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

    Abstract

    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 [1], claudins

    [2], and junction adhesion molecule [3], 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 [1]. 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

    [57]. 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 [8]. Expression of occludin mutants lack-

    ing the C-terminal tail results in the disruption of TJ [9].

    A signicant body of evidence indicates that the intra-

    cellular C-terminal tail interacts with ZO-1, ZO-2, ZO-3,

    and F-actin [57]. 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 [10].

    Tyrosine kinase activity is essential for both the dis-

    assembly [1115] and the assembly [1618] 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 [14] 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 [15]. These studies suggested that tyrosine

    Biochemical and Biophysical Research Communications 302 (2003) 324329

    www.elsevier.com/locate/ybbrc

    BBRC

    * Corresponding author. Fax: 1-901-448-7126.

    E-mail address: rkrao@physio1.utmem.edu (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 [5]. 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 [5].

    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-

    munoblot analysis.

    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).

    Results

    Tyrosine phosphorylation of C-terminal tail of occludin

    Recombinant non-phosphorylated GST-C-occlu-

    din(NP) was puried as described before [5]. 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)

    (Fig. 1B).

    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 [5], 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

    immunoblot analysis.

    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).

    Discussion

    It has been demonstrated that signaling pathways

    involving both tyrosine phosphorylation [1115] and

    serine/threonine phosphorylation [2327] regulate TJ

    permeability in dierent epithelia. Tyrosine kinase ac-

    tivity plays a crucial role in the regulation of both theassembly [1618] and the disassembly [1115] 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 [1115].

    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 [15]. 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 [1], thus suggesting a possible role for Src family

    kinases in the regulation of TJ. Recent reports suggest

    that c-Yes interacts with occludin [10] and is likely toplay a role in the assembly of TJ [17]. 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 [5]. 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 [24] 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 [2527].

    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.

    Acknowledgments

    This study was supported by Grants from National Institute of

    Diabetes, Digestive, and Kidney Diseases, R01-DK55532, and Na-

    tional Institute for Alcoholism and Alcohol Abuse, R01-AA12307.

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    G. Kale et al. / Biochemical and Biophysical Research Communications 302 (2003) 324329 329

    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

    DiscussionAcknowledgementsReferences

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