T H E J O U R N A L O F C E L L B I O L O G Y
© The Rockefeller University Press $30.00
The Journal of Cell Biology, Vol. 179, No. 2, October 22, 2007 247–254
Cerebral cavernous malformations (CCMs) are vascular mal-
formations mostly found within the central nervous system. CCMs
can occur in sporadic or autosomal dominant inherited forms, the
latter of which map to three loci, KRIT-1/CCM1, MGC4607/
OSM/CCM2, and PDCD10/CCM3 (Dubovsky et al., 1995; Craig
et al., 1998; Labauge et al., 2007; Liquori et al., 2007). KRIT-1
protein was detected in endothelial cells by Western blot, immuno-
fl uorescence, and immunohistochemistry (Gunel et al., 2002;
Guzeloglu-Kayisli et al., 2004a,b), but this work was challenged
because KRIT-1 mRNA was not detected in the endothelium
(Petit et al., 2006). However, CCM lesions are composed of
endothelial cells (Wong et al., 2000; Clatterbuck et al., 2001)
and can occur outside the brain (Eerola et al., 2000). Further-
more, mice lacking KRIT-1 die because of defective vascular
development but have apparently normal brain development
(Whitehead et al., 2004), all of which suggest that the primary
defect in CCM lesions is in the endothelial compartment.
KRIT-1 possesses four ankyrin repeats, a band 4.1/ ezrin/
radixin/moesin (FERM) domain, and multiple NPXY sequences,
one of which is essential for integrin cytoplasmic domain-
associated protein-1α (ICAP1α) binding (Zawistowski et al.,
2002) and all of which mediate binding of CCM2 (Zawistowski
et al., 2005; Zhang et al., 2007). KRIT-1 was identifi ed as a Rap1-
binding protein in yeast two hybrid experiments (Serebriiskii
et al., 1997), and the FERM domain of KRIT-1 binds with 10-fold
higher affi nity to Rap1 than to H-Ras (Wohlgemuth et al., 2005).
Although the interaction of Rap1 and full-length KRIT-1 has
been disputed (Zhang et al., 2001), we have confi rmed the asso-
ciation by coimmunoprecipitation (unpublished data). Rap1
regulates cell–cell junctions in both endothelial and epithelial
cells (Knox and Brown, 2002; Price et al., 2004; Cullere et al.,
2005). The disruption of endothelial cell junctions in CCM
suggests that KRIT-1 may have a role in the capacity of Rap1
to regulate endothelial cell–cell junctions. Here, we report that
KRIT-1 is expressed in endothelial cells where it is present in
cell–cell junctions and associated with junctional proteins.
The junctional localization of KRIT-1 is mediated by its FERM
domain and regulated by Rap1 activity. Furthermore, we fi nd
that KRIT-1 is required for the stabilizing effect of Rap1 on
endothelial cell–cell junctions. Together, these data establish
that KRIT-1 is a Rap1-binding protein that regulates endothelial
junction integrity and may provide a molecular explanation for
aspects of the CCM phenotype.
KRIT-1/CCM1 is a Rap1 effector that regulates
endothelial cell–cell junctions
Angela Glading, Jaewon Han, Rebecca A. Stockton, and Mark H. Ginsberg
Department of Medicine, University of California, San Diego, La Jolla, CA 92093
mutations that cause loss of KRIT-1 protein function, though
how the loss of KRIT-1 leads to CCM is obscure. KRIT-1
binds to Rap1, a guanosine triphosphatase that maintains
the integrity of endothelial junctions. Here, we report
that KRIT-1 protein is expressed in cultured arterial and
venous endothelial cells and is present in cell–cell junc-
tions. KRIT-1 colocalized and was physically associated
with junctional proteins via its band 4.1/ezrin/radixin/
erebral cavernous malformation (CCM), a dis-
ease associated with defective endothelial junc-
tions, result from autosomal dominant CCM1
moesin (FERM) domain. Rap1 activity regulated the junc-
tional localization of KRIT-1 and its physical association
with junction proteins. However, the association of the
isolated KRIT-1 FERM domain was independent of Rap1.
Small interfering RNA–mediated depletion of KRIT-1
blocked the ability of Rap1 to stabilize endothelial junc-
tions associated with increased actin stress fi bers. Thus,
Rap1 increases KRIT-1 targeting to endothelial cell–cell
junctions where it suppresses stress fi bers and stabilizes
A. Glading, J. Han, and R.A. Stockton contributed equally to this paper.
Dr. Han died on 4 July 2006.
Correspondence to M.H. Ginsberg: email@example.com
Abbreviations used in this paper: 8-pCPT-2′-O-Me-cAMP, 8-pCPT-2′-O-methyl-
adenosine-3′,5′-cAMP; BAEC, bovine aortic endothelial cell; CCM, cerebral
cavernous malformation; CS, calf serum; FERM, band 4.1/ezrin/radixin/moesin;
FN, fi bronectin; GAP, GTPase activating protein; GAPDH, glyceraldehyde
3-phosphate dehydrogenase; HUVEC, human umbilical vein endothelial cell; ICAP1α,
integrin cytoplasmic domain-associated protein-1α; NGS, normal goat serum.
The online version of this article contains supplemental material.
JCB • VOLUME 179 • NUMBER 2 • 2007 248
Results and discussion
Immunoprecipitation with an mAb anti–KRIT-1 (15B2) followed
by Western blotting with an affi nity-purifi ed pAb (Rb6832) was
performed to increase the sensitivity of detection of endogenous
KRIT-1. We detected a band of ?80 kD in bovine aortic endo-
thelial cells (BAECs; Fig. 1, A and B) and human umbilical
vein endothelial cells (HUVECs; Fig. 1 B). The mobility of
this band is consistent with the calculated molecular mass of
KRIT-1 (81 kD). Moreover, CHO cells also exhibited this 80-kD
band, and when transfected with authentic HA-tagged human
KRIT-1 exhibited a second intense band of slightly lower mo-
bility consistent with the presence of the HA tag (Fig. 1 A).
An unrelated antibody (glyceraldehyde 3-phosphate dehydrog-
enase [GAPDH]) did not immunoprecipitate KRIT-1, nor did
nonimmune mouse IgG. We used siRNA-mediated knock-
down of endogenous KRIT-1 to confi rm that the endogenous
80-kD polypeptide was authentic KRIT-1. Transfection with
KRIT-1–specifi c siRNA (530; Fig. 1 B) completely eliminated
this band from BAECs and reduced it by 70% in HUVECs, but
had no effect on the expression of a variety of cell–cell junc-
tion proteins (Fig. 1 C). An irrelevant (Fig. 1 C, GAPDH) and
a negative control siRNA (NC) had no effect on the expression
of this polypeptide.
Having shown the antibodies’ specificity in immuno-
precipitation blotting experiments, we then used the mAb anti–
KRIT-1 to detect endothelial cell KRIT-1 by immunofl uorescence.
KRIT-1 staining was enriched at sites of cell–cell junctions and
the nucleus in confl uent bovine aortic endothelial monolayers
(Fig. 2 A), but was absent from free membranes (not depicted),
suggesting cell junction localization consistent with the reported
localization of recombinant KRIT-1 in transfected HEK293
cells (Zawistowski et al., 2005). Staining specifi city was con-
fi rmed by its marked reduction after transfection with the KRIT-1
siRNA 530 (Fig. 2 A). Similar staining was seen in HUVECs
and was also observed with the affi nity-purifi ed pAb anti–KRIT-1.
No staining was seen with nonimmune rabbit or mouse IgG
(unpublished data). Thus, KRIT-1 protein is expressed in both
arterial and venous endothelial cells in culture, which is consis-
tent with its reported presence in endothelium in vivo (Guzeloglu-
Kayisli et al., 2004a).
Confocal microscopy demonstrated that KRIT-1 staining
was enriched in cell junctions and colocalized with β-catenin, a
cell–cell junction protein (Fig. 2 A; Vestweber, 2000). In KRIT-1
siRNA 530–treated cells, β-catenin localization to the cell–
cell junction was disrupted but global expression of junctional
proteins was unchanged (Figs. 1 C and 2 A). β-catenin also
physically associated with KRIT-1, as did p120-catenin and the
junctional scaffold AF-6 (also called afadin; Miyahara et al.,
2000), as judged by their presence in KRIT-1 immunopreci p-
itates (Fig. 2 B). The coimmunoprecipitation was done under
conditions that garnered near complete recovery of KRIT-1 from
the cell lysate. From this, we estimate that KRIT associates with
?2% of total cellular β-catenin and p120-catenin and 6% of
total AF-6. Nevertheless, the intensities of the KRIT-1 bands were
similar to those observed for the junctional proteins, suggesting
that KRIT-1 is not an abundant protein. This idea is supported
by the requirement for prior immunoprecipitation to visualize
KRIT-1 by immunoblotting (Fig. 2 B). The association of KRIT-1
with AF-6 is noteworthy as AF-6 can inhibit Rap1 activity
(Su et al., 2003; Zhang et al., 2005) through recruitment of Rap
GTPase activating proteins (GAPs). Thus, the interaction of
KRIT-1 with AF-6 could act as a negative feedback modulator
of Rap activity. Also present in coprecipitates, but not shown
here, were vascular endothelial cadherin and α-catenin. In contrast,
ZO-1, a tight junction marker, and talin, a focal adhesion protein
absent from adherens junctions (Geiger et al., 1985), were not
detected in KRIT-1 immunoprecipitates (Fig. 2 B).
Figure 1. Monoclonal anti–KRIT-1 (15B2)
recognizes authentic KRIT-1 protein in endo-
thelial cells. (A) Immunoprecipitation using mAb
anti–KRIT-1 (15B2) and immunoblotting with pAb
anti–KRIT-1 (Rb6832) yields a band of ?80 kD
in endothelial and CHO cells. An irrelevant
antibody, anti-GAPDH, and nonimmune mouse
IgG did not immunoprecipitate the KRIT-1 band.
Asterisk indicates that the input level of the
lane was changed with Photoshop due to high
background to show the absence of a band.
(B) Antibody reactivity requires the presence of
KRIT-1. siRNA against KRIT-1 (siRNA 530) elimi-
nated the KRIT-1 band immuno precipitated
from BAECs and reduced that in HUVECs by
70%. Negative control siRNA and siRNA di-
rected against GAPDH had no effect on KRIT-1
expression. Blots are representative; BAEC,
n = 3; HUVEC, n = 2. (C) KRIT-1 depletion using
anti–KRIT-1 siRNA 530 has no effect on the ex-
pression of junctional proteins. BAECs treated
with anti–KRIT-1 siRNA (530) or control siRNA
(NC) were Western blotted for junctional proteins.
Although KRIT-1 knockdown of >80% was
observed by immunoprecipitation and blotting
of KRIT (bottom), no effect on the expression
of junction proteins was seen. Blots are repre-
sentative; n = 3. Black lines indicate that inter-
vening lanes have been spliced out.
KRIT-1 AND CELL JUNCTIONS • GLADING ET AL. 249
KRIT-1 associates with Rap1 small GTPase and exhibits
a preference for Rap versus Ras in vitro (Serebriiskii et al., 1997;
Wohlgemuth et al., 2005). We therefore asked whether Rap1 could
regulate the localization of KRIT-1 to the junctions and its asso-
ciation with junctional proteins. Transfection of cells with an acti-
vated Rap1 (Rap1A-G12V, referred to as RapV12) increased the
association of endogenous KRIT-1 with β-catenin and AF-6
(Fig. 3 A). Expression of the inhibitor of Rap activity (Rap1GAP)
inhibited the association of KRIT-1 with β-catenin and AF-6 to a
greater extent than the increase elicited by activated Rap1A.
The strong effect of Rap1GAP suggests the presence of basal Rap
activity in BAECs. Neither RapV12 nor Rap1GAP expression
affected KRIT-1 expression (Fig. 3 A). Furthermore, thrombin
treatment led to a dramatic loss of KRIT-1 from the junctions
(Fig. 3 B) but did not alter KRIT-1 expression (Fig. S1 A, avail-
able at http://www.jcb.org/cgi/content/full/200705175/DC1).
Thrombin’s effect was counteracted by the addition of an activator
(Enserink et al., 2002) of the RapGEF Epac-1, 8-pCPT-2′-O-
methyladenosine-3′,5′-cAMP (8-pCPT-2′-O-Me-cAMP; Fig. 3 B) .
RapV12 also reversed the thrombin-stimulated loss of KRIT from
the junctions, supporting the idea that effect of 8-pCPT-2′-O-Me-
cAMP is caused by the activation of Rap1 and that KRIT-1 associa-
tion with cell–cell junctions is increased by Rap1 activation.
To explore the mechanism of KRIT-1 targeting of endo-
thelial cell–cell junctions, we examined the capacity of different
domains of the protein to mediate junctional localization.
The isolated KRIT-1 FERM domain (GFP-KRIT-F123; Fig. 4 A)
colocalized with β-catenin in cell–cell junctions (Fig. 4 B).
Furthermore, the KRIT-1 FERM domain was physically associated
with β-catenin (Fig. 4 C). In sharp contrast to the intact protein,
the interaction of GFP-KRIT-F123 with β-catenin was insensi-
tive to the activation state of Rap1 (Fig. 4 D), suggesting that
active Rap1 may increase the association of KRIT-1 with junc-
tional proteins by increasing the availability of this domain.
In contrast to the FERM domain, the KRIT-1 N terminus (Fig. 4 A)
did not colocalize with either junctional β-catenin (Fig. 4 B)
or ZO-1. Furthermore, the N terminus did not associate with
β-catenin in coimmunoprecipitation experiments (Fig. 4 C).
Moreover, the KRIT-F23 fragment of the FERM domain failed
to localize to junctions, suggesting that the F1 region is required
for localization to the junction. Thus, KRIT-1 localization to
endothelial junctions is mediated by the KRIT-1 FERM domain
and regulated by Rap1.
CCM lesions display characteristics typical of a loss
of endothelial cell–cell junctions (Clatterbuck et al., 2001).
As noted previously, KRIT-1 is a relatively specifi c Rap1 effector
(Serebriiskii et al., 1997; Wohlgemuth et al., 2005), and Rap1
regulates the integrity of cell–cell junctions in endothelial and
epithelial cells (Knox and Brown, 2002; Price et al., 2004;
Cullere et al., 2005), leading us to examine the role of KRIT-1 in
Figure 2. KRIT-1 localizes to cell junctions and associates with junction proteins. (A) KRIT-1 staining localizes to cell junctions (top left). mAb anti–KRIT-1
(15B2) recognizes endogenous KRIT-1 by immunofl uorescence staining in untransfected and negative control siRNA–transfected BAECs (negative control
siRNA–transfected cells are shown). Staining specifi city was demonstrated by knockdown of KRIT-1 using siRNA 530 (bottom left). Confocal microscope
images demonstrate a colocalization between KRIT-1 and β-catenin immunofl uorescence (top right). In KRIT-1–depleted cells, β-catenin localization to the
cell–cell junction is disrupted (bottom right). In top panels, the confocal imaging plane did not contain the nucleus, which does stain for KRIT-1 (not
depicted); in bottom panels (with siRNA), the imaging plane slices through the nucleus. Confocal images are representative; n = 3. Bar, 50 μm. (B) KRIT-1
coimmunoprecipitates with β-catenin, AF-6, and p120-catenin but not with ZO-1 or talin. Lysates of BAECs were immunoprecipitated using monoclonal
anti–KRIT-1 (15B2) or nonimmune mouse IgG (IgG). WCL, 10% of input whole cell lysate. Blots are representative; n = 4. All lanes were from the same
membrane and black lines indicate that intervening lanes have been spliced out, or in the case of the p120 blot, reordered for clarity.
JCB • VOLUME 179 • NUMBER 2 • 2007 250
Rap1-induced stabilization of endothelial junctions. We noted
that there was extensive redistribution of β-catenin out of cell–
cell junctions in KRIT-1–depleted BAECs (Fig. 1 A), suggest-
ing that KRIT-1 might be required for the stability of cell–cell
junctions in these cells. As a test for the function of these junctions,
we examined the permeability of endothelial cell monolayers to
HRP (Stockton et al., 2004). KRIT-1 siRNA transfection caused
an approximately twofold increase in permeability (Fig. 5 A)
that was reversed by reconstitution with recombinant KRIT-1.
Furthermore, treatment of KRIT-1–depleted cells with 8-pCPT-2′-
O-Me-cAMP had little effect on permeability (Fig. 5 A).
In sharp contrast, 8-pCPT-2′-O-Me-cAMP reversed the increase
in permeability caused by thrombin as expected (Rangarajan
et al., 2003; Cullere et al., 2005). Thrombin treatment caused a
loss of KRIT-1 from the junctions (Fig. 3 B) and a concomitant
redistribution of β-catenin away from the junction (Fig. S1 B);
however, thrombin treatment did not disrupt the interaction
of KRIT-1 and β-catenin, suggesting that they remain asso-
ciated as they redistribute away from the junction (Fig. S1 A).
Thrombin increased permeability further in KRIT-1–depleted
cells. Furthermore, overexpression of KRIT-1 partially reversed
the increased permeability induced by expression of Rap1GAP
(Fig. 5 B). Thus, KRIT-1 regulates endothelial permeabil-
ity and is required for Rap1-mediated stabilization of endo-
We have shown here that Rap1 activation leads to associa-
tion of KRIT-1 with endothelial cell–cell junction proteins.
The KRIT-1 N terminus has binding sites for ICAP1α and CCM2
(Zawistowski et al., 2002, 2005; Zhang et al., 2007). Con-
sequently, recruitment of KRIT-1 through its FERM domain has
the potential to bring these proteins to the junction. Both
ICAP1α and CCM2 are involved in the regulation of Rho family
GTPases (Degani et al., 2002; Zawistowski et al., 2005), which
are known to control the integrity of endothelial junctions via
the actin cytoskeleton (Wojciak-Stothard and Ridley, 2002).
Furthermore, activation of Rap1 by 8-pCPT-2′-O-Me-cAMP
inhibits thrombin-induced RhoA activation in endothelial cells
(Cullere et al., 2005). Hence, we examined F-actin distribution
after depletion of KRIT-1 with siRNA. In control cells, actin
was distributed around the circumference of each cell, and few
stress fi bers were observed. This contrasts strongly with KRIT-1
siRNA–treated cells, which had abundant stress fi bers. Over-
expression of HA-KRIT in control cells increased cortical actin
staining, and HA-KRIT reexpression restored cortical actin mor-
phology in KRIT-1 siRNA–treated cells (Fig. 5 C). As changes
in actin morphology are linked to altered permeability (Stockton
et al., 2004; Kooistra et al., 2005; Hayashi et al., 2006), this
result suggests that KRIT-1 may act as a scaffold that regulates
endothelial junctions by recruiting modulators of the Rho family
GTPases that control the actin cytoskeleton.
CCM lesions are composed of a bed of leaky capillaries
(Wong et al., 2000; Clatterbuck et al., 2001). Thus, our fi nding
that the loss of KRIT-1 increases endothelial permeability pro-
vides a direct link between the pathogenesis of CCM and KRIT-1
function. KRIT-1 is a relatively specifi c effector of Rap1, which
is a regulator of endothelial permeability and the stability of cell–
cell junctions (Knox and Brown, 2002; Cullere et al., 2005).
We now fi nd that the capacity of Rap1 to stabilize endothelial
cell junctions depends on KRIT-1 and that Rap1 regulates the
junctional localization of the KRIT-1 protein and increases the
association of KRIT-1 with junctional proteins. The KRIT-1
FERM domain mediates its association with junctional proteins
and the isolated FERM domain associates with junctional
proteins in a Rap1-independent manner. This suggests that the
Figure 3. KRIT-1 is a Rap1 effector protein
and Rap1 regulates KRIT-1 junctional association.
(A) Expression of RapV12 increases coimmuno-
precipitation of β-catenin and AF-6 with KRIT-1,
whereas expression of Rap1GAP reduces as-
sociation. Transfection of BAECs with RapV12
or Rap1GAP does not affect the expression of
KRIT-1 (top) or β-catenin and AF-6 as shown by
Western blot of input lysate (bottom). Black lines
indicate that intervening lanes have been spliced
out; all lanes were cut from the same membrane.
Blots are representative; n = 4. (B) Rap1
activation counteracts thrombin-mediated re-
duction of junctional KRIT-1. 2 U/ml throm-
bin treatment reduced KRIT-1 localization to
cell–cell junctions. Treatment with 8-pCPT-2′-
O-Me-cAMP before thrombin treatment pre-
vented the loss of KRIT-1 from junctions. KRIT-1
localization in BAECs transfected with RapV12
was relatively resistant to thrombin treatment.
Confocal images are representative; n = 3.
Bar, 50 μm.
KRIT-1 AND CELL JUNCTIONS • GLADING ET AL.251
availability of the KRIT-1 FERM domain may be regulated by
the binding of active Rap1. This would allow KRIT-1 to associate
via the FERM domain with the junction complex, thus stabilizing
cell–cell junctions, perhaps through modifying the activity of
Rho family GTPases. In summary, Rap1 stimulates the junctional
localization of KRIT-1 via KRIT-1’s FERM domain, and KRIT-1
is required for Rap1 stabilization of endothelial junctions. These
data provide a molecular linkage between KRIT-1 protein func-
tion and the CCM phenotype and identify a Rap effector that
regulates cell–cell junctions.
Figure 4. KRIT-1 junctional localization is regulated by Rap1 and requires the FERM domain. (A) Schematic of KRIT-1 constructs. All constructs include an
N-terminal GFP tag not depicted here. (B) GFP-tagged KRIT-1 FERM domain (GFP-KRIT-F123) colocalized with β-catenin in bovine aortic endothelial cell–cell
junctions. A KRIT-1 FERM domain truncation (GFP-F23) does not localize to cell junctions nor does a KRIT-1 N-terminal construct containing the ICAP1α and
CCM2 binding sites. Minor colocalization with β-catenin is seen in the cytoplasm of all treatments. Confocal images are representative; n = 5. Bars, 50 μm.
(C) Full-length GFP–KRIT-1 (KRIT-1) and GFP-KRIT-F123 (F123) coimmunoprecipitate with β-catenin, whereas the GFP-labeled N terminus (N-term) does not.
BAECs expressing GFP-labeled KRIT-1 constructs were immunoprecipitated with rabbit anti-GFP and blotted with anti–β-catenin (left). Blotting with mouse
anti-GFP demonstrated similar expression of each construct. GFP alone did not coimmunoprecipitate β-catenin, nor did immunoprecipitation with
nonimmune rabbit IgG of lysates of cells transfected with GFP–KRIT-1 (right). 10% of input lysate (WCL) from GFP-KRIT-F123–transfected cells was blotted
to assess expression of β-catenin. Lanes were cut from the same membrane and reordered using Photoshop. (D) Association of GFP-KRIT-F123 and β-catenin
is insensitive to Rap1 activation. BAECs were transfected with GFP–KRIT-1, GFP-KRIT-F123, or GFP alone in the presence of either RapV12 or RapGAP.
Anti-GFP immunoprecipitates were immunoblotted for β-catenin. Rap1GAP reduced the association of full-length KRIT-1with β-catenin ?75% (as evaluated
by densitometry). This decrease was not seen in precipitates from GFP-KRIT-F123–expressing cells (top). In parallel, anti-GFP immunoprecipitates were blotted
with mouse anti-GFP and 10% of input lysate was blotted for β-catenin to assess expression of the interacting proteins (bottom). Blots are representative;
n = 3. Black lines indicate that intervening lanes have been spliced out.
JCB • VOLUME 179 • NUMBER 2 • 2007 252
Materials and methods
BAECs were a gift of S. Shattil (University of California, San Diego, La
Jolla, CA). BAECs were cultured in DME with 10% calf serum (CS) and 1%
penicillin/streptomycin (Invitrogen). HUVECs were obtained from Cam-
brex and cultured in endothelial growth medium 2 (Cambrex). CHO cells
were obtained from American Type Culture Collection. CHO cells were
cultured in DME with 10% fetal bovine serum plus 1% penicillin/streptomycin,
1% L-glutamine, and 1% nonessential amino acids (Invitrogen).
siRNA and cDNA
siRNA directed against human KRIT was designed by Ambion. Three pre-
designed siRNA sequences (146530, 146531, and 214883) were tested
and all were found to inhibit expression of KRIT protein in human cells.
siRNA 146530 (530) was found to have activity in BAECs, as would be
expected from its 100% identity in the target sequence, and was used in
all subsequent experiments. Control siRNA was obtained from Ambion, in-
cluding anti-GAPDH and negative control siRNA 1. All siRNA was used at
cDNA encoding HA- and GFP-tagged full-length KRIT, a GFP-tagged
KRIT FERM domain (GFP-F123), and a GFP-tagged FERM domain trunca-
tion (GFP-F23) was constructed from full-length KRIT-1 cDNA provided by
H. Dietz (Johns Hopkins University, Baltimore, MD). Domain boundaries
were estimated using protein sequence alignment with other FERM-contain-
ing proteins: KRIT-1 FERM (F123) extended from amino acids 410–737,
F23 amino acids 512–737, and KRIT-1 N terminus amino acids 1–274.
A GFP-tagged N-terminal truncation of KRIT was made by subcloning the
N terminus from a GST construct into the pEGFP-C1 vector (CLONTECH
Laboratories, Inc.). Mammalian expression constructs for HA-Rap1A-G12V
(RapV12) and HA-Rap1GAP (RapGAP) were gifts from J. Bos (Utrecht
University, Utrecht, Netherlands).
CHO cells were transfected with Lipofectamine Plus (Invitrogen) according
to the manufacturer’s instructions. Cells were plated at 80% confl uence 24 h
before transfection and media was replaced 3 h after transfection.
For Western blot detection of siRNA effi ciency, siRNA was transfected
into BAECs and HUVECs using HiPerfect transfection reagent (QIAGEN)
according to the manufacturer’s suggested instructions. Cells were plated
at 60–70% confl uence and 25 nM siRNA was incubated with the cells for
24–48 h with no difference in knockdown effi ciency.
For permeability, immunofl uorescence, and cotransfection studies,
BAECs were transfected using a nucleofection device (Amaxa). In brief,
0.5 × 106 cells per transfection were suspended in Basic endothelial cell
solution (Amaxa) together with 25 nM siRNA with or without 1 μg DNA.
The cells were then nucleoporated using program M-003 (Amaxa). After
recovery at 37°C for 10 min, the cells were plated as required. This method
garnered transfection effi ciencies from 70 to 90%.
Anti–KRIT-1 antibody production and characterization
Polyclonal anti–KRIT-1 6832 was developed against the recombinant KRIT-1
FERM (F123) domain coupled to keyhole limpet hemocyanin. Monoclonal
anti–KRIT-1 antibodies were also developed using the recombinant KRIT-1
FERM (F123) domain as the antigen. Mice were immunized with the KRIT-1
FERM domain in incomplete Freund’s adjuvant. Mouse sera were titered by
ELISA against GST-F123 before fusion of splenic cells with myeloma cells.
After fusion, single hybridoma cells were plated by limiting dilution, and
antibody production was assayed in the hybridoma supernatant by ELISA.
Titers were done in parallel on GST-coated plates to assess background
binding. Hybridomas with high titer were selected for subcloning, and the
process was repeated twice. Selected hybridoma supernatants were further
purifi ed by affi nity chromatography on a protein G–Sepharose column.
Figure 5. KRIT-1 regulates endothelial cell permeability. (A) Treatment of
BAECs with 2 U/ml thrombin increased endothelial permeability to HRP,
whereas treatment with 8-pCPT-2′-O-Me-cAMP inhibited thrombin-stimulated
permeability, as did overexpression of recombinant KRIT-1 (in control
siRNA–treated cells). Knockdown of KRIT-1 expression by KRIT-1 siRNA
530 caused an approximately twofold increase in permeability. Thrombin
treatment further increased permeability in KRIT-1–depleted cells. However,
8-pCPT-2′-O-Me-cAMP had little effect on permeability in these cells.
The increased permeability was almost completely rescued by reexpression of
recombinant KRIT-1. Negative control siRNA had no effect on permeability.
Data shown is the mean increase in permeability ± SEM; n = 5. *, P < 0.05
compared with control siRNA plus vehicle. (B) KRIT-1 overexpression
reverses Rap1GAP-induced increased endothelial permeability. Data shown
is the mean increase in permeability ± SEM; n = 4. *, P < 0.05 com-
pared with vector control. (bottom) A representative blot of Rap1GAP
expression in the cells used for permeability experiments. (C) Depletion of
KRIT-1 increases stress fi ber formation. The actin cytoskeleton of anti–KRIT-1
siRNA–treated and HA–KRIT-1–reconstituted cells was stained with rhoda-
mine-phalloidin to assess changes in the distribution of F-actin as a con-
sequence of KRIT-1 depletion. Epifl uorescence images are representative;
n = 3. Bar, 50 μm.
KRIT-1 AND CELL JUNCTIONS • GLADING ET AL.253
Immunoprecipitation and Western blotting
For endogenous coimmunoprecipitation experiments, cells expressing KRIT-1
were scraped into 500 μl of lysis buffer containing 50 mM Tris, pH 7.4,
150 mM NaCl, 0.5% NP-40, and 5 mM MgCl2 plus a protease inhibitor
cocktail (Roche). After resting 5 min on ice, the lysate was incubated for 15 min
at 4°C with rocking. The lysate was spun down at 14,000 rpm for 10 min
and the protein concentration of the supernatant was determined using a
bicinchoninic acid assay (Pierce Chemical Co.). 500 μg of precleared total
cell protein was added to 2 μg of immunoprecipitating antibody and incu-
bated at 4°C with continuous rocking for 2 h. 10 μl of a 50% slurry of pro-
tein G–Sepharose beads (GE Healthcare) was then added and the rocking
of the samples was continued overnight. The immunoprecipitated samples
were washed three times with lysis buffer and solubilized with 10 μl SDS-PAGE
sample buffer. Samples were resolved on 4–20% polyacrylamide gels
(Invitrogen) in SDS-PAGE buffer and transferred to nitrocellulose membranes.
Anti–KRIT-1 15B2 was used for immunofl uorescence at a dilution of
1:1,000. Rabbit anti-GFP (Invitrogen) was used at a dilution of 1:200 for
immunoprecipitation of GFP-tagged proteins. Nonimmune mouse IgG was
obtained from Santa Cruz Biotechnology, Inc. Rabbit anti–human–KRIT-1 6832
serum was used for immunoblotting at a dilution of 1:1,000. Mouse anti–
human AF-6 (BD Biosciences), mouse anti–human ZO-1 (Zymed Laboratories),
rabbit anti–human VE-cadherin (CD144; Serotec), mouse anti-murine p120-
catenin (Sigma-Aldrich), and rabbit anti–human GAPDH (Santa Cruz Bio-
technology, Inc.) were used for immunoblotting at dilutions of 1:1,000. Mouse
anti–chicken β-catenin (6F9; Sigma-Aldrich), mouse anti-GFP (Invitrogen), and
mouse anti–chicken talin (8d4; Sigma-Aldrich) were used for immuno-
blotting at dilutions of 1:5,000, 1:2,500, and 1:3,000, respectively. Anti–
β-catenin was used in immunofl uorescence at a dilution of 1:1,000. Blots were
probed with the appropriate secondary antibody conjugated to Alexa 680
(Invitrogen) or IRDye 800 (Rockland Immunochemicals) and imaged using
an infrared imaging system (Odyssey; Li-Cor). Blots were processed using
Photoshop (Adobe) and all lanes were adjusted equally.
BAECs were grown to confl uence on fi bronectin (FN)-coated glass cover-
slips. Slips were fi xed with 4% formaldehyde for 20 min, permeabilized
for 10 min with 0.2% Triton X-100 (Sigma-Aldrich), and blocked for 1 h
with 10% normal goat serum (NGS; Sigma-Aldrich) in PBS. Anti–β-catenin
(1:1,000 in NGS) was added and incubated at RT for 1 h in a humidifi ed
chamber. Slips were washed three times with PBS plus 0.001% Triton
X-100 (PBS-TX100), and then goat anti–mouse IgG Alexa 568, goat anti–
mouse IgG Alexa 488, or goat anti–rabbit IgG Alexa 568 (Invitrogen) in
NGS was added at 1:1,000 and incubated for 1 h at RT. Control stain was
performed with second antibody only at 1:1,000. Slips were washed six
times in PBS-TX100 and mounted on glass slides using 10 μl of fl uorescent
mounting medium (DakoCytomation) and dried overnight. Images were
obtained using a confocal microscopy system (TCS SP2 AOBS; Leica
DMRE microscope with an HCX PL APO 63× 1.32 oil objective). Images
were processed using Photoshop. Colocalization images were created
using ImageJ software (National Institutes of Health).
Concomitant to the leak assay, a portion of the transfected cells was
plated on FN-coated glass coverslips and grown to confl uence (?24 h) in
low-glucose DME/10% CS. Cells were serum starved (0.5% CS) for 4 h then
treated for 30 min with 100 μM 8-pCPT-2′-O-Me-cAMP or the vehicle alone
(control), and indicated cells were treated with 2 U/ml thrombin for 60 min.
Slips were fi xed with 3.5% formaldehyde for 2 h, permeabilized for 10 min
with 0.15% Triton X-100 and washed with TBST. Slips were blocked with
10% NGS for 60 min at RT and washed again. 200 μl/slip of primary anti-
body was incubated overnight at 4°C in a humidifi ed chamber. The control
stain was mouse IgG at 1:1,000. After washing, donkey anti–mouse IgG
Alexa 488 (Invitrogen) was added at a 1:1,000 dilution overnight at 4°C.
Coverslips were washed six times in alternating PBS/TBST rinses and
mounted on 10 μl Prolong Gold mounting medium (Invitrogen) and photo-
graphed using a confocal microscopy system. Images were processed using
Photoshop. For actin staining (Fig. 5 C), cells were incubated with a 1:200
dilution of Alexa 488 or Alexa 568–phalloidin (6 μM in methanol; Invitro-
gen) overnight at 4°C. Coverslips were washed and mounted as above and
imaged using a microscope (DMLS; Lecia NPlan 40× 0.65 objective) with
a camera (SPOT RT Color-2000; Diagnostic Instruments).
BAEC monolayer leak assay
The permeability of the endothelial monolayer was evaluated using a leak
assay originally described in Stockton et al. (2004). In brief, BAECs were
grown to semiconfl uence. Cells were transfected with 25 nM of negative con-
trol siRNA or KRIT-1 siRNA 530 with or without 1 μg pcDNA3.1 HA–KRIT-1.
Transfected cells in phenol-free DME/10% CS were plated into 3-μm-pore
polyester FN-coated transwell fi lters (Corning). Filter-plated cells were incu-
bated for 48 h at 37°C to full confl uence. Cells were then incubated in serum-
free, phenol-free DME for 2 h. As indicated in the fi gure legends, in some
conditions cells were treated with 100 μM 8-pCPT-2′-O-Me-cAMP for 30 min,
and then half were treated with 2 U/ml thrombin (GE Healthcare) for 30 min.
50 μl of phenol-free DME containing 1.5 ?g/ml HRP (Sigma-Aldrich) was
added to upper wells for an additional 30 min. Filters were removed from
outer wells and fi xed in 3.5% formaldehyde and later stained with 0.25%
Coomassie blue and examined by phase-contrast microscopy to reconfi rm
the integrity of cell monolayers.
The HRP content of the lower chamber medium was measured using
a microplate peroxidase colorimetric assay. 100-μl-per-well guaiacol/
sodium phosphate assay buffer (1:1) was added to 25 μl of each sample
in triplicate. 25 μl of freshly made 0.6-mM H2O2 in ddH2O was added to
each well for ?15 min or until color developed. Reaction was stopped with
10-μl-per-well 2N H2SO4. A490 was acquired and raw absorbance values
were normalized as a percentage of control untransfected (vehicle) cell
sample absorbance. Data was analyzed for statistical signifi cance using
analysis of variance and SigmaStat software (Jandel).
Online supplemental material
Fig. S1 shows the effect of thrombin treatment on KRIT-1 expression, associa-
tion with β-catenin, and β-catenin localization. Online supplemental material is
available at http://www.jcb.org/cgi/content/full/200705175/DC1.
This work was supported by grants from the National Institutes of Health
(AR27214, HL 078784, and HL31950). A. Glading is a postdoctoral fellow
of the American Cancer Society and J. Han was an arthritis investigator of the
Submitted: 29 May 2007
Accepted: 19 September 2007
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