T H E J O U R N A L O F C E L L B I O L O G Y
© 2008 Ma et al.
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 181 No. 3 439–446
Correspondence to Edward F. Plow: firstname.lastname@example.org
Abbreviations used in this paper: CT, cytoplasmic tail; HUVEC, human umbilical
vein endothelial cell; talin-H, talin head domain.
The online version of this paper contains supplemental material.
Integrin activation, the rapid transition from a low to a high af-
fi nity state for ligand, regulates the numerous cellular responses
consequent to integrin engagement by extracellular matrix pro-
teins or counter-receptors on other cells ( Hynes, 2002 ). This
transformation is tightly controlled by the integrin cytoplasmic
tails (CTs) ( Qin et al., 2004 ; Ma et al., 2007 ). Mutational and
structural analyses suggest that the ? 3 CT can be divided two
regions, and both infl uence integrin activation. The membrane-
proximal region of the ? 3 CT is primarily ? -helix, which inter-
acts with the membrane-proximal helix of the ? subunit through
several electrostatic and hydrophobic bonds ( Vinogradova
et al., 2002 ). Unclasping of the complex is a critical event in
integrin activation ( Hughes et al., 1996 ; Kim et al., 2003 ; Ma
et al., 2006 ). The membrane-distal region of the ? 3 CT contains
two NXXY turn motifs, NPLY 747 and NITY 759 , which are sepa-
rated by a short helix containing a T/S cluster, the TS 752 T region
( Fig. 1 A ). The head domain of talin (talin-H) docks at the
NPLY 747 motif through its F 3 domain and also interacts with the
membrane-proximal region, perturbing the membrane clasp and
leading to at least partial integrin activation ( Vinogradova et al.,
2002 ; Tadokoro et al., 2003 ; Wegener et al., 2007 ). The T/S
cluster and the NITY motif are also critical for integrin activa-
tion ( Chen et al., 1994 ; O ’ Toole et al., 1995 ; Xi et al., 2003 ; Ma
et al., 2006 ). However, the mechanisms underlying their effects
remain unresolved. In this study, we found that kindlin-2, a
widely distributed PTB domain protein, interacts with the C ter-
minus of ? 3 CT at the TS 752 T and NITY 759 motifs and markedly
enhances talin-induced integrin activation. Thus, kindlin-2 is
identifi ed as a coactivator of integrins.
Results and discussion
To address the functional signifi cance of the membrane-distal re-
gion of the ? 3 CT, we considered whether it might interact with
intracellular regulator(s). A CHO cell line stably expressing ? IIb ? 3
was transfected with cDNAs encoding for wild-type or mutated
? 3 CT based on the rationale that these expressed constructs
would compete for integrin binding partners. A similar strategy
had been used previously to screen the ? CT binding partners es-
sential for integrin activation ( Fenczik et al., 1997 ). In our stud-
ies, these ? 3 CT were expressed as chimeric constructs containing
the extracellular domain of PSGL-1 so that expression levels of
the various ? 3 CT could be verifi ed. As assessed by fl ow cytome-
try (FACS), PSGL-1 expression differed by less than 10%. The
effects of the various ? 3 CT on ? IIb ? 3 -mediated cell spreading on
immobilized fi brinogen were evaluated. Compared with cells ex-
pressing PSGL-1 alone, expression of the wild-type ? 3 CT chi-
mera totally abolished ? IIb ? 3 -mediated cell spreading ( Fig. 1 B ).
As a specifi city control, Y 747 A mutation, which would interfere
with talin binding, resulted in a loss of inhibitory activity. Other
mutations in the membrane-distal region in ? 3 CT chimera, S 752 P
and Y 759 A, beyond the talin interactive sites and which perturb
short cytoplasmic tails (CTs). It is widely accepted that the
head domain of talin (talin-H) can mediate integrin acti-
vation by binding to two sites in integrin ? ’ s CT; in integrin
? 3 this is an NPLY 747 motif and the membrane-proximal
region. Here, we show that the C-terminal region of inte-
grin ? 3 CT, composed of a conserved TS 752 T region and
NITY 759 motif, supports integrin activation by binding to a
ntegrin activation is essential for dynamically linking
the extracellular environment and cytoskeletal/signal-
ing networks. Activation is controlled by integrins ’
cytosolic binding partner, kindlin-2, a widely distributed
PTB domain protein. Co-transfection of kindlin-2 with
talin-H results in a synergistic enhancement of integrin ? IIb ? 3
activation. Furthermore, siRNA knockdown of endog-
enous kindlin-2 impairs talin-induced ? IIb ? 3 activation in
transfected CHO cells and blunts ? v ? 3 -mediated adhesion
and migration of endothelial cells. Our results thus iden-
tify kindlin-2 as a novel regulator of integrin activation; it
functions as a coactivator.
Kindlin-2 (Mig-2): a co-activator of ? 3 integrins
Yan-Qing Ma , 1 Jun Qin , 1 Chuanyue Wu , 2 and Edward F. Plow 1
1 Department of Molecular Cardiology, Cleveland Clinic, Cleveland, OH 44195
2 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
JCB • VOLUME 181 • NUMBER 3 • 2008 440
A reasonable synthesis of the data in Fig. 1 (B and C) is
that the membrane-distal region of the ? 3 CT regulates integrin
activation and does so by interacting with a cytoplasmic binding
partner that cooperates with talin but binds to distinct sites.
Molecules reported to bind to the membrane-distal conservative
regions of ? 3 CT include fi lamin, which binds to the T/S cluster,
and ? 3 -endonexin, which binds to the NITY 759 motif. Both have
been suggested as regulators of integrin activation (fi lamin,
a negative regulator, and ? 3 -endonexin, a positive regulator)
( Eigenthaler et al., 1997 ; Kiema et al., 2006 ). To assess their
roles in integrin activation, fi lamin A Ig-like domain 21 (FLNa21,
the ? CT binding region) or ? 3 -endonexin was transfected or
cotransfected together with talin-H into ? IIb ? 3 -CHO cells. Neither
modulated talin-induced integrin activation or directly mediated
integrin activation ( Fig. 2, A and B; and Fig. S1 A, available at
excluding them as the hypothetical coactivator of integrins.
It should be noted that these data are not inconsistent with the
proposed role of fi lamin A as a negative regulator of integrin
activation ( Kiema et al., 2006 ); suppressive effects of FLNa21
may not be evident in the presence of high talin-H levels.
Recently, we identifi ed another ? 3 CT binding protein,
kindlin-2 ( Shi et al., 2007 ), one of a three-member kindlin family
that are characterized by bearing a FERM domain ( Wick et al.,
1994 ; Siegel et al., 2003 ; Weinstein et al., 2003 ; Ussar et al., 2006 ).
key structural features in this region, the short helix and the turn
motif, respectively, also led to loss of competitive activity. This
loss was not observed with Y 747 F, S 752 A, or Y 759 F substitutions,
which would sustain the secondary structural features of the
Cell spreading is a complex response and we sought to
confi rm the role of membrane-distal residues in integrin activa-
tion more directly. ? IIb ? 3 containing a point mutation of R 995 D
in ? IIb or D 723 R in ? 3, which disrupts a salt bridge formed by R 995
and D 723 , is a particularly sensitive reporter of talin-H – induced
activation in a CHO cell system as assessed with the ligand mi-
metic mAb, PAC1 ( Hughes et al., 1996 ; Tadokoro et al., 2003 ;
Ma et al., 2006 ). Disrupting either of the two NXXY turn motifs,
NPLY 747 or NITY 759 , with a Y 747 A or a Y 759 A mutation dramati-
cally impairs integrin activation caused by R 995 D ( Fig. 1 C ).
However, conservative substitutions that should be structurally
silent, Y 747 F or Y 759 F, have no signifi cant effect on integrin acti-
vation. Consistent with previous data, disruption of the short
helix between two NXXY motifs with the naturally occurring
S 752 P ( Chen et al., 1992, 1994 ) suppresses integrin activation
whereas the S 752 A substitution, which maintains the helix ( Ma
et al., 2006 ), does not affect activation. Although the above de-
scription focuses on the ? 3 CT, most of the key sequences are
shared by other integrin ? subunits ( Fig. 1 A ), and the potential
to be activated extends to multiple integrin subfamilies.
Figure 1. Sequences of the membrane-distal region of ? 3 CT have essential roles in integrin ? IIb ? 3 activation. (A) Alignment of integrin ? CT sequences,
highlighting (red) the conserved regions, the two NXXY/F motifs and one T/S cluster. (B) Suppression of integrin ? IIb ? 3 -mediated cell spreading by
expressed ? 3 CT depends on conserved sequences in its membrane-distal region. After transient transfection with plasmids encoding the indicated
? 3 CT-containing chimera ( ? 3 CT/PSGL-1), adhesion of the ? IIb ? 3 -CHO cells to fi brinogen was examined. The adherent cells were fi xed and stained with
the anti-PSGL-1 mAb, KPL-1, for visualization by fl uorescence microscopy (10 × objective). Bar, 20 μ m. (C) Conserved residues in the membrane-distal
region support ? IIb ? 3 activation. Plasmids encoding ? IIb and ? 3 or its mutants were transiently transfected to CHO cells. The transfected cells were stained
with 2G12 to assess ? IIb ? 3 expression or PAC1 to assess ? IIb ? 3 activation. FACS was used to measure the mean fl uorescence intensity (MFI) of 2G12 or
PAC1 binding, and relative MFI of PAC1 binding was normalized to integrin expression levels based on 2G12 staining ( Ma et al., 2006 ). The error bars
represent means ± SD of three independent experiments.
441KINDLIN-2 SUPPORTS ? 3 INTEGRIN ACTIVATION • Ma et al.
umbilical vein endothelial cells (HUVECs). As shown in Fig. 3 A ,
wild-type ? 3 CT interacts with kindlin-2 but GST alone did not,
ascribing specifi city to the interactions. The Y 747 A mutation ab-
rogates talin-H but not kindin-2 binding to ? 3 CT. In contrast,
the S 752 P and Y 759 A mutations still support talin-H binding but
dramatically reduce kindlin-2 association ( Fig. 3 A ). Thus, the
binding requirements for talin-H and kindlin-2 on the ? 3 CT are
distinct and both bind to sites known to regulate integrin activa-
tion. Consistent with our observations ( Fig. 2 A and Fig. S1 A),
overexpression of FLNa21 or ? 3 -endonexin, two C-terminal bind-
ing proteins of ? 3 CT, failed to suppress endogenous kindlin-2
binding to ? 3 CT in CHO cells (Fig. S1 B), indicating a privi-
leged interaction of kindlin-2 with the ? 3 CT among these
binding partners. As a point of emphasis, endogenous kindlin-2
coprecipitates with endogenous ? 3 integrin subunit in both
? IIb ? 3 -CHO cells and HUVECs (Fig. S2, available at http://
Peptides corresponding to Y 747 -T 762 or a variant peptide
containing the S 752 P and Y 759 A substitutions were synthesized
( Fig. 3 B ). When added as competitors (200 μ M), wild-type
Y 747 -T 762 peptide inhibited kindlin-2 coprecipitation with the
Kindlin-2 contributes to the maturation of focal adhesions
during cell shape changes through recruitment of migfi lin and
fi lamin ( Tu et al., 2003 ). Targeted disruption of the kindlin-2
gene results in embryonic lethality in mice and causes multi-
ple, severe abnormalities in zebrafi sh ( Dowling et al., 2008 ).
Distinct from talin, its interaction site on ? 3 CT is not dependent
on the NPLY 747 motif ( Shi et al., 2007 ). When expressed in
? IIb ? 3 -CHO cells, kindlin-2 induces statistically signifi cant but
very weak integrin activation compared with talin-H ( Shi et al.,
2007 ). To consider the role of kindlin-2 a coactivator with talin-H,
both were transfected into ? IIb ? 3 -CHO cells. As shown in Fig. 2
(C and D) , kindlin-2 dramatically enhanced talin-H – mediated
? IIb ? 3 activation. This enhancement was not simply additive but
represented functional synergism. We assessed the expression
levels in different transfectants by Western blots to exclude that
coexpression of kindlin-2 enhanced talin-H expression or vise-
versa; expression of talin-H in single and double transfectants
was similar ( Fig. 2 E ).
To further assess the role of kindlin-2 as a coactivator,
GST-fused ? 3 CT proteins were used to coprecipitate endoge-
neous kindlin-2 in lysates of CHO cells, platelets, and human
Figure 2. Kindlin-2 enhances talin-induced integrin
? IIb ? 3 activation. EGFP-fused ? 3 CT binding proteins were
transiently transfected into ? IIb ? 3 -CHO cells. Their effects on
? IIb ? 3 activation were evaluated by PAC1 binding (A and D)
and expression levels were measured by Western blot-
ting with anti-GFP antibody (B and E). Representative
FACS histograms of PAC1 binding to talin-H and/or
kindin-2 (kind-2) positive cells (C). Error bars (A and D)
represent means ± SD ( n = 3). *, P < 0.05; **, P < 0.01
JCB • VOLUME 181 • NUMBER 3 • 2008 442
kindlin-2 into several fragments, and their ? 3 CT-binding capac-
ities were evaluated by pull-down assays ( Fig. 4 B ). Deletion of
the N-terminal region of kindlin-2, at N217 or at E345, the border
of the PH domain insertion, ablated interaction with the ? 3 CT.
In addition, truncation of kindlin-2 to delete the second part
of its F 2 and F 3 subdomains also disrupted ? 3 CT interaction
( Fig. 4 B ). These deletions were more disruptive than the QW 615
mutation. However, with deletion of PH domain alone, the mutant
GST- ? 3 CT ( Fig. 3 C ); the inhibition was ? 70% by densitometry.
A lower concentration of peptide (100 μ M) was still inhibitory
but produced only 50% inhibition (unpublished data), suggesting
a dose-dependent inhibitory effect. Introduction of S 752 P and
Y 759 A mutations into the peptide totally abolished its competitive
activity ( Fig. 3 C ). As control, both peptides had no effect on
talin-H association with the GST- ? 3 CT. It is noteworthy that intro-
duction of similar peptides into endothelial cells ( Liu et al., 1996 )
and platelets ( Hers et al., 2000 ) signifi cantly perturbed ? v ? 3 and
? IIb ? 3 mediated responses, respectively. Thus, our results may
provide a molecular explanation for these prior observations.
Like talin-H, kindlin-2 contains a FERM domain; its F 2
subdomain is bisected by a PH domain, but its F 3 (PTB) sub-
domain is intact ( Fig. 4 A ). Our previous experiments had shown
that a QW 615 /AA mutation in F 3 , a site predicted by molecular
modeling to be involved in ? CT engagement, did, in fact, dis-
rupt its association with ? CT ( Shi et al., 2007 ). We segmented
Figure 3. Distinct binding sites for kindlin-2 and talin in ? 3 CT. (A) Lysates
of CHO cells, HUVECs, or out-dated platelets were incubated with GST
or GST-fused ? 3 CT bearing the indicated mutations in the presence of
glutathione-Sepharose. After washing, the precipitates were analyzed by
SDS-PAGE. The loading of the GST proteins was assessed by Coomassie
blue staining. The associated kindlin-2 or talin-H was detected in Western
blots with anti-kindlin-2 or anti-talin-H. (B) Amino acid sequences of ? 3 CT
C-terminal peptide corresponding to Y 747 -T 762 and a mutant peptide with
two loss-of-function mutations, S 752 P and Y 759 A. (C) The pull-down assay
was performed in the presence of indicated peptides. The infl uence of
these peptides on kindlin-2 or talin-H binding to ? 3 CT was evaluated by
SDS-PAGE and Western blotting.
Figure 4. Both the N and C terminus of kindlin-2 are required for ? 3 CT
association and support of talin-induced integrin activation. (A) Organiza-
tion of predicated domains of kindlin-2 protein. The FERM domain is shown
in yellow, in which the F 2 subdomain is split by the PH domain. Deletion
mutations from N terminus ( ? N) or C terminus ( ? C) are indicated. (B) The
lysates of CHO cells transfected with EGFP-kindlin-2 with indicated muta-
tions were used for pull-down assays. After incubating with GST fusion ? 3
CT (wild-type) and glutathione-Sepharose, kindlin-2 protein bound to the
? 3 CT was evaluated by SDS-PAGE and Western blotting using anti-GFP
antibody. Kindlin-2 expression levels in lysates are also shown. (C) CHO
cells expressing ? IIb ? 3 were transiently transfected with empty EGFP vector
or cDNA encoding the indicated proteins. Binding of PAC1 to the different
transfectants was assessed by FACS and relative MFI of PAC1 binding
were calculated as described in Materials and methods. Error bars repre-
sent means ± SD ( n = 3). **, P < 0.01 (versus talin-H).
443 KINDLIN-2 SUPPORTS ? 3 INTEGRIN ACTIVATION • Ma et al.
appears to depend on binding of kindlin-2 through both its N- and
C-terminal F 3 (PTB) domains. As to why the C-terminal F 3 (PTB)
of kindlin-2 recognizes the NITY 759 rather than the NPLY 747 re-
gion of ? 3 CT will require high resolution structures.
The colocalization of ? 3 integrin and kindlin-2 was also
tested in living cells. We found they dynamically associate with
each other in HUVECs during ? 3 integrin mediated cell spread-
ing on the ? 3 ligand ( Fig. 5 A ). At the early stage of spreading
(30 min), ? 3 (green) and kindlin-2 (red) colocalized in the lamelli-
podia at the edges of spreading cells ( Fig. 5 A , top). Over time,
both ? 3 integrin and kindlin-2 moved into focal adhesion sites
( Fig. 5 A , bottom, 60 min). The merged images in Fig. 5 A (right)
kindlin-2 still retained its capacity to bind the ? 3 CT. The effects
of these mutants on the coactivator activity of kindlin-2 were
tested. When cotransfected with talin-H, deletion of either the
N- or C-terminal region of kindlin-2 resulted in loss of coactiva-
tor activity ( Fig. 4 C ). The mutant with its PH domain deletion
still retained some coactivator activity, although it was less po-
tent than intact kindlin-2. Also, the QW 615 mutant lacked coacti-
vator activity, verifying that this site is involved not only in
binding but also in coactivator function. We cannot exclude that
some of these mutations may affect global folding of kindlin-2.
However, it should be noted that FERM subdomains tend to fold
independently into functional units. Thus, coactivator activity
Figure 5. Endogenous kindlin-2 supports ? 3
integrin function in cells. (A) Subcellular local-
izations of kindlin-2 and ? 3 integrin. HUVECs
spread on fi brinogen for 30 or 60 min were
stained with the anti-kindlin-2 mAb and anti- ? 3
subunit polyclonal antibody followed by Alexa-
Fluor 568 anti – mouse IgG and AlexaFluor 488
anti – rabbit IgG. Bar, 10 μ m. (B) RNAi sup-
pression of kindlin-2 expression in CHO cells.
Expression of kindlin-2 in parental CHO cells
(non-T), kindlin-2 siRNA (SiKind-2), or control
RNA (SiControl) transfectants was analyzed
by Western blotting with kindlin-2 or actin anti-
bodies. (C) CHO cells expressing ? IIb ? 3 were
transiently transfected with vector or talin-H,
together with control RNAs (SiControl) or
siRNAs targeting kindlin-2 (SiKind-2). The bind-
ing of PAC1 to the different transfectants was
assessed by FACS and MFI of PAC1 binding
was calculated. The error bars are means ± SD
( n = 3). (D) RNAi suppression of kindlin-2 ex-
pression in HUVECs. (E and F) Non-transfected
(Non-T) or HUVECs transfected with control
RNAs (SiControl) or targeted siRNAs for Kind-
lin-2 (SiKind-2) were used in adhesion assays
(E) or migration assays (F). The adherent or mi-
grated cells were fi xed, stained, and counted
(10 × objective). The error bars are means ± SD
of three independent experiments. (G) HUVECs
transfected with control RNAs (SiControl) or
targeted siRNAs for kindlin-2 (SiKind-2) were
stimulated with PMA, and adhesion to fi brino-
gen was measured. (H) Kindlin-2 as an inte-
grin coactivator. Integrin activation depends
on interaction of talin-H with the NPLY 747 motif
and the membrane-proximal clasping region.
Kindlin-2 facilitates activation by associating
with the C-terminal regions of ? 3 CT, involving
the TS 752 T and NITY 759 motifs.
JCB • VOLUME 181 • NUMBER 3 • 2008 444
In summary, we found that kindlin-2 is a coactivator of
talin in supporting ? 3 integrin activation. As such, kindlin-2 is
the fi rst of the postulated coactivators of integrins ( Ma et al.,
2007 ) to be identified. Our data support a model ( Fig. 5 H )
in which kindlin-2 binds to the C terminus of ? 3 CT beyond
of the talin-binding sites. Functionally, kindlin-2 synergisti-
cally enhances talin-induced integrin activation. Kindlin-2
may associate with membrane via its PH domain, an inter-
action commonly associated with these domains, and interacts
with ? 3 CT via its N terminus and C-terminal PTB domain.
Anchoring ? 3 CT by kindlin-2 could reduce the fl exibility of
? 3 CT in the cytosol, positioning it more favorably for inter-
action with talin and might also displace other ? 3 CT binding
partners. Due to the variable expression of kindlin-2 in different
tissues and cells, e.g., its levels are quite low in human plate-
lets versus HUVECs (Fig. S3 B), one must consider the possi-
bilities that kindlin-2 may exert a “ catalytic ” effect on integrin
activation, where one molecule coactivates multiple integ-
rins, or whether this coactivator activity of kindlin-2 is shared
or compensated by other kindlin family members or by other
integrin binding partners.
Materials and methods
Plasmid construction and mutagenesis
The cDNA of human ? IIb and ? 3 subunits were inserted into the mammalian
expression vector pcDNA3.1 (Invitrogen). The mouse talin head domain
(1 – 429 amino acids), human kindlin-2, ? 3 -endonexin, and fi lamin A Ig-like
domain 21 (2235 – 2330 amino acids) were cloned into pEGFP vectors
(Clontech Laboratories, Inc.). For the construct of GST-tagged ? 3 cytoplas-
mic tail, the fragment of ? 3 tail (716 – 762 amino acids) was amplifi ed by
PCR and inserted into pGST-parallel-1 vector ( Sheffi eld et al., 1999 ). The
PSGL-1/ ? 3 chimera was constructed in pcDNA3.1 vector in which N termi-
nus (1 – 91 amino acids) of human PSGL-1 was fused onto C terminus
(468 – 762 amino acids) of human ? 3 subunit. All the indicated mutations were
introduced into the respective constructs using QuikChange site-directed
mutagenesis kit (Stratagene) and confi rmed by gene sequencing.
Integrin ? IIb ? 3 activation assay
The integrin ? IIb ? 3 activation was evaluated with PAC1, a mAb which spe-
cifi cally recognizes active ? IIb ? 3 . For testing how the membrane-distal
regions of ? 3 CT regulate ? IIb ? 3 activation, the ? 3 subunit bearing different
mutations was cotransfected with ? IIb subunit, with or without R 995 D muta-
tion, into CHO-K1 cells using Lipofectamine 2000 (Invitrogen). 24 h after
transfection, the cells were collected and PAC1 binding was assessed as
described previously ( Ma et al., 2006 ). In brief, PAC1 binding was fi rst
normalized by ? IIb ? 3 expression level on the cell surfaces measured by
mAb 2G12, which is against ? IIb ? 3 complex independent of activation sta-
tus. The values of normalized PAC1 binding on different transfectants were
compared to determine relative integrin activation, defi ning the basal acti-
vation of wild-type ? IIb ? 3 as 1.0.
For determining the regulatory roles of different ? 3 -binding partners
in ? IIb ? 3 activation, individual EGFP-fused candidate binding partners or
combinations of binding partners were transfected into CHO cells stably
expressing wild-type ? IIb ? 3 ( ? IIb ? 3 -CHO). PAC1 binding to the different
transfectants was analyzed by fl ow cytometry, gating only on the EGFP-
positive cells. Mean fl uorescence intensities (MFI) of PAC1 binding were
normalized based on the basal level of PAC1 binding to cells transfected
with the EGFP vector alone to obtain relative MFI values.
Monomeric PSGL-1 (mPSGL-1) or PSGL-1/ ? 3 chimera (PSGL1N- ? 3 C) was
transfected into ? IIb ? 3 -CHO cells. The mPSGL-1 was obtained by substitu-
tion of a single extracellular cysteine at the junction of the transmembrane
domain with A to disturb the disulfi de bond essential for PSGL-1 homodimer
formation ( McEver and Cummings, 1997 ). The transiently transfected cells
were allowed to adhere and spread on immobilized fi brinogen in Laboratory-
Tek II chambers (Nalge Nunc International). After incubation at 37 ° C for 2 h,
verify the colocalization of kindlin-2 and ? 3 integrin. ? 3 integrin
and talin also colocalize in spreading HUVECs with a similar
pattern (Fig. S3 A, available at http://www.jcb.org/cgi/content/full/
jcb.200710196/DC1). These observations place talin and kindlin-2
together, consistent with their cooperativity in function.
To determine if endogenous kindlin-2 supports ? 3 integrin
function, RNA-mediated interference experiments were per-
formed. Small interfering RNAs targeting kindlin-2 (siKind-2)
or irrelevant RNAs as control (siControl) were introduced into
? IIb ? 3 -CHO cells, and kindlin-2 expression levels were analyzed
by Western blot. Transfection of siKind-2 but not siControl ef-
fectively inhibited the expression of kindlin-2 ( Fig. 5 B ). The de-
crease in kindlin-2 protein expression was 70% by densitometry.
Neither the siKind-2 nor the siControl changed actin expression,
establishing selectivity of the siKind-2 on kindlin-2 expression.
Talin-H can induce ? IIb ? 3 activation in transfected ? IIb ? 3 -CHO
cells as shown by others ( Tadokoro et al., 2003 ) and in this study.
However, talin-H – mediated integrin activation was signifi cantly
blunted when kindlin-2 levels were reduced with siKind-2 but
not siControl ( Fig. 5 C ), indicating that endogenous kindlin-2
supports talin-H – induced ? IIb ? 3 activation in these cells.
We also tested the function of kindlin-2 knock-down in
cells that express an integrin naturally. HUVECs express and use
? v ? 3 to mediate cell adhesion and migration on fi brinogen or
vitronectin ( Plow et al., 2000 ). Endogenous kindlin-2 could be
knocked down in HUVEC using siRNA ( Fig. 5 D ), and the defi -
ciency of kindlin-2 dramatically suppressed HUVEC adhesion
on the ? 3 integrin ligands, fi brinogen or vitronectin ( Fig. 5 E ).
In addition, knockdown of kindlin-2 in HUVECs signifi cantly
inhibited VEGF-induced cell migration ( Fig. 5 F ). Under the con-
ditions used, VEGF induced HUVEC migration on fi brinogen or
vitronectin is dependent on ? v ? 3 activation ( Byzova et al., 2000 ),
and there is little cell proliferation ( < 50% increase) in serum-
free medium (unpublished data). Interestingly, we previously
found that overexpression of kindlin-2 also inhibited migration
for some cancer cells ( Shi et al., 2007 ). These two distinct obser-
vations suggest that the supportive role of kindlin-2 in integrin
activation might be cell type and/or integrin specifi c or depends
on specifi c experimental conditions such as ligand concentration
( Huttenlocher et al., 1996 ; Palecek et al., 1997 ). Furthermore,
knocking down kindlin-2 signifi cantly suppressed PMA-induced
HUVEC adhesion on fi brinogen ( Fig. 5 G ), which is also an ? v ? 3
activation-dependent process. In concert, these results suggest
that kindlin-2 plays an important role in supporting ? 3 integrin
functions dependent on activation.
Nonetheless, kindlin-2 is unlikely to be a direct activator
of integrin; overexpression of kindlin-2 alone only had a mild
effect on integrin activation compared with talin-H ( Fig. 2 D ).
Even though kindlin-2 also bears a FERM-like domain as does
talin-H, the binding sites of kindlin-2 on ? 3 CT are solely local-
ized at its C terminus beyond of the talin-H recognition sites
( Fig. 3 ), which allows kindlin-2 and talin to bind to the ? 3 CT
together. This possibility has been established by the synergistic
role of talin-H and kindlin-2 in integrin activation ( Fig. 2, C and D )
and further verifi ed by the fi nding that knockdown of endog-
enous kindlin-2 signifi cantly suppressed talin-H – induced integrin
activation ( Fig. 5, B and C ).
445 KINDLIN-2 SUPPORTS ? 3 INTEGRIN ACTIVATION • Ma et al.
expression in human platelets. Online supplemental material is available at
We thank Ka Chen, Zhen Xu, Kamila Bledzka, and Mitali Das for technical
This work was supported by NIH grants P01HL073311 (to E.F. Plow
and J. Qin), GM62823 (to J. Qin), and GM65188 to C. Wu.
Submitted: 29 October 2007
Accepted: 1 April 2008
Byzova , T.V. , C.K. Goldman , N. Pampori , K.A. Thomas , A. Bett , S.J. Shattil , and
E.F. Plow . 2000 . A mechanism for modulation of cellular responses to
VEGF: activation of the integrins. Mol. Cell . 6 : 851 – 860 .
Chen , Y.-P. , I. Djaffar , D. Pidard , B. Steiner , A.-M. Cieutat , J.P. Caen , and J.-P.
Rosa . 1992 . Ser-752 → Pro mutation in the cytoplasmic domain of integrin
? 3 subunit and defective activation of platelet integrin ? IIb ? 3 (GPIIb-IIIa)
in a variant of Glanzmann ’ s thrombasthenia. Proc. Natl. Acad. Sci. USA .
89 : 10169 – 10173 .
Chen , Y.P. , T.E. O ’ Toole , J. Ylanne , J.P. Rosa , and M.H. Ginsberg . 1994 . A point
mutation in the integrin beta 3 cytoplasmic domain (S752 → P) impairs
bidirectional signaling through alpha IIb beta 3 (platelet glycoprotein IIb-
IIIa). Blood . 84 : 1857 – 1865 .
Dowling , J.J. , E. Gibbs , M. Russell , D. Goldman , J. Minarcik , J.A. Golden , and
E.L. Feldman . 2008 . Kindlin-2 is an essential component of intercalated
discs and is required for vertebrate cardiac structure and function . Circ.
Res . 102 : 423 – 431 .
Eigenthaler , M. , L. Hofferer , S.J. Shattil , and M.H. Ginsberg . 1997 . A con-
served sequence motif in the integrin beta3 cytoplasmic domain is re-
quired for its specifi c interaction with beta3-endonexin. J. Biol. Chem.
272 : 7693 – 7698 .
Fenczik , C.A. , T. Sethi , J.W. Ramos , P.E. Hughes , and M.H. Ginsberg . 1997 .
Complementation of dominant suppression implicates CD98 in integrin
activation. Nature . 390 : 81 – 85 .
Hers , I. , J. Donath , P.E.M.H. Litjens , G. Van Willigen , and J.W.N. Akkerman .
2000 . Inhibition of platelet integrin ? IIb ? 3 by peptides that interfere
with protein kinases and the ? 3 tail. Arterioscler. Thromb. Vasc. Biol.
20 : 1651 – 1660 .
Hughes , P.E. , F. Diaz-Gonzalez , L. Leong , C. Wu , J.A. McDonald , S.J.
Shattil , and M.H. Ginsberg . 1996 . Breaking the integrin hinge. A de-
fi ned structural constraint regulates integrin signaling. J. Biol. Chem.
271 : 6571 – 6574 .
Huttenlocher , A. , M.A. Ginsberg , and A.F. Horwitz . 1996 . Modulation of cell
migration by integrin-mediated cytoskeletal linkages and ligand-binding
affi nity. J. Cell Biol. 134 : 1551 – 1562 .
Hynes , R.O. 2002 . Integrins: bidirectional, allosteric signaling machines. Cell .
110 : 673 – 687 .
Kiema , T. , Y. Lad , P. Jiang , C.L. Oxley , M. Baldassarre , K.L. Wegener , I.D. Campbell ,
J. Ylanne , and D.A. Calderwood . 2006 . The molecular basis of fi lamin bind-
ing to integrins and competition with talin. Mol. Cell . 21 : 337 – 347 .
Kim , M. , C.V. Carman , and T.A. Springer . 2003 . Bidirectional transmembrane
signaling by cytoplasmic domain separation in integrins. Science .
301 : 1720 – 1725 .
Liu , K.Y. , S. Timmons , and J. Hawiger . 1996 . Identifi cation of a functionally
important sequence in the cytoplasmic tail of integrin ? 3 by using cell-
permeable peptide analogs. Proc. Natl. Acad. Sci. USA . 93 : 11819 – 11824 .
Ma , Y.Q. , J. Yang , M.M. Pesho , O. Vinogradova , J. Qin , and E.F. Plow . 2006 .
Regulation of integrin alpha(IIb)beta(3) activation by distinct regions of
its cytoplasmic tails. Biochemistry . 45 : 6656 – 6662 .
Ma , Y.Q. , J. Qin , and E.F. Plow . 2007 . Platelet integrin alpha(IIb)beta(3): activa-
tion mechanisms. J. Thromb. Haemost. 5 : 1345 – 1352 .
McEver , R.P. , and R.D. Cummings . 1997 . Perspectives series: cell adhesion in
vascular biology. Role of PSGL-1 binding to selectins in leukocyte re-
cruitment. J. Clin. Invest. 100 : 485 – 491 .
O ’ Toole , T.E. , J. Ylanne , and B.M. Culley . 1995 . Regulation of integrin affi nity
states through an NP X Y motif in the ? subunit cytoplasmic domain.
J. Biol. Chem. 270 : 8553 – 8558 .
Palecek , S.P. , J.C. Loftus , M.H. Ginsberg , D.A. Lauffenburger , and A.F. Horwitz .
1997 . Integrin-ligand binding properties govern cell migration speed
through cell-substratum adhesiveness. Nature . 385 : 537 – 540 .
Plow , E.F. , T.A. Haas , L. Zhang , J. Loftus , and J.W. Smith . 2000 . Ligand binding
to integrins. J. Biol. Chem. 275 : 21785 – 21788 .
Qin , J. , O. Vinogradova , and E.F. Plow . 2004 . Integrin bidirectional signaling: a
molecular view. PLoS Biol. 2 : e169 .
the chambers were washed three times with PBS and the adherent cells
were fi xed by 4% paraformaldehyde. To identify PSGL-1 – expressing cells,
the fi xed cells were stained by anti-PSGL-1 mAb, KPL-1 (BD Biosciences),
followed by goat anti – mouse IgG conjugated with AlexaFluor 488 (Invitro-
gen). As controls, nontransfected cells with the same treatment were in-
cluded in each experiment and always showed no PSGL-1 staining. The
positively stained (green) cells were observed using a fl uorescence micro-
scope (model DMR; Leica) with a 10X objective and recorded with a cooled
CCD camera (Retiga Exi; Q-Imaging). Data were analyzed with ImagePro
Plus Capture and Analysis software (Media Cybernetics).
GST pull-down assays and Western blotting
Glutathione- S -transferase (GST) fusion proteins were expressed in Rosetta2
(DE3) cells, purifi ed by glutathione-affi nity chromatography using a GST-
PrepFF column (GE Healthcare), and quantifi ed by spectrophotometry
using calculated extinction coeffi cients and Coomassie blue staining of
SDS-PAGE. The cell lysates of transfected CHO cells, out-dated platelets,
and HUVECs were prepared in the lysis buffer (50 mM Tris-HCl, pH 7.4,
150 mM NaCl, and 1% Triton X-100) containing protease inhibitors and
centrifuged at 15,000 g for 12 min. For the GST pull-down assays, gluta-
thione-Sepharose 4B (GE Healthcare) and the indicated GST fusion pro-
teins were added to the aliquots of lysate supernatants and incubated at
4 ° C for 8 h. The antibodies used for Western blotting were anti-kindlin-2
( Tu et al., 2003 ), anti-GFP (Santa Cruz Biotechnology, Inc.), and anti – human
talin (Chemicon International).
Immunofl uorescence and confocal microscopy
To observe the distributions of kindlin-2 and ? 3 integrins, HUVECs were al-
lowed to spread on immobilized fi brinogen at 37 ° C for 30 min or 60 min.
The spread cells were fi xed with 4% paraformaldehyde, permeabilized with
0.1% Triton X-100, and stained with mAb anti-kindlin-2 and polyclonal Ab
anti- ? 3 (Chemicon International) followed by AlexaFluor 568 anti – mouse
IgG and AlexaFluor 488 anti – rabbit IgG (Invitrogen). The images were re-
corded by a confocal microscope with a 63X objective (Leica).
To knock down endogenous kindlin-2, irrelevant control RNAs or designed
siRNAs targeting kindlin-2 (from Dharmacon) were transfected to CHO
cells using Lipofectamine 2000 (based on the protocol for siRNA transfec-
tion from Invitrogen) and HUVECs using targetfect-HUVEC (Targeting Sys-
tems) according to the manufacturers ’ protocols. The extent of suppression
and specifi city for kindlin-2 were evaluated by Western blotting with anti-
kindlin-2 and actin antibodies as controls.
HUVEC adhesion and migration
For cell adhesion assays, the nontransfected or transfected HUVECs, with
targeting or control siRNAs, were incubated with the immobilized integrin
ligand, fi brinogen (20 μ g/ml for coating) or vitronectin (5 μ g/ml for coat-
ing), for 30 min at 37 ° C. After washing, the adhered cells were fi xed by
70% methanol and stained with 1% totuidine blue, and the cell number
was counted under a microscope in several randomly selected fi elds.
To test the effect of kindlin-2 defi ciency on integrin ? v ? 3 activation, we
stimulated transfected HUVECs with PMA and measured cell adhesion as
previously described ( Byzova et al., 2000 ). The PMA-induced cell adhe-
sion was calculated as the increase based on the background without
PMA treatment. Cell migration was performed in Transwell plates (8 μ m
pore size). In brief, the HUVEC suspensions were added to the upper
chamber, which was precoated with fi brinogen or vitronectin and allowed
to migrate for 8 – 12 h in the presence of 20 ng/ml recombinant human
VEGF (R & D Systems) at 37 ° C in a 5% CO 2 humidifi ed incubator. After migra-
tion, the cells on the upper surface of the fi lter were removed; and the
migrated cells on the bottom surface of the fi lter were fi xed with methanol,
stained with 1% totuidine blue, and quantifi ed by performing microscopic
Quantitative data were compared using a two-tailed t test. P values to de-
termine statistical signifi cance are indicated in the text. For the experiments
to observe adherent or migrated cells, 10 – 20 fi elds or confocal cell images
were randomly taken in at least three independent experiments.
Online supplemental material
Fig. S1 shows that neither FLNa21 nor ? 3 -endonexin has direct effect on in-
tegrin ? IIb ? 3 activation and ? 3 CT/kindlin-2 association. Fig. S2 shows the
interaction of endogenous ? 3 integrin subunit and kindlin-2. Fig. S3 shows
the dynamic colocalization of ? 3 integrin and talin in HUVECs and kindlin-2
JCB • VOLUME 181 • NUMBER 3 • 2008 446
Sheffi eld , P. , S. Garrard , and Z. Derewenda . 1999 . Overcoming expression and
purifi cation problems of RhoGDI using a family of “ parallel ” expression
vectors. Protein Expr. Purif. 15 : 34 – 39 .
Shi , X. , Y.Q. Ma , Y. Tu , K. Chen , S. Wu , K. Fukuda , J. Qin , E.F. Plow , and C. Wu .
2007 . The MIG-2/integrin interaction strengthens cell-matrix adhesion
and modulates cell motility. J. Biol. Chem. 282 : 20455 – 20466 .
Siegel , D.H. , G.H. Ashton , H.G. Penagos , J.V. Lee , H.S. Feiler , K.C. Wilhelmsen ,
A.P. South , F.J. Smith , A.R. Prescott , V. Wessagowit , et al . 2003 . Loss of
kindlin-1, a human homolog of the Caenorhabditis elegans actin-extra-
cellular-matrix linker protein UNC-112, causes Kindler syndrome. Am. J.
Hum. Genet. 73 : 174 – 187 .
Tadokoro , S. , S.J. Shattil , K. Eto , V. Tai , R.C. Liddington , J. M.de Pereda , M.H.
Ginsberg , and D.A. Calderwood . 2003 . Talin binding to integrin ? tails: a
fi nal common step in integrin activation. Science . 302 : 103 – 106 .
Tu , Y. , S. Wu , X. Shi , K. Chen , and C. Wu . 2003 . Migfi lin and Mig-2 link focal
adhesions to fi lamin and the actin cytoskeleton and function in cell shape
modulation. Cell . 113 : 37 – 47 .
Ussar , S. , H.V. Wang , S. Linder , R. Fassler , and M. Moser . 2006 . The Kindlins:
subcellular localization and expression during murine development. Exp.
Cell Res. 312 : 3142 – 3151 .
Vinogradova , O. , A. Velyvis , A. Velyviene , B. Hu , T.A. Haas , E.F. Plow , and J.
Qin . 2002 . A structural mechanism of integrin ? IIb ß 3 “ inside-out ” activa-
tion as regulated by its cytoplasmic face. Cell . 110 : 587 – 597 .
Wegener , K.L. , A.W. Partridge , J. Han , A.R. Pickford , R.C. Liddington , M.H.
Ginsberg , and I.D. Campbell . 2007 . Structural basis of integrin activation
by talin. Cell . 128 : 171 – 182 .
Weinstein , E.J. , M. Bourner , R. Head , H. Zakeri , C. Bauer , and R. Mazzarella .
2003 . URP1: a member of a novel family of PH and FERM domain-
containing membrane-associated proteins is signifi cantly over-expressed
in lung and colon carcinomas. Biochim. Biophys. Acta . 1637 : 207 – 216 .
Wick , M. , C. Burger , S. Brusselbach , F.C. Lucibello , and R. Muller . 1994 .
Identifi cation of serum-inducible genes: different patterns of gene regula-
tion during G0 → S and G1 → S progression . J. Cell Sci . 107 (Pt 3):preced-
ing table of contents.
Xi , X. , R.J. Bodnar , Z. Li , S.C. Lam , and X. Du . 2003 . Critical roles for the
COOH-terminal NITY and RGT sequences of the integrin beta3 cyto-
plasmic domain in inside-out and outside-in signaling. J. Cell Biol.
162 : 329 – 339 .