Domain analysis of supervillin, an F-actin bundling plasma membrane protein with functional nuclear localization signals

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 funct ional nuclear targeting signals and localizes at or near vinculin-containin g focal adhesion plaques in COS7-2 and CV1 cells. Overexpression of full-le ngth supervillin in these cells disrupts the integrity of focal adhesion pl aques and results in increased levels of F-actin and vinculin, Localization studies of chimeric proteins containing supervillin sequences fused with t he enhanced green fluorescent protein indicate that: (1) the amino terminus promotes F-actin binding, targeting to focal adhesions, and limited nuclea r localization; (2) the dominant nuclear targeting signal is in the center of the protein; and (3) the carboxy-terminal villin/gelsolin homology domai n of supervillin does not, by itself, bind tightly to the actin cytoskeleto n 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 cytoarchit ecture in the nucleus, as well as at the plasma membrane.