Regulation of Actin Dynamics by Tyrosine Phosphorylation:  Identification of Tyrosine Phosphorylation Sites within the Actin-Severing Domain of Villin

Abstract

We have previously shown that villin, an epithelial cell actin-binding protein, is tyrosine phosphorylated both in vitro and in vivo and that villin's actin-modifying functions are regulated by phosphorylation. Here as a first step toward understanding the role of villin tyrosine phosphorylation, we sought to identify the major phosphorylation site(s) in human villin and study its role in actin filament assembly. We generated a series of carboxyl-terminal truncation mutants of villin and cloned them in the prokaryotic expression vector pGEX-2T. Full-length villin and the truncation mutants were expressed in TKX1 cells, which carry an inducible tyrosine kinase gene. Using this approach, we identified a region in the amino-terminal actin-severing domain of villin as the site of phosphorylation (amino acids 1-261). Five phosphorylation sites were identified by direct mutation of candidate tyrosines (Y) to phenylalanine (F), namely, Y46, -60, -64, -81, and -256. Changing all of these sites to phenylalanine resulted in a villin mutant that neither was phosphorylated in TKX1 cells nor was a substrate for c-src kinase in an in vitro kinase assay. Using a pyrene actin-based fluorescence assay, we mapped the various phosphorylated tyrosine residues with the actin-nucleating and -depolymerizing functions of villin. Phosphorylation of any one of the identified sites inhibited the actin-nucleating function of villin, whereas phosphorylation at Y46 and/or Y60 increased the actin-severing activity of villin. Since there is significant homology between the amino-terminal end of villin and other actin-severing proteins, the results provide a structural basis for the actin-severing mechanism and help understand the relationship of phosphorylation with this function.

Regulation of Cell Motility by Tyrosine Phosphorylated Villin

“…The bottom panel in Figure 3B is an SDS-PAGE of VIL/WT and VILT/WT stained with GelCode Blue, showing the expression of the two recombinant proteins VILT/WT and VIL/WT. Consistent with our previous studies Zhai et al, 2002), tyrosine phosphorylation of villin (in response to wounding or treatment with HGF/EGF) is inhibited by PP2 but not PP3 ( Figure 3C). By using this approach, no basal tyrosine phosphorylation of villin in the absence of wounding or EGF/HGF treatment could be detected in these cells with either phospho-villin antibody (VP-70782) or phospho-tyrosine antibody .…”

“…Previous studies from our laboratory have demonstrated that villin is a substrate in vitro for the src kinase(s) Zhai et al, 2002). In addition, our studies have suggested that villin phosphorylation and the resulting changes in the actin rearrangements might serve to promote the migratory phenotype of epithelial cells Zhai et al, 2002).…”

“…In addition, our studies have suggested that villin phosphorylation and the resulting changes in the actin rearrangements might serve to promote the migratory phenotype of epithelial cells Zhai et al, 2002). To confirm this, we examined the migratory capacity of cells stably expressing wild-type villin in the absence or presence of the src kinase inhibitor PP2 and its inactive analog PP3.…”

“…Mutation of any of these phosphorylation sites inhibits the ability of villin protein to polymerize actin filaments in vitro (Zhai et al, 2002). Based on our previous studies, phosphorylated villin could regulate cell motility by modifying actin assembly by distinct mechanisms, including lowering the binding affinity for Factin, inhibiting polymerization of existing actin nuclei, and/or cutting preexisting filaments to generate new actin nuclei (Zhai et al, , 2002. In this study, we investigated the role of each tyrosine phosphorylation site of villin in actin reorganization and cell migration by establishing HeLa cell lines stably expressing wild-type or each of the phosphorylation site mutants (tyrosine to phenylalanine) of villin.…”

“…Four major sites of phosphorylation were identified in this region by direct mutation of candidate tyrosines (Y) to phenylalanine (F), namely, Y-46, -60, -81, and -256 (Zhai et al, 2002). Mutation of any of these phosphorylation sites inhibits the ability of villin protein to polymerize actin filaments in vitro (Zhai et al, 2002). Based on our previous studies, phosphorylated villin could regulate cell motility by modifying actin assembly by distinct mechanisms, including lowering the binding affinity for Factin, inhibiting polymerization of existing actin nuclei, and/or cutting preexisting filaments to generate new actin nuclei (Zhai et al, , 2002.…”