Villin is a tissue-specific actin modifying protein that is associated with actin filaments in the microvilli and terminal web of epithelial cells. It belongs to a large family of actin-binding proteins which includes actin-capping, -nucleating and/orsevering proteins such as gelsolin, severin, fragmin, adseverin/ scinderin and actin crosslinking proteins such as dematin and supervillin. Studies done in epithelial cell lines and villin knock-out mice have demonstrated the function of villin in regulating actin dynamics, cell morphology, epithelial-to-mesenchymal transition, cell migration and cell survival. In addition, the ligand-binding properties of villin (F-actin, G-actin, calcium, phospholipids and phospholipase C-c 1 ) are mechanistically important for the crosstalk between signaling pathways and actin reorganization in epithelial cells.
Temporal and spatial regulation of the actin cytoskeleton is vital for cell migration. Here, we show that an epithelial cell actin-binding protein, villin, plays a crucial role in this process. Overexpression of villin in doxycyline-regulated HeLa cells enhanced cell migration. Villin-induced cell migration was modestly augmented by growth factors. In contrast, tyrosine phosphorylation of villin and villin-induced cell migration was significantly inhibited by the src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) as well as by overexpression of a dominant negative mutant of c-src. These data suggest that phosphorylation of villin by c-src is involved in the actin cytoskeleton remodeling necessary for cell migration. We have previously shown that villin is tyrosine phosphorylated at four major sites. To further investigate the role of tyrosine phosphorylated villin in cell migration, we used phosphorylation site mutants (tyrosine to phenylalanine or tyrosine to glutamic acid) in HeLa cells. We determined that tyrosine phosphorylation at residues 60, 81, and 256 of human villin played an essential role in cell migration as well as in the reorganization of the actin cytoskeleton. Collectively, these studies define how biophysical events such as cell migration are actuated by biochemical signaling pathways involving tyrosine phosphorylation of actin binding proteins, in this case villin. INTRODUCTIONVillin, an epithelial cell-specific protein belongs to a family of actin-binding proteins that contain segments that display internal homology with each other (Arpin et al., 1988). The amino terminal core of villin retains the actin-capping and -severing functions of villin, whereas the carboxyl terminal headpiece enables villin to cross-link actin filaments. The actin-modifying properties of villin are regulated in vitro by calcium (Northrop et al., 1986), phosphoinositides (Janmey and Matsudaira, 1988; and tyrosine phosphorylation . It has been assumed for several years that villin's actin-bundling and not actin-severing functions are important because nonphysiologically high Ca 2ϩ concentrations (200 M) are required to activate villin's actin-severing activity. However, studies done with the villin knockout mice suggest that in the absence of villin, the actin-bundling properties associated with villin can be substituted by other proteins in the microvilli (Pinson et al., 1998); on the other hand, the actin-severing activity of the microvilli is lost (Ferrary et al., 1999). In recent years, we have demonstrated that villin's actin-modifying functions can be regulated in vitro by tyrosine phosphorylation and phosphatidylinositol bisphosphate (PIP 2 ), suggesting that villin has the potential to function as a link between receptor activation and actin cytoskeleton reorganization even in the absence of high calcium (Arora and McCulloch, 1996;Zhai et al., 2001;. In addition, we have recently demonstrated that the autoinhibited conformation of villin can be released by tyrosine phosp...
Na؉ /H ؉ exchanger isoform 3 (NHE3), an epithelial brush border isoform of the Na ؉ /H ؉ exchanger gene family, plays an important role in reabsorption of Na ؉ in the small intestine, the colon, and the kidney. In several cell types, phorbol 12-myristate 13-acetate (PMA) acutely inhibits NHE3 activity by changes in V max , but the mechanism of this inhibition is unknown. We investigated the role of subcellular redistribution of NHE3 in the PMA-induced inhibition of endogenous brush border NHE3 in a model human colon adenocarcinoma cell line, Caco-2. Subcellular localization of NHE3 was examined by confocal morphometric analysis complemented with cell surface biotinylation and compared with NHE3 activity evaluated by fluorometric measurement of intracellular pH. PMA inhibited NHE3 activity by 28% (p < 0.01), which was associated with a decrease of the ratio of the brush border/subapical cytoplasmic compartment of NHE3 from ϳ4.3 to ϳ2.4. This translocation resulted in 10 -15% of the total cell NHE3 being shifted from the brush border pool to the cytoplasmic pool. These effects were mediated by protein kinase C, since they were blocked by the protein kinase C inhibitor H7. We conclude that inhibition of NHE3 by protein kinase C in Caco-2 cells involves redistribution of the exchanger from brush border into a subapical cytoplasmic compartment, and that this mechanism contributes ϳ50% to the overall protein kinase C-induced inhibition of the exchanger.
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