The LPP gene is the preferred translocation partner of the HMGIC gene in a subclass of human benign mesenchymal tumors known as lipomas. Here we have characterized the LPP gene product that shares 41% of sequence identity with the focal adhesion protein zyxin. LPP localizes in focal adhesions as well as in cell-to-cell contacts, and it binds VASP, a protein implicated in the control of actin organization. In addition, LPP accumulates in the nucleus of cells upon treatment with leptomycin B, an inhibitor of the export factor CRM1. The nuclear export of LPP depends on an N-terminally located leucine-rich sequence that shares sequence homology with well-defined nuclear export signals. Moreover, LPP displays transcriptional activation capacity, as measured by GAL4-based assays. Altogether, these results show that the LPP protein has multifunctional domains and may serve as a scaffold upon which distinct protein complexes are assembled in the cytoplasm and in the nucleus.

Zyxin contains a proline-rich N-terminal domain that is similar to the C-terminal domain in the ActA protein of the bacteria, Listeria monocytogenes. We screened the entire amino acid sequence of human zyxin for Menainteracting peptides and found that, as with ActA, proline-rich sequences were the sole zyxin sequences capable of binding to Ena/vasodilator-stimulated phosphoprotein (VASP) family members in vitro. From this information, we tested zyxin mutants in which the proline-rich sequences were altered. The reduction in Mena/VASP binding was confirmed by peptide tests, immunoprecipitation, and ectopic expression of zyxin variants at the surface of mitochondria. By transfection assays we showed that zyxin interaction with Mena/ VASP in vivo enhances the production of actin-rich structures at the apical surface of cells. Microinjection into cells of peptides corresponding to the first prolinerich sequence of zyxin caused the loss of Mena/VASP from focal contacts. Furthermore, these peptides reduced the degree of spreading of cells replated after trypsinization. We conclude that zyxin and proteins that harbor similar proline-rich repeats contribute to the positioning of Mena/VASP proteins. The positioning of Ena/VASP family members appears to be important when the actin cytoskeleton is reorganized, such as during spreading.

The localization of proteins to particular intracellular compartments often regulates their functions. Zyxin is a LIM protein found prominently at sites of cell adhesion, faintly in leading lamellipodia, and transiently in cell nuclei. Here we have performed a domain analysis to identify regions in zyxin that are responsible for targeting it to different subcellular locations. The N-terminal proline-rich region of zyxin, which harbors binding sites for ␣-actinin and members of the Ena/VASP family, concentrates in lamellipodial extensions and weakly in focal adhesions. The LIM region of zyxin displays robust targeting to focal adhesions. When overexpressed in cells, the LIM region of zyxin causes displacement of endogenous zyxin from focal adhesions. Upon mislocalization of full-length zyxin, at least one member of the Ena/VASP family is also displaced, and the organization of the actin cytoskeleton is perturbed. Zyxin also has the capacity to shuttle between the nucleus and focal adhesion sites. When nuclear export is inhibited, zyxin accumulates in cell nuclei. The nuclear accumulation of zyxin occurs asynchronously with approximately half of the cells exhibiting nuclear localization of zyxin within 2.3 h of initiating leptomycin B treatment. Our results provide insight into the functions of different zyxin domains.Cells change shape, migrate, proliferate, and differentiate in response to extracellular cues. Such sensitivity to environmental conditions is necessary for multiple processes including development, cell-mediated immunity, and organ regeneration (1, 2). Although much has been learned about a cell's capacity to recognize and respond to extracellular cues, in many cases we do not understand how engagement of a particular cell surface receptor triggers changes in cell behavior. One present challenge, for example, is to understand how engagement of integrin receptors for extracellular matrix can stimulate cell motility and changes in gene expression. In the past few years, several proteins that may contribute to these processes have been identified. For example, zyxin, a protein that is co-localized with integrins at sites of membrane-substratum adhesion, has properties that suggest it could participate in both localized actin polymerization and communication to the nuclear compartment.Zyxin is an elongate phosphoprotein composed of three C-terminal LIM domains, a proline-rich N-terminal region, and at least one nuclear export signal (NES) 1 (Fig. 1A) (3-6). Two binding partners for the LIM region of zyxin, H-warts/LATS1 and members of the cysteine-rich protein (CRP) family, have been identified (5, 7). H-warts/LATS1 is a tumor suppressor that is postulated to be an important regulator of mitotic progression (7). CRPs are thought to stabilize actin-rich structural elements in muscle (8,9). The N-terminal proline-rich region of zyxin displays docking sequences for several proteins implicated in actin assembly and organization including ␣-actinin and members of the Ena (enabled)/VASP (vasodilator-stimulate...