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¹ Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390
² Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390
³ Institute of Medical Biochemistry, Division of Molecular Biology, Vienna Biocenter, A-1030 Vienna, Austria

Address correspondence to Estelle Sontag, Dept. of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9073. Tel.: (214) 648-2327. Fax: (214) 648-2077. E-mail: Estelle.Sontag{at}UTSouthwestern.edu

	Abstract

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Tight junctions (TJs) play a crucial role in the establishment of cell polarity and regulation of paracellular permeability in epithelia. Here, we show that upon calcium-induced junction biogenesis in Madin-Darby canine kidney cells, ABC, a major protein phosphatase (PP)2A holoenzyme, is recruited to the apical membrane where it interacts with the TJ complex. Enhanced PP2A activity induces dephosphorylation of the TJ proteins, ZO-1, occludin, and claudin-1, and is associated with increased paracellular permeability. Expression of PP2A catalytic subunit severely prevents TJ assembly. Conversely, inhibition of PP2A by okadaic acid promotes the phosphorylation and recruitment of ZO-1, occludin, and claudin-1 to the TJ during junctional biogenesis. PP2A negatively regulates TJ assembly without appreciably affecting the organization of F-actin and E-cadherin. Significantly, inhibition of atypical PKC (aPKC) blocks the calcium- and serum-independent membrane redistribution of TJ proteins induced by okadaic acid. Indeed, PP2A associates with and critically regulates the activity and distribution of aPKC during TJ formation. Thus, we provide the first evidence for calcium-dependent targeting of PP2A in epithelial cells, we identify PP2A as the first serine/threonine phosphatase associated with the multiprotein TJ complex, and we unveil a novel role for PP2A in the regulation of epithelial aPKC and TJ assembly and function.

Key Words: PP2A; aPKC; ZO-1; occludin; claudin

	Introduction

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Tight junctions (TJs)^* serve as occluding barriers, maintain polarity and homeostasis, and regulate the permeability characteristics of the paracellular space in epithelia (Mitic et al., 2000). ZO-1, a member of the MAGUK family of proteins, acts as a scaffold to organize transmembrane TJ proteins and recruit various signaling molecules and the actin cytoskeleton to TJs (Gonzalez-Mariscal et al., 2000). Occludin binds to ZO-1 and regulates TJ paracellular permeability (Furuse et al., 1993; Wong and Gumbiner, 1997). Several lines of evidence point to proteins of the claudin family as key components of TJ fibrils and the primary seal-forming proteins responsible for mediating TJ's physiological barrier (Tsukita and Furuse, 2000). Although TJs serve as organizing centers for numerous other proteins involved in trafficking and signaling (Mitic et al., 2000), little is known about the molecular mechanisms involved in the dynamic regulation of these multiprotein complexes. Translocation of occludin and ZO-1 from the cytoplasm to the membrane during Ca²⁺-induced TJ biogenesis is accompanied by their phosphorylation (Stuart and Nigam, 1995; Wong, 1997; Sakakibara et al., 1997; Farshori and Kachar, 1999). However, so far, how dephosphorylation events are involved in the structural/functional regulation of TJs remains obscure.

Protein phosphatase (PP)2A enzymes are major Ser/Thr protein phosphatases. The core enzyme is a dimer containing a catalytic subunit (C) and a regulatory subunit (A), which can associate to a regulatory subunit (B). Several families of B subunits have been identified and modulate PP2A catalytic activity and substrate specificity (Sontag, 2001). Distinct B subunits contribute to targeting PP2A to defined intracellular domains and recruiting PP2A to signaling complexes, thereby ensuring its functional specificity (Sim and Scott, 1999; Sontag, 2001). Notably, the holoenzyme containing the B subunit is a major PP2A isoform involved in cell growth and cytoskeletal regulation in numerous cell types (Sontag, 2001). Here, we chose to undertake a detailed analysis of ABC behavior in epithelial cells, a cell type for which PP2A properties and functions are poorly documented. We show that ABC is targeted to the TJ complex and identify a novel role for PP2A in TJ regulation.

	Results

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Ca²⁺-dependent recruitment of ABC to regions of cell–cell contact
In Madin-Darby canine kidney (MDCK) cells, Ca²⁺ depletion from the culture medium results in disruption of intercellular junctions and cell rounding; conversely, the formation of functional junctional complexes can be triggered upon transferring cells cultured in low Ca²⁺ (LC) medium to normal Ca²⁺ (NC) medium (Gonzalez-Mariscal et al., 1990; Cereijido et al., 2000). Because the B subunit is always complexed to the AC core enzyme (Sontag, 2001), immunofluorescent microscopy and immunoblotting with anti-B antibodies were used to assess the behavior of ABC during Ca²⁺ switch experiments in confluent MDCK cells. Ca²⁺ deprivation of cells resulted in progressive cell retraction and disappearance of the peripheral membrane staining for ABC (Fig. 1 A). When cells were Ca²⁺ starved overnight and then switched to NC medium to induce junction biogenesis, a pool of ABC gradually reconcentrated at cell–cell contact sites (Fig. 1 B). Significant amounts of ABC copurified with the membrane fraction from cells cultured in NC medium, whereas most of the holoenzyme was present in the cytosolic fraction from cells transferred to LC medium (Fig. 1 C). The proportion of membrane-bound ABC increased considerably during junction biogenesis and remained unchanged 24 h after the Ca²⁺ switch, at which time TJs were completely reformed (Fig. 1 D).

View larger version (114K): [in this window] [in a new window]   Figure 1. Ca2+-dependent membrane localization of ABC in MDCK cells. (A) Confluent MDCK cells grown on glass coverslips in NC medium were switched for 5 min, 30 min, or 2 h to LC medium containing 1 mM EGTA to induce rapid TJ disassembly (NC to LC), and then processed for indirect immunofluorescence with rabbit anti-B 237 antibody. (B) MDCK cells were Ca2+ starved overnight then switched to NC medium to induce TJ biogenesis (LC to NC). Cells were fixed 15 min, 45 min, and 5 h after initiation of cell–cell contact and analyzed as described in A. Bars, 10 µm. (C) MDCK cells cultured in NC medium (NC) were switched overnight to LC medium (LC). Equivalent amounts of proteins from cytosolic (Cy) and membrane (M) fractions were analyzed by immunoblotting with anti-B antibodies. (D) MDCK cells were switched from LC to NC medium for 0, 5, or 24 h. Equivalent amounts of proteins from cytosolic (Cy) and membrane (M) fractions were analyzed by immunoblotting for the presence of PP2A B and C subunits.

Ca²⁺-dependent interaction of ABC with the TJ complex

View larger version (129K): [in this window] [in a new window]   Figure 2. Analysis of the distribution of ABC at junctional complexes in polarized MDCK cells and in human colon. (A–C) Analysis of polarized cells by confocal microscopy. Arrows in x-z images indicate the thickness of the monolayers. Bars, 10 µm. (A) Representative apical x-y sections and transversal x-z views obtained in MDCK cells labeled with either 237 (1), P9 (2), or 2G9 (3) anti-B antibodies, and in MDCK-B cells labeled with anti-HA antibody (anti-HA). (B) Representative x-z sections of ZO-1, occludin, claudin-1, or E-cadherin staining in MDCK cells. (C) MDCK cells were double labeled with rabbit anti-B and either rat anti–ZO-1 or mouse anti–E-cadherin antibodies. Representative apical (for B/ZO-1) and basolateral (for B/E-cadherin) x-y sections, and corresponding x-z views are shown. (D) Immunogold labeling was performed with the anti-B 237 antibody (diluted 1:80) on frozen ultrathin sections obtained from a normal human adult colon tissue biopsy. Note the concentration of gold particles in the TJ region beneath the lumen of the microvilli. Similar results were obtained with the P9 antibody; no TJ labeling was obtained with corresponding preimmune sera (not depicted). Bar, 150 nm. D, desmosome.

View larger version (72K): [in this window] [in a new window]   Figure 3. Ca2+-dependent association of ABC with membrane-associated TJ proteins. (A) Immunoblots of total cell lysates prepared from MDCK, MDCK-Wt C, or MDCK-B cells and immunoprecipitated with anti–HA-coupled affinity matrix. (B) Immunoblots of detergent-insoluble fractions prepared from MDCK cells and immunoprecipitated with either anti–ZO-1, –occludin, –claudin-1, or –E-cadherin antibodies (+), or no antibody (-). (C) Immunoblots of homogenates prepared from rat colon or kidney tissue and immunoprecipitated with (+) or without (-) anti-occludin antibody. (D) Immunoblots of cytosolic (Cy) and membrane (M) fractions prepared from MDCK, MDCK-B, and MDCK-Wt C cells cultured in NC medium (NC) then switched overnight to LC medium (LC), and immunoprecipitated with either anti–ZO-1 or anti–HA antibodies.

View larger version (60K): [in this window] [in a new window]   Figure 4. Effects of deregulating PP2A activity on the phosphorylation state of TJ proteins and TJ barrier properties of MDCK cells cultured in NC medium. (A–E) Confluent cells were grown in NC medium. (A) Polarized MDCK-Wt C cells were analyzed by confocal microscopy with anti-HA antibody for the distribution of expressed Wt C subunit. Representative apical x-y and x-z views are shown. Bar, 10 µm; arrow, thickness of the cell monolayer. (B) ZO-1, occludin, and claudin-1 were immunoprecipitated from membrane fractions prepared from control MDCK cells (lanes 1 and 4), MDCK cells incubated for 1 h with 200 nM OA before harvesting (lanes 2 and 5), or stable MDCK-Wt C cells (lanes 3 and 6), before (lanes 1–3) or after (lanes 4–6) preincubation with sodium butyrate. The samples were analyzed by SDS-PAGE and immunoblotting with anti-phosphoserine antibody. The blots were reprobed with anti–ZO-1, occludin, or claudin-1 antibodies. In parallel, aliquots of membrane fractions from OA-treated cells were left untreated (lane 7) or treated (lane 8) with alkaline phosphatase, and analyzed by immunoblotting with anti–ZO-1, occludin, or claudin-1 antibodies. Note that the upward band shift caused by OA results from phosphorylation, as it can be abolished after alkaline phosphatase treament. Arrows and brackets (]*) indicate the position of dephosphorylated and phosphorylated TJ protein species, respectively. (C) TER was measured in control MDCK (1–2) and MDCK-Wt C cells expressing distinct levels of transfected Wt C subunit (3–5), before and after preincubation with sodium butyrate. Subsets of cells were incubated for 1 h with (+ OA) or without (- OA) 100 nM OA. Results are expressed as the mean TER ± SD of duplicate determinations performed in four separate experiments with monolayers of equivalent cell density. The representative blot in the bottom panel shows the relative amounts of expressed Wt C subunits detected with anti-HA antibody in total extracts prepared from the cell lines analyzed for TER. (D) Paracellular diffusion of [3H]-mannitol and [3H]-inulin was determined in identical subsets of MDCK-Wt C monolayers treated with or without 100 nM OA, before and after preincubation with sodium butyrate. Results are expressed as percent of the relative tracer flux measured in untreated cells before addition of sodium butyrate, and are the mean ± SD of triplicate determinations performed in four distinct stable MDCK-Wt C populations. (E) Paracellular diffusion of [3H]-mannitol and [3H]-inulin was determined in MDCK cells incubated with increasing concentrations of OA. Results are expressed as percent of the relative tracer flux measured in untreated cells, and are the mean ± SD of triplicate determinations from six separate experiments.

Inhibition of PP2A promotes TJ protein phosphorylation and TJ assembly
To explore the potential role of PP2A in TJ assembly, the distribution of ZO-1, occludin, and claudin-1 was compared by immunofluorescent microscopy and immunoblotting during Ca²⁺ switch experiments performed in untreated or OA-treated control MDCK cells, and in MDCK-Wt C cells (Figs. 5 and 6). Cells were Ca²⁺-starved overnight to induce TJ downregulation, resulting in internalization/redistribution of TJ proteins from the cell periphery to the cytosol, and then transferred to NC medium to induce TJ biogenesis. The Ca²⁺ switch initiates a rapid sorting of TJ proteins from the cytosol to the membrane; however, complete TJ stabilization and resealing, as measured by TER restoration, is only achieved >20 h after the calcium switch (Stuart and Nigam, 1995; Farshori and Kachar, 1999). 1 h after the Ca²⁺ switch, a portion of total TJ proteins had already migrated to the cell periphery in control MDCK cells, but this redistribution was largely inhibited after expression of Wt C (Fig. 5 A). The peripheral membrane staining for TJ proteins appeared more continuous after incubation of MDCK cells with OA. The critical role played by PP2A during early TJ biogenesis was even more clearly demonstrated during Ca²⁺ switch experiments performed in the absence of serum (Fig. 5 B). OA promoted, whereas expression of Wt C severely prevented the accumulation of TJ proteins at cell-cell contact sites. The inhibitory effect of expressed Wt C could be partially reversed by OA, reinforcing the idea that PP2A negatively regulates the initial sorting of TJ proteins to the membrane. 4 h after the Ca²⁺ switch, a complete junctional distribution of ZO-1 was achieved over the entire circumference of MDCK cells (Fig. 6 A). OA accelerated the formation of the TJ network. Expression of Wt C noticeably delayed the accumulation of TJ proteins at junctional areas, as revealed by the predominant cytosolic concentration and fragmentary staining of ZO-1 at cell–cell boundaries. 24 h after the Ca²⁺ switch and thereafter, all the cells displayed the typical chicken wire staining pattern of ZO-1. However, cytosolic pools of TJ proteins were still visible at that stage in MDCK-Wt C cells. Significantly, in contrast to TJ proteins, changes in PP2A activity did not affect the redistribution of E-cadherin to areas of cell–cell contact during AJ formation.

View larger version (85K): [in this window] [in a new window]   Figure 5. Effects of deregulating PP2A activity on the initial redistribution of TJ proteins during Ca2+-induced TJ biogenesis. Control MDCK or MDCK-Wt C cell monolayers were incubated overnight in LC medium, then switched for 1 h to NC medium (A) or for 2 h to DC medium (B), in the absence or presence of 100 nM OA. Cells were analyzed by immunofluorescence for the distribution of ZO-1, occludin, and claudin-1. Bars, 10 µM.

View larger version (81K): [in this window] [in a new window]   Figure 6. Effects of deregulating PP2A activity on the distribution, detergent solubility, and phosphorylation state of junctional proteins, and development of TER during junctional assembly. (A) Control MDCK or MDCK-Wt C cell monolayers were switched for 4 or 24 h from LC to NC medium, and analyzed by immunofluorescence for the distribution of ZO-1 or E-cadherin. Subsets of control cells were incubated for 1 h with 100 nM OA before staining. The distribution of ZO-1 was comparable to those of occludin and claudin-1 (not depicted). Bars, 10 µM. (B) Control MDCK or MDCK-Wt C cells were switched for 5 or 24 h from LC to NC medium. Control cells were incubated for 2 h with (OA) or without (-) 100 nM OA before harvesting. Equivalent amounts of proteins (20 µg) from detergent-soluble (S) and -insoluble (I) fractions were prepared from the cells and simultaneously analyzed by Western blotting for the presence of ZO-1, occludin, claudin-1, or E-cadherin. Arrows indicate the position of dephosphorylated proteins. (C) Polarized control MDCK and MDCK-Wt C cells were incubated for 90 min in LC medium containing 1 mM EGTA to induce TJ opening (t = 0; TER < 30 .cm2), then switched to NC or DC medium for the indicated time. Results are expressed as the mean TER ± SD of duplicate determinations from seven separate experiments performed with seven distinct stable cell populations.

TJ assembly also correlates with the development of TER. When MDCK-Wt C cells were switched from LC to NC medium to induce TJ resealing, they developed TER with much slower kinetics than control cells (Fig. 6 C). This delay in TER development was exacerbated when the Ca²⁺ switch was performed under serum-free conditions (LC to DC), validating our hypothesis that PP2A activity negatively regulates TJ assembly.

Effects of PP2A deregulation on F-actin
The complete reestablishment of the actin cytoskeleton architecture plays a crucial role during Ca²⁺-mediated junctional biogenesis, and the contraction of perijunctional F-actin critically regulates the TJ permeability barrier (Denker and Nigam, 1998). Thus, we addressed the hypothesis that PP2A regulates TJs via F-actin remodeling. As shown in Fig. 7, we could not observe any significant difference in the organization of F-actin in MDCK cells subjected to a Ca²⁺ switch, whether they were treated with OA, or transiently or stably expressing HA-tagged Wt C. Notably, subconfluent MDCK cells expressing high levels of transfected Wt C subunits exhibited a flattened, irregular shape with occasional lamellipodia, that was somewhat reminiscent of the morphology of cells expressing dominant-negative mutants of atypical protein kinase C (aPKC) (Suzuki et al., 2001). Overall, OA treatment or Wt C expression did not dramatically affect the appearance of the cortical F-actin bundles (see "apical" sections) and stress fibers (see "basal" sections) present in confluent cells cultured in NC medium. However, now and then, the pattern of stress fibers appeared interwoven or less dense in confluent MDCK-Wt C cells.

View larger version (75K): [in this window] [in a new window]   Figure 7. Effects of PP2A deregulation on the organization of F-actin. (A) Immunofluorescent localization of F-actin in control MDCK or MDCK-Wt C cells switched for 2 h from LC to NC medium or cultured in NC medium. Subsets of control cells were incubated for 1 h with 100 nM OA before staining. (B) MDCK cells were transiently transfected with the pcDNA3.1 vector encoding HA-tagged Wt C, and seeded at confluency on coverslips 24 h posttransfection. After plating, cells were either incubated overnight in LC medium then switched to DC or NC medium for the indicated time, or cultured in NC medium. Cells were double stained for the HA epitope and F-actin. For A and B, representative apical and basal x-y sections are shown for cells cultured in NC medium. Bars, 10 µM.

View larger version (77K): [in this window] [in a new window]   Figure 8. aPKC-dependent regulation of TJ proteins by PP2A. (A) In vitro PKC-phosphorylated TJ proteins were incubated with 100 nM ABC, 100 nM ABC plus 1 µM OA, or the phosphorylation buffer alone. The samples were resolved by SDS-PAGE and analyzed by autoradiography. Only major radioactive bands corresponding to PKC-phosphorylated ZO-1, occludin, and claudin-1 could be detected on the autoradiographs. (B) Immunofluorescent localization of TJ proteins in confluent MDCK cells incubated overnight in LC medium, or switched for 2 h from LC to DC medium. Subsets of cells were preincubated for 1 h in the absence or presence of 20 µM myristoylated PKC/ pseudosubstrate (ZI), then incubated for 1 h with 50 nM OA before staining. Bars, 10 µM. (C) Confluent MDCK cells were switched for 1 h from LC to DC medium containing or not 50 µM ZI, then incubated for 2 h with or without 100 nM OA. Equivalent amounts of proteins from detergent-insoluble fractions were prepared from the cells and analyzed by Western blotting for the presence of TJ proteins.

View larger version (108K): [in this window] [in a new window]   Figure 9. PP2A associates with and regulates aPKC. (A) Representative apical x-y sections of polarized MDCK cells double labeled with rabbit anti-B and rat anti-PKC antibodies. Bar, 10 µM. (B) Representative immunoblots of MDCK, MDCK-B, and MDCK-Wt C cell extracts immunoprecipitated with anti-HA antibody, of MDCK cell detergent-insoluble fractions immunoprecipitated with anti-PKC (+) or no (-) antibody, and of cytosolic (Cy) and membrane (M) fractions prepared from MDCK-Wt C or MDCK-B cells cultured in NC medium (NC) or LC medium (LC), and immunoprecipitated with anti-HA antibody. IgG, immunoglobulins. (C) Control and MDCK-Wt C cells were incubated overnight in LC medium and switched for the indicated time to DC or NC medium. Subsets of control cells were incubated for 2 h in the absence or presence of 20 µM ZI, then for 1 h with 50 nM OA before fixation and staining with rat anti-PKC antibody. Bars, 10 µM. (D) Control and MDCK-Wt C cells were switched from LC to DC medium in the absence or presence of 100 nM OA. PKC immunoprecipitates were prepared from membrane fractions 2 h after the Ca2+ switch and assayed for aPKC kinase activity. aPKC activity was completely abolished when kinase assays were performed in the presence of 10 µM ZI (not depicted). For (A–D), similar results were obtained when using either mouse anti-PKC antibody or rabbit anti-PKC (C-20) antibody, which recognizes both PKC and PKC (not depicted). (E) Control MDCK or MDCK-Wt C cells were switched for 1 h to DC medium with or without 50 µM ZI, then incubated for another 2 h with or without 100 nM OA. Detergent-insoluble fractions were prepared from the cells and equivalent amounts of proteins (100 µg) were analyzed by immunoblotting for the presence of phosphorylated PKC using anti–p-nPKC (Thr410) antibody. A duplicate aliquot of OA-treated cell fraction was incubated with alkaline phosphatase (+ AP). Note the disappearance of the signal for phosphorylated PKC after AP treatment.

	Discusssion

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So far, the evidence for a role for Ser/Thr phosphatases in TJ regulation is fragmentary, and derives entirely from studies utilizing OA and calyculin A, which inhibit PP2A/PP1 enzymes in vivo in a dose- and time-dependent manner. Interestingly, these inhibitors are naturally occurring toxins that modulate intestinal paracellular permeability and are responsible for diarrheic shellfish poisoning in humans (Tripuraneni et al., 1997; Okada et al., 2000). As reported previously in other epithelial cells (Singer et al., 1994; Tripuraneni et al., 1997; Okada et al., 2000), we observed that high concentrations of OA or prolonged incubation of MDCK cells with this inhibitor induced cell rounding and TJ leakiness. Under these conditions, not only PP2A but also PP1 enzymes became inhibited, and a direct role for PP2A in TJ regulation cannot be meaningfully evaluated. Instead, lower OA concentrations more selective for PP2A did not appreciably alter the organization of the TJ network and the resistance and paracellular permeability of MDCK cells grown in NC medium, in agreement with previous studies (Pasdar et al., 1995; Tripuraneni et al., 1997; Okada et al., 2000). At first sight, this may suggest that PP2A does not regulate TJs once formed. However, enhanced PP2A activity induced dephosphorylation of membrane-associated TJ proteins, leading to decreased TER and increased TJ permeability. Based on previous studies pointing to an important role for occludin dephosphorylation in promoting TJ opening (Farshori and Kachar, 1999; Clarke et al., 2000; Simonovic et al., 2000), reduced levels of TJ-associated, phosphorylated occludin may contribute in part to enhanced TJ leakiness in MDCK-Wt C cells. The fact that OA can inhibit the effects of expressed Wt C suggests that PP2A-dependent changes in the phosphorylation state of TJ-bound proteins modulate the dynamic opening/closing of mature TJs. Many signaling molecules mediate changes in TJ barrier properties via remodeling of the actin cytoskeleton (Denker and Nigam, 1998). Yet, we were unable to demonstrate that changes in PP2A activity induce clear-cut effects on F-actin organization under our experimental conditions. Likewise, OA does not significantly affect F-actin distribution in carcinoma cells (Strnad et al., 2001). However, the effects of OA on F-actin are dose- and time-dependent (Leira et al., 2001); high OA concentrations consistent with PP1 inhibition disrupt F-actin (Fiorentini et al., 1996).

OA promoted the phosphorylation and recruitment of TJ proteins to TJs during junctional assembly. Reciprocally, expressed Wt C induced the accumulation of soluble, dephosphorylated forms of TJ proteins, which correlated with a severe delay in the sorting of TJ proteins from the cytosol to TJs, and retardation in TER development. The observation that OA induces the membrane translocation of TJ proteins in LC medium and reverses the effects of Wt C in the absence of serum is consistent with the hypothesis that inhibition of PP2A promotes TJ assembly. Thus, the phosphorylation state of TJ proteins is controlled by PP2A and is closely linked to their ability to redistribute to the membrane during junctional biogenesis. It has been proposed that the nucleation of AJ assembly by cadherin/catenin complexes controls TJ assembly (Gumbiner et al., 1988). Although PP2A C subunit associates with and stabilizes the ß-catenin/E-cadherin complex in immature blastocysts (Gotz et al., 2000), we were unable to colocalize or coimmunoprecipitate ABC and E-cadherin in polarized MDCK cells. The accumulation of E-cadherin at regions of cell–cell contact also proceeded normally despite PP2A deregulation, suggesting that PP2A-dependent defects in TJ assembly do not occur secondary to abnormalities in AJ formation. Together with the observations that AB56C, but not ABC, regulates ß-catenin in the Wnt signaling pathway (Li et al., 2001), and that ABC, but not AB56C, associates with TJ proteins, our findings suggest that the regulation of TJs by PP2A likely involves an E-cadherin–independent pathway (Stuart and Nigam, 1995) controlled by ABC. However, the hierarchical regulation of junctional complexes is not absolute but rather multifaceted, as inhibition of cadherin adhesion can exert both negative and positive effects on TJ biogenesis in MDCK cells (Troxell et al., 2000). It is thus possible that distinct PP2A holoenzymes differentially affect separate signaling pathways that converge on TJs.

The results from our inhibitor studies indicate that aPKC participates in the regulation of ZO-1, occludin, and claudin-1 by PP2A. OA induced a 2.7-fold activation of membrane-bound aPKC, in agreement with the finding that membrane-associated PKC activity more than doubles during TJ assembly (Stuart and Nigam, 1995). Our conclusions support those of previous reports showing the importance of aPKC-mediated TJ protein phosphorylation for Ca²⁺-induced TJ assembly (Stuart and Nigam, 1995; Suzuki et al., 2001). PP2A associated with both soluble and membrane-bound aPKC and modulated aPKC activity and distribution. Thus, PP2A may be part of and directly regulate the aPKC/Par-3 signaling complex that is involved in regulation of TJ formation and cell polarity. The aPKC/Par-3 complex is tethered to TJs via JAM. It is noteworthy that, as with Wt C, overexpression of a JAM mutant disrupts the localization of aPKC and ZO-1 without significantly affecting the localization of E-cadherin (Ebnet et al., 2001).

In conclusion, we demonstrate that ABC is recruited to and is a novel component and regulator of the TJ signaling complex. The regulation of TJs by PP2A is dependent on functional aPKC. We establish PP2A as a novel regulator of aPKC and identify the TJ proteins, ZO-1, occludin, and claudin-1, as targets of PP2A/aPKC signaling in epithelial cells.

	Materials and methods

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Plasmids and antibodies
The pcDNA3.1 vector encoding HA-tagged Wt C has been described previously (Ogris et al., 1997; Goedert et al., 2000). The pcDNA3.1 vector (Invitrogen) encoding HA-tagged B was prepared from pcDNA1/Amp-HA-B (Yu et al., 2001) via NdeI/XhoI restriction sites. Antibodies included: anti-HA (Covance); anti-ZO-1 (Chemicon International, Inc.); anti–claudin-1, -occludin, and -phosphoSer (Zymed Laboratories, Inc.); anti-C, anti–E-cadherin, and anti-PKC (Transduction Laboratories); anti-PKC (Alexis Corporation); anti-PKC (C-20), anti–p-nPKC (Thr410) (Santa Cruz Biotechnology, Inc.); anti-PP1 (Calbiochem); anti-B56 subunit (Tehrani et al., 1996); and affinity-purified FITC- and Texas red–coupled secondary antibodies (Jackson ImmunoResearch Laboratories). The rabbit anti-B 237 antibody was raised against a fusion protein containing a 6 x His tag fused to amino acids 238–447 of the rat B subunit. The rabbit anti-B P9 and monoclonal 2G9 antibodies were raised against a synthetic peptide corresponding to amino acids 398–411 of the rat B sequence. Antibodies raised against B may crossreact with the ß, , or isoforms of the B subunit family, but expression of Bß and B is restricted to testis and/or brain, and B is a cytosolic subunit almost undetectable in kidney (for review see Sontag, 2001).

Cell culture and characterization of stable MDCK cell lines
All experiments were performed in the highly polarized MDCK strain II D5 clonal cell line (Brewer and Roth, 1991). Cells were maintained on plastic dishes in DME (Gibco BRL) containing 10% FBS (Hyclone). For most experiments, cells were plated at high density on Transwell filters (Costar) and cultured until complete polarization, as verified by measuring TER. Cells were transfected using Lipofectamine Plus reagent (Gibco BRL). Stable clones were obtained after selection with 800 µg/ml geneticin (Gibco BRL), analyzed for expression of the transfectant, and pooled together to minimize the effects of clonal variations. Seven separate pooled populations of stable transfectants were utilized throughout our studies. The expression level of transfected proteins was constantly monitored by immunofluorescence and immunoblotting. Immunoblotting indicated that HA-tagged Wt C subunit was expressed at 30–50% of the levels of endogenous C in stable MDCK cell lines, as previously reported in NIH 3T3 cells (Ogris et al., 1997). HA-tagged B subunits were expressed at 20% of the levels of endogenous B. Stable MDCK cells transfected with the pcDNA3.1 vector were used as controls (control MDCK cells) and behaved like nontransfected cells in our experiments. Sodium butyrate (2.5–5 mM; Sigma-Aldrich) was added in the culture medium for 16 h before each experiment to enhance expression of the transfected proteins, and had no detrimental effects on any of the parameters measured in our studies, as reported previously (Brewer and Roth, 1991).

Calcium switch experiments
To induce rapid TJ disassembly, monolayers of MDCK cells grown in normal Ca²⁺ (NC) medium (DMEM + 10% FBS; 1.8 mM Ca²⁺) were incubated in LC medium (Ca²⁺-free S-MEM; Gibco BRL) containing 1 mM EGTA. For prolonged Ca²⁺ removal, cells were incubated overnight in LC medium containing 1% dialyzed FBS. For the Ca²⁺ switch, Ca²⁺-starved cells were transferred to either NC or DC (DMEM + 1% dialyzed FBS) medium. If indicated, OA (Alexis Biochemicals) or the vehicle (100% DMSO) was added to the medium during the Ca²⁺ switch.

Transepithelial resistance measurements
Cells were plated at confluency and grown on sixwell Transwell filters in NC medium. TER values were measured in duplicate wells using an Endohm^TM voltohmmeter (World Precision Instruments). TER values (.cm²) were normalized to the area of the monolayer (filter), and calculated by subtracting the blank values (18 .cm²) from the filter and the bathing medium. All cell culture media were supplemented with 25 mM Hepes, pH 7.4, and the integrity and cell density of the monolayers were carefully monitored during TER measurement studies.

Paracellular diffusion measurement
Cells were grown on 6.5-mm Transwell filters until complete polarization. TJ leakiness was assessed by measuring the diffusion of [³H]-inulin (Amersham Biosciences) and [³H]-mannitol (NEN^TM Life Science Products, Inc.) across the membrane in monolayers of equivalent cell density (Wong and Gumbiner, 1997). The tracer diffusion was examined by replacing the apical compartment medium by fresh NC medium containing the tracer (2.5 µCi/ml), and the basal compartment medium with the same medium without tracer. When indicated, OA or the vehicle (DMSO) was added in both basal and apical bathing media. The compartment media were collected 60 min afterwards, and counted by liquid scintillation.

Confocal microscopy
Cells grown on Transwell filters or on glass coverslips were fixed with methanol for 5 min at -20°C, and labeled sequentially for 1 h with primary and secondary antibodies at a 1:100 dilution (Sontag et al., 1995). For the visualization of F-actin, cells were fixed for 20 min with 4% paraformaldehyde, permeabilized for 5 min with 0.1% Triton X-100, and labeled for 30 min with FITC-labeled phalloidin (Sigma-Aldrich). The samples were mounted using Fluoromount-G (Fisher) and examined on a Leica TCS SP confocal microscope using a 63x objective. Images (16 x-y or x-z sections across cells) were directly captured, saved and transferred to Adobe Photoshop^® 5.5 for printing. The specificity of the labeling was verified by omitting first or second antibodies during the staining procedures, and by using pre-immune sera and antibodies that had been preadsorbed with the corresponding antigen.

Electron microscopy
Cryosections (80 nm) from normal human adult colon fixed in 1% glutaraldehyde/4% paraformaldehyde were picked up using a 1.8-mm loop with a droplet of frozen 2.3 M sucrose, thawed, moved onto Formvar-covered and carbon-coated glow-discharged 100 mesh copper grids and stored on buffer. Immunogold labeling of the cryosections was performed by the Tokuyasu method (Tokuyasu, 1986) by which the grids are moved through series of droplets containing blocking agents, antibodies and are ultimately coated with a layer of 0.2% uranyl acetate in 2% methylcellulose. The cryosections were incubated for 30 min each with serial dilutions of the primary antibody in PBS containing 5% milk, and then with protein A gold (10 nm diameter; 1:70 dilution; a gift from G. Posthuma, Utrecht University Medical Center, The Netherlands). The cryosections were examined using a JEOL 1200EX Transmission Electron Microscope operating at 120kV. Electron micrographs were taken using Eastman Kodak SO-163 electron image film.

Cell fractionation and Western blotting
To prepare detergent-soluble and -insoluble fractions, confluent cells were washed in PBS, harvested in 400 µl buffer 1 (25 mM Tris, 150 mM NaCl, 1% NP-40, 4 mM EDTA, 25 mM sodium fluoride, 1 mM sodium orthovanadate, 1 µM OA, pH 7.4) containing a cocktail of protease inhibitors (Roche) and incubated for 30 min at 4°C. The extracts were then centrifuged for 30 min at 4°C in a microfuge to yield a soluble fraction (supernatant), after which the remaining insoluble fraction (pellet) was harvested in the same buffer, sonicated to disrupt protein aggregates, then recentrifuged to eliminate insoluble material. To prepare membrane fractions, cells were washed in PBS and incubated for 5 min at room temperature in buffer S (0.25 M sucrose, 1 mM imidazole, 5 mM MgCl₂). The medium was aspirated and cells were harvested in buffer S containing 1 µM OA, 1 mM DTT and a cocktail of protease inhibitors, incubated on ice for 15 min then dounce homogenized. Cells were centrifuged at 800 g at 4°C to pellet nuclei, after which the supernatant was centrifuged at 4°C for 45 min at 100,000 g in a Beckman TL-100.3 rotor. The supernatant (cytosol) was collected and the remaining pellet (membrane fraction) was resuspended in buffer 1 and sonicated. Equivalent aliquots of proteins from the cell fractions determined using Bio-Rad protein assay kit were resolved by SDS-PAGE on 5% (for ZO-1), 8% (for occludin, E-cadherin, and aPKC) and 12% (for PP2A subunits and claudin-1) polyacrylamide gels, and analyzed by immunoblotting for the presence of PP2A subunits or junctional proteins. Immunoreactive proteins were detected using SuperSignal Chemiluminescence substrates (Pierce Chemical Co.).

Immunoprecipitation
Cytosolic/membrane fractions were normalized for protein concentration and volume, after which the buffer was adjusted to 150 mM NaCl and 1% NP-40. After preclearing, total, detergent-soluble/insoluble, or cytosolic/membrane fractions were incubated overnight at 4°C with either the indicated antibodies (7 µl antibody/ml cell extract), or no antibody to assess nonspecific binding. The immunoprecipitates were collected using either protein A Sepharose or G PLUS-agarose beads (Santa Cruz Biotechnology), washed extensively in buffer 1, and resuspended in Laemmli sample buffer. In some experiments, the immunoprecipitations were carried out with HA-tagged antibody-coupled affinity matrix (Covance) and control nontransfected cells were used in parallel assays to verify the specificity of the immunoprecipitations (Goedert et al., 2000). Equivalent aliquots of the immunoprecipitates were analyzed by SDS-PAGE on 4–15% gradient Ready gels (Bio-Rad Laboratories) and transferred to nitrocellulose. The blots were cut into strips and simultaneously immunoblotted with antibodies directed against PP2A subunits, PP1, and junctional proteins to allow for comparative analysis of the relative amounts of immunoprecipitated material in each condition. In other experiments, the colon and kidney from a 10-wk old rat was minced, homogenized in buffer 1 with a tissue grinder and centrifuged at 13,000 g for 15 min to remove insoluble material. Aliquots of the supernatants were immunoprecipitated with mouse anti-occludin antibody and analysed as described above.

Analysis of TJ protein phosphorylation
MDCK cell detergent-insoluble fractions were immunoprecipitated as described above with anti–ZO-1, -occludin, or -claudin-1 antibodies. The immunoprecipitates were washed and resuspended in P buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl₂, 0.5 mM EGTA, 1 mM DTT, 1 mM sodium fluoride, 100 µM sodium orthovanadate). Phosphorylation of TJ proteins was performed for 1 h at 30°C by adding 10 µg phosphatidylserine, a gift from P. Sternweis (UT Southwestern, Dallas TX), 1 µg recombinant human aPKC (Calbiochem), and 100 µM [-³²P]ATP (5 µCi) per reaction. The tubes containing phosphorylated TJ proteins were transferred on ice and carefully divided into 15-µl aliquots. Either 100 nM purified ABC (Goedert et al., 2000), 100 nM ABC preincubated on ice for 20 min with 1 µM OA, or buffer alone, were added into the reaction mixtures. The samples (25 µl) were incubated for another 30 min at 30°C, after which the reactions were terminated by addition of 3x sample buffer for SDS-PAGE. The samples were boiled for 5 min then simultaneously analyzed by SDS-PAGE on 4-20% gradient gels (Bio-Rad Laboratories), followed by autoradiography. In other experiments, ZO-1, occludin, and claudin-1 were immunoprecipitated from membrane fractions, resolved by SDS-PAGE and analysed by immunoblotting using rabbit anti-phosphoserine antibody. The blots were reprobed with anti-ZO-1, occludin, and claudin-1 antibodies. Phosphorylation of PKC was examined by immunoblotting using p-nPKC (Thr410) antibody (Standaert et al., 1999a). In parallel, aliquots of cell fractions were resuspended in AP buffer (50 mM Tris, pH. 7.4, 50 mM NaCl, 1 mM MgCl₂, 1 mM DTT, 0.1% NP-40, and cocktail of protease inhibitors) and incubated for 1 h at 30°C with or without alkaline phosphatase (20 U/sample; Roche) before being analyzed by immunoblotting.

aPKC activity assays
aPKC activity was measured in immunoprecipitates as described previously (Standaert et al., 1999a). After washing in P buffer, aPKC immunoprecipitates were incubated for 15 min at 30°C with 50 µl of P buffer containing 50 µM ATP, 1 µCi [-³²P]ATP, and 5 µg serine analogue of PKC-pseudosubstrate (BioSource International), a selective aPKC substrate. The reactions were performed in the presence or absence of 10 µM PKC/ pseudosubstrate (BioSource International), as described previously (Sontag et al., 1997). The reaction mixtures were spotted onto P-81 phosphocellulose paper and washed with 75 mM phosphoric acid. Incorporation of ³²P was determined by liquid scintillation counting.

PP2A activity assays
Aliquots of cell extracts were assayed for PP2A activity for 6 min at 30°C using the RRREEE(pT)EEE phosphopeptide (Biosynthesis, Inc.) as a substrate. Dephosphorylation of the peptide was determined by measuring the release of P_i using a quantitative colorimetric assay (Sontag et al., 1999).

	Footnotes

 
T. Machleidt's present address is RW Johnson Pharmaceutical Research Institute, La Jolla, CA 92121.

^* Abbreviations used in this paper: AJ, adherens junction; aPKC, atypical PKC; JAM, junctional-adhesion molecule; LC, low Ca²⁺; MDCK, Madin-Darby canine kidney; NC, normal Ca²⁺; OA, okadaic acid; PP, protein phosphatase; TER, transepithelial resistance; TJ, tight junction; ZI, PKC/ pseudosubstrate.

	Acknowledgments

 
We thank Dr. M. Roth (Uiversity of Texas Southwestern Medical Center, Dallas, TX) for MDCK cells and technical advice, Dr. D. Pallas (Emory University School of Medicine, Atlanta, GA) for PP2A plasmids, Dr. E. Bigio (University of Texas Southwestern Medical Center) for tissue samples, Dr. C. Kamibayashi (Uiversity of Texas Southwestern Medical Center) for anti-B56 antibodies, and Sivapong Satumtira for technical assistance.

E. Ogris serves as a consultant to Upstate Biotechnology, Inc. This work was supported by National Institutes of Health grant AG18883 to E. Sontag, and a grant from the Austrian Science Foundation (FWF, P13707-MOB) to E. Ogris.

Submitted: 26 June 2002

Revised: 22 July 2002

Accepted: 22 July 2002

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