Actin structures are dynamic and are affected by extracellular signals. Although the organization of the actin cytoskeleton in a moving cell has been described reasonably well, it is still unclear how the cytoskeleton is directed by external signals to coordinate its activity to provide directional movement. In the present report, we provide evidence that tyrosine phosphorylation of villin, which could occur in response to receptor activation or during wound healing results in both rearrangement of the microfilament structure as well as regulation of cell migration. In this study, we further demonstrated that overexpression of villin enhances cell migration. This is consistent with previous observations made with proteins of the villin family, including cofilin (Aizawa et al., 1996
), CapG, and gelsolin (Sun et al., 1995
). Data presented in our study suggest that an epithelial cell also could use villin as an intrinsic signal to stimulate actin assembly and increase cell migration in the absence of an external signal. Furthermore, our studies demonstrate that this change in cell motility by villin can be enhanced in response to environmental cues such as chemoattractants. For our studies we used HGF, a cytokine that is known to coordinate changes in cell morphology associated with the induction of cell motility during epithelial-mesenchyme transition (Boyer et al., 1996
) and EGF, another powerful motogen. Together, these studies suggest that villin and other members of its family may function in vivo to provide a signaling mechanism for translating cell surface receptor-mediated biochemical reactions into cell locomotion.
Tyrosine kinase activity has been shown to be necessary for intestinal epithelial cell migration (Calalb et al., 1995
; Cary et al., 1996
; Parsons and Parsons, 1997
; Polk, 1998
; Reiske et al., 1999
). Activation of c-src
kinase has been reported in preneoplastic colonic adenomas and in colon carcinomas. In addition overexpression of pp60c-src
has been demonstrated to increase the invasive behavior of intestinal epithelial cells (Pories et al., 1998
). Thus, both tyrosine phosphorylation and activation of c-src
kinase have been associated with the motile properties of intestinal epithelial cells. We have previously demonstrated that villin is phosphorylated in vitro by c-src
kinase (Zhai et al., 2002
). In the present study we provide evidence that in migrating cells, villin is a substrate for c-src
kinase as well. Further, the potent and specific src kinase inhibitor PP2 inhibits the villin-induced increase in cell migration. Overexpression of the dominant negative c-src likewise inhibits villin-induced cell migration. These data suggest that villin is a substrate for src
kinases in vivo just as it is in vitro and furthermore that src kinase phosphorylation of villin is important for villin-mediated cell motility.
We have previously identified the tyrosine phosphorylation sites in villin and mapped phosphorylation at these sites with the actin-modifying activities of villin (Zhai et al., 2002
). Human villin contain four major tyrosine phosphorylation sites, namely, Y-46, -60, -81, and -256. In the current study, we assessed the effects of tyrosine phosphorylation of villin as well as mapped the phosphorylation sites that regulate villin-induced increase in cell migration. Our studies revealed that phosphorylation at Y60, Y81, and Y256 is required for cell migration. Both Y46F and wild-type villin enhance cell migration and demonstrate similar intracellular distribution, including redistribution of actin to the cell perimeter, loss of stress fibers, and colocalization with F-actin, suggesting that actin cytoskeletal reorganization by villin is necessary for its role in cell migration. This is supported by the observation that the villin mutants VIL/Y60F, VIL/Y81F, and VIL/Y256F migrate like the VIL/NULL cells and demonstrate a different subcellular distribution of villin as well as microfilament organization pattern. The most obvious difference between villin-induced migrating and villin-null or phosphorylation site mutant-villin cells is that villin that can be tyrosine phosphorylated at residues 60, 81, and 256 as well as the phosphomimetics localize at or near the cell surface. In contrast villin that cannot be tyrosine phosphorylated at these sites, show an intracellular distribution and are not very well distributed in cell surface structures. One possibility is that by altering the ability of villin to be tyrosine phosphorylated, we may have modified the ligand-binding properties of villin that determine its intracellular distribution and localization.
Recombinant tyrosine phosphorylated villin does not associate with PIP2
, whereas nonphosphorylated villin does (Panebra et al., 2001
). Furthermore, we have demonstrated that tyrosine phosphorylation of villin decreases villin's binding affinity for F-actin (Zhai et al., 2001
). This would suggest that within the cell at or near the leading edge, there could be two separate pools of villin: tyrosine-phosphorylated villin that does not associate with the plasma membrane and has decreased affinity for F-actin, and nonphosphorylated villin that could bind both PIP2
, and F-actin. Consistent with this observation, we have previously reported that tyrosine-phosphorylated villin redistributes to a Triton X-100–soluble fraction of intestinal villus cells, whereas nonphosphorylated villin remains associated with the F-actin filaments in a Triton-insoluble fraction (Khurana et al., 1997
). Because phosphorylated villin does not associate with PIP2
, we speculate that this Triton-soluble pool may represent the cellular G-actin pool, whereas the Triton X-100–insoluble pool is the F-actin pool in live cells. This is a reasonable speculation because we also have reported that tyrosine phosphorylation of villin releases an autoinhibited conformation, allowing it to sever actin filaments at physiological Ca2+
concentrations (Kumar and Khurana, 2004
). Furthermore, we have identified the biochemical properties of the individual phosphorylation sites in villin. Phosphorylation at Y60 enhances the actin-severing activity of villin, suggesting that this site could be involved in generating new barbed ends (Zhai et al., 2002
). In contrast, phosphorylation at Y81 and Y256 inhibits the ability of villin to polymerize actin filaments, thus altering the F-actin dynamics in vivo. These sites may have additional biochemical properties such as to decrease the binding affinity of villin for F-actin, decrease the actin cross-linking activity of villin and/or induce conformational changes in villin, resulting in constitutively active villin that can sever actin filaments at nanomolar Ca2+
. We have previously shown all these functions of villin to be regulated by tyrosine phosphorylation (Zhai et al., 2001
; Kumar and Khurana, 2004
). Alternatively, in vivo these tyrosine phosphorylation sites may be ligand-binding sites for second messengers that may regulate either tyrosine phosphorylation of villin such as c-src kinase or yet unidentified tyrosine phosphatases, as well as the SH2 domain of PLC-γ1
(Panebra et al., 2001
), all of which would modify villin-induced cell migration.
The integrity of these tyrosine residues is required for actin nucleation and depolymerization by villin, because phosphorylation of any one site impairs villin's capacity to nucleate actin, and likewise mutation at Y60 to F severely impairs phosphorylated villin's ability to sever actin filaments (Zhai et al., 2002
). In other words, villin's ability to regulate the actin dynamics is dependent on its phosphorylation. Inhibition of cell migration by villin mutants suggests that a decrease in the actin-severing activity of villin is a negative regulator of villin-induced increase in cell migration. In other words, increased actin depolymerization by villin could be the signal for the enhanced cell migration.
The phosphomimetics lend further support to the idea that phosphorylation of villin at these three sites is necessary for the regulation of villin-induced migration. Interestingly, the villin mutant Y46E inhibited cell migration. The significance of this observation is not clear at this point, except it suggests that although phosphorylation at Y46 is not required for cell migration (VIL/Y46F behaves like VIL/FL), maintaining this site in a dephosphorylated form may be necessary for the villin-induced effects on cell migration. This also suggests that regulation of cell migration by villin may require both kinases and phosphatases maintaining the Y-60, -81, and -256 sites in a phosphorylated state and the Y46 site in a dephosphorylated state for efficient cell migration.
Based on our observations, we propose the following model for villin-induced cell migration. We believe that spatially restricted accumulation of signaling molecules could determine the activation of one or more of villin's actin regulatory properties, thus establishing villin's function in regulating cell motility. One of these signals could be a tyrosine kinase and/or phosphatase that could determine the phosphorylation state of villin. In the unphosphorylated state, the actin cross-linking property of villin could be important. For instance, it is known that cross-linking of actin filaments is essential to convert the force of polymerization into forward movement of the membrane and the cell. Although Arp 2/3 is considered the major cross-linking component, it is possible that in epithelial cells villin plays a significant role in cross-linking the actin filaments and generating stable lamellae. The cross-linking functions of villin could determine the rate and extent of lamellipodia formation and/or regulate the amount of F-actin incorporated into newly formed lamellipodia. Unphosphorylated villin also could bind the membrane phospholipid PIP2 and the villin-PIP2 complex could favor persistent growth of the barbed ends by preventing capping of the barbed ends, thus favoring both filament growth as well as allowing actin to push the membrane forward.
Phosphorylated villin could have other functions. Phosphorylation of villin could increase actin depolymerization, which could replenish the actin monomer pool thereby allowing the cell to maintain high concentration of unpolymerized actin far from the equilibrium. Depolymerization of actin by villin also could produce new barbed ends that may be used for the formation of new filaments. Phosphorylation of villin and activation of villin's actin-capping function could help cap barbed ends. Capping of barbed ends could help maintain the length of actin filaments, thus resulting in short filaments that can generate propulsive force and effectively push the membrane forward. Tyrosine phosphorylation of villin could regulate directed cell movement. It is possible that some of the phosphorylation site mutants exhibit lower migration rates because of lack of directed and coordinated movement. Because phosphorylated villin also disassembles actin filament bundles, cytoskeletal disassembly associated with disruption of actin bundles by tyrosine phosphorylated villin could generate pulling forces that may even be involved in the rearward retraction of a moving cell. Examples of such solation-contraction include microtubules that segregate chromosomes during mitosis (Mogilner and Oster, 2003
), and sperm of the nematode Ascaris suum
(Miao et al., 2003
). Cell migration is intimately linked to cytoskeleton dynamics and our study demonstrates that tyrosine phosphorylation that affects cytoskeleton dynamics also affects cell migration. Our studies suggest that filament turnover in cells may be defined by the regulated action of actin-binding proteins interacting with signaling molecules.