with 79.2% identity of the NH2-terminus and conservation of three of the four nuclear localization signals found in bovine supervillin. The COOH-terminus is even more conserved, with 95.1% amino acid identity overall and 100% conservation of the villin-like headpiece. Supervillin mRNAs are expressed in all human tissue tested, bu are most abundant in muscle, bone marrow, thyroid gland, and salivary gland; comparatively little message is found in brain. Human supervillin mRNA is approximately 7.5 kb; this message is especially abundant in HeLa S3 cervical carcinoma, SW480 adenocarcinoma, and A549 lung carcinoma cell lines. The human supervillin gene (SVIL) is localized to a single chromosomal locus at 10p11.2, a region that is deleted in some prostate tumors.

Actin-binding membrane proteins are involved in both adhesive interactions and motile processes. We report here the purification and initial characterization of p205, a 205-kD protein from bovine neutrophil plasma membranes that binds to the sides of actin filaments in blot overlays. p205 is a tightly bound peripheral membrane protein that cosediments with endogenous actin in sucrose gradients and immunoprecipitates. Amino acid sequences were obtained from SDS-PAGE-purified p205 and used to generate antipeptide antibodies, immunolocalization data, and cDNA sequence information. The intracellular localization of p205 in MDBK cells is a function of cell density and adherence state. In subconfluent cells, p205 is found in punctate spots along the plasma membrane and in the cytoplasm and nucleus; in adherent cells, p205 concentrates with E-cadherin at sites of lateral cell-cell contact. Upon EGTA-mediated cell dissociation, p205 is internalized with E-cadherin and F-actin as a component of adherens junctions "rings." At later times, p205 is observed in cytoplasmic punctae. The high abundance of p205 in neutrophils and suspension-grown HeLa cells, which lack adherens junctions, further suggests that this protein may play multiple roles during cell growth, adhesion, and motility. Molecular cloning of p205 cDNA reveals a bipartite structure. The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin. The NH2 terminus is novel, but contains four potential nuclear targeting signals. Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.