Tyrosine phosphorylation of occludin attenuates its interactions with ZO-1, ZO-2, and ZO-3
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Tyrosine phosphorylation of occludin attenuates its interactionswith ZO-1, ZO-2, and ZO-3
G. Kale, A.P. Naren, P. Sheth, and R.K. Rao*
Department of Physiology, University of Tennessee Health Science Center, 894 Union Avenue, Memphis, TN 38163, USA
Received 14 January 2003
Occludin, the transmembrane integral protein of the tight junction, plays a crucial role in the molecular organization and
function of tight junction. While the homotypic interaction of extracellular loops of occludin appears to determine the barrier
function of tight junction, the intracellular C-terminal tail, C-occludin, interacts with other tight junction proteins such as ZO-1,
ZO-2, and ZO-3 and with the actin laments of cytoskeleton. In the present study we phosphorylated GST-fused C-occludin on
tyrosine residues, in TKX1 Epicurian coli or by active c-Src in vitro. c-Src binds to occludin and phosphorylates it on tyrosine
residues. The eect of tyrosine phosphorylation of C-occludin on its ability to bind ZO-1, ZO-2, ZO-3, and F-actin was evaluated.
Results show that the amounts of ZO-1, ZO-2, and ZO-3 bound to tyrosine phosphorylated C-occludin were several fold less than
the amounts bound to non-phosphorylated C-occludin. However, the amount of tyrosine phosphorylated C-occludin bound to F-
actin was not signicantly dierent from the amount of non-phosphorylated C-occludin bound to F-actin. These results demonstrate
that tyrosine phosphorylation of occludin reduces its ability to bind ZO-1, ZO-2, and ZO-3, but not F-actin. Results also suggest
that c-Src-mediated disruption of tight junction may involve tyrosine phosphorylation of occludin.
2003 Elsevier Science (USA). All rights reserved.
The tight junction (TJ) forms a barrier for the
movement of substances through the paracellular space.
As a fence it limits the crossover of membrane proteins
between the apical and basolateral membranes. Three
dierent transmembrane proteins, occludin , claudins
, and junction adhesion molecule , have been
identied at the TJ. Occludin appears to be the most
important and relatively well-characterized transmem-brane protein of the TJ. A number of other proteins,
including zonula occludens (ZO)-1, ZO-2, and ZO-3, are
localized at the TJ . ZO-1, ZO-2, and ZO-3 bind to
the intracellular C-terminal tail of occludin [4,5] and the
interactions between these proteins are crucial for the
assembly of TJ and the maintenance of barrier function
. Occludin is approximately 65 kDa protein, which
spans the plasma membrane four times to form twoextracellular loops and one intracellular loop. The short
N-terminal tail (65 amino acids) and the long C-terminal
tail (255 amino acids) extend into the intracellular
compartment . Expression of occludin mutants lack-
ing the C-terminal tail results in the disruption of TJ .
A signicant body of evidence indicates that the intra-
cellular C-terminal tail interacts with ZO-1, ZO-2, ZO-3,
and F-actin . A recent study, using the bait peptide
method, determined the interaction of occludin C-ter-
minus with signaling proteins, such as protein kinase
C-f, c-Yes, and regulatory subunit of PI-3 kinase, inaddition to ZO-1 and connexin-26 .
Tyrosine kinase activity is essential for both the dis-
assembly  and the assembly  of TJ in
dierent epithelial monolayers. While protein tyrosine
phosphorylation is associated with the disruption of TJ
in MDCK and Caco-2 cell monolayers [19,20], tyrosine
kinase inhibitors prevent oxidative stress- [12,13,15] and
acetaldehyde-induced  disruption of the TJ in Caco-2cell monolayers. In a recent study we showed that oxi-
dative stress-induced disassembly of TJ in Caco-2 cell
monolayer is associated with the tyrosine phosphoryla-
tion of occludin, ZO-1, E-cadherin, and b-catenin, anddissociation of occludin from ZO-1 and the actin cyto-
skeleton . These studies suggested that tyrosine
Biochemical and Biophysical Research Communications 302 (2003) 324329
* Corresponding author. Fax: 1-901-448-7126.
E-mail address: firstname.lastname@example.org (R.K. Rao).
0006-291X/03/$ - see front matter 2003 Elsevier Science (USA). All rights reserved.doi:10.1016/S0006-291X(03)00167-0
phosphorylation of TJ-proteins results in the destabili-zation of TJ. However, there is no evidence for the in-
uence of tyrosine phosphorylation on the direct
interaction of occludin with ZO proteins or F-actin.
In this study, we used non-phosphorylated and ty-
rosine-phosphorylated recombinant C-terminal tail of
occludin to determine the role of tyrosine phosphory-
lation in the regulation of interactions between occludin
and ZO-1, ZO-2, ZO-3, or F-actin. We demonstrate forthe rst time that the tyrosine phosphorylation of C-
terminal tail of occludin prevents its interactions with
ZO-1, ZO-2, and ZO-3, while the interaction with F-
actin was not altered.
Materials and methods
Preparation of non-phosphorylated and tyrosine phosphorylated
C-occludin. C-terminal tail of occludin, C-occludin, as a GST fusion
protein was prepared in non-phosphorylated form, GST-C-occlu-
din(NP), in Escherichia coli DH5a cells, and puried using GSHagarose as described before . cDNA for C-terminal tail of occludin
(amino acids 354503) was a gift from Dr. Bruce Stevenson, University
of Alberta, Edmonton, Canada.
Tyrosine phosphorylated GST-C-occludin, GST-C-occludin(pY),
was prepared in TKX1 Epicurian coli cells (Stratagene, La Jolla, CA)
by transformation with pGEX2TC-occludin. GST-C-occludin was in-
duced by IPTG (0.1mM) for 2 h, followed by induction of a non-
specic tyrosine kinase by incubating cells in tryptophan-decient
medium in the presence of indoleacetic acid for additional 2 h. GST-C-
occludin(pY) was puried from the cell lysate using GSHagarose as
described before .
Tyrosine phosphorylation of C-occludin by c-Src. GST-C-occlu-
din(NP) on GSHagarose was incubated with 80U active c-Src (Up-
state Biotechnology, Lake Placid, NY), 25lM ATP, and 18mMMnCl2 in a total volume of 200ll at 30 C for 4 h on a rocker platform.For control, similar incubation was carried in the presence of heat-
inactivated c-Src. After the incubation, agaroseGST-C-occludin was
washed in PBS containing 0.25mM sodium orthovanadate. Agarose-
GST-C-occludin(NP) incubated with active and inactivated c-Src were
used for pull down assay and actin binding assay. For actin binding
assay, GST-C-occludin(NP) and GST-C-occludin(pY) were eluted
from GSHbeads with GSH elution buer and washed free of GSH by
3 exchanges of PBS using Microcon (10 kDa cut-o) centri-lter tubes
(Millipore, Bedford, MA).
In vitro transcription and translation. ZO-1 cDNA cloned in ex-
pression vector, pBluescript SK+ with T7 promoter upstream of
coding sequence, was transcribed and translated in vitro in a coupled
system of T7-rabbit reticulocyte lysate system using TNT Quick cou-
pled Transcription/Translation System kit (Promega, Madison, WI).
Human ZO-1 cDNA was a kind gift from Dr. Anderson (Yale Uni-
versity, New Haven, CT). Prior to transcription the cloned DNA was
puried from 1% agarose gel as a single band and 0.5 lg DNA wasused for translation in TNT Quick coupled master-mix. The newly
synthesized protein was separated from un-incorporated amino acids
using gel ltration column and conrmed by immunoblot analysis.
ZO-1 protein fractions were used for C-occludin binding assays.
Pull down assay for occludin binding of ZO-1, ZO-2, and ZO-3. Cell
extracts were prepared from Caco-2 cells, obtained for American Type
Cell Culture, grown on plastic dishes in DMEM containing 10% fetal
bovine serum under standard cell culture conditions. Conuent cell
monolayers were washed three times with PBS. Cells were lysed in
0.2% Triton X-100 in PBS containing 1mM sodium orthovanadate,
100mM sodium uoride, and 10mM sodium pyrophosphate (2ml/
plate). Cell lysates were centrifuged at 3000g for 15min and superna-
tant was used for pull down assay. Cell lysate (0.6ml) was incubated
with 320lg GST-C-occludin(NP) or GST-C-occludin(pY) and 20llGSHagarose at 4 C for 16 h on an inverter. Agarose beads werewashed three times with PBS and proteins were extracted by heating at
100 C for 5min in 25ll Laemmlis sample buer. The amounts of ZO-1, ZO-2, and ZO-3 present in these samples were determined by im-
Actin binding assay. Actin binding of occludin was determined
using the actin binding kit (Cytoskeleton, Denver, CO) according to
the vendors instructions with minor modications. Briey, 210lgGST-C-occludin(NP) or GST-C-occludin(pY) in 10 ll actin buer wasincubated with 40 ll of 23 lM in vitro polymerized F-actin for 30minat 24 C. F-actin was pelletted down by centrifugation at 100,000g for1.5 h at 24 C. Pellet and supernatant were mixed with Laemmlissample buer and the distribution of occludin in these fractions was
determined by immunoblot analysis.
Immunoblot analysis. Proteins in dierent samples were separated
by SDSolyacrylamide gel (412% gradient) electrophoresis and elec-
tro-blotted into PVDF membranes. Membranes were probed for ZO-1,
ZO-2, ZO-3, or occludin using rabbit polyclonal anti-ZO-1, anti-ZO-3,
and mouse monoclonal anti-ZO-2 and anti-occludin antibodies. Blots
were developed using ECL chemiluminescence kit (Amersham, Ar-
lington Heights, IL). Densitometric analysis of immunoblots was
performed using a high-resolution scanner (UMAX powerlook III,
Umax Technologies, Dallas, TX) and UN-SCAN-IT densitometry
software (Silk Scientic Incorporated, Orem, UT).
Chemicals. Cell culture media and related reagents were purchased
from Gibco-BRL (Grand Island, NY). IPTG, ampicillin, kanamycin,
tetracycline, glutathioneagarose, reduced glutathione, Triton X-100,
Tween 20, ATP, indoleacetic acid, aprotinin, leupeptin, bestatin, and
PMSF were from Sigma Chemical (St. Louis, MO). All other
Fig. 1. Preparation of recombinant GST-C-occludin. (A) Non-phos-
phorylated GST-C-Occludin, GST-C-Occludin(NP) was expressed in
E. coli DH5a cells and tyrosine phosphorylated GST-C-Occludin(pY)was prepared in TKX1 Epicurian coli which expresses a tyrosine ki-
nase. Puried, GST-C-Occludin(NP) and GST-C-Occludin(pY) were
immunoblotted for occludin and phosphotyrosine. Results show the
dierence in tyrosine phosphorylation. (B) Same as in A; GST-C-Oc-
cludin(NP) was incubated with active c-Src or heat-inactivated c-Src as
described in Materials and methods and immunoblotted for occludin,
phosphotyrosine, and c-Src.
G. Kale et al. / Biochemical and Biophysical Research Communications 302 (2003) 324329 325
chemicals were of analytical grade purchased either from Sigma
Chemical, or Fisher Scientic (Tustin, CA).
Tyrosine phosphorylation of C-terminal tail of occludin
Recombinant non-phosphorylated GST-C-occlu-
din(NP) was puried as described before . Tyrosine
phosphorylated GST-C-occludin(pY) was generated inTKX1 E. coli. Immunoblot analysis for occludin and
phosphotyrosine shows that GST-C-occludin(pY) pro-
duced in TKX1 cells, but not GST-C-occludin(NP)
produced in E. coli DH5a cells, was tyrosine phos-phorylated (Fig. 1A). In vitro incubation of GST-C-
occludin(NP) with activated c-Src, but not inactivated
c-Src, resulted in the tyrosine phosphorylation of C-occludin (Fig. 1B). Active c-Src was also found to be
associated with the resulting GST-C-occludin(pY)
Tyrosine phosphorylation prevents the binding of C-
occludin to ZO-1, ZO-2, and ZO-3
Since it has been shown that recombinant C-occludin
binds to ZO-1, ZO-2, and ZO-3 , we compared GST-C-occludin(pY) with GST-C-occludin(NP) for binding
to ZO-1, ZO-2, and ZO-3 using pull down assay. Incu-
bation of GST-C-occludin(NP) with Triton-soluble
protein extracts from Caco-2 cells co-precipitated ZO-1,
ZO-2, and ZO-3 in a concentration-dependent manner
(Figs. 2A and B). As a control, incubation with GST did
not precipitate ZO-1 (Fig. 2C). The amounts of ZO-1,
ZO-2, and ZO-3 binding to GST-C-occludin(pY) wereseveral fold lower than the amounts co-precipitated with
GST-C-occludin(NP) (Figs. 2A and B). Binding of ZO-
1, prepared by in vitro transcription and translation, to
GST-C-occludin(pY) was also several fold lower than
that bound to GST-C-occludin(NP) (Fig. 2D). Our re-
Fig. 2. Eect of tyrosine phosphorylation on occludin binding to ZO
proteins. (A) Varying concentrations of GST-C-Occludin(NP) and
GST-C-Occludin(PY) were incubated with Triton-soluble protein ex-
tracts from Caco-2 cells. Pulled down beads were examined for the
presence of ZO-1, ZO-2, and ZO-3 by immunoblot analysis. (B)
Densitometric analysis of ZO-1, ZO-2, and ZO-3 bound to GST-C-
Occludin(NP) and GST-C-Occludin(PY) in A. Values (arbitrary den-
sity units) are average of three independent experiments. (C) GST or
GST-C-Occludin(NP) was incubated with Triton-soluble protein ex-
tracts from Caco-2 cells and precipitated with GSHagarose as de-
scribed in Materials and methods. Pulled down beads were assessed for
ZO-1 by immunoblot analysis. (D) Full length ZO-1 was produced by
in vitro transcription/translation. Varying amounts (515ll of trans-lation assay mixture) were incubated with 10 lg GST-C-Occludin(NP)or GST-C-Occludin(pY). ZO-1 bound to occludins was detected by
Fig. 3. Tyrosine phosphorylation of occludin by c-Src reduces the
binding to ZO proteins. (A) GST-C-occludin(NP) was phosphorylated
on tyrosine residues by incubation with active c-Src as described in Fig.
1B. Varying concentrations of GST-C-Occludin(NP) that was prein-
cubated with active or inactive c-Src were incubated with Triton-sol-
uble protein extracts from Caco-2 cells and precipitated with
GSHagarose. Pulled down beads were assessed for ZO-1, ZO-2, and
ZO-3 by immunoblot analysis. (B) Densitometric analysis of ZO-1,
ZO-2, and ZO-3 bound to GST-C-Occludin(NP) and GST-C-Occlu-
din(PY) in A. Values (arbitrary density units) are average of values
from two independent experiments.
326 G. Kale et al. / Biochemical and Biophysical Research Communications 302 (2003) 324329
cent study showed that c-Src is involved in the oxidativestress-induced tyrosine phosphorylation of TJ-proteins
and the disruption of TJ. Therefore, we tyrosine phos-
phorylated GST-C-occludin(NP) by incubation with
active c-Src and evaluated its binding of ZO-1, ZO-2,
and ZO-3. The binding of ZO-1, ZO-2, and ZO-3 to
GST-C-occludin was reduced upon phosphorylation by
active c-Src (Figs. 3A and B) compared to those bound
to GST-C-occludin(NP) incubated with inactive c-Src.
Tyrosine phosphorylation does not aect the binding of
C-occludin to F-actin
C-terminal tail of occludin binds to F-actin (Fig. 4A).
This binding has been reported to be important for the
assembly of TJ [21,22]. There was no signicant dier-
ence between the amounts of GST-C-occludin(NP) and
GST-C-occludin(pY) bound to F-actin (Figs. 4A and
B). Albumin and GST did not bind F-actin, and servedas negative controls. On the other hand, a-actinin, awell-known actin binding protein, that bound F-actin
(Fig. 4C) was used as a positive control. Similar to the
recombinant GST-C-occludin(pY), the actin binding of
GST-C-occludin(pY), that was phosphorylated by
active c-Src, was also similar to the actin binding of
GST-C-occludin(NP) (Figs. 5A and B).
It has been demonstrated that signaling pathways
involving both tyrosine phosphorylation  and
serine/threonine phosphorylation  regulate TJ
permeability in dierent epithelia. Tyrosine kinase ac-
tivity plays a crucial role in the regulation of both theassembly  and the disassembly  of TJ.
Evidence suggests that there is a correlation between
tyrosine phosphorylation of occludin and ZO-1, and the
disruption of TJ. However, there is no evidence avail-
able to show an eect of phosphorylation on direct
interactions between occludin, ZO-1, ZO-2, ZO-3, and
F-actin. The present study provides evidence for the rst
time that tyrosine phosphorylation of C-terminal tail ofoccludin results in a reduced interaction with ZO-1, ZO-
2, and ZO-3, suggesting that tyrosine phosphorylation
Fig. 4. Eect of tyrosine phosphorylation occludin on actin binding.
(A) Three micrograms of GST-C-Occludin(NP) or GST-C-Occlu-
din(pY) was incubated without or with F-actin as described in Mate-
rials and methods. C-occludin present in F-actin pellet (P) and
supernatant (S) was determined by immunoblot analysis. (B) Varying
concentrations of GST-C-Occludin(NP) and GST-Occludin(pY) were
incubated with F-actin, and occludin distribution in F-actin pellet (P)
and supernatant (S) was determined by immunoblot analysis. Occludin
in P and S were evaluated by densitometric analysis and occludin bound
to P was calculated as percent of total occludin [P=P S 100].Values are average of values from three independent experiments. (C)
GST, bovine serum albumin (BSA) and a-actinin, 10 lg each, wereincubated with F-actin to determine control actin bindings. Binding of
GST and BSA was used as negative controls, and the binding of a-actinin as a positive control.
Fig. 5. Eect of c-Src-mediated tyrosine phosphorylation of C-occlu-
din on actin binding. (A) GST-C-Occludin(NP) was phosphorylated
on tyrosine residues by incubation with active c-Src as described in Fig.
1B. GST-C-Occludin(NP) (10lg) preincubated with active c-Src orinactive c-Src was incubated with F-actin as described in the Methods.
C-occludin present in F-actin pellet (P) and supernatant (S) was de-
termined by immunoblot analysis. (B) Occludin in P and S in the above
blot (Fig. 5A) was evaluated by densitometric analysis and the occlu-
din bound to P was calculated as percent of total occludin
[P=P S 100]. Values are average of values from two independentexperiments.
G. Kale et al. / Biochemical and Biophysical Research Communications 302 (2003) 324329 327
of occludin C-terminus may prevent the assembly ordestabilize the TJ. In previous studies we showed that
tyrosine kinase inhibitors prevented oxidative stress-
and acetaldehyde-induced disruption of TJ .
Disruption of TJ by oxidative stress was associated with
tyrosine phosphorylation of TJ-proteins and dissocia-
tion of occludin-ZO-1 complex in the Caco-2 cell
monolayer . The present study suggests that tyrosine
phosphorylation of occludin may be involved in themechanism of oxidative stress-induced disruption of TJ
in Caco-2 cells.
In a recent study we demonstrated that c-Src plays a
crucial role in oxidative stress-induced disruption of the
TJ and increase in paracellular permeability in Caco-2
cell monolayers. It is therefore suggestive of a role of c-
Src in tyrosine phosphorylation of occludin and
disruption of TJ. The present study shows that the C-terminal tail of occludin binds c-Src and phosphorylates
occludin on tyrosine residues. Tyrosine phosphorylation
induced by c-Src also resulted in reduced anity of
occludin for binding to ZO-1, ZO-2, and ZO-3. It was
previously shown that c-Src and c-Yes are localized at
the TJ , thus suggesting a possible role for Src family
kinases in the regulation of TJ. Recent reports suggest
that c-Yes interacts with occludin  and is likely toplay a role in the assembly of TJ . However, the
present study demonstrates a direct interaction of c-Src
with C-occludin and prevents the assembly of TJ.
Therefore, the role of Src family kinases in the assembly
or disassembly of TJ may be determined by the specic
isoform of Src kinases involved.
The binding of ZO-1, ZO-2, and ZO-3 to the in-
tracellular C-terminal tail of occludin anchors the TJprotein complex to the actin cytoskeleton [4,5]. A re-
cent study demonstrated that C-occludin also interacts
directly with F-actin . The present study shows that
tyrosine phosphorylation does not aect this interac-
tion of C-occludin with F-actin. It is possible that the
interaction between occludin and F-actin may be
regulated by phosphorylation of occludin on Sr/Thr
residues. It was shown that occludin is hyperphosph-orylated on Ser/Thr residues  and that hyper-
phosphorylated occludin is predominantly associated
with the actin cytoskeleton [23,24], suggesting that
phosphorylation of occludin on Ser/Thr residues is es-
sential for its interaction with F-actin. In support of
this view studies also show that disruption of TJ is
associated with the dephosphorylation of occludin on
Ser/Thr residues and its release from the actin cyto-skeleton .
In summary, the present study demonstrates that
tyrosine phosphorylation of occludin results in the at-
tenuation of its interaction with ZO-1, ZO-2, and ZO-3,
without aecting its interaction with F-actin. Our study
also suggests that c-Src is likely to be involved in the
tyrosine phosphorylation of occludin in vivo. Further
studies using this approach may advance our under-standing of the regulation of interactions among dier-
ent TJ-associated proteins and their regulation by
intracellular signaling pathways.
This study was supported by Grants from National Institute of
Diabetes, Digestive, and Kidney Diseases, R01-DK55532, and Na-
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Tyrosine phosphorylation of occludin attenuates its interactions with ZO-1, ZO-2, and ZO-3Materials and methodsResultsTyrosine phosphorylation of C-terminal tail of occludinTyrosine phosphorylation prevents the binding of C-occludin to ZO-1, ZO-2, and ZO-3Tyrosine phosphorylation does not affect the binding of C-occludin to F-actin