Molecular Biology of the Cell
Vol. 15, 4234–4247, September 2004
The Phosphorylation of Vinculin on Tyrosine
Residues 100 and 1065, Mediated by Src Kinases,
Affects Cell Spreading
Zhiyong Zhang,* Gonzalo Izaguirre,* Siang-Yo Lin,* Hwa Young Lee,*
Erik Schaefer,†and Beatrice Haimovich*‡
*Department of Surgery and the Cancer Institute of New Jersey, Robert Wood Johnson Medical
School-University of Medicine and Dentistry of New Jersey, New Brunswick, NJ 08903; and†BioSource
International, Hopkinton, MA 01748
Submitted March 29, 2004; Accepted June 18, 2004
Monitoring Editor: Mark Ginsberg
Vinculin is a conserved actin binding protein localized in focal adhesions and cell-cell junctions. Here, we report that
vinculin is tyrosine phosphorylated in platelets spread on fibrinogen and that the phosphorylation is Src kinases
dependent. The phosphorylation of vinculin on tyrosine was reconstituted in vanadate treated COS-7 cells coexpressing
c-Src. The tyrosine phosphorylation sites in vinculin were mapped to residues 100 and 1065. A phosphorylation-specific
antibody directed against tyrosine residue 1065 reacted with phosphorylated platelet vinculin but failed to react with
vinculin from unstimulated platelet lysates. Tyrosine residue 1065 located in the vinculin tail domain was phosphorylated
by c-Src in vitro. When phosphorylated, the vinculin tail exhibited significantly less binding to the vinculin head domain
than the unphosphorylated tail. In contrast, the phosphorylation did not affect the binding of vinculin to actin in vitro.
A double vinculin mutant protein Y100F/Y1065F localized to focal adhesion plaques. Wild-type vinculin and single
tyrosine phosphorylation mutant proteins Y100F and Y1065F were significantly more effective at rescuing the spreading
defect of vinculin null cells than the double mutant Y100F/Y1065F. The phosphorylation of vinculin by Src kinases may
be one mechanism by which these kinases regulate actin filament assembly and cell spreading.
Cell spreading is a complex process requiring a bidirectional
transmembrane linkage between the extracellular matrix
and the actin cytoskeleton. Most cell-substrate interactions
are mediated by members of the integrin superfamily of
transmembrane adhesion receptors (Hynes, 2002). Integrin-
activating ligands trigger changes in receptor conformation
and receptor clustering, which in turn activate signaling
events leading to the assembly of focal complexes and actin
filament networks (Critchley, 2000; Emsley et al., 2000; Liu et
al., 2000; Geiger et al., 2001; Kim et al., 2003; Ridley et al.,
2003). Vinculin is one of the first actin binding proteins
recruited into focal complexes and subsequently focal adhe-
sion plaques (Geiger et al., 1980; Zaidel-Bar et al., 2003).
Vinculin is composed of a large 95-kDa globular head and a
30-kDa tail linked by a short proline-rich sequence (Coutu
and Craig, 1988). The three regions serve as docking sites for
several proteins. Actin and paxillin bind to sequences lo-
cated in the vinculin tail domain (Menkel et al., 1994; Wood
et al., 1994; Huttelmaier et al., 1997; Goldmann et al., 1998).
Talin and ?-actinin bind to the vinculin head domain,
whereas members of the Ena/VASP and ponsin/ArgBP52/
vinexin families, as well as the Arp 2/3 complex, interact
with the proline-rich region in vinculin (Kawabe et al., 1999;
Mandai et al., 1999; Critchley, 2000; DeMali et al., 2002).
Biochemical and structural studies established that vincu-
lin exists in at least two distinct conformations dependent
upon an intramolecular interaction between its head and tail
domains (Johnson and Craig, 1994; Winkler et al., 1996;
Bakolitsa et al., 1999, 2004; Borgon et al., 2004; Izard et al.,
2004). Binding of the head to the tail domain results in a
“closed conformation” that limits accessibility of vinculin-
binding proteins to their docking sites. It is currently
thought that acidic phospholipids, which bind to the vincu-
lin tail domain, transiently disrupt head–tail interactions
and enable the binding of ligands such as talin, VASP, and
protein kinase C? (PKC?), to their respective sites (Gilmore
and Burridge, 1996; Weekes et al., 1996; Huttelmaier et al.,
1998; Steimle et al., 1999; Ziegler et al., 2002; Bakolitsa et al.,
2004). PKC? phosphorylates vinculin on at least two serine
residues in positions 1033 and 1045; the role of the phos-
phorylation is currently not fully understood (Ziegler et al.,
2002). Izard et al. (2004) recently reported that binding of
talin derived peptides to vinculin triggered marked confor-
mational changes in vinculin and head-tail displacement.
These observations raised the possibility that various inputs
might either activate specific repertories of vinculin-depen-
dent signaling events or determine the duration of down-
Vinculin null embryos did not survive past embryonic
day 10, demonstrating that vinculin plays a key role during
embryonic development (Xu et al., 1998a). The role of vin-
culin relative to cell spreading, however, is currently not
fully understood. Vinculin null fibroblasts assembled focal
Article published online ahead of print. Mol. Biol. Cell 10.1091/
mbc.E04–03–0264. Article and publication date are available at
‡Corresponding author. E-mail address: email@example.com.
4234© 2004 by The American Society for Cell Biology
adhesion plaques, and yet, spread poorly on fibronectin (Xu
et al., 1998b), suggesting that vinculin might regulate the
dynamics of actin filament assembly. The interaction be-
tween vinculin and Arp 2/3 uncovered by DeMali and
Burridge (DeMali et al., 2002) provided insight into one
mechanism by which vinculin may execute its regulatory
function. More recently Subauste et al. (2004) reported that
vinculin modulates the interaction between paxillin and fo-
cal adhesion kinase (FAK), which in turn affected extracel-
lular signal-regulated kinase signaling required for cell sur-
vival and motility. These findings revealed a previously
unappreciated layer of interactions involving vinculin.
Vinculin was one of the first identified tyrosine phosphor-
ylation substrates of v-Src, the transforming oncogene of
Rouse sarcoma virus (Sefton et al., 1981). The v-Src–depen-
dent phosphorylation sites in vinculin were not mapped (Ito
et al., 1983). Tyrosine phosphorylation of vinculin was ob-
served in platelets stimulated by Ca2?(Vostal and Shulman,
1993) and more recently in leukotriene D(4)-treated intesti-
nal epithelial cells (Massoumi and Sjolander, 2001). In an
effort to characterize a protein of 120 kDa that is heavily
tyrosine phosphorylated in spread platelets, we identified
the protein as vinculin. We also found that the phosphory-
lation of vinculin in platelets is regulated by Src kinases. The
phosphorylation of vinculin was reconstituted in COS-7
cells cotransfected with c-Src and the tyrosine phosphoryla-
tion sites were mapped to residues 1065 and 100. A vinculin
double mutant protein, Y100F/Y1065F, was less effective
than wild-type vinculin in rescuing the spreading defect of
vinculin null cells on fibronectin. These findings suggest that
the phosphorylation of vinculin by Src kinases might affect
early events in cell spreading.
MATERIALS AND METHODS
Antibodies and Cell Lines
Monoclonal antibodies (mAbs) 4G10 and PY-20 were purchased from Upstate
Biotechnology (Lake Placid, NY) and BD Transduction Laboratories (San
Diego, CA). The mAbs to human vinculin (hVin-1 and V9131) or to chicken
vinculin (VIN-115) were from Sigma-Aldrich (St. Louis, MO). The mAb
against 6-His was purchased from QIAGEN (Valencia, CA). The vinculin-null
mouse embryo fibroblasts (Vin ?/?) (Xu et al., 1998a) were a gift from E.
Adamson (Burnham Institute, La Jolla, CA). The Vin ?/? clone used in this
study was identical to the clone used by DeMali and Burridge (DeMali et al.,
2002). The COS-7 and the NIH 3T3 cells were from the American Tissue Type
Culture Collection (Manassas, VA).
Human platelets were isolated by gel filtration as described previously
(Haimovich et al., 1993). Platelets were lysed immediately or were adhered for
1 h to fibrinogen (100 ?g/ml; Sigma-Aldrich) coated onto the surface of
electrically untreated polystyrene plates as described previously (Haimovich
et al., 1993). Where indicated, platelets were treated for 30 min with 5 ?M PP2
(Calbiochem, San Diego, CA) before exposure to the fibrinogen-coated plates.
Unstimulated and adherent platelets were lysed with 2% SDS in buffer
containing 66 mM Tris, pH 7.4, 1 mM vanadate, and 1 mM phenylmethyl-
sulfonyl fluoride (PMSF).
Isolation and Characterization of a ?120-kDa Platelet
Platelets were purified by gel-filtration from 5 U of outdated platelets (pur-
chased from the New Jersey Blood Bank, New Brunswick, NJ). To activate the
platelets and to induce aggregation, the platelet suspension was stirred vig-
orously for 20 min with 10 nM of phorbol 12-myristate 13-acetate (PMA). The
resulting platelet aggregates were collected by 5-min centrifugation at 800 ?
g. The platelet pellet was resuspended in 6 ml of lysis buffer containing 50 mM
Tris-HCl, pH 8.0, 0.02% Triton X-100, 1 mM EGTA, 1 mM Na3VO4, and 1 mM
PMSF. The cell suspension was sonicated briefly to disrupt the aggregates. All
subsequent steps were carried out at 4°C. The lysate was cleared by 20-min
centrifugation at 17,000 ? g, and the resulting supernatant was loaded onto a
Sephacryl-300 HR column (1.6 ? 70 cm). Fractions were collected at a constant
flow of 0.32 ml/min. Proteins were resolved by SDS-PAGE and transferred to
a nitrocellulose membrane. The pattern of tyrosine phosphorylated proteins
in each fraction was analyzed by immunoblotting and probing with mono-
clonal antibody (mAb) 4G10. Fractions 6–11 were combined and dialysis-
concentrated against 50 mM Tris buffer, pH 8.0, containing 1 mM vanadate.
The pooled fraction (4 ml) was loaded onto a DEAE-Sepharose column (1.2 ?
14 cm) preequilibrated with 50 mM Tris buffer, pH 8.0, containing 1 mM
vanadate. The column was washed with 30 ml of loading buffer, and proteins
were eluted off the column with 40 ml of 50 mM Tris buffer, pH 8.0, containing
a linear gradient of NaCl concentration from 0 to 0.5 M and 1 mM vanadate.
Fractions containing the 120-kDa protein were identified as described above.
Fractions 3–5 (total volume 4.5 ml) were pooled, concentrated (final concen-
tration ?1 mg/ml), and analyzed by two-dimensional (2-D) gel electrophore-
sis and Coomassie Blue staining. The gel segment containing the 120-kDa
protein was cut out from the Coomassie Blue-stained gel and sent to the W.M.
Keck Foundation Biotechnology Resource Laboratory (Yale University, New
Haven, CT) for analysis and identification by matrix-assisted laser desorption
ionization/mass spectrometry (MALDI-MS). The mass of 18 peptides ob-
tained from a tryptic digest of the sample matched the predicted mass of
Western Blotting Analysis
Proteins resolved by SDS-PAGE were blotted onto either a polyvinylidene
diflouride (PVDF) or a nitrocellulose membrane. The membranes were
probed sequentially with mAb 4G10 and the mAb to human- or chicken-
vinculin. Secondary antibody was horseradish peroxidase-conjugated rabbit
anti-mouse (Bio-Rad, Hercules, CA). Immunoreactive bands were visualized
bychemiluminescence using enhanced
(PerkinElmer Life and Analytical Sciences, Boston, MA).
2-D Gel Electrophoresis
Suspended or fibrinogen-adherent platelets were lysed in boiling buffer con-
taining 2% SDS and 66 mM Tris, pH 8.0. Isoelectric focusing of samples
containing 100 ?g of protein per sample was carried out by Kendrick Labs
(Madison, WI) as described previously (Izaguirre et al., 1999).
Immunoprecipitation of Phosphorylated Vinculin from
Fibrinogen adherent platelets were lysed in radioimmunoprecipitation assay
buffer (1% Triton X-100, 1% deoxycholic acid, 10 mM Tris-HCl, pH 7.2, 158
mM NaCl, 0.1% SDS,1 mM Na3VO4, and 1 mM PMSF). The lysates were
precleared for 1 h with 40 ?l of protein A/G agarose beads followed by an
overnight incubation with mAb PY-20. Antibody–antigen complexes were
precipitated with 50 ?l of protein A/G agarose beads, eluted with sample
buffer, and analyzed by SDS-PAGE.
Proteolytic Digestion of Platelet Vinculin
Activated platelet lysates were resolved by SDS-PAGE on a 7.5% T acryl-
amide gel (18 cm in length, 1.5 mm in thickness). One-half of the gel was
blotted onto nitrocellulose. To verify the phosphorylation of vinculin and its
position on the gel, the membrane was probed sequentially with the mAbs to
vinculin and to phosphotyrosine. The second half of the gel was stained with
Coomassie Blue R-250 for 2 h and destained overnight. The gel was rehy-
drated in water until it regained its original dimension. The area of the gel
containing vinculin was excised and placed in a microcentrifuge tube. Each
gel piece was washed for 5 min with 300 ?l of 50% acetonitrile and then for
30 min each with the same volumes of 50% acetonitrile, 50 mM NH4HCO3,
pH 8.0, followed by 50% acetonitrile, 10 mM NH4HCO3, pH 8.0. After the last
wash, the gel pieces were dried by lyophilization and stored below 4°C. In gel
proteolytic digestions of vinculin were carried out with carboxylpeptidase Y
(0.22 ?g/?l; Sigma-Aldrich) in 50 mM citrate buffer, pH 6.0. The dehydrated
gel pieces were first rehydrated on ice for 30 min with 20 ?l of buffer plus
enzyme (twofold concentrated). The volume was increased to 40 ?l by adding
buffer, and the samples were incubated at 37°C for the indicated time. The
digestion was terminated by the addition of 50 ?l of Laemmli’s loading buffer
(4?) and by heating for 2 min at 100°C. The samples (including the gel pieces)
were loaded onto a SDS-PAGE gel. Proteins resolved on the gel were trans-
ferred to nitrocellulose and analyzed by Western blotting and immunodetec-
Molecular Cloning and Mutagenesis
The complete cDNA encoding for chicken vinculin (Coutu and Craig, 1988)
kindly provided by Susan W. Craig (John Hopkins University, Baltimore,
MD) was subcloned in frame into the EcoRI site of a pQE-31 vector (QIAGEN),
resulting in the addition of a 6-His tag to the carboxy-terminal end of vinculin.
The gene was then subcloned into the EcoRI site of pcDNA3.1(?) (Invitrogen,
San Diego, CA) for mammalian expression. The tyrosine residues at positions
100, 160, 537, 822, and 1065 were replaced by phenylalanines by single base
substitution (TAT to TTT) by using QuikChange site-directed mutagenesis
(Stratagene, La Jolla, CA). The Y100F/Y1065F double mutant was generated
by two cycles of mutagenesis. The introduction of the expected mutations was
confirmed by DNA sequencing. The same approach was used to introduce the
Tyrosine Phosphorylation of Vinculin
Vol. 15, September 20044235
Y100F, Y1065F, and Y100F/Y1065F substitutions in vinculin cloned into the
pEGFP vector (kindly provided by Susan W. Craig). To introduce glutamic
acid substitutions in place of the tyrosine residues at position 100 and 1065,
the TAT in the appropriate positions were changed to GAG by site-directed
mutagenesis. The double mutant Y100E/Y1065E was generated using two
cycles of mutagenesis.
Expression of Recombinant Proteins in COS-7 Cells
Cells were transfected using the Lipofectamine Plus reagents (Invitrogen)
following the protocol recommended by the vendor. Briefly, cells cultured in
10-cm dishes were cotransfected with 3 ?g of wild-type or mutant vinculin
cDNAs and 1 ?g of a constitutively active c-Src cDNA (Y529F) (kindly
provided by David Schlaepfer, Scripps Research Institute, La Jolla, CA). Van-
adate (1 mM) prepared as described previously (Izaguirre et al., 2001) was
added to the cell culture medium 48 h after transfection. The cells were
cultured in the presence of vanadate for 24 h before analysis. Proteins were
immunoprecipitated as described previously (Izaguirre et al., 2001).
Generation of Antibodies Specific for the Phosphorylated
Y1065 Residue in Vinculin
A rabbit polyclonal antibody against the phosphorylated tyrosine residue
1065 (anti-vinculin [pY1065]) was generated by BioSource International (Hop-
kinton, MA) by using the peptide VRKTPW[pY]Q. To verify the antibody
specificity, COS-7 cells were transfected with recombinant wild-type and
mutant vinculin cDNAs as described above, and lysates containing 500
?g/ml protein were subjected to immunoprecipitation with an antibody to
the His tag. The immunoprecipitated proteins were resolved by SDS-PAGE
and were Western blotted with the anti-vinculin[pY1065] antibody at a final
concentration of 0.2 ?g/ml. For Western blotting of total platelet lysates, the
antibodies were first preabsorbed against unstimulated platelet lysates re-
solved by SDS-PAGE and then subjected to Western blotting as indicated
above. Antibody specificity was also verified by peptide competition for
which the anti-vinculin [pY1065] antibody (6 ?g) was first incubated for 1 h
at room temperature with the phosphopeptide immunogen (60 ?g) (Bio-
Source International) in a final volume of 70 ?l. The treated antibody was then
used in Western blotting of platelet lysates.
In Vitro Src Kinase Assay
The expression plasmids encoding the His-tagged chicken vinculin head
(V1-851) and tail (V884-1066) domains were described previously (Johnson
and Craig, 2000) and were kindly provided by Susan W. Craig. Fusion
proteins were produced in Escherichia coli BL21(DE3) cells treated with iso-
propyl ?-d-thiogalactoside (IPTG) and purified as described previously (John-
son and Craig, 2000). His tagged-vinculin tail or head domains (1 ?g each)
were incubated with 7.5 U/sample of constitutively active c-Src kinase (Up-
state Biotechnology) in the presence of 10 ?Ci of [?-33P]ATP in 25 ?l of
phosphorylation buffer (20 mM MOPS, pH 7.4, 5 mM MnCl2, 5 mM MgCl2, 1
mM dithiothreitol [DTT], 0.5 mM sodium vanadate). Phosphorylation was
carried out for 15 min at 37°C. Reactions were terminated by the addition of
Laemmli’s sample buffer and heating for 5 min at 100°C. Proteins were
resolved by SDS-PAGE and transferred to a PVDF membrane. Incorporation
of [?-33P]ATP was visualized by autoradiography. To immunodetect the
recombinant vinculin tail and head domains, respectively, membranes were
probed with the mAbs to His and to vinculin.
Expression and Purification of Glutathione S-Transferase
The plasmid encoding the glutathione S-transferase (GST)-tagged chicken
vinculin head domain (GST/V1-855) (Johnson and Craig, 1994) was a gener-
ous gift from Susan W. Craig. The GST-tagged protein was expressed and
purified as described previously (Johnson and Craig, 1994).
Vinculin’s Head–Tail Interaction
Purified His-vinculin-tail (His-Tail) (0.75 ?g/sample) was incubated in the
presence or absence of c-Src, and the in vitro kinase assay was carried out as
described above. After the kinase reaction, the total volume of the sample was
adjusted to 450 ?l with TEEAN buffer containing 0.5% CHAPS, 0.1 mM
Na3VO4, and 1 mM PMSF. The Ni2?-NTA agarose beads were collected by a
brief centrifugation and washed twice to remove the c-Src kinase. The result-
ing phosphorylated and unphosphorylated His-tail proteins immobilized on
Ni2?-NTA agarose beads were incubated for 3 h at 25°C with purified
GST/V1-head at the indicated concentration. Incubations (400 ?l) were per-
formed in TEEAN buffer supplemented with 0.5 mM ?-mercaptoethanol and
1% bovine serum albumin. The samples were collected by brief centrifugation
and washed four times in TEEAN. To elute the bound proteins, the Ni2?-NTA
agarose pellets were heated in electrophoresis sample buffer. Eluted proteins
were subjected to SDS-PAGE. Phosphorylation of the vinculin tail domain
was analyzed by autoradiography and immunoblotting.
Vinculin-Actin Cosedimentation Assay
To obtain unphosphorylated- and phosphorylated-vinculin, respectively,
COS-7 cells were cotransfected with cDNAs encoding for wild-type vinculin
and c-Src and were either untreated or treated with vanadate as described
above. The His-tagged recombinant proteins in the phosphorylated and un-
phosphorylated form were purified on Ni2?-agarose columns as described
previously (Izaguirre et al., 2001). Actin (1.6 ?M; Cytoskeleton, Denver, CO)
and vinculin (0.05 ?M) were incubated for 2 h at room temperature in a final
volume of 250 ?l containing 1.5 mM Tris, pH 8.0, 0.04 mM CaCl2, 0.1 mM
DTT, 100 mM KCl, 2 mM MgCl2, 1 mM ATP, 1 mM PMSF, and 0.1 mM
Na3VO4. An aliquot (50 ?l) representing the total protein content was re-
moved from each sample before the sedimentation. The samples (200 ?l each)
were sedimented for 30 min at 95,000 ? g (Steimle et al., 1999) in a Discovery
M150 SE Micro ultraspeed centrifuge (Sorvall, Newton, CT) by using the
S120AT3 rotor. The supernatants were separated from the pellets, and the
pellets were resuspended in 50 ?l of SDS-PAGE sample buffer. Samples (35 ?l
each) representing a fraction of the initial reaction mix and the supernatant,
and the entire pellet, were resolved by SDS-PAGE and Western blotting with
the indicated antibodies.
Vinculin Null Cells Spreading Assay
The vinculin-null mouse embryo fibroblasts (Vin ?/?) were maintained in
DMEM supplemented with 10% fetal bovine serum and cultured in 5% CO2
at 37°C. Control cells were only transfected with a vector encoding for a
puromycin-resistant gene (pPUR; BD Biosciences Clontech, Palo Alto, CA).
All other groups were cotransfected with the pPUR vector plus green fluo-
rescent protein (GFP) tagged-vinculin constructs at a ratio of 4:1. The cells
were treated with puromycin at a concentration of 2 ?g/ml starting at 24 h
posttransfection. The puromycin-resistant cultures (we did not select clones)
were propagated and expanded for ?2 wk. To determine the level of expres-
sion of the recombinant proteins, lysates containing 20 ?g of protein was
subjected to Western probing with the mAb to human vinculin.
To determine the effect of reconstituted vinculin on cell spreading, puro-
mycin-resistant cell populations were trypsinized and replated onto fibronec-
tin (10 ?g/ml) (Calbiochem) coated surfaces for 2 h. The cultures were fixed
for 20 min with 3.7% formaldehyde, washed three times with phosphate-
buffered saline (PBS), and examined by light microscopy by using a Nikon
Eclipse TE200 microscope equipped with a 20?/0.45 objective. For each cell
population, a total of at least 500 cells distributed in six randomly selected
fields were scored as either spread or round. Experiments were repeated three
times. Data represent the average ? S.D. The data were analyzed using an
unpaired t test. The statistical analysis was performed using the StatView
software from Abacus Concepts (Berkeley, CA).
NIH 3T3 cells were transfected with the GFP-tagged vinculin constructs by
using the Lipofectamine Plus reagents. The cells were trypsinized at 24 h
posttransfection and were replated for 24 h onto fibronectin (20 ?g/ml)
precoated glass coverslips. The cells were fixed, permeabilized for 10 min
with 0.1% Triton-X-100 in PBS, and stained with Texas red-phalloidin (1:100;
Molecular Probes, Eugene, OR) in PBS containing 1% bovine serum albumin.
The cells were visualized using an LSM 510 confocal microscope (Carl Zeiss,
Thornwood, NY) equipped with a 60?/1.4 water objective.
Vinculin Is Tyrosine Phosphorylated in Spread and/or
Numerous proteins are tyrosine phosphorylated in activated
platelets. Stable phosphorylation of several of these proteins,
including phosphoproteins of 101 and 120 kDa, as well as
?-actinin (105 kDa) (Figure 1A, lane 2), are only detected
when platelets are fully spread on fibrinogen or when they
are stimulated with PMA to form large aggregates. Our
initial goal was to identify and characterize the phosphopro-
tein of 120 kDa (pp120), which we partially purified from
PMA-stimulated outdated platelets (Izaguirre et al., 1999).
The activated platelets were lysed in buffer containing 0.02%
Triton X-100, and soluble proteins were fractionated by se-
quential chromatography on a Sephacryl 300-HR and a
DEAE-Sepharose column. Western blotting and probing
with mAb 4G10 identified fractions containing pp120 (Fig-
ure 1, B and C). Fractions eluted off the DEAE-column that
contained pp120 were combined and analyzed by Coomas-
sie Blue staining (Figure 1D). The section of the gel contain-
ing pp120 was excised and analyzed by MALDI-MS. The
Z. Zhang et al.
Molecular Biology of the Cell4236
mass of 18 peptides obtained from a tryptic digest of the
sample matched the predicted mass of human vinculin-
derived peptides (unpublished data), indicating that pp120
is platelet vinculin.
Because phosphorylated vinculin was not immunopre-
cipitated using commercially available mAbs to vinculin
(unpublished data), we took a reversed approach and im-
munoprecipitated vinculin with a generic mAb to phospho-
tyrosine, PY-20. As shown in Figure 1E, a protein of 120 kDa
immunoprecipitated by mAb PY-20 was recognized by the
mAb to vinculin by Western blotting.
To further confirm that vinculin is tyrosine phosphory-
lated in activated platelets, lysates of unstimulated platelets
(Figure 2A) and fibrinogen-adherent platelets (Figure 2B)
were analyzed by two-dimension gels and Western blotting.
The samples were resolved on parallel gels and transferred
to PVDF membranes. The membranes were stained with
Coomassie Blue to visualize the general pattern of resolved
proteins, destained, and probed with mAb 4G10 and the
mAb to human vinculin. The region of the membrane con-
taining vinculin is marked by a rectangle. Platelet activation
did not significantly alter the migration profile of most pro-
teins detected by staining of the membranes with Coomassie
Blue (Figure 2). Vinculin migrated between pI 6.6 and 7.6.
The broad migration pattern of vinculin reflects the multiple
vinculin isoforms (five) identified in platelets (Bruin et al.,
1991; Gravel et al., 1995). Platelet spreading on fibrinogen
did not significantly alter the electrophoretic mobility of
tyrosine phosphorylated protein from acti-
vated platelets. (A) Gel-filtered platelets
were unstimulated (UN) or were adhered
and spread on fibrinogen for 1 h (FBGN),
and lysates were probed on Western blots
with mAb 4G10. Arrowheads mark the po-
sition of the tyrosine phosphorylated pro-
teins of 101 kDa, 105 kDa/?-actinin, 120
kDa (pp120), and 125 kDa. The location of
the molecular weight standards is indicated
on the left. PMA-activated platelet lysates
were used as the source material for the
purification of pp120. Soluble proteins were
fractionated by gel filtration on a Sephacryl
300-HR (B) and a DEAE-Sepharose (C) col-
umn. The pattern of tyrosine phosphory-
lated proteins in each fraction was deter-
mined by Western blotting with mAb 4G10.
Details are described in Materials and Meth-
ods. (D) A final fraction containing pp120
was analyzed by SDS-PAGE and Coomassie
Blue staining. The 120-kDa protein band
was identified by MALDI-MS as vinculin.
(E) Activated platelet lysates were immuno-
precipitated with mAb PY-20 (lane 1). Con-
trol samples included a lysate incubated
without the primary antibody (lane 2) or
with the primary antibody alone (lane 3).
Western blot analysis with mAb 4G10 (anti-
pTyr) and the mAb to vinculin.
Partial purification of a 120-kDa
Tyrosine Phosphorylation of Vinculin
Vol. 15, September 20044237
vinculin. As expected, and previously shown (Izaguirre et
al., 1999), platelet activation resulted in a significant increase
in the number and intensity of tyrosine phosphorylated
proteins. The region of the membrane containing vinculin in
the unstimulated platelets revealed only trace reactivity
with mAb 4G10 (Figure 2A). In contrast, two tyrosine-phos-
phorylated proteins were noted in the same region of the
membrane containing the fibrinogen-adherent platelet ly-
sate (Figure 2B). One tyrosine phosphorylated protein colo-
calized to the lower region of vinculin. A second tyrosine
phosphorylated protein migrated immediately above and at
the basic end of vinculin (Figure 2, B and C). These findings
established that vinculin is the only protein of 120 kDa
detected by this method of analysis exhibiting robust ty-
rosine phosphorylation in spread platelets.
Treatment of Platelets with an Inhibitor of Src Kinases,
PP2, Prevents Spreading and Tyrosine Phosphorylation of
Vinculin was one of the first proteins found to be phosphor-
ylated on tyrosine by the transforming gene product of Rous
sarcoma virus, pp60src(v-Src) (Sefton et al., 1981; Ito et al.,
1983). Platelets express a high concentration of pp60c-src
(c-Src), the cellular homologue of v-Src (Golden et al., 1986)
as well as several other Src kinase family members (Huang
et al., 1991). To examine whether Src kinases regulate the
and vinculin. Platelets were unstimulated (A) or spread on a fibrin-
ogen-coated surface (B) for 1 h. Lysates containing 100 ?g of protein
were resolved on parallel gels and transferred onto PVDF mem-
branes. The membranes were stained with Coomassie Blue,
destained, and probed sequentially with mAb 4G10 and the mAb to
human vinculin. The position of albumin derived from the buffer in
which the isolated platelets were lysed is indicated in A. The rectan-
gles mark parallel areas of the membranes. (C) Vinculin and the
tyrosine phosphorylated proteins migrating with, or adjacent to, vin-
culin shown within the areas marked by rectangles in B were traced,
and the traces were superimposed as shown. The data demonstrate
that vinculin and pp120 colocalize.
Two-dimension analysis reveals colocalization of pp120
Z. Zhang et al.
Molecular Biology of the Cell4238
phosphorylation of vinculin in platelets, platelets were
treated with the inhibitor of Src kinases PP2 (Hanke et al.,
1996) before adhesion to a fibrinogen-coated surface. Con-
sistent with the results reported by Obergfell et al. (2002), we
also found that PP2 inhibited platelet spreading on fibrino-
gen (unpublished data). Lysates of untreated platelets and
PP2-pretreated platelets were analyzed by Western blotting.
As shown in Figure 3A, the phosphorylation of vinculin was
significantly reduced by PP2. In contrast, the phosphoryla-
tion of the 125-kDa tyrosine phosphorylated protein migrat-
ing immediately above vinculin was only minimally affected
by the inhibitor. These findings were confirmed by analysis
of the PP2-pretreated platelet sample by 2-D gels. To enable
accurate comparison between the PP2-treated and the un-
treated samples, the gel shown in Figure 3B and those
shown in Figure 2 were run in parallel and were processed
at the same time. PP2 eliminated the reactivity of mAb 4G10
with pp120/vinculin but had no effect on the antibody re-
activity with the 125-kDa protein (Figure 3B). These results
established that pp120/vinculin and pp125 are two distinct
proteins. These findings also provided a strong indication
that the phosphorylation of vinculin on tyrosine in platelets
is Src kinases dependent.
Vinculin Is Tyrosine Phosphorylated on Residues 100 and
Because platelets are not amenable to genetic manipulations,
we sought to establish a model cell system in which the phos-
phorylation of vinculin could be further investigated. As
COS-7 cells are more resistant to vanadate than other cell types
(e.g., NIH 3T3 cells), we constructed and expressed a recombi-
nant His-tagged vinculin (His-vinculin) cDNA in these cells.
His-vinculin was successfully expressed in COS-7 cells, but the
protein was not phosphorylated either in the absence or the
presence of vanadate (Figure 4). However, coexpression of
His-vinculin with a constitutively active form of c-Src (Y529F)
resulted in robust phosphorylation of vinculin. The phosphor-
ylation of vinculin was only detected in cells treated with
vanadate, indicating that in the absence of vanadate vinculin is
rapidly dephosphorylated by an endogenous phosphatase. En-
dogenous vinculin immunoprecipitated with the mAb to vin-
culin also was phosphorylated in vanadate-treated cells co-
transfected with c-Src (Figure 4C). The phosphorylation of the
endogenous vinculin was somewhat variable, and in some
experiments (Figure 5), it was barely detectable. Based on these
findings, the phosphorylation of vinculin was examined in all
subsequent experiments in COS-7 cells that were cotransfected
with c-Src and treated with vanadate.
Vinculin contains 8 tyrosine residues (Figure 5A). Protease
V8 cleaves vinculin in the proline-rich region (amino acid
residues 837–879) to produce two major vinculin fragments:
a 95-kDa head domain and a 30-kDa tail domain (Johnson
and Craig, 1994). Seven of the eight tyrosine residues found
in vinculin are located in the head domain. The only tyrosine
residue located in the vinculin tail domain is positioned at
residue 1065, one amino acid residue away from its carboxy-
terminal end (Figure 5A). To begin to map the phosphory-
lation site(s) in vinculin, fibrinogen-adherent platelet lysates
were resolved by SDS-PAGE, and the region of the gel
containing phosphorylated vinculin was excised and sub-
jected to in-gel digestion with carboxypeptidase Y, a proteo-
lytic enzyme that removes amino acids from the carboxy-
terminal end. Carboxypeptidase Y eliminated the reactivity
of mAb 4G10 with the phosphorylated vinculin within min-
utes (unpublished data). These data provided the first indi-
cation that vinculin is phosphorylated on tyrosine residue
1065 in platelets.
To further investigate the phosphorylation sites in vincu-
lin, the tyrosine in position 1065 was changed to phenylal-
anine by site-directed mutagenesis (Figure 5). The mutant
adhered, and spread on fibrinogen for 1 h (FBGN), or treated with 5 ?M PP2 for 1 h before adherence to fibrinogen (FBGN ? PP2). Samples
containing equal protein amounts (15 ?g) were probed on Western blots with mAb 4G10 and the mAb to vinculin. (B) Platelets treated with
PP2, as described above, were lysed and analyzed on a two-dimension gel. The rectangles mark parallel areas of the membranes. The data
shown demonstrate that PP2 greatly reduced the phosphorylation of vinculin on tyrosine. PP2 did not affect the phosphorylation of the
Phosphorylation of vinculin on tyrosine is affected by the inhibitor of Src kinases, PP2. (A) Platelets were unstimulated (UN),
Tyrosine Phosphorylation of Vinculin
Vol. 15, September 20044239
protein Y1065F immunoprecipitated from COS-7 cells by
using the mAb to His exhibited significantly less phosphor-
ylation than the wild-type protein, suggesting that residue
1065 is a phosphorylation site (Figure 5B). However, West-
ern blotting of the proteins immunoprecipitation by using
the mAb to vinculin, revealed no difference in the level of
phosphorylation between the recombinant wild-type vincu-
lin and the Y1065F mutant (Figure 5D). The simplest inter-
pretation of these data was that vinculin is phosphorylated
on more than one site. We speculate that the mAb to vincu-
lin has a low affinity or does not recognize the population
phosphorylated on residue 1065. This could explain why the
mAb to vinculin failed to immunoprecipitate phosphory-
lated vinculin from activated platelets. To identify a second
phosphorylation site in vinculin five additional tyrosine res-
idues were individually changed to phenylalanines by site
directed mutagenesis. The tyrosine residues chosen for site
directed mutagenesis included residue 822 implicated as a
vanadate treated COS-7 cells coexpressing c-Src.
COS-7 cells were untransfected (control, lane 1),
transfected with wild-type His-vinculin alone (wt;
lane 2), or cotransfected with wild-type His- vinculin
and a constitutively active c-src (Y529F) cDNA (wt ?
c-Src; lanes 3 and 4). At 48 h posttransfection, the
cells were either untreated (lane 4) or treated for 24 h
with vanadate (lanes 1–3). (A) Lysates containing
equal protein amounts were subjected to immuno-
precipitation with a mAb to His, and the immuno-
precipitates were probed on Western blots as indi-
cated. (B) Lysates containing equal protein amounts
(15 ?g/sample) were analyzed by Western blotting
with a mAb to vinculin. (C) COS-7 cells transfected
and treated as described above were subjected to
immunoprecipitation with a mAb to vinculin. The
immunoprecipitates were probed by Western blot-
ting as indicated.
Vinculin is tyrosine phosphorylated in
vinculin. The black-and-white areas represent, respectively, the vinculin head and tail domains. (B–D) COS-7 cells were not transfected
(control) or were transfected with cDNAs encoding for wild-type His-vinculin (wt), the His-vinculin point mutants Y100F, Y822F, and
Y1065F, or the His-vinculin double mutant Y100F/Y1065F. The cells were transfected with the His-vinculin construct alone (B, lane 2) or were
cotransfected with a cDNA encoding for a constitutively active c-Src. The cultures were treated with vanadate for 24 h before lysis. Lysates
containing equal protein amounts were immunoprecipitated with the mAb to His (B and C) or the mAb (D) to vinculin. The immunopre-
cipitates were probed with the indicated antibodies.
Vinculin is tyrosine phosphorylated on residues 100 and 1065. (A) Schematic diagram of the eight tyrosine residues found in
Z. Zhang et al.
Molecular Biology of the Cell4240
phosphorylation site by other investigators (Goldmann and
Ingber, 2002), and residues 100, 160, 537, and 692. Pinna and
Ruzzene (1996) proposed that Src kinase family members
require a hydrophobic residue at n-1 for optimal phosphor-
ylation. Because a charged residue precedes the tyrosine
residues in positions 107 and 144, these residues were not
included in the first round of mutagenesis. The mutant
proteins were coexpressed with c-Src in COS-7 cells. As
shown in Figure 5, B and D, the Y100F mutant exhibited less
phosphorylation than wild-type vinculin. In contrast, the
phosphorylation of Y822F (Figure 5), Y160F, Y537F, or Y692F
mutants (unpublished data) did not significantly differ from
that of wild-type vinculin. Building on these data, we next
generated a Y100F/Y1065F double mutant. The Y100F/
Y1065F double mutant protein immunoprecipiated by the
mAbs to either His or vinculin was not phosphorylated
(Figure 5, C and D), indicating that tyrosine residues 100 and
1065 are the primary, if not the only, tyrosine phosphoryla-
tion sites in vinculin.
To confirm that platelet vinculin is phosphorylated on
tyrosine residue 1065, a phosphorylation site-specific rabbit
polyclonal antibody was generated against tyrosine residue
1065. To verify the antibody specificity, recombinant wild-
type and mutant His-vinculin proteins expressed in COS-7
cells were immunoprecipitated with an antibody to His and
were analyzed by Western blotting and probing with the
anti-vinculin [pY1065] antibody. The antibody reacted with
phosphorylated wild-type vinculin and the Y100F mutant
protein but failed to react with either the Y1065F or Y100F/
Y1065F mutant proteins (Figure 6A). These data established
that the anti-vinculin [pY1065] antibody is phosphotyrosine
1065 site specific. Unstimulated and fibrinogen adherent
platelet lysates were next subjected to Western blotting anal-
ysis with the same antibody. As shown in Figure 6B, the
antiserum reacted with vinculin extracted from platelets
adherent to fibrinogen but did not react with vinculin ex-
tracted from unstimulated platelets. Furthermore, the anti-
body reactivity was completely blocked by the phosphopep-
tide immunogen. These findings established that platelet
vinculin is phosphorylated on tyrosine residue 1065. It re-
mains to be determined whether platelet vinculin is phos-
phorylated on tyrosine residue 100 and/or any other site.
c-Src Phosphorylates Vinculin on Residue 1065 In Vitro
To examine whether tyrosine residues 100 or 1065 are bona
fide c-Src substrates, His-tagged vinculin tail (V884-1066)
and head (V1-851) domain proteins expressed in E. coli were
purified on Ni2?-agarose columns and subjected to in vitro
kinase assays in the presence of recombinant c-Src. The
vinculin tail domain, but not the vinculin head domain, was
phosphorylated by c-Src in vitro (Figure 7). These findings
established that c-Src can phosphorylate vinculin on residue
1065, which is the only tyrosine residue in the tail domain.
The mechanism by which vinculin is phosphorylated on
residue 100 remains presently unclear. We also have found
that intact vinculin was not phosphorylated by c-Src in vitro,
raising the possibility that the tail residue is inaccessible to
the kinase (unpublished data).
The Phosphorylation of Vinculin on Tyrosine Residue 1065
Affects Head–Tail Interaction
Biochemical studies were the first to suggest that the vincu-
lin head and tail domains are involved in intramolecular
interactions (Johnson and Craig, 1994). To examine whether
the phosphorylation of vinculin on its tail domain affected
head–tail interactions, GST-tagged head (GST-head) and
His-tagged tail (His-tail) domain proteins were expressed
and purified from IPTG-induced BL21 E. coli. The His-tail
was immobilized on Ni2?-NTA beads. Uncoated- and His-
tail-coated beads were subsequently incubated with GST-
head. As shown in Figure 8, A and B, the GST-head did not
bind to the beads in the absence of His-tail. In the presence
of His-tail, however, the GST-head was pulled-down in a
concentration-dependent manner, reaching a plateau at a 1:1
ratio of His-tail to GST-head. These data indicated that this
assay could quantify head–tail interactions when the His-tail
is used in excess relative to the GST-head.
In the next set of experiments, the His-tail was either not
phosphorylated or was phosphorylated by c-Src in vitro.
Unphosphorylated or phosphoryated His-tail proteins im-
mobilized on Ni2?-NTA agarose beads were incubated with
GST-head at a ratio of 3:1 (0.75 ?g of His-tail: 0.25 ?g of
GST-head). As shown in Figure 8B, although the GST-head
was effectively pulled down by the unphosphorylated His-
tail, the GST-head was not pulled down by the phosphory-
lated His-tail. These data indicated that the phosphorylation
of vinculin on residue 1065 affects head–tail interaction.
culin when phosphorylated on tyrosine residue 1065 reacts with
platelet vinculin. (A) COS-7 cells were cotransfected with a consti-
tutively active c-Src (Y529F) cDNA and with cDNAs encoding for
wild-type His-vinculin (wt; lane 1), the His-vinculin point mutants
Y100F (lane 2) and Y1065F (lane 3), or the His-vinculin double
mutant Y100F/Y1065F (lane 4). The cells were treated and lysed as
described in the legend to Figure 3. Lysates containing equal protein
amounts were subjected to immunoprecipitation with a mAb to His,
and the immunoprecipitates were analyzed by Western blotting and
probing with a rabbit polyclonal antiserum raised against a phos-
phopeptide immunogen mimicking the phosphorylated tyrosine
residue 1065 in vinculin (anti-vinculin[pY1065]). The blot was
stripped and reprobed with the antibody to vinculin. (B) Unstimu-
lated (UN) and fibrinogen (FBGN)-adherent platelet lysates (15
?g/sample) were probed by Western blotting with the anti-vincu-
lin[pY1065] antiserum that was not treated (lanes 1 and 2) or that
was blocked with the phosphopeptide immunogen (lanes 3 and 4).
The blot was stripped and reprobed with the antibody to vinculin.
Phosphorylation site-specific antibody recognizing vin-
Tyrosine Phosphorylation of Vinculin
Vol. 15, September 20044241
The Phosphorylation of Vinculin on Tyrosine Residues 100
and 1065 Does Not Affect the Interaction between Vinculin
and Actin In Vitro
An intramolecular association between the vinculin head
and tail domains masks the actin binding site in vinculin
(Johnson and Craig, 1995) and prevents the cosedimentation
of intact vinculin with actin filaments. We considered the
possibility that the phosphorylation of vinculin on tyrosine
residues 100 and 1065 may alter the actin binding properties
of intact vinculin. To test for this possibility recombinant
vinculin in either their phosphorylated and unphosphory-
lated form were purified and subjected to cosedimentation
with polymerized actin. The data shown in Figure 9 dem-
onstrate that as previously shown for unphosphorylated
vinculin, phosphorylated vinculin also failed to cosediment
with actin filaments suggesting that the phosphorylation of
vinculin by Src did not trigger a conformational change that
was sufficient to expose the actin-binding site in vinculin.
Vinculin Phosphorylation Mutants Localize to Focal
To examine whether the phosphorylation of vinculin affects
its cellular localization, we used GFP-tagged wild-type vin-
culin as a template to generate Y100F, Y1065F, and Y100F/
Y1065F mutant constructs. The proteins were expressed in
NIH 3T3 cells. The GFP-tagged wild-type and mutant vin-
culin proteins localized to focal adhesion plaques, indicating
that the cellular localization of vinculin is not affected by its
state of phosphorylation on tyrosine (Figure 10). Further
studies are required to determine whether the phosphory-
lation affects cell migration and/or the rate of assembly or
turnover of focal adhesion plaques.
The Phosphorylation of Vinculin on Tyrosine Residues 100
and 1065 Affects Cell Spreading
To determine whether the phosphorylation of vinculin on
tyrosine residues 100 and/or 1065 affects cell spreading,
GFP-tagged wild-type and vinculin mutant proteins were
expressed in vinculin null cells (Vin ?/?) (Xu et al., 1998b).
Effort to identify vinculin reconstituted cells based on the
fluorescence of the GFP tag was unsuccessful due to the fact
that the fluorescence signal was very weak. To circumvent
this problem, the vinculin null cells were cotransfected with
GFP-vinculin constructs and a vector encoding for a puro-
mycin resistant gene (Gu et al., 1999). A control group was
transfected with only the puromycin-encoding vector. Puro-
mycin-resistant populations were propagated and expanded
Purified, recombinant His-vinculin tail (His-tail) and -head (His-
head) domain proteins were incubated for 15 min in kinase buffer
containing 10 ?Ci of [?-33P]ATP without (lanes 1 and 3) or with
c-Src (7.5 U/reaction) (lanes 2 and 4). Reaction products were re-
solved by SDS-PAGE and transferred to a PVDF membrane. Incor-
poration of [?-33P]ATP was detected by autoradiogaphy (Autorad).
The membrane was subsequently probed with the mAb to vinculin
and to His to confirm, respectively, equal loading of the head and
tail domain proteins.
Vinculin tail domain is phosphorylated in vitro by c-Src.
head-tail interactions. (A) GST-tagged vinculin head domain pro-
tein (V1–855) at the indicated concentration was incubated for 3 h at
25°C with the His-tagged vinculin tail domain protein (V884-1066;
0.75 ?g/sample). Complexes were pulled down with Ni2?-NTA
agarose beads and were analyzed by Western blotting with the
indicated antibodies. (B) His-tail domain protein (V884-1066; 0.75
?g/sample) was either not phosphorylated or was phosphorylated
by c-Src in vitro by using the kinase assay described above. The tail
proteins were immobilized onto Ni2?-NTA agarose beads as de-
scribed in Materials and Methods. The tail–bead complexes were
incubated for 3 h at 25°C with the GST-head domain protein (V1-
855; 0.065 ?g/sample). Bound proteins were eluted off the beads
and analyzed by autoradiography (Autorad) and by Western blot-
ting with the indicated antibodies. Results are representative of
Phosphorylation of tyrosine residue 1065 affects vinculin
Z. Zhang et al.
Molecular Biology of the Cell4242
for 2 wk. The cultures transfected with the various vinculin
constructs plus puromycin expressed equal levels of wild-
type and mutant vinculin proteins as determined by a West-
ern blotting analysis of equal protein amounts (Figure 11A).
To compare the spreading ability of vinculin null cells to
cells expressing wild-type vinculin or vinculin mutant pro-
teins, the puromycin resistant cell populations were plated
on fibronectin-coated surfaces for 2 h, fixed, and examined
by light microscopy. As previously reported and shown in
Figure 11, B and C, few (13 ? 2%) of the control cells
expressing only the puromycin-resistant gene were spread,
whereas 60 ? 7% of the cells expressing wild-type vinculin
were spread on fibronectin within 2 h. The number of spread
cells expressing Y100F or Y1065F mutant proteins was sta-
tistically indistinguishable from that of cells expressing
wild-type vinculin (56 ? 65 and 53 ? 64%, respectively). In
contrast, 36 ? 5% of the cells expressing the Y100F/Y1065F
double mutant protein were spread, representing a ?40%
decrease in the number of spread cells relative to the three
other cell populations. The difference between the cells ex-
pressing the double mutant protein and the cells expressing
wild-type vinculin, or the mutant proteins Y100F and
Y1065F, was statistically significant (p ? 0.001) for all three
We reasoned that if the phosphorylation of vinculin on
tyrosine residues 100 and 1065 positively regulates cell
spreading, then the introduction of negatively charged
amino acid residues in the same positions may enhance cell
spreading. To test this hypothesis, single and a double ty-
rosine to glutamic acid substitutions were introduced to
generate Y100E, Y1065E, and Y100E/Y1065E mutants. The
proteins were expressed in the vinculin null cells as de-
scribed above. The expression of the recombinant proteins
was verified by Western blotting (unpublished data). We
observed no statistically significant difference in spreading
among the cells expressing wild-type vinculin (51 ? 7%)
compared with cells expressing the single point mutant
proteins Y100E and Y1065E (50 ? 4 and 44 ? 4%, respec-
tively). In contrast, 65 ? 3% of the cells expressing the
double mutant protein were spread on a fibronectin coated
surface by 2 h, representing a 25% increase in the number of
spread cells as compared with the other three cell popula-
1065 does not affect its binding to actin. Recombinant phosphory-
lated and unphosphorylated vinculin were purified from trans-
fected COS-7 cells. The purified proteins (0.05 ?M) were incubated
for 2 h with actin (1.6 ?M) in polymerization buffer. An aliquot was
removed from each sample before the sedimentation (lane 1; total).
The samples then were subject to centrifugation at 100,000 ? g for 30
min. The supernatants were removed and the pellets were resus-
pended in sample buffer. Equal volumes from each sample were
analyzed by SDS-PAGE and Western blotting with the indicated
Phosphorylation of vinculin on tyrosine residue 100 and
with either a GFP-wild-type vinculin cDNA (wt) or a GFP-Y100F/Y1065F double mutant cDNA. The cells were fixed, permeabilized, and
stained with the F-actin binding drug Texas Red-phalloidin. The cells were imaged by confocal microscopy. Bar, 20 ?m.
Double phosphorylation mutant protein Y100F/Y1065F is localized to focal adhesion plaques. NIH 3T3 cells were transfected
Tyrosine Phosphorylation of Vinculin
Vol. 15, September 20044243
tions. The difference between the cells expressing the
Y100E/Y1065E mutant protein and the cells expressing
wild-type vinculin, Y100E or Y1065E mutant proteins was
statistically significant (p ? 0.001 for all three comparisons).
Cell adhesion to the extracellular matrix triggers a rapid and
dynamic assembly of multiprotein complexes that is driven
by integrins. The protein complexes serve as a platform for
coupling integrins to the actin polymerization and assembly
machinery. The earliest identified multiprotein complexes,
referred to as “focal complexes” contain integrin(s), talin,
paxillin, ?-actinin, and low levels of vinculin and FAK
(Zaidel-Bar et al., 2003). The focal complexes are also rich in
tyrosine phosphorylated proteins, indicating that kinases
are among the first proteins to be recruited and/or activated
at these sites. In platelets, the ?IIb?3-integrin receptor con-
stitutively interacts with Src kinases; the kinases are acti-
vated as a consequence of ?IIb?3-activation and ligation
(Kralisz and Cierniewski, 1998; Obergfell et al., 2002; Arias-
Salgado et al., 2003). Platelets pretreated in vitro with an
inhibitor of Src kinases, PP2, and murine platelets harvested
from Src family kinase-deficient mice, failed to spread on
fibrinogen, the primary integrin ?IIb?3extracellular matrix
ligand (Obergfell et al., 2002). Similarly, triple Src-, Yes-, and
Fyn-kinase–deficient fibroblasts exhibited impaired cell mi-
gration and spreading on fibronectin (Klinghoffer et al., 1999;
Cary et al., 2002). These findings established that Src kinases
are key, early components, in the signal transduction path-
way(s) from integrins to the cytoskeleton. The tyrosine ki-
nase Syk and its downstream effector substrates Vav1, Vav3,
and SLAP-130 were implicated as one signal transduction
pathway linking ?IIb?3and Src kinases to the cytoskeleton
(Obergfell et al., 2002). Our results indicating that vinculin is
tyrosine phosphorylated by Src kinases in platelets, and in
reconstituted COS-7 cells, open the door to the possibility
that Src kinases also may use vinculin as a vehicle to relay
integrin-dependent signals to the actin cytoskeleton.
The data reported in this study demonstrate that endog-
enous vinculin is phosphorylated on tyrosine residue 1065 in
spread platelets. The phosphorylation was not detected in
unstimulated platelets or in platelets treated with the inhib-
itor of Src kinases, PP2. The phosphorylation of vinculin on
tyrosine residue 1065 was reconstituted in vanadate-treated
cells cotransfected with wild-type vinculin and a constitu-
tively active c-Src kinase. Tyrosine residue 1065 was also
phosphorylated by c-Src in vitro, thus establishing that this
residue is a bona fide Src kinases substrate. Using the recon-
stituted COS-7 system, we identified a second phosphoryla-
tion site on residue 100. Unlike residue 1065, residue 100 was
not phosphorylated by c-Src in vitro, raising the possibility
a puromycin resistant gene alone (control) or were cotransfected with cDNAs encoding for wild-type vinculin (wt) or the vinculin mutant
proteins Y100F, Y1065F, or Y100F/Y1065F. (A) Lysates of puromycin resistant cultures were probed on Western blots with the mAb to
vinculin. (B) Puromycin-resistant cells were allowed to spread on fibronectin-coated surfaces (10 ?g/ml) for 2 h. The cells were fixed and
examined by light microscopy. The images shown are representative of each cell population. Bar, 40 ?m. (C) Cells in six random microscopic
fields were scored as either spread or round for each population. The number of spread cells was expressed as a percentage relative to the
total number of cells per field. Each data point represents the average ? SD of six fields. Approximately 500 cells were examined for each
population in each experiment. Results are representative of three experiments. The difference in spreading between cells expressing the
double mutant protein Y100F/Y1065F compared with cells expressing wt, Y100F, or Y1065F vinculin proteins was statistically significant (p ?
0.001) for all three comparisons.
Effect of wild-type and vinculin mutant proteins on cell spreading. Vinculin null cells were transfected with a cDNA encoding
Z. Zhang et al.
Molecular Biology of the Cell4244
that the phosphorylation of vinculin may be regulated by
two, or more, distinct kinases. Importantly, we also found
that although the single point mutant proteins (Y100F and
Y1065F) were as effective as wild vinculin in rescuing the
spreading defect of vinculin ?/? cells on fibronectin, the
double mutant protein (Y100F/Y1065F) was significantly
less effective than wild-type vinculin, or the single point
mutants. Furthermore, a second double mutant protein car-
rying negative charges in place of the tyrosine residues in
position 100 and 1065 (Y100E/Y1065E) was significantly
more effective than wild-type vinculin in rescuing the
spreading defect of vinculin ?/? cells on fibronectin. It is
therefore possible that the activity of vinculin is optimal only
when both sites are phosphorylated.
Biochemical and crystal structure data demonstrated that
vinculin exists in closed and open conformations. Vinculin is
held in an inactive, closed conformation by intramolecular
interactions between its head and tail domains (Johnson and
Craig, 1994; Bakolitsa et al., 1999, 2004; Borgon et al., 2004;
Izard et al., 2004). The actin binding site in vinculin is
masked when vinculin is in its closed conformation (Johnson
and Craig, 1995). Binding of acidic phospholipids to the
vinculin tail domain affects head–tail interactions and con-
verts vinculin to an active, ligand-binding competent form
(Gilmore and Burridge, 1996; Weekes et al., 1996). Crystal
structures recently resolved by Izard et al. (2004) further
revealed that binding of talin derived peptides to the vincu-
lin head domain induces a marked conformation change in
the vinculin head domain, resulting in tail displacement.
Binding of ?-actinin to vinculin similarly affected vinculin’s
head–tail interaction (Izard et al., 2004). These observations
established that the conversion of vinculin from a closed to
an open conformation is regulated by several, alternative
mechanisms. Based on the observation that the phosphory-
lation of vinculin on tyrosine residue 1065 reduced head–tail
interaction in vitro, we speculated that the phosphorylation
of vinculin by Src kinases also could positively regulate the
activation state of vinculin. In at least one analogous situa-
tion, the valosin-containing protein (VCP) was found to be
phosphorylated on tyrosine residue 805, the penultimate
residue in the protein (Egerton et al., 1992). The phosphor-
ylation of VCP on residue 805 disrupted intramolecular
head–tail interactions, which in turn exposed a nuclear-
targeting sequence in the VCP head domain (Madeo et al.,
1998). Similarly, the phosphorylation of ezrin on a threonine
residue caused conformational changes that unmasked both
membrane and actin binding sites in the protein (Fievet et al.,
2004). Here, we show that phosphorylated vinculin did not
cosediment with polymerized actin, suggesting that the ac-
tin binding site in vinculin remained masked despite the
phosphorylation. It thus seems that the primary function of
the phosphorylation of vinculin on serine residues 1033 and
1045 (Ziegler et al., 2002) or tyrosine residues 100 and 1065 is
not to modulate the interaction between vinculin and actin.
The change(s) in conformation brought about by the phos-
phorylation might impact the interaction of vinculin with
other proteins, and/or determines how long vinculin is in an
open conformation. The recent study by Subauste et al.
(2004) suggesting that the vinculin tail domain could mod-
ulate the interaction between paxillin and FAK highlights
one mechanism by which a change in the tail conformation
may affect cellular responses.
Vinculin is a well-established constituent of focal adhe-
sion plaques. Recent studies revealed that the localization of
some focal adhesion components is regulated by their state
of phosphorylation. Interestingly, FAK and ?-actinin, two of
the proteins thought to directly interact with integrins (Otey
et al., 1990; Schaller et al., 1995), were excluded from focal
adhesion plaques when phosphorylated on tyrosine resi-
dues (Katz et al., 2003; von Wichert et al., 2003). These ob-
servations raised the possibility that the density of proteins
such as FAK and ?-actinin within the plaque is regulated.
We have no evidence at the present time that the localization
of vinculin to focal adhesion plaques is similarly affected by
its state of phosphorylation; in fact, two lines of evidence
argue against this possibility. First, we found that the vin-
culin double mutant Y100F/Y1065F and Y100E/Y1065E (un-
published data) localized to focal adhesion plaques. In ad-
dition, Volberg et al. (2001) reported that vinculin localized
to focal adhesion plaques in Src, Yes, and Fyn triple null
cells. These findings, however, do not exclude the possibility
that the phosphorylation of vinculin by Src kinases affects
the dynamics of its recruitment and/or residency time
within the early focal complexes, where it may play an
important role in regulating the assembly of actin filaments,
possibly through an Arp 2/3-dependent mechanism (De-
Mali et al., 2002).
Ziegler et al. (2002) proposed that the phosphorylation
of vinculin by protein kinase C may regulate the incorpo-
ration of vinculin into nascent cell adhesion complexes. In
fact, it is possible that both Src and protein kinase C
enzyme families regulate the dynamics of vinculin assem-
bly into plaques. Indeed, treatment of platelets with PMA,
a protein kinase C activator, triggered robust phosphory-
lation of vinculin, whereas treatment of the platelets with
bisindolylmaleimide, an inhibitor of protein kinase C
(Toullec et al., 1991), inhibited platelet spreading on fi-
brinogen as well as the phosphorylation of vinculin on
tyrosine 1065 (Zhang, Lin, and Haimovich, unpublished
data). It is also possible that as shown in T cells, protein
kinase C regulates the activity of a Src kinase family
member(s) (Niu et al., 2003). Further studies are required
to determine whether platelet vinculin is phosphorylated
on serine residues 1033 and 1045 and whether the phos-
phorylation on these sites is a prerequisite for the phos-
phorylation on tyrosine residue 1065.
The crystal structure of chicken vinculin tail domain
(residues 879-1066), the human vinculin head (residues
1–258), and tail (residues 879-1066) domain complex, and
that of intact vinculin were resolved previously (Bakolitsa
et al., 1999, 2004; Borgon et al., 2004; Izard et al., 2004). In
the intact vinculin, residue 100 is fully exposed, whereas
residue 1065 is occluded by a stretch of amino acids
derived from the proline-rich region (Bakolitsa et al.,
2004). The fact that residue 1065 is buried explains why
intact vinculin was not phosphorylated by c-Src in vitro
(Zhang, Lin, and Haimovich, unpublished data) and is
consistent with the possibility that the phosphorylation of
vinculin by c-Src requires a priming input. The atomic
structure of the vinculin tail domain revealed a bundle of
five helices connected by short loops and packed in an
antiparallel orientation with the N- and C-terminal ends
emerging from the same side of the bundle (Bakolitsa et
al., 1999). Extending from the last helix is a stretch of 21
amino acids, referred to as the C-terminal arm (1045–
1066). Bakoltisa et al. (1999) identified three potential re-
gions within the C-terminal arm: a flexible loop (1047–
1052), a ? clamp (1053–1061), and a hydrophobic hairpin
(1062–1066). A mutant protein lacking residues 1052–1066
failed to cosediment with acidic phospholipid vesicles at
a physiological pH (Bakolitsa et al., 1999). This observa-
tion, the hydrophobic nature of the vinculin hairpin tail,
and the possibility that tryptophans may orient proteins
toward membranes, or perhaps play a role in the bilayer
Tyrosine Phosphorylation of Vinculin
Vol. 15, September 2004 4245
insertion process (Wallace and Janes, 1999), led Bakolitsa
et al. (1999) to propose that the last five amino acid resi-
dues in vinculin, Thr-ProTrp-Tyr-Gln (TPWYQ), are in-
serted into membranes. Whether this observation holds
true or not for the intact protein is a question that needs
to be addressed in more detail because Johnson et al.
(1998) have shown that native vinculin does not sponta-
neously associate with acidic phospholipid vesicles under
physiological conditions. The phosphorylation of vinculin
on residue 1065 may generate a transient conformation
that is more favorable for membrane recognition than the
unphosphorylated tail, particularly if, as our data suggest,
this phosphorylation affects head–tail interactions. One
intriguing possibility is that the phosphorylation may
help unmask residues 916–970 shown to interact with,
and insert into, acidic phospholipids (Johnson et al., 1998).
On the other hand, it is also possible that the phosphor-
ylation creates a deliberate obstacle for the membrane
insertion step and thus serves to regulate the membrane
binding activity of vinculin, and/or the targeting of vin-
culin to specific sites, where it may help facilitate the
assembly of actin filaments. As a follow-up to this notion,
it is also possible to envision that the recruitment of
vinculin to specific sites is regulated by a phosphatase
that can rapidly dephosphorylate vinculin thus rendering
the phosphorylation a transient event. This may explain
why the phosphorylation of vinculin in COS-7 cells is not
detected unless the cells are pretreated with vanadate.
Such a model would also imply that the activation state of
vinculin is tightly regulated by Src kinases and a coun-
?-Actinin (Izaguirre et al., 1999) and as shown here,
vinculin, undergo robust tyrosine phosphorylation in
spread platelets. The significance of these tyrosine phos-
phorylation events is clearly not limited to platelets (von
Wichert et al., 2003) but rather is easier to detect in plate-
lets than in other types. Why is that the case? It seems that
platelet spreading is an “all-out” process. Because platelet
spreading is an irreversible process, platelets can afford to
unleash robust tyrosine phosphorylation of proteins that
are only transiently or sparsely phosphorylated in other
cell types thus limiting their detection. Considering the
large number of proteins that are phosphorylated on ty-
rosine in spread platelets this system should prove to be
informative in efforts to dissect events regulating focal
We are indebted to Susan W. Craig for sharing constructs with us and for
many stimulating discussions. We thank Eileen D. Adamson for providing
the vinculin null cells. The study was supported by grant HL54104 from the
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