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Originally published In Press as doi:10.1074/jbc.M604323200 on August 18, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31972-31986, October 20, 2006
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Interaction of Phospholipase C-{gamma}1 with Villin Regulates Epithelial Cell Migration*Formula

Alok Tomar, Sudeep George, Pallavi Kansal, Yaohong Wang, and Seema Khurana1

From the Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163

Received for publication, May 5, 2006 , and in revised form, July 31, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tyrosine-phosphorylated villin regulates actin dynamics, cell morphology, and cell migration. Previously, we identified four tyrosine phosphorylation sites in the amino-terminal domain of villin. In this study we report six new sites in the carboxyl-terminal region of the villin core. With this study we document all phosphorylatable tyrosine residues in villin and map them to functions of villin. In this study, we identify for the first time the functional relevance of the carboxyl-terminal domains of the villin core. Expression of the carboxyl-terminal phosphorylation site mutant, as well as the villin truncation mutant S1-S3, inhibited cell migration in HeLa and Madin-Darby canine kidney Tet-Off cells, confirming the role of the carboxyl-terminal phosphorylation sites in villin-induced cell migration. The carboxyl-terminal phosphorylation sites were found to be critical for the interaction of villin with its ligand phospholipase C-{gamma}1 and for its localization to the developing lamellipodia in a motile cell. The results presented here elucidate the molecular basis for tyrosine-phosphorylated villin-induced changes in cell motility.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In intestinal epithelial cells as well as in the opossum kidney cell line, tyrosine phosphorylation of villin regulates ion transport (1, 2). In addition, rearrangement of the microvillar cytoskeleton and concomitant redistribution of villin have been reported in studies with enteroinvasive bacteria (3), in intestinal restitution (4, 5), in renal ischemia, in the ischemia of the small intestine (6-9), and in colonic adenocarcinomas (10). It may be noted that villin expression is maintained in carcinomas derived from renal and intestinal epithelial cells. Villin is also expressed in other adenocarcinomas even though it is absent from normal tissue such as in Barrett's metaplasia and gastric cardia adenocarcinomas. More recently, chronic Helicobacter pylori infection has been shown to induce endogenous villin expression in the stomach (11). Consistent with this and other studies, it has been suggested that villin expression arises in these tissues in response to chronic injury and may be a marker of pre-neoplastic lesions and may even participate in the altered genetic program that results in intestinal metaplasia (12). Studies done with the villin knockout mice show that villin is required for intestinal cell migration as well as Shigella infection (13, 14). Abnormalities in villin gene expression have also been associated with progressive cholestatic liver disease (15).

Villin is a member of a conserved family of actin-associated proteins widely expressed from slime molds to humans. Severin from Dictyostelium discoideum, fragmin from Physarum polycephalum, and the vertebrate proteins villin, gelsolin, adseverin, and scinderin belong to the group of actin-severing proteins that contain 3-6 repeats of a conserved domain. Villin contains six such domains (S1-S6). Like several proteins of its family, villin caps, nucleates, and severs actin filaments, and these functions are confined to the villin core (S1-S6). In addition, villin contains a carboxyl-terminal domain (S7) called the headpiece, which provides villin with the ability to cross-link actin filaments (16). We have demonstrated previously that villin is tyrosine-phosphorylated in vitro and in vivo by c-Src kinase (18, 19).2 Likewise, gelsolin, fragmin, and CapG have been shown to be tyrosine-phosphorylated in vitro by c-Src (20). Furthermore, proteomic analysis of phosphotyrosyl proteins in human lumbar cerebrospinal fluid has been shown to include tyrosine-phosphorylated gelsolin (21). These reports and our own data point to a more general mechanism involving tyrosine phosphorylation of this family of proteins, thus giving new properties to these proteins and adding another level of regulation that will be recognized by future studies involving the identification of the phosphorylated tyrosine residues and functional assays. Epithelial cells of the intestine and kidney express more than one protein of this family (villin, gelsolin, and adseverin) (22). We and others have reported previously that although these proteins share structural homology they are not functionally identical (18, 23-26). The identification of the tyrosine phosphorylation sites and their molecular characterization in these proteins will facilitate our understanding of their functional diversity. Furthermore, such studies will help elucidate why some cells express more than one protein of this family and whether these proteins have identical, overlapping, or distinct functions in these tissues.

Tyrosine phosphorylation of villin releases its auto-inhibited conformation allowing it to sever actin at physiologically relevant calcium concentrations (24). We have also reported that tyrosine phosphorylation regulates villin functions, specifically, the ability of villin to modify the actin cytoskeleton, redistribution of F-actin in cells, and villin-induced changes in cell shape and cell motility (19). In addition, tyrosine phosphorylation of villin modifies the ligand-binding properties of villin, including its association with phosphatidylinositol 4,5-bisphosphate (PIP2),3 phospholipase C-{gamma}1 (PLC-{gamma}1), and F-actin (1, 18, 27). Regulation of the functions of villin by tyrosine phosphorylation, which is often a consequence of receptor activation, suggests that villin may function to communicate cell surface activation to the cytoskeletal machinery.

In an effort to comprehend the role of tyrosine phosphorylation to the function of villin, we have elected to identify the tyrosine phosphorylation sites in human villin and map the functions of villin to these sites. We have previously identified four major phosphorylated tyrosine residues in the villin core (19, 28). In this study we identify six additional sites in the carboxyl-terminal villin core (domains S3-S6). With this study we have identified all the tyrosine residues in human villin that can be phosphorylated, and we mapped the functions of villin regulated by these phosphorylation sites. Specifically, we have characterized the role of these sites in regulating F-actin reorganization, cell morphology, and cell motility by transfecting full-length, truncation, or phosphorylation site mutants of villin in the tetracycline-regulated HeLa and MDCK cells. In this study, we demonstrate for the first time that the carboxyl-terminal tyrosine phosphorylation sites in villin are required for its association with PLC-{gamma}1, thus determining their significance in villin-induced cell migration. Because there is considerable structural and functional homology between villin and other proteins of its family, the results presented here help with understanding the relationship of phosphorylation with the role of these proteins in cell migration (14, 19, 29-31).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Epicurian coli TKX1 and BL21 competent cells and the QuikChange site-directed mutagenesis kit were from Stratagene. Glutathione-Sepharose 4B Fastflow was from Amersham Biosciences. GelCode Blue was from Pierce. Monoclonal antibodies to phosphotyrosine (clone PY-20) were from ICN; monoclonal antibodies to villin were from Transduction Laboratories, and polyclonal phospho-villin antibody VP-70782 used in these studies has been described previously (19). Monoclonal antibodies to phospholipase C-{gamma}1 were purchased from Upstate%20Biotechnology">Upstate Biotechnology, Inc. Monoclonal antibody to the influenza A virus hemagglutinin (HA) tag was purchased from Roche Applied Science. Alexa Fluor 488-phalloidin was purchased from Molecular Probes, and Cy3-conjugated affinity-purified donkey anti-mouse IgG was purchased from Jackson ImmunoResearch. Lysophosphatidic acid (LPA) and epidermal growth factor (EGF) were purchased from Sigma. Cell Death Detection ELISA Plus kit was purchased from Roche Applied Science. HOECHST 33258 was purchased from Sigma. HeLa Tet-Off cells stably expressing the tetracycline-controlled transactivator, G418, hygromycin, doxycycline, the eukaryotic expression vectors pTRE-6xHN, pTRE-HA, and the selection vector pTK-Hyg were purchased from Clontech. MDCK Tet-Off cells were a kind gift from Dr. Keith Mostov (University of California, San Francisco). LipofectamineTM 2000 was purchased from Invitrogen. 35-mm glass bottom culture dishes were purchased from Mat Tek Corp. The muscle actin polymerization kit was purchased from Cytoskeleton (Denver, CO). All other chemicals were from Sigma or Invitrogen.

Tyrosine Phosphorylation of Villin in TKX1 Cells—Full-length or mutant villin cDNA cloned in pGEX-4T1 were expressed in E. coli TKX1 cells as described previously (18). Briefly, TKX1 cells carry a plasmid with the elk tyrosine kinase (tk) gene controlled by the trp promoter. The TKX1 cells were transformed with wild-type or mutant villin plasmids. A two-step protocol was followed to first induce the villin gene (by addition of isopropyl beta-D-thiogalactopyranoside) followed by induction of the tk gene (by addition of indoleacrylic acid (IAA)), which generated glutathione S-transferase (Gst)-tagged tyrosine-phosphorylated villin protein(s). TKX1 cells transformed with the villin gene and cultured in the absence of IAA were used to obtain nonphosphorylated villin controls. Tyrosine-phosphorylated proteins were detected by Western analysis using a phosphotyrosine monoclonal antibody (PY-20). Densitometric analysis was carried out using Scion Image software.

Amino-terminal Truncation Mutants of Villin—To identify the villin phosphorylation site(s), we created amino-terminal truncation mutants of villin using full-length human villin cDNA cloned in the prokaryotic expression vector pGEX-4T1. Briefly, PCR was used to introduce EcoRI (Forward) and XhoI (Reverse) restriction sites using the following primers: CT, 5' (Forward)-AGGGAATTCGCCACACGGCCACTGACA, 5' (Reverse)-CTTTCTCGAGGAATAGGTACATTATTA; CT1, 5' (Forward)-GTGGAATTCGTGAAGTTCGATGCCACA, 5' (Reverse)-CTTTCTCGAGGAATAGGTACATTATTA; CT2, 5' (Forward)-TTTGAATTCCCAGCGCGGGCCAATTTC, 5' (Reverse)-CTTTCTCGAGGAATAGGTACATTATTA; CT3, 5' (Forward)-AGGGAATTCGCCACACGGCCACTGACA, 5' (Reverse)-TTTGGCCCTCGAGCCCACAGTGTG; CT4, 5' (Forward)-GTGGAATTCGTGAAGTTCGATGCCACA, 5' (Reverse)-TGGACCTCGAGCAGCCGTGTGGAG; CT5, 5' (Forward)-GTGGAATTCGTGAAGTTCGATGCCACA, 5' (Reverse)-CAGGTAAGGTACTCGAGCTTCTAGCCGAT; and CT6, 5' (Forward)-ATCGGCGAATTCCAGCATTCCTG, 5' (Reverse)-TGGACCTCGAGCAGCCGTGTGGAG. The PCR amplicons were digested using EcoRI and XhoI and directionally cloned in pGEX-4T1. The cloning of the specific truncation mutants was confirmed by sequencing.

Substitution of Tyrosine with Phenylalanine in Villin Truncation Mutants—The putative phosphorylatable tyrosine residues in the truncation mutants (CT2-CT6) were changed to phenylalanine by designing complementary primers in which a Tyr codon was replaced with a Phe codon. Tyrosines at positions 286, 296, 324, 422, 427, 431, 433, 441, 444, 461, 470, 555, 604, 681, and 725 were replaced with phenylalanine using the QuikChange site-directed mutagenesis kit to make single-base change from TAT and TAC to TTT and TTC respectively. The mutation primers were as follows: Y286F, 5' (Forward)-TCACGAGGACTGGTTTCATCCTGGACCAGG and 5' (Reverse)-CCTGGTCCAGGATGAAACAGTCCTCGTGA; Y296F, 5' (Forward)-GGCCTGAAGATCTTCGTGTGGAAAGGG and 5' (Reverse)-CCCTTTCCACACGAACATCTTCAGGCC; Y324F, 5' (Forward)-ATCAAAGCCAAGCAGTTCCCACCAAGCACACCAG and 5' (Reverse)-CTGTGTGCTTGGTGGGAACTGCTTGGCTTTGAT; Y422F 5' (Forward)-CTAGGCCACTTCTTTGGGGGCGACTGC and 5' (Reverse)-GCAGTCGCCCCCAAAGAAGTGGCCT-AG; Y427F, 5' (Forward)-GGGGGCGACTGCTTCCTGCTGCTCTAC and 5' (Reverse)-GTAGAGCAGCAGGAAGCAGCAGTCGCCCCC; Y431F, 5' (Forward)-TACCTGCTGCTCTTCACCTACCTCATC and 5' (Reverse)-GATGAGGTAGGTGAAGAGCAGCAGGTA; Y433F, 5' (Forward)-CTGCTCTACACCTTCCTCATCGGCGAG and 5' (Reverse)-CTCGCCGATGAGGAAGGTGTAGAGCAG; Y441F, 5' (Forward)-GGCGAGAAGCAGCATTTCCTGCTCTACGTTTGG and 5' (Reverse)-CCAAACGTAGAGCAGGAAATGCT-GCTTCTCGCC; Y444F, 5' (Forward)-CATTACCTGCTCTTCGTTTGGCAGGGC and 5' (Reverse)-GCCCTGCCAAACGAAGAGCAGGTAATG; Y461F 5' (Forward)-ACAGCATCAGCTTTCAAGCCGTCATC and 5' (Reverse)-GATGACGGCTTGAAAAGCTGATGCTGT; Y470F, 5' (Forward)-ATCCTGGACATCCGGACCAGAAGTTCAATGGTGAACCAGTC and 5' (Reverse)-GACTGGTTCACCATTGAACTTCTGGTCCAGGAT; Y555F, 5' (Forward)-ACCCAGTCTTGCTGCTTTCTATGGTGTGGGAAG and 5' (Reverse)-CTTCCCACACCATAGAAAGCAGCAAGACTGGGT; Y604F, 5' (Forward)-TGGGAAGGCCCCCTTTGCCAACACCAAGAG and 5' (Reverse)-CTCTTGGTGTTGGCAAAGGGGGCCTTCCCAC; Y681F, 5' (Forward)-ACCACTGCACAGGAATTCCTCAAGACCCATCCC and 5' (Reverse)-GGGATGGGTCTTGAGGAATTCCTGTGCAGTGGT; Y725F, 5' (Forward)-TAACACCAAATCCTTTGAGGACCTGAAGG and 5' (Reverse)-CCTTCAGGTCCTCAAAGGATTTGGTGTTA. A villin mutant lacking all 10 identified tyrosine phosphorylation sites, namely Tyr-46, -60, -81, -256, -286, -324, -461, -555, -604 and -725 (substituted with phenylalanine), was made to confirm the tyrosine phosphorylation status of recombinant villin expressed in TKX1 cells (VILT/WT (AYFM)).

Urea Denaturation Assay—To determine the effects of specific mutations on the overall stability of the villin molecules, fluorescence-monitored urea denaturation was performed on each recombinant protein as described previously (25). Fluorescence measurements were taken at an excitation wavelength of 280 nm, and an emission scan was performed from 335 to 360 nm.

Measurement of Actin Polymerization and Depolymerization by Phosphorylated Wild Type and Point Mutants of Villin—The kinetics of actin polymerization were determined using a muscle actin polymerization kit according to the instructions of the manufacturer and as described previously (26). The ability of villin to nucleate actin assembly or to depolymerize actin filaments was determined by its effect on the rate and extent of increase or decrease, respectively, of fluorescence of pyrene-labeled actin. Fluorescence measurements were performed at 25 °C using the FluoroMax 3 spectrofluorometer. The excitation wavelength was set at 365 nm, and the emission wave-length was set at 407 nm.

Measurement of Actin Uncapping by Phosphorylated Wild Type and Point Mutants of Villin—The actin-capping activity of wild-type and mutant villin proteins was measured essentially as described by Northrop et al. (32) using pyrene-labeled actin as described by Schafer et al. (33). 290 nM villin-actin seeds were used as nuclei for polymerization with pyrene-labeled G-actin (1.4 µM) in a reaction volume of 200 µl. The increase in fluorescence was measured over time as described before (26). The concentration of calcium (2.5 µM) used in the assays has been shown to be saturating for capping but not severing of actin filaments by villin.

Transfection of HeLa and MDCK Tet-Off Cells with Full-length and Mutant Villin cDNA—Full-length villin and the point mutants of villin (Y286F, Y324F, Y461F, Y555F, Y604F, and Y725F) were cloned in the eukaryotic expression vector pTRE-6xHN by amplification using PCR of the coding sequences cloned in pGEX-4T1 as described before (19). Three additional constructs were made as follows: (i) VIL/ANFM, in which all four amino-terminal tyrosine phosphorylation sites, namely Tyr-46, Tyr-60, Tyr-81, and Tyr-256, were mutated to phenylalanine; (ii) VIL/ACFM, in which all six carboxyl-terminal tyrosine phosphorylation sites, namely Tyr-286, Tyr-324, Tyr-461, Tyr-555, Tyr-604, and Tyr-725, were mutated to phenylalanine; and (iii) VIL/AYFM, in which all 10 phosphorylatable tyrosine residues were mutated to phenylalanine. Yellow fluorescent protein-tagged version of full-length villin was made by using superenhanced yellow fluorescent protein (SEYFP) subcloned into the SalI site of full-length villin cloned into pTRE-HA between the HA tag and villin (SEYFP/FL) in collaboration with Dr. Ian Macara (University of Virginia School of Medicine, Charlottesville, VA) (19). To check if villin and SEYFP were in-frame, the following primer was used, 5'-CATGGTCCTGCTGGAGTTCGTCA; and to check if SEYFP was inframe with the HA tag, the following sequencing primer was used, 5'-CGCCTCCAGACGCCATCCACGCT. Deletion mutant S1-S3 (SEYFP/S1-S3) was made by designing complementary primers to introduce a stop codon at position 338 of SEYFP/VIL using the QuikChange site-directed mutagenesis kit, as recommended by the manufacturer. The following primers were used: 5' (Forward)-TGGGGCTGAGTAGGCCGTCTTTCAGC and 5' (Reverse)-GCTGAAAGACGGCCTACTCAGCCCCA. The introduction of the stop codon was verified by sequencing. SEYFP-tagged versions of VIL/ANFM, VIL/ACFM, and VIL/AYFM were cloned by digesting VIL/ANFM, VIL/ACFM, and VIL/AYFM cloned in pTRE-HA with SalI. Likewise the 0.7-kb SEYFP fragment was obtained by digestion of SEYPF/FL with SalI. The recombinant constructs VIL/ANFM, VIL/ACFM, or VIL/AYFM were ligated with the SEYFP fragment using nondirectional sticky end cloning. The cloning of the SEYFP insert was verified by sequencing, and the SEYFP fragment was found upstream of VIL/ANFM, VIL/ACFM, and VIL/AYFM sequences and in the correct reading frame with the HA tag. HeLa Tet-Off cells were stably cotransfected with the wild-type or mutant villin plasmids and a selection plasmid carrying the hygromycin resistance gene. Villinnull HeLa and MDCK Tet-Off cells were transiently transfected with SEYFP/FL, SEYFP/ACFM, or SEYFP/S1-S3 plasmids, and cells were analyzed 16-24 h post-transfection. Alternatively, MDCK Tet-Off cells were stably transfected with SEYFP/FL, SEYFP/ANFM, SEYPF/ACFM, or SEYFP/AYFM.

Cell Motility Assay—HeLa Tet-Off cells transfected with wild-type or mutant villin proteins were seeded in 6-well plates and cultured in the absence (VIL+) or presence (VIL-) of doxycycline. Confluent monolayers were scraped with a plastic pipette tip to generate wounds essentially as described before (19). Images were obtained at the initial time of wounding and at various time intervals up to 24 h post-wounding. Data are expressed as a percentage of original wound width. Wound width measurements were averaged from two regions of the same well, and the mean was treated as a single data point. Comparisons between mean values were made using one-way repeated measures analysis of variance and Tukey's modified t test (Benferroni criteria) with p < 0.05.

Immunofluorescence Microscopy—HeLa Tet-Off cells expressing wild-type and mutant villin proteins were cultured on coverslips and fixed in 3.7% formaldehyde and permeabilized by incubation in phosphate-buffered saline containing 0.2% Triton X-100 and 0.5% normal goat serum. Cells were incubated with villin monoclonal antibody (1:100), and Alexa Fluor 488-phalloidin was included to record the distribution of F-actin. The secondary antibody was Cy3-conjugated affinity-purified donkey anti-mouse IgG (1:100). The fluorescence was examined by confocal laser scanning microscopy (LSM 5 PASCAL; Carl Zeiss, Thornwood, NY).

For time-lapse microscopy, cells were incubated on 35-mm glass bottom dishes. MDCK/HeLa cells transiently transfected with SEYFP/FL or SEYFP/S1-S3 were used for these studies 16-24 h post-transfection. Alternatively MDCK Tet-Off cells stably transfected with SEYPF/FL, SEYFP/ANFM, or SEYFP/ACFM were used for these studies. Cells were washed twice with phosphate-buffered saline, and 50 ng/ml epidermal growth factor (EGF) or lysophosphatidic acid (LPA 1 µM) were added to the cells 5 min before time-lapse images were captured. Time-lapse images were acquired with a x40 objective on a confocal microscope (LSM 5 PASCAL, Carl Zeiss, Thornwood, NY). Images were captured every 2 min for a maximum of 60 min.

Measurement of Lamellipodial Protrusion Rate—Lamellipodia were characterized as thin regions 3-10 µm wide located at the cell margin. Using time-lapse imaging, lamellipodia protrusion rate was measured as the increase in total cell area after growth factor (LPA (1 µM) or EGF (50 ng/ml))-stimulated cell migration using time-lapse images essentially as described before (14, 34). The mean relative area was plotted as a function of time to measure rate of protrusive activity after EGF/LPA treatment. Approximately 10 cells were examined, and for all cells the rate remained approximately constant during the 20 min of observation.

Coimmunoprecipitation Analysis—HeLa or MDCK Tet-Off cells transfected with full-length villin (VIL/FL) or phosphorylation site mutants of villin, namely VIL/ANFM, VIL/ACFM, or VIL/AYFM, were extracted with a solution containing 1% Triton X-100, 20 mM HEPES, pH 7.2, 150 mM NaCl, 1 mM sodium orthovanadate, 50 mM NaF, and a mixture of protease inhibitors for 15 min at 4 °C. PLC-{gamma}1 and tyrosine-phosphorylated villin were immunoprecipitated from the soluble extracts as described previously (1, 19).

Separation of Triton-soluble and -insoluble Pool—For fractionation of Triton X-100-soluble and -insoluble pool, MDCK Tet-Off cells expressing VIL/FL, VIL/ANFM, VIL/ACFM, or VIL/AYFM were lysed in buffer containing 1% Triton X-100, 20 mM HEPES, pH 7.2, 150 mM NaCl, 2 mM EDTA, 1 mM sodium orthovanadate, 50 mM NaF, and a mixture of protease inhibitors for 15 min at 4 °C. After centrifugation, the supernatants were collected and represented the Triton-soluble pool. The pellets were resuspended in buffer containing 15 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 mM EDTA, 1 mM dithiothreitol, 1 mM sodium orthovanadate, and a mixture of protease inhibitors. Samples were vortexed well and incubated on ice for 20 min. The pellet fraction was centrifuged, and the resulting supernatant represented the Triton-insoluble pool. Triton-soluble and -insoluble proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane, and Western analysis was done with villin monoclonal antibodies.

Identification of Apoptotic Cells Using Hoechst 33258 Staining—16 h post-transfection, MDCK Tet-Off cells expressing SEYFP-tagged S1-S3 villin truncation were incubated with Hoechst 33258 at a concentration of 10 µg/ml for 20 min at 37 °C. Apoptotic cells were distinguished from viable cells by nuclear condensation and DNA fragmentation seen as bright blue fluorescence in the nuclei. Morphological changes corresponding to nonviable cells were determined using an inverted Nikon fluorescence microscope with a CoolSnap FX charge coupled device camera.

Quantitative DNA Fragmentation Assay—An enzyme-linked immunosorbent assay was used to quantitate DNA fragmentation in MDCK cells transfected with S1-S3 villin cloned in pTRE-HA and in cells transfected with vector alone, as described previously (35). S1-S3 expressing MDCK cells were cultured in 12-well plates, lysed, and centrifuged to remove nuclei. An aliquot of nuclei-free supernatant was incubated with immunoreagents (anti-histone biotin plus anti-DNA peroxidase-conjugated antibody) in 96-well streptavidin-coated plates for 2 h with shaking. After washing with PBS, 100 µl of peroxidase substrate, 2,2'-azino-di(3-ethylbenzothiazoline sulfonate) was added to each well and incubated for an additional 10 min at room temperature. The absorbance was recorded at 405 nm using a microplate reader. Triplicates of samples were used to quantify protein concentration using the bicinchoninic acid (BCA) kit from Pierce. DNA fragmentation was measured as absorbance units per mg of protein per min and expressed as fold increase in apoptotic cell death in VIL/S1-S3 expressing cells compared with cells transfected with vector alone (control).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Carboxyl Terminus of Villin Is Tyrosine-phosphorylated— We have identified previously four major tyrosine phosphorylation sites (Tyr-46, -60, -81, and -256) in the amino terminus of villin (28). Substitution of all four of these sites with phenylalanine (ANFM) allowed us to determine that there were additional phosphorylation sites in the carboxyl terminus of villin that were tyrosine-phosphorylated in vitro (Fig. 1A). Likewise, expression of this amino-terminal phosphorylation site mutant of villin (VIL/ANFM) in MDCK Tet-Off cells allowed us to confirm that there were additional phosphorylation sites in the carboxyl-terminal domain of villin (Fig. 1B). For these studies, cell extracts of MDCK Tet-Off cells expressing wild-type villin (VIL/FL) or the amino-terminal phosphorylation site mutant of villin (VIL/ANFM) were immunoprecipitated with a phosphovillin antibody (VP-70782) and probed with a phosphotyrosine antibody (PY-20) essentially as described before (19). Together these studies demonstrate that both in vitro as well as in MDCK cells, the carboxyl-terminal domain of villin contributes to the tyrosine phosphorylation of villin. In addition, the identification of at least five tyrosine phosphorylation sites in human plasma gelsolin, including a major phosphorylation site in domain S4, led us to speculate that villin may likewise have tyrosine-phosphorylated residues in the carboxyl-terminal domain(s) (36). To identify these sites in villin as well as to determine the functional significance of these sites, truncation mutants of villin were constructed as shown in Fig. 1C. The constructs consisted of amino-terminal deletions of human villin cDNA engineered into the prokaryotic expression vector pGEX-4T1. Five truncation mutants (CT-CT4) were generated as Gst-tagged fusion proteins in TKX1 cells. The expression of full-length villin and the truncation mutants of villin were assessed by SDS-PAGE and staining with GelCode Blue (Fig. 1D). The apparent molecular masses were in agreement with those predicted for each deletion mutant. To assess tyrosine phosphorylation of these mutants, the proteins were analyzed by Western analysis using a phosphotyrosine antibody (PY-20). All five truncation mutants were tyrosine-phosphorylated (Fig. 1D). These data suggested to us that each of the truncation mutants contained one or more tyrosine-phosphorylated residues. To identify these phosphorylation sites, smaller truncation mutants were generated (Fig. 1C, CT5 and CT6). Individual tyrosine residues in these shorter truncation fragments (CT2, CT3, CT5, and CT6) were substituted with phenylalanine, and the effect of these mutations on the levels of phosphorylation was determined essentially as described before (28).


Figure 1
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  		FIGURE 1.
Tyrosine phosphorylation of wild type and truncation mutants of villin. A, carboxyl terminus of villin contains additional tyrosine phosphorylation sites. Four of the tyrosine phosphorylation sites identified in previous studies (Tyr-46, -60, -81, and -256) were mutated to phenylalanine (ANFM) (28). The mutant villin protein cloned in the pGEX-4T1 was expressed as a nonphosphorylated protein (VIL/WT (ANFM)) by expression in TKX1 cells in the absence of IAA (see "Experimental Procedures") or as a phosphorylated protein by expression in TKX1 cells in the presence of IAA (VILT/WT (ANFM)). Lower panel is an SDS-PAGE stained with GelCode Blue. Upper panel is a Western blot using a phosphotyrosine antibody (PY-20). B, amino-terminal phosphorylation site mutant of villin is tyrosine-phosphorylated in MDCK Tet-Off cells. Cell extracts from MDCK Tet-Off cells stably transfected with full-length villin (VIL/FL) or amino-terminal phosphorylation site mutant (VIL/ANFM) were immunoprecipitated (IP) with phospho-villin antibody (VP-70782) described previously (19) and Western analysis done with phosphotyrosine antibody. C, schematic representation of wild-type and amino-terminal truncation mutants of villin that were expressed as Gst-tagged phosphorylatable proteins in TKX1 cells. D, expression and tyrosine phosphorylation of wild-type (VILT/WT) and truncation mutant (CT-CT4) villin proteins. The left panel is an SDS-PAGE analysis of recombinant, phosphorylated wild-type (VILT/WT) and amino-terminal truncation mutants of villin. The gels were stained with GelCode Blue. The right panel is a Western blot of VILT/WT and truncation mutants of villin probed with phosphotyrosine monoclonal antibody (PY-20). Data are representative of four experiments with similar results.

 
Identification of One Major and Two Minor Phosphorylation Sites in CT2—The truncation mutant of villin CT2 contains four tyrosine residues (Tyr-555, -604, -681, and -725) between amino acids 533 and 827 of human villin that could be phosphorylated. We used site-directed mutagenesis to substitute each of these tyrosine residues with phenylalanine in the truncation mutant CT2. The mutants were expressed in TKX1 cells, and the phosphorylation status of the mutant proteins was analyzed by Western analysis using an anti-phosphotyrosine antibody (Fig. 2A). Densitometric analysis was done by determining the ratio of phosphorylated protein to total protein for each sample (supplemental Fig. 1A). Mutation of Tyr-725 to Phe significantly decreased the level of tyrosine phosphorylation in the truncation mutant CT2 (54.3 ± 3.3%, n = 4, p < 0.001). In contrast mutation of Tyr-555 or Tyr-604 to Phe decreased the level of tyrosine phosphorylation of the villin construct CT2 to a lesser extent (25.0 ± 5.7% and 37.5 ± 4.1% respectively, n = 4, p < 0.001). This suggested that CT2 contains one major (Tyr-725) and two minor (Tyr-555 and Tyr-604) tyrosine-phosphorylated residues. To confirm this observation, double mutants of these sites were generated in CT2. Western analysis of the double mutants using the phosphotyrosine antibody indicated essentially a complete loss of tyrosine phosphorylation with double mutants T725F,Y604F (91%, n = 4, p < 0.001) and Y725F,Y555F (81%, n = 4, p < 0.001) (Fig. 2B, supplemental Fig. 1B). In contrast, the double mutant T604F,Y555F resulted in a relatively smaller decrease in the total phosphorylation level in the truncation mutant CT2 (33.0 ± 5.3%, n = 4, p < 0.001). These results indicate the presence of one major and two minor sites in human villin protein between amino acids 533 and 827.


Figure 2
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  		FIGURE 2.
Identification of the tyrosine phosphorylation sites in the carboxyl-terminal domain of human villin. A, mutant villin proteins were generated by substitution of tyrosine residues at 555, 604, 681, or 725 with phenylalanine in villin truncation mutant CT2 (CT2(Y555F); CT2(Y604F); CT2(Y681F); CT2(Y725F)). B, villin mutant was generated by substituting two of the three identified tyrosine phosphorylation sites in different combinations (CT2 (Y604F;Y555F); CT2 (Y725F;Y604F); CT2 (Y725F;Y555F). C, mutant villin proteins were generated by substitution of tyrosine residues at 286, 296, or 324 with phenylalanine in villin truncation mutant CT3 (CT3 (Y286F); CT3 (Y296F); CT3 (Y324F)). D, mutant villin proteins were generated by substitution of tyrosine residues at 444, 461, or 470 with phenylalanine in villin truncation mutant CT6 (CT6 (Y444F); CT6 (Y461F); CT6 (Y470F)). E, Tyr-461 in villin truncation mutant CT4 was substituted with phenylalanine (CT4 (Y461F). The villin mutants (A-E) were expressed as Gst-fused phosphorylatable proteins in TKX1 cells. The samples were normalized for protein content and subjected to SDS-PAGE and Western analysis using phosphotyrosine antibody (PY-20). The protein content of the gels was determined by separating the proteins by SDS-PAGE and staining the gels with GelCode Blue. Densitometric analysis of these Western blots are shown in supplemental Fig. 1, A-E.

 
Identification of Two Major Phosphorylation Sites in Villin Truncation Mutant CT3—Villin truncation mutant CT3 contains three putative phosphorylatable tyrosine residues, Tyr-286, Tyr-296, and Tyr-324. We mutated each of these tyrosine residues to a phenylalanine and determined the effect of the mutation on the levels of phosphorylation in the truncation mutant CT3. Equal amounts of the proteins were separated by SDS-PAGE and Western analysis done with a phosphotyrosine antibody (PY-20). As shown in Fig. 2C and supplemental Fig. 1C, mutation of Tyr-286 as well as Tyr-324 significantly decreased the levels of tyrosine phosphorylation in the truncation mutant CT3 (99.0 ± 1.0% and 65.9 ± 3.7%, respectively, n = 4, p < 0.001). On the other hand, mutation of Tyr-296 to phenylalanine had no effect on the phosphorylation levels of the truncation mutant CT3. These data demonstrate that in addition to Tyr-555, Tyr-604, and Tyr-725, the carboxyl-terminal domain of villin contains two additional tyrosine residues that can be phosphorylated in vitro, namely Tyr-286 and Tyr-324.

Tyr-461 Is the Major Phosphorylation Site in the Villin Truncation Mutant CT4—As shown in Fig. 1D, the villin truncation CT4 is also tyrosine-phosphorylated, suggesting that this truncation contains one or more tyrosine residues that can be phosphorylated in vitro. Human villin contains 7 tyrosine residues within this domain that could be phosphorylated in vitro. To determine these sites of phosphorylation, we generated two additional truncation mutants, CT5 and CT6, that encompass the amino acid residues between 373 and 533 (similar to CT4, see Fig. 1C). CT5 contains four tyrosine residues, namely Tyr-422, -427, -431, and -433. Mutation of either of these sites to phenylalanine resulted in less than a 5% decrease in the phosphorylation levels suggesting that these are minor sites of phosphorylation, if at all, in the villin truncation CT4 (data not shown). The truncation mutant CT6 contains three tyrosine residues, namely Tyr-461, -444, and -470, and mutation of one of these residues, Tyr-461, completely (99%, n = 5, p < 0.001) abolished the phosphorylation in the truncation CT6 (Fig. 2D and supplemental Fig. 1D). To confirm that Tyr-461 was the only major phosphorylation site in CT4, we also generated a point mutant, Y461F, in CT4 (Fig. 2E and supplemental Fig. 1E). The expression and analysis of this mutant demonstrated that Tyr-461 was the single key phosphorylation site in CT4 (amino acids 373-534 of human villin). In conclusion, in this study we have identified six tyrosine phosphorylation sites in the carboxyl-terminal domain of villin between amino acids 271-827 of human villin.


Figure 3
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  		FIGURE 3.
Human villin protein contains 10 phosphorylatable tyrosine residues. A, sequence alignment of villin and related proteins of the villin superfamily. The top panel shows alignment of villin sequences from human, mouse, and chicken. The bottom panel shows alignment of homologous sequences in gelsolin, adseverin, and CapG. B, substitution of all 10 tyrosine residues at positions 46, 60, 81, 256, 286, 324, 461, 555, 604, and 725 with phenylalanine (VILT/WT (AYFM)) eliminates tyrosine phosphorylation in full-length recombinant villin protein. Tyrosine phosphorylation of full-length recombinant villin protein (VILT/WT) was compared with villin mutant VILT/WT(AYFM) using a phosphotyrosine antibody (PY-20) or a phospho-villin antibody (VP-70782) described previously (19). The right panel is a Western blot of VILT/WT and VILT/WT (AYFM) probed with villin monoclonal antibody. This is a representative of three experiments with similar results. C, mutation of all 10 identified tyrosine residues abolishes tyrosine phosphorylation of villin in MDCK Tet-Off cells. Cell extracts from MDCK Tet-Off cells stably transfected with full-length villin (VIL/FL) or a villin mutant lacking all 10 identified tyrosine phosphorylation sites (VIL/AYFM) were immunoprecipitated with phospho-villin antibody (VP-70782) and Western analysis done with phosphotyrosine antibody (PY-20). D, cell extracts from VIL/FL, VIL/ANFM, VIL/ACFM, and VIL/AYFM were immunoprecipitated with phosphotyrosine antibody (PY-20), and Western analysis was done with a villin monoclonal antibody (upper panel). Middle panel is a Western blot with anti-villin monoclonal antibody, and the bottom panel is a Western blot with anti-actin antibody, which was performed in parallel as quantitative controls. This is a quantitative immunoprecipitate, showing 30.6, 56.79, and 98.52% decrease in the phosphorylation of VIL/ANFM, VIL/ACFM, and VIL/AYFM, respectively, compared with VIL/FL. The Western blot is representative of four other experiments with similar results. Densitometric analysis of this Western blot is shown in supplemental Fig. 1F.

 
Human Villin Contains 10 Phosphorylatable Tyrosine Residues—The six tyrosine phosphorylation sites identified in this study are well conserved in villin across different species. Four of these sites are also conserved among other proteins of the villin family, including gelsolin, adseverin, and CapG (Fig. 3A). Two of these sites, Tyr-324 and Tyr-604, in human villin have been identified as minor sites that are phosphorylated in human plasma gelsolin (Tyr-576 and Tyr-624, respectively, in gelsolin) (36). Mutation of all 10 sites (VIL(T/W)T(AYFM)) in villin, four previously identified (Tyr-46, -60, -81, and -256) as well as the six sites identified in this study (Tyr-286, -324, -461, -555, -604, and -725), resulted in the complete loss of tyrosine phosphorylation in full-length recombinant villin protein (Fig. 3B) (28). These data were confirmed in MDCK Tet-Off cells stably transfected with this villin mutant lacking all 10 identified tyrosine phosphorylation sites (VI(L/A)YFM) (Fig. 3C). Collectively with our previous study, we have now identified 10 tyrosine residues in human villin that are phosphorylated in recombinant villin protein. Furthermore, mutation of these sites in MDCK cells results in the complete loss of villin phosphorylation.

We also examined the tyrosine-phosphorylated villin in MDCK cells expressing full-length villin (VIL/FL), villin mutant lacking the four amino-terminal phosphorylation sites previously identified (VIL/ANFM), villin mutant lacking the six carboxyl-terminal phosphorylation sites identified in this study (VIL/ACFM), and a villin mutant lacking all 10 identified tyrosine phosphorylation sites (VIL/AYFM). MDCK cells stably expressing wild-type and mutant villin proteins were lysed and immunoprecipitated using anti-phosphotyrosine antibody PY-20. Because the commercially available villin monoclonal antibody cannot be used for immunoprecipitation and because the phospho-villin antibody VP-70782 binds to selective tyrosine-phosphorylated epitopes of villin, we reasoned that a phosphotyrosine antibody would bind phosphorylated villin proteins with comparable affinity. We have demonstrated previously that the phosphotyrosine antibody (PY-20) immunoprecipitates tyrosine-phosphorylated villin protein (1, 19). Quantitative immunoprecipitation studies demonstrated that in MDCK cells VIL/FL, VIL/ANFM, and VIL/ACFM were tyrosine-phosphorylated, whereas villin mutant VIL/AYFM was not (Fig. 3D). Densitometric analysis revealed that mutation of the amino-terminal phosphorylation sites or the carboxyl-terminal phosphorylation sites resulted in a 30.6 ± 12.22 and 56.79 ± 10.96% decrease, respectively, in the phosphorylation levels of mutant villin proteins compared with wild-type villin protein (supplemental Fig. 1F). Mutation of all 10 tyrosine phosphorylation sites (VIL/AYFM) resulted in the complete loss of phosphorylation of the villin protein in MDCK cells (98.52 ± 0.47% decreased compared with VIL/FL). These studies demonstrate a net reduction in phosphorylation of villin mutants VIL/ANFM, VIL/ACFM, and VIL/AYFM that is proportional to the number of sites identified in the recombinant villin protein.

Functional Significance of Tyrosine Phosphorylation Sites Identified in the Carboxyl Terminus of Villin—Each of the six tyrosine phosphorylation sites identified in this study were mutated individually in full-length human villin cDNA to phenylalanine (VILT/WT(Y286F); VILT/WT(Y324F); VILT/WT(Y461F); VILT/WT(Y555F); VILT/WT(Y604F); and VILT/WT(Y725F)), expressed in TKX1 cells, and purified as Gst-tagged tyrosine-phosphorylated proteins. All six mutants were tyrosine-phosphorylated but lacked one of the identified tyrosine residues (Fig. 4A). In order to assess whether the single point mutants maintained the conformation of the wild-type villin protein, the unfolding profiles of wild-type and mutant villin proteins expressed in the absence of IAA (nonphosphorylated proteins) as a function of the urea concentration were recorded by measuring the intrinsic tryptophan fluorescence emission spectrum essentially as described before (25). The results indicate that single point mutants express similar unfolding transitions as wild-type villin, thus maintaining a comparable overall conformation as the wild-type recombinant villin protein (Fig. 4B).

Full-lengthnonphosphorylatedvillin(VIL/WT),phosphorylated villin (VILT/WT), and the phenylalanine-substituted single point mutants were used in a pyrene-based actin polymerization and depolymerization assay as described before (28). As shown in Fig. 4C, the addition of nonphosphorylated full-length villin (VIL/WT) increased the initial rate of actin polymerization compared with the polymerization kinetics of actin alone (control). In contrast, tyrosine-phosphorylated full-length villin (VILT/WT) resulted in a lag phase and decreased the rate of actin polymerization. This is consistent with our previously published observation that tyrosine phosphorylation inhibits the actin-nucleating function of villin (24, 27, 28). Mutation of any of the six identified phosphorylation sites individually from Tyr to Phe did not rescue the actin-nucleating property of nonphosphorylated full-length villin protein. This observation is similar to what we determined earlier with the amino-terminal phosphorylation sites (28). Together, these studies suggest that the entire villin core and multiple tyrosine phosphorylation sites in the villin core could regulate actin nucleation by villin. Trypsin cleaves villin into two fragments, 44T (amino-terminal domain) and 51T (carboxyl-terminal domain), both of which bind G-actin in the presence of calcium (37). It has been reported previously that actin nucleation by 44T is significantly less than full-length villin protein (37, 38). A similar observation was made using a villin variant (V1-3 + HP) that lacks domains S4-S6 (39). Consistent with this observation, actin nucleation by gelsolin requires the domains S4-S6 (or G4-G6) (40, 41). Our study suggests that some of the phosphorylation sites identified by us, both in the amino and the carboxyl-terminal domain, could regulate the actin nucleating activity of villin, consistent with these previous observations that demonstrate that the actin-nucleating activity of villin is determined by the entire villin core.

Next we examined the effect of tyrosine phosphorylation of villin on F-actin depolymerization. Nonphosphorylated full-length villin protein (VIL/WT) increased the depolymerization of F-actin compared with control samples (Fig. 4D). Furthermore, phosphorylation of villin enhanced its actin-depolymerizing property, similar to our previous reports (24, 27, 28). Mutation of Tyr-286 to Phe resulted in actin severing by villin comparable with that of nonphosphorylated full-length villin protein, whereas all other mutants (Y324F, Y461F, Y555F, Y604F, or Y725F) behaved like VILT/WT. These data suggest that Tyr-286 is the major phosphorylation site that regulates the actin-depolymerizing function of villin. Taken together with our previous study, we have now determined three tyrosine residues, namely Tyr-46, Tyr-60, and Tyr-286, in the amino-terminal domain (S1-S3) of villin that regulate its actin-depolymerizing activity (28).

The ability of VIL/WT, VILT/WT, and the phosphorylation site mutants of villin to bind to the plus or barbed end of actin filaments was tested by following the polymerization kinetics of pyrene-labeled G-actin from barbed ends under polymerization conditions where there is little or no growth from the pointed end. The concentration of calcium (2.5 µM) used in this assay has been shown to be saturating for capping but not severing of actin filaments by villin (32). As shown in Fig. 4E, addition of pyrene-labeled G-actin in the presence of F-actin seeds results in significant and rapid actin polymerization over time. VIL/WT shows decreased rates of polymerization in the presence of 2.5 µM calcium, consistent with capped barbed ends. VILT/WT as well as the phosphorylation site mutants of villin likewise capped the barbed ends of actin. The inhibition of F-actin polymerization in the presence of 20 nM cytochalasin D confirms the fact that capping of the barbed end is indeed the cause of abolished actin polymerization. These data show that phosphorylated villin caps actin filaments very similar to nonphosphorylated villin protein. Furthermore, these results demonstrate that the actin capping activity of villin is not regulated by tyrosine phosphorylation. Taken together, in this study we identified six new tyrosine phosphorylation sites in villin and mapped these sites with the actin-modifying functions of recombinant villin in vitro (Fig. 4F).

Regulation of Villin-induced Cell Migration by Carboxyl-terminal Phosphorylation Site Mutants—As we have shown previously that tyrosine phosphorylation of villin regulates cell migration, we examined the ability of the carboxyl-terminal phosphorylation site mutants of villin to regulate this function (19). For these studies, HeLa Tet-Off cells were stably transfected with wild-type or the individual phosphorylation site mutants of villin. Clones expressing comparable levels of wild-type and phosphorylation site mutant proteins were selected for these studies (supplemental Fig. 2A). As shown in supplemental Fig. 2B, panels b1 to b3, and as reported previously, overexpression of wild-type villin results in loss of stress fibers and redistribution of F-actin at or near the cell surface compared with untransfected cells that appear more flat and exhibit stress fibers (supplemental Fig. 2B, panels a1 to a3) (19). Mutation of any of the six carboxyl-terminal phosphorylation sites individually did not result in any significant changes in either the distribution of villin or F-actin (supplemental Fig. 2B, panels c-h). Intracellular distribution of F-actin and villin was also examined in HeLa cells expressing the villin mutants VIL/ACFM as well as VIL/ANFM. Clones expressing comparable levels of VIL/ACFM and VIL/ANFM were selected for these studies (supplemental Fig. 2B). There was no significant change in either the cell morphology or the intracellular distribution of villin or F-actin in cells expressing VIL/ACFM compared with VIL/FL (compare supplemental Fig. 2B, panels j1 to j3, and supplemental Fig. 2B, panels b1 to b3). In contrast, the villin mutant VIL/ANFM demonstrated a cell shape change consistent with villin-null cells (supplemental Fig. 2B, panels i1 to i3, and supplemental Fig. 2B, panels a1 to a3). This is similar to the observation made with individual amino-terminal phosphorylation site mutants (19). Together with our previous studies, these data suggest that changes in the microfilament structure and cell morphology as well as the intracellular distribution of villin are regulated by the amino-terminal phosphorylation sites (19). Next, we examined the effects of these point mutants on villin-induced cell migration. Consistent with our previous report, HeLa Tet-Off cells overexpressing wild-type (VIL+) villin migrated faster than villin null cells (VIL-) (19). The carboxyl-terminal phosphorylation site point mutants behaved like HeLa cells expressing wild-type villin and individually did not regulate villin-induced cell migration (supplemental Fig. 2C). To test the possibility that these sites may collectively be important to the function of villin in cell migration, we examined the villin mutant lacking all six carboxyl-terminal phosphorylation sites (VIL/ACFM). To our surprise, mutation of all six carboxyl-terminal tyrosine residues to phenylalanine significantly inhibited the villin-induced increase in cell migration (Fig. 5A). The villin mutant VIL/ACFM migrated significantly slower than VIL/FL and more like the villin-null cells (p < 0.01, n = 24). Likewise, mutation of all four amino-terminal phosphorylation sites (VIL/ANFM) significantly (p < 0.01, n = 24) inhibited villin-induced cell migration, consistent with our previous report (19). Together with our previous studies, these data demonstrate that multiple tyrosine phosphorylation sites contained within the core are required for the function of villin in cell migration.


Figure 4
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  		FIGURE 4.
Regulation of actin dynamics by the carboxyl-terminal tyrosine phosphorylation sites in villin. A, expression of full-length villin proteins with point mutations at position 286, 324, 461, 555, 604, or 725. Tyrosine residues at these positions were substituted with phenylalanine in full-length villin and expressed as tyrosine-phosphorylated recombinant proteins in TKX1 cells. The mutant proteins were characterized by Western analysis with anti-villin or anti-phosphotyrosine (PY-20) monoclonal antibodies. Data are representative of three experiments with similar results. B, urea-induced, equilibrium-unfolding transition of wild-type and mutant villin proteins. Urea-induced denaturation of wild-type and mutant villin proteins was measured by the change in tryptophan fluorescence maxima at an excitation wavelength of 280 nm and an emission scan from 300 to 420 nm. Fluorescence data points are the average of three independent experiments. The sample concentration used was 2 µM. C, effect of wild-type and phosphorylation site mutant villin proteins on the kinetics of actin polymerization. Pyrene-labeled G-actin (6 µM) was incubated with nonphosphorylated (VIL/WT), tyrosine-phosphorylated (VILT/WT), or mutant villin proteins (120 nM) in polymerization-inducing buffer, and fluorescence intensity was measured over time. Control represents the polymerization of actin in the absence of villin. Fluorescence was recorded every 15 s as described under "Experimental Procedures." D, effect of wild-type and phosphorylation site mutant villin proteins on the kinetics of actin depolymerization. Pyrene-labeled F-actin (1 µM) in the presence of nonphosphorylated (VIL/WT), phosphorylated (VILT/WT), or mutant villin proteins (60 nM) was diluted to 0.1 µM in actin depolymerizing buffer, and the decrease in fluorescence intensity was followed over time. Control represents the depolymerization of actin in the absence of villin. E, effect of wild-type and phosphorylation site mutant villin proteins on the kinetics of actin capping. Polymerization of 1.4 µM G-actin at barbed ends was nucleated by F-actin (290 nM) seeds in the absence (control) or presence of nonphosphorylated (VIL/WT), phosphorylated (VILT/WT), or mutant villin proteins. Cytochalasin D at 20 nM capped the actin filaments at barbed ends and decreased actin polymerization. Values represent the mean of three independent experiments. F, schematic representation of the actin-modifying activities of villin mapped to the carboxyl-terminal tyrosine phosphorylation sites. Asterisk indicates that mutation of any of the six tyrosine residues individually to phenylalanine did not restore the actin-nucleating activity of nonphosphorylated villin protein (VIL/WT), and thus two or more of these sites could regulate actin-nucleation by villin.

 
To further explore the mechanisms underlying the observations described above, we generated a villin truncation mutant, S1-S3 (amino acids 1-338), that lacks the second half of the villin core (S4-S6), as well as the headpiece (S7), but retains the amino-terminal tyrosine phosphorylation sites. Full-length villin (VIL), villin mutant lacking the carboxyl-terminal phosphorylation sites (ACFM), and S1-S3 truncation mutants were transiently expressed as SEYFP-tagged fusion proteins. 16 h post-transfection cells expressing SEYFP/VIL, SEYFP/ACFM, and SEYFP/S1-S3 were treated with LPA (1 µM) or EGF (50 ng/ml), and time-lapse images were recorded every 2 min for 35 min. MDCK cells overexpressing full-length villin and treated with EGF (50 ng/ml) show association of villin with the lamellipodia (Fig. 5B). The association of villin with these cell surface structures was tightly coupled to the growth of the lamellipodia and the forward extension of the cell body (supplemental Video 1). In contrast, MDCK Tet-Off cells overexpressing either SEYFP/ACFM or SEYFP/S1-S3 villin did not form lamellipodia and demonstrated little if any cell spreading during the 35 min of LPA/EGF treatment. In addition, unlike the full-length villin protein, the truncation mutant S1-S3 was localized diffusely to the cytosol (Fig. 5B; Video 3 and Video 4). A similar observation was made in cells treated with LPA (data not shown). Consistent with our previous report, time-lapse images of MDCK Tet-Off cells transfected with the villin mutant lacking all four amino-terminal phosphorylation sites, SEYFP/ANFM, also show no lamellipod formation in response to EGF treatment (supplemental Fig. 3A and Video 4) (19). We quantitated these morphological changes by measuring the lamellipodial protrusion rate in MDCK Tet-Off cells expressing wild-type or mutant villin proteins essentially as described before (14, 34). MDCK cells expressing SEYFP/VIL demonstrated a significant increase in the rate of lamellipodia formation (0.29 ± 0.008 µm/min) compared with cells expressing SEYFP/ACFM (0.07 ± 0.003 µm/min) or SEYFP/S1-S3 (0.03 ± 0.044 µm/min) (Fig. 5C). Similar observations were made in HeLa Tet-Off cells transiently transfected with these plasmids (data not shown). These data suggest that optimal lamellipodia formation and a maximum increase in cell migration following EGF/LPA treatment occurred in cells expressing full-length villin and not in cells expressing the amino-terminal phosphorylation site mutant, the carboxyl-terminal phosphorylation site mutant, or the carboxyl-terminal truncation mutant of villin. Together these data confirm our observation that like the amino-terminal domain, the carboxyl-terminal domain of villin is also required for villin-induced cell migration.


Figure 5
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  		FIGURE 5.
Collectively the carboxyl-terminal tyrosine residues regulate villin-induced cell migration. A, villin mutants lacking all four amino-terminal phosphorylation sites (Tyr-46, Tyr-60, Tyr-81, and Tyr-256; VIL/ANFM) or villin mutant lacking all six carboxyl-terminal phosphorylation sites (Tyr-286, Tyr-324, Tyr-461, Tyr-555, Tyr-604,and Tyr-725; VIL/ACFM) were cloned in pTRE-6xHis and transfected in HeLa Tet-Off cells. HeLa Tet-Off cells expressing equal amounts of wild-type (VIL/FL) or amino- and carboxyl-terminal phosphorylation site mutants (VIL/ANFM, VIL/ACFM) were used in a wound-healing assay, as described under "Experimental Procedures." Cells expressing wild-type villin migrate significantly faster than villin-null (VIL-) cells. The amino- and carboxyl-terminal phosphorylation site mutants, VIL/ANFM and VIL/ACFM, migrate like villin-null cells. The error bars are the measured S.E., and the asterisk denotes statistically significant values (p < 0.01, n = 24, compared with VIL/FL). B, the carboxyl-terminal phosphorylation sites in villin regulate villin-induced lamellipodia formation. MDCK Tet-Off cells transfected with SEYFP-tagged full-length villin (SEYFP/VIL), villin mutant lacking the amino-terminal phosphorylation sites (SEYFP/ANFM), or villin truncation mutant lacking the carboxyl-terminal domain (SEYFP/S1-S3) were treated with EGF (50 ng/ml), and time-lapse images were recorded 5 min after the addition of EGF for a total of 36 min. MDCK cells expressing full-length villin form lamellipodia in response to EGF treatment, and villin localizes to the lamellipodia. In contrast cells expressing either SEYFP/ACFM or SEYFP/S1-S3 do not form lamellipodia. Bars,10µm. C, lamellipodial protrusion rates were determined as described under "Experimental Procedures." 10 cells were examined, and for all cells the rate remained approximately constant during the 20 min of observation. Average velocity of lamellipod formation is denoted in µm/min. The errorbars are the measured S.E., and the asterisk denotes statistically significant values (p < 0.001, n = 10, compared with VIL/FL).

 
Tyrosine Phosphorylation of Carboxyl-terminal Domain of Villin Is Required for Its Association with PLC-{gamma}1—Because the carboxyl-terminal phosphorylation site of mutants did not result in any significant changes in cell shape or actin redistribution (supplemental Fig. 2), we speculated that the carboxyl-terminal phosphorylation sites in villin may determine its interaction with ligands that are crucial to cell migration. We have demonstrated previously that tyrosine-phosphorylated villin associates with PLC-{gamma}1 and that PLC-{gamma}1 is a major binding partner of tyrosine-phosphorylated villin in detergent-soluble fractions of cells (1, 18). Furthermore, we know from previous studies that PLC-{gamma}1 is required for villin-induced increase in cell migration.2 Based on these previous studies, we hypothesized that the COOH-terminal phosphorylation sites in villin may determine the association of villin with PLC-{gamma}1. To test this hypothesis, we examined the coimmunoprecipitation of villin with PLC-{gamma}1 in MDCK Tet-Off cells ectopically expressing VIL/FL, VIL/ACFM, VIL/ANFM, or VIL/AYFM. By using immunoprecipitated PLC-{gamma}1, we observed that tyrosine-phosphorylated full-length villin associated with PLC-{gamma}1 in MDCK cells expressing wild-type villin protein (Fig. 6A). These data are consistent with our previous reports demonstrating the association of tyrosine-phosphorylated villin with PLC-{gamma}1 (1, 18).2 In cells expressing VIL/ANFM, PLC-{gamma}1 formed a complex with the mutant villin protein, indicating that amino-terminal phosphorylation sites are not involved in PLC-{gamma}1 binding. In contrast, in cells expressing either VIL/ACFM or VIL/AYFM, PLC-{gamma}1 did not form a complex with villin. All individual point mutants of the carboxyl-terminal phosphorylation sites can associate with PLC-{gamma}1 (supplemental Fig. 3B). It may be noted that for these coimmunoprecipitation studies PLC-{gamma}1 was immunoprecipitated from the Triton X-100-soluble fraction of cells. These data show that collectively the carboxyl-terminal tyrosine phosphorylation sites determine the association of villin with PLC-{gamma}1. In concurrence with these immunoprecipitation studies, analysis of the detergent-soluble and -insoluble pools indicated that wild-type villin protein was distributed both in the Triton-soluble and Triton-insoluble fractions of the cell, as reported earlier (1). Likewise, VIL/ANFM protein was distributed both in the detergent-soluble and -insoluble fractions of the cell. In contrast, in cells expressing either of the phosphorylation site mutants, VIL/ACFM or VIL/AYFM, the villin mutant proteins were almost exclusively distributed to the Triton X-100-insoluble fraction, and none of the mutant proteins were associated with the detergent-soluble cell fraction (Fig. 6B). This is noteworthy, because we have demonstrated previously that the majority of the PLC-{gamma}1 present in rabbit ileal brush border is associated with the Triton-soluble fraction and not the detergent-insoluble fraction (1). Furthermore, we have also shown that the tyrosine-phosphorylated villin associates with PLC-{gamma}1 in the detergent-soluble cell fraction (1). Taken together, these data demonstrate that the carboxyl-terminal tyrosine phosphorylation sites in villin determine the distribution of villin to the detergent-soluble cell fraction, where phosphorylation of these sites allows villin to form a complex with PLC-{gamma}1 that is required for a villin-induced increase in cell migration.


Figure 6
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  		FIGURE 6.
Collectively, the carboxyl-terminal tyrosine phosphorylation sites are required for redistribution of villin to the detergent-soluble fraction and for its association with PLC-{gamma}1. A, this figure shows coimmunoprecipitation (IP) of villin with PLC-{gamma}1 from the Triton X-100-soluble cell extracts. B, distribution of villin in detergent-soluble and -insoluble cell fractions. Triton X-100-soluble fraction (DS) and Triton X-100-insoluble (DI) fractions from MDCK cells transfected with VIL/FL, VIL/ANFM VIL/ACFM, or VIL/AYFM were immunoblotted with villin monoclonal antibodies. VIL/FL and VIL/ANFM distribute to the detergent-soluble and -insoluble fraction, whereas villin mutants, VIL/ACFM as well as VIL/AYFM, distribute exclusively to the detergent-insoluble fraction. The Western blots are representative of three other experiments with similar results.

 
While studying the SEYFP/S1-S3 mutant that lacks the second half of villin core (S4-S6) as well as headpiece (S7), we made an interesting observation. We determined that longer transfection times (>20 h) resulted in significant cell death in HeLa/MDCK Tet-Off cells expressing the truncation mutant S1-S3 as noted by cell rounding, loss of fluorescence, chromatin condensation, and an increase in DNA fragmentation (supplemental Fig. 5 and Video 5). For the same reason, stable cell lines expressing S1-S3 could not be established for these studies. We speculate that like gelsolin, the S1-S3 domain of villin results in unregulated actin severing and apoptotic cell death (42). This may be important to the role of villin as an anti-apoptotic protein; and if villin is a substrate for caspases and/or other proteases, the cleavage of villin into this fragment may have a regulatory function.4 Hence the carboxyl-terminal domain of villin may also be required to maintain a biologically relevant conformation of villin that may be important to its functions as an anti-apoptotic protein. Most importantly, the data presented in this study demonstrate that the carboxyl-terminal half of the villin core is not redundant as has been suggested by some, but is essential to the function of villin in cell migration and perhaps cell death.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Metastases is the most lethal property of cancer cells, and critical to this property is the increased migratory state of tumor cells. The current thinking is that cancer cells switch to a more motile state thus enhancing the metastatic potential of a tumor cell. The molecular mechanisms that propel invasive growth and metastases are also found during embryonic development and in adult tissue maintenance such as wound repair. Most human cancers originate from epithelial cells and include the major killers, namely colon, breast, and lung cancer. Epithelial cell migration differs from that of fibroblasts in that epithelia migrate as a sheet such as during wound closure or as a tube such as during kidney development in vertebrates. More recent studies have established that examining epithelial cell migration in the context of proteins that are uniquely expressed in epithelial cells, including the actin-binding protein, villin is important to our understanding of this rather complex process (14).2 We have demonstrated that overexpression of villin enhances cell migration (19). Likewise Athman et al. (14) have demonstrated that villin enhances hepatocyte growth factor (HGF)-induced actin remodeling in epithelial cells. Tyrosine phosphorylation of villin is essential to its function in cell migration (14, 19). We have reported previously that three tyrosine residues (Tyr-60, Tyr-81, and Tyr-256) in the amino-terminal domain of villin are required for the villin-induced increase in cell migration (19). Phosphorylation at these three sites was shown to regulate the intracellular distribution of villin, the F-actin redistribution and remodeling, and the villin-induced increase in cell migration (19). In this study we determined that in addition to these three amino-terminal residues, the carboxyl-terminal tyrosine phosphorylation sites of villin are equally important to its function in cell migration. Although the tyrosine phosphorylation sites in the amino-terminal half regulate villin-induced cell migration by regulating the actin reorganization and cell morphology, the tyrosine phosphorylation sites in the carboxyl-terminal half regulate the ligand-binding properties of villin, namely its association with PLC-{gamma}1. Mutation of all six carboxyl-terminal phosphorylation sites in villin prevents the association of villin with PLC-{gamma}1. The role of PLC-{gamma}1 in cell migration is undisputed. Overexpression of PLC-{gamma}1 has been shown to result in malignant transformation and has been implicated in the pathophysiology of tumorigenesis (43-46). Overexpression of PLC-{gamma}1 is associated with progression of colorectal tumors from normal mucosa to adenoma and then to carcinoma (47). Growth factor-mediated PLC-{gamma}1 signaling is also required for enhanced cell motility (48-50). For instance, activation of PLC-{gamma}1 downstream of HGF receptor activation is required for the morphogenetic effects of HGF during epithelial-mesenchymal transition (51, 52). Redistribution of PLC-{gamma}1 to the leading edge and the activation of the phosphoinositide signal cascade at the leading edge is likewise well documented (46, 53, 54). Our study suggests that villin keeps the signal equilibrium of actin cytoskeleton organization, cell morphology, and cell migration by utilizing distinct domains and tyrosine phosphorylation sites within these domains in a differential and cooperative way.


Figure 7
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  		FIGURE 7.
Schematic representation of the role of the carboxyl-terminal tyrosine phosphorylation sites in villin-induced cell migration. The left panel shows resting cells in which villin associates with phosphatidylinositol 4,5-bisphosphate at the plasma membrane (see Ref. 18; and S. George, L. Chatman, A. Tomar, T. Wang, S. Mathew, and S. Khurana, submitted for publication). In response to receptor activation, including EGF treatment, villin is tyrosine-phosphorylated (by c-Src kinase) and dissociates from PIP2 and binds PLC-{gamma}1 through its carboxyl-terminal phosphorylation sites (18, 19).2 The association of villin with PLC-{gamma}1 at the plasma membrane results in the hydrolysis of PIP2 into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3).2 Inositol 1,4,5-trisphosphate releases calcium (Ca2+) from intracellular stores such as the endoplasmic reticulum (ER). Both tyrosine phosphorylation and an increase in intracellular calcium induce actin-severing by villin resulting in actin remodeling, change in cell morphology, and increase in cell migration (19, 24, 27).2 Villin headpiece is denoted by the letter H.

 
In this study we determined that a villin mutant lacking the carboxyl-terminal phosphorylation sites does not associate with the detergent-soluble fraction of the cell thus preventing the association of phospho-villin with PLC-{gamma}1. Likewise, time-lapse video demonstrates that mutation of the carboxyl-terminal phosphorylation sites prevents the formation of lamellipodia in growth factor-stimulated cells. A very important step in cell locomotion is the development of a polarized phenotype with the formation of a leading edge in the direction of cell movement. It has been suggested that in addition to phosphatidylinositol 3-kinase, PLC-{gamma}1 also determines the polarized phenotype of a motile cell by its recruitment to the leading edge, thus regulating the first step of cell migration, namely the formation of a protrusion (46, 53, 54). In fact, such an observation has been made with cofilin, where inhibition of PLC inhibited the effects of cofilin during the early stages of cell migration by delaying the initiation of lamellipodia formation and inhibiting the ability of the cells to sense EGF gradients (55). In these studies, the authors determined that both cofilin and PLC activation were required for the initial but not late changes in the generation of free barbed ends, thus regulating cell migration. Our own studies with PLC{gamma}-/-1 and PLC{gamma}1+/+ cells have demonstrated that villin-induced cell migration requires PLC-{gamma}1.2 Likewise, we have demonstrated that down-regulation of endogenous PLC-{gamma}1 using small interfering hairpin RNA prevents villin-induced cell migration.2 Together, these studies suggest that the villin-induced increase in cell migration is likely regulated by a rapid increase in actin polymerization at the leading edge, which is enhanced by tyrosine phosphorylation of villin and by the catalytic activation of PLC-{gamma}1 by phosphovillin (18).2 Thus, the synergistic interaction between tyrosine-phosphorylated villin and PLC-{gamma}1 may drive the EGF-stimulated lamellipod formation seen in our studies. Together with our previous reports, data presented in this study show that tyrosine phosphorylation sites in the carboxyl-terminal domain of villin are important for the ligand binding properties of villin, specifically for its association with PLC-{gamma}1, thus regulating villin-induced cell migration (Fig. 7).

Another interesting observation made in this study was the significance of the carboxyl-terminal domains of villin in maintaining a functionally relevant protein conformation. Transient expression of S1-S3 fragment of villin in HeLa/MDCK Tet-Off cells resulted in cell death (supplemental Fig. 4 and Video 5). These studies suggest that the S1-S3 fragment of villin might be functionally similar to the S1-S3 fragment of gelsolin, which has been shown to be pro-apoptotic resulting in cell death (42, 56). Furthermore, these studies suggest that the carboxyl-terminal half of the villin core may be functionally similar to gelsolin domains S4-S6, in that these domains may be required to maintain an auto-inhibited conformation that prevents unregulated actin depolymerization. It is also likely that releasing this conformation may be biologically relevant to the functions of these proteins in vivo. Although a caspase-3 cleavage site has been identified in gelsolin that generates the pro-apoptotic S1-S3 fragment, a similar caspase-3 cleavage site or caspase-3-mediated cleavage of villin is lacking, even though the villin fragment S1-S3 is apoptotic4 (57). The pro-apoptotic functions of these two proteins could thus be regulated differently as well. For instance gelsolin could be a substrate for caspase-3, whereas villin could be a substrate for other proteases. Such proteolytic cleavage of villin has been reported in enteric cells infected with Entamoeba histolytica (17). It may also be noted that in villin-gelsolin, where the S4-S6 domains of villin were substituted with the second half of the gelsolin core, chimeras failed to function like full-length villin protein, suggesting that although these two proteins share significant structural and functional homology, they are not identical (23). This lends support to our view that proteins of this family may have overlapping and yet distinct function(s) in tissues where more than one member of this family is expressed, such as in the intestine and kidney.

Our in vitro studies allowed us to identify the carboxyl-terminal phosphorylation sites in villin. In addition, we determined that phosphorylation of multiple carboxyl-terminal domain phosphorylation sites could regulate actin-nucleation by villin, whereas phosphorylation of Tyr-286 enhanced actin-depolymerization by villin. Furthermore, we determined that tyrosine phosphorylation of villin does not regulate its actin capping activity. Together with our previous studies, these data confirm our observation that tyrosine phosphorylation regulates all but one actin regulatory activity of villin (19, 27, 28). Although the carboxyl-terminal phosphorylation sites regulated actin kinetics in vitro, mutation of these sites did not result in any significant change in actin redistribution in the cell. The significance of these changes is unclear at this point.

In addition to revealing new tyrosine phosphorylation sites as well as identifying all phosphorylatable tyrosine residues in villin, this study established the domain functions of villin by investigating the ability of a series of mutants of villin to rescue actin organization, cell morphology, and cell migration. These studies determined that the amino-terminal half of the core regulates actin reorganization and cell shape, whereas the entire villin core is required for cell migration. In this study we demonstrate for the first time the functional relevance of the carboxyl-terminal tyrosine phosphorylation sites in villin, namely in the distribution of villin to the detergent-soluble fraction; in the association of villin with its ligand PLC-{gamma}1 in this fraction; in growth factor-induced lamellipodia formation, hence in defining the directionality of a polarized motile cell; and in the synergistic interaction with PLC-{gamma}1 to initiate the early steps involved in actin polymerization and assembly of the lamellipodia in a moving cell. Elucidating the molecular mechanisms linking the actin regulatory activities modified by phosphorylation of these sites with the function(s) of villin will be the subject of future studies.


   FOOTNOTES
 
* This work was supported by NIDDK Grants DK-65006 and DK-54755 from the National Institutes of Health (to S. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-4 and Videos 1-5. Back

1 To whom correspondence should be addressed: Dept. of Physiology, the University of Tennessee Health Science Center, Nash 402, 894 Union Ave., Memphis, TN 38163. Tel.: 901-448-3410; Fax: 901-448-3505; E-mail: skhurana{at}utmem.edu.

2 Y. Wang, A. Tomar, S. George, L. Chatman, and S. Khurana, submitted for publication. Back

3 The abbreviations used are: PIP2, phosphatidylinositol 4,5-bisphosphate; EGF, epidermal growth factor; LPA, lysophosphatidic acid; PLC-{gamma}1, phospholipase C-{gamma}1; SEYFP, superenhanced yellow fluorescent protein; IAA, indoleacrylic acid; MDCK, Madin-Darby canine kidney cells; Gst, glutathione S-transferase; HA, hemagglutinin; WT, wild type. Back

4 Y. Wang and S. Khurana, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Shi Jin for assistance with the DNA fragmentation assay.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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