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AMP-activated Protein Kinase (AMPK) Activation andGlycogen Synthase Kinase-3� (GSK-3�) Inhibition InduceCa2�-independent Deposition of Tight Junction Componentsat the Plasma Membrane*□S �

Received for publication, September 20, 2010, and in revised form, February 21, 2011 Published, JBC Papers in Press, March 7, 2011, DOI 10.1074/jbc.M110.186932

Li Zhang‡, Francois Jouret§1, Jesse Rinehart¶, Jeff Sfakianos‡�, Ira Mellman‡�, Richard P. Lifton¶, Lawrence H. Young§**,and Michael J. Caplan‡§2

From the Departments of ‡Cell Biology, §Cellular and Molecular Physiology, and ¶Genetics and the **Section of CardiovascularMedicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520 and �GenentechInc., South San Francisco, California 94080

Extracellular Ca2� is essential for the development of stableepithelial tight junctions. We find that in the absence of extra-cellular Ca2�, AMP-activated protein kinase (AMPK) activationand glycogen synthase kinase (GSK)-3� inhibition indepen-dently induce the localization of epithelial tight junction com-ponents to the plasma membrane. The Ca2�-independent de-position of junctional proteins induced by AMPK activation andGSK-3� inhibition is independent of E-cadherin. Furthermore,the nectin-afadin system is required for the deposition of tightjunction components induced by AMPK activation, but it is notrequired for that induced by GSK-3� inhibition. Phosphoryla-tion studies demonstrate that afadin is a substrate for AMPK.These data demonstrate that two kinases involved in regulatingcell growth and metabolism act through distinct pathways toinfluence the deposition of the components of epithelial tightjunctions.

Polarized epithelial cells possess highly specialized intercel-lular junctions, including adherens junctions and tight junc-tions. These cell junctions regulate the paracellular diffusion ofsmall molecules and restrain the movement of proteinsbetween the apical and the basolateral membrane compart-ments (1). A number of studies have demonstrated that extra-cellular Ca2� is essential for both the development of new junc-tions (2–4) and the stabilization of mature junctions (5–10).The cell adhesion molecule E-cadherin (11) enriched at adher-

ens junctions (12) plays a critical role in epithelial cell adhesionand junction assembly (11, 13–17). The dependence of junctionassembly on Ca2� is likely attributable to the capacity of Ca2� tostabilize E-cadherin in its adhesive state (18). Antibodies blockingthe adhesion of E-cadherin also inhibit the formation of cell junc-tions (11, 19). Depletion of E-cadherin expression disrupts the

establishment but not the maintenance of cell junctions (20).The cytoplasmic tail of E-cadherin contains a binding site for�-catenin (21), which subsequently binds �-catenin (22, 23)and then the actin cytoskeleton (24, 25).Nectins belong to another family of cell adhesion molecules

that consists of four members (26). The intercellular interac-tions of nectinmolecules are Ca2�-independent. TheC terminiof nectins bind the PDZ domain of afadin (27), which thenbinds F-actin (28). Together, the nectins and afadin constituteanother system that organizes adherens junctions coopera-tively with the cadherin-catenin system in epithelial cells. Thenectin-afadin system can also independently support the for-mation of weak cell-cell junctions when cadherin-based celljunctions are absent (29). A recent study further demonstratedthat nectin junctions appear during the earliest stages of epithe-lial cell morphogenesis and that expression of a dominant neg-ative form of nectin causes failure of cell polarization and dis-ruption of tight junction assembly (30).Tight junction membrane proteins can be recruited to the

cadherin- or nectin-based adhesion sites via the interactionsbetween a tight junction scaffolding protein, zonula occludens(ZO)3-1, and either �-catenin (31) or afadin (32), respectively.The association between tight junction components and adhe-rens junctions mediates the deposition of tight junction pro-teins to the cell surface in the early steps in tight junctionassembly (33).Our previous study using Mardin-Darby canine kidney

(MDCK) epithelial cells revealed that AMPK plays a role in theregulation of epithelial tight junction assembly (34). AMPK is aserine-threonine kinase involved in the regulation of cell metabo-lism. Its activity increases with increasing AMP:ATP ratios (35).ElevatedAMP levels renderAMPKsusceptible to activatingphos-phorylation by upstream kinases such as LKB1 (36–38).Activation of LKB1 induces a series of epithelial cell polar-

ization events, including the deposition of junctional proteinsto a belt surrounding an apical like region. These events appear

* This work was supported, in whole or in part, by National Institutes of HealthGrants DK17433 and 072614.

� This article was selected as a Paper of the Week.□S The on-line version of this article (available at http://www.jbc.org) contains

supplemental Figs. S1–S6 and Table S1.1 Fellow of the Belgian American Educational Foundation.2 To whom correspondence should be addressed: Dept. of Cellular and

Molecular Physiology, Yale University School of Medicine, P. O. Box208026, New Haven, CT 06520-8026. Tel.: 203-785-7316; Fax: 203-785-4951; E-mail: [email protected].

3 The abbreviations used are: ZO, zonula occludens; AICAR, aminoimidazolecarboxamide ribonucleotide; AMPK, AMP-activated protein kinase; GSK,glycogen synthase kinase; HCM, high calcium medium; LCM, low calciummedium; MDCK, Mardin-Darby canine kidney; MRLC, myosin regulatorylight chain; SILAC, stable isotope labeling by amino acids in cell culture;MEM, minimal essential medium; MRLC, myosin regulatory light chain.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 19, pp. 16879 –16890, May 13, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

MAY 13, 2011 • VOLUME 286 • NUMBER 19 JOURNAL OF BIOLOGICAL CHEMISTRY 16879

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to be independent of cell-cell contact and thus of intercellularinteractions of adhesion (39). Interestingly, two recent studiesfound that localized activation of LKB1 represents an early sig-nal for axon initiation in undifferentiated neurons (40, 41),which constitutes a critical step in neuronal polarization (42).The signal initiated by LKB1 activation induces inactivation ofGSK-3�, and this event appears to be essential for axon elonga-tion (43). GSK-3� is a serine-threonine kinase found ubiqui-tously in eukaryotes (44, 45), which plays important roles inmany biological processes, ranging from the Wnt signalingpathway to astrocyte migration (46, 47).In this study, we report for the first time that GSK-3� is also

involved in regulating the deposition of epithelial cell junctioncomponents. We determined that AMPK activation andGSK-3� inhibition exert their effects on Ca2�-independentdeposition of tight junction proteins via independent pathways.We also identified afadin as a substrate for AMPK. Thus, ourstudy demonstrates that alterations in the activities of twokinase pathways can lead independently to the deposition oftight junction components without the participation of extra-cellular Ca2� or of the Ca2�-dependent epithelial cell adhesionmachinery.

EXPERIMENTAL PROCEDURES

Plasmids and Construct—pSUPER plasmids containingshort hairpin interfering RNA sequences targeting canineE-cadherin, cadherin-6, and luciferase (served as a transfec-tion control) were kindly provided by Dr. Ian Macara, Uni-versity of Virginia. Detailed information about the prepara-tion of these constructs was reported previously (20). For theshRNA construct targeting afadin, the E-cadherin RNAisequences in the pSUPER plasmid were substituted by afa-din RNAi sequences 5�-GCATGGATGCTGAGACTTA-3�(clone 2) and 5�-GACAATCCTGCTGTCTACC-3� (clone5). Details of afadin shRNA construct preparation are avail-able upon request.Antibodies—Sheep anti-�1-AMPK was kindly provided by

Dr. D. Grahame Hardie (University of Dundee). Rabbit anti-PATJ and rabbit anti-PAR3 were kindly provided by Dr. BenMargolis (University of Michigan). Rabbit anti-pACC (Ser79),anti-pan-�-AMPK, anti-pAMPK (Thr172), and anti-p-�-catenin (Ser33/Ser37/Thr41) were purchased from Cell Signal-ing. Rabbit anti-ZO-1, anti-occludin, and anti-claudin-1 werepurchased from Zymed Laboratories Inc.. Mouse anti-E-cad-herin clone 36 was purchased from BD Biosciences. Rhoda-mine-conjugated goat anti-rabbit IgG was purchased fromChemicon. Alexa Fluor 488-conjugated goat anti-mouse IgGand Alexa Fluor 488-conjugated goat anti-rabbit IgG werepurchased from Molecular Probes. HRP-conjugated goat anti-mouse IgG andHRP-conjugated goat anti-rabbit IgGwere pur-chased from Jackson ImmunoResearch. Rabbit anti-human-1-afadin and HRP-conjugated donkey anti-sheep IgG werepurchased from Sigma. All commercially available antibodieswere used according to the manufacturers’ instructions.Cell Culture and Transfection—MDCK cells were main-

tained in �-MEM (Invitrogen) supplemented with 10% fetalbovine serum (Invitrogen), 2 mM L-glutamine (Invitrogen), 50units/ml penicillin (Invitrogen), and 50 �g/ml streptomycin

(Invitrogen). Cells were grown in a humidified incubator at37 °C and 5% CO2 atmosphere.

To transiently transfect pSUPER plasmids encoding shRNAsequences targeting E-cadherin, cadherin-6, afadin, and lucif-erase into MDCK cells, an Amaxa nucleofector was utilized.The detailed protocols for transfecting MDCK cells using anAmaxa device have been previously reported (48).Establishment of AMPK Knockdown Stable Cell Lines—Se-

quences targeting the canineAMPK�1 subunitwere subclonedinto pLH1, a derivative of the pSUPER plasmid. Plasmids weretransfected with pVSVG into HEK-G2 cells for lentivirus pack-aging. Subconfluent MDCK cells were then infected with theresultant lentivirus. Selection and maintenance of stableMDCK cell clones were performed in �-MEM containing 4�g/ml puromycin (Sigma). Clones were screened for reducedexpression levels of AMPK�1 subunit byWestern blot analysis.An empty pLH1 plasmid was also packaged into lentivirus, anda pooled control MDCK cell line was established by infectionwith this lentivirus. The sequence chosen for targeting AMPK�1 subunit was 5�-GCAGAAGTTTGTAGGGCAATT-3�.Calcium Switch—MDCK cells were grown on plastic (for

Western blot analysis) or coverslips (for immunofluorescence)in �-MEM containing 1.8 mM calcium (high calcium medium,HCM) until they formed a confluent monolayer. Cells werethen washed twice with minimum essential medium for sus-pension culture (S-MEM, Invitrogen) and incubated in S-MEMsupplementedwith 5% dialyzed fetal bovine serum (Invitrogen)(low calciummedium, LCM). Cells were incubated in LCM for16 h before being switched back to HCM for the indicatedtimes.Immunofluorescence and Quantification of ZO-1 Staining—

Cells on coverslips were washed three times with cold PBS andfixed in 100% methanol at �20 °C for 5 min. Cells were thenpermeabilized in 0.3% Triton X-100, 0.15% BSA (permeabiliza-tion buffer) in PBS for 15min at room temperature and blockedin goat serum dilution buffer (GSDB, 16% goat serum (Invitro-gen), 20 mM sodium phosphate, pH 7.4, 450 mM NaCl, 0.3%TritonX-100) for 30min at room temperature. Cells were incu-bated in primary antibody diluted 1:200 in GSDB for 1 h atroom temperature, then immersed three times in permeabili-zation buffer, and then incubated for 1 h in secondary antibodydiluted 1:200 in GSDB. Cells were then rinsed three times inPBS before mounting in Vectashield (Vector Laboratories).Cells were visualized on a Zeiss Axiophot fluorescence micro-scope equippedwith aZeissAxioCamHRmCCDcamera. Con-trast and brightness settings were chosen so that all pixels werein the linear range. Pictures were taken in the focal plane inwhich the most strands of tight junction components werevisualized.To quantify the mean ZO-1 length per cell, four fields were

randomly selected from each coverslip, and the total length ofZO-1 at the cell periphery in each field was manually outlined,followed by length measurement with ImageJ software. Cellnumbers were counted for each field, and the mean ZO-1length per cell was calculated. Statistical analysis was per-formed using the two-tailed Student’s t test. In each case, thedata presented are representative of at least two or three inde-pendent experimental repetitions.

Calcium-independent Junction Deposition

16880 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 19 • MAY 13, 2011

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Western Blot—Cells were lysed on ice in kinase lysis buffer(250 mM sucrose, 20 mM Tris-HCl, pH 7.4, 50 mMNaCl, 50 mM

NaF, 5 mM sodium pyrophosphate, 1 mM Na3VO4, 2 mM freshDTT, 1% Triton X-100) for 30 min. Cell lysate was centrifugedat 15,000 rpm at 4 °C for 10 min. Supernatant was then col-lected for Western blot. Proteins were resolved with 8% SDS-PAGE using standard protocols. The protein was electropho-retically transferred to nitrocellulose membranes (Bio-Rad)and blocked with milk solution (150 mM NaCl, 20 mM Tris, 5%milk (w/v), 0.1% Tween (v/v), pH 7.5) to quench nonspecificprotein binding. The blocked membranes were probed withprimary and secondary antibodies diluted in the milk solution,and the bands were visualized with the enhanced chemilumi-nescence kit (Amersham Biosciences).AMPK in Vitro Phosphorylation Assay—Each AMPK in vitro

phosphorylation assay included a pre-determined amount ofimmunoprecipitated proteins of interest immobilized on pro-tein A-agarose beads (no more than 20 �l of beads), 1 �Ci of[�-32P]ATP (freshly purchased from PerkinElmer Life Sci-ences), 50 �MATP (Sigma), 100 �MAMP (Sigma), and 50 ng ofrecombinant AMPK �1�1�2 protein complex (Cell Signaling).The assaymixtures were then incubated for 15min at 37 °C in asolution containing 5 mM MOPS, pH 7.2, 2.5 mM �-glycero-phosphate, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, and 50�M DTT. The reactions were terminated by incubating mix-tures at 100 °C for 5 min. The proteins of interest were theneluted from the beads by incubating with SDS sample buffer at60 °C for 5 min. The proteins were separated by SDS-PAGE,after which the gel was dried, and the radioactive signals on thegel were revealed by autoradiography.SILAC Labeling and LC-MS/MS Analysis—MDCK cell lines

were passaged at 10% confluence onto 10-cm plates in 10 ml ofheavy (0.1 mg/ml L-lysine-13C6 and 0.025 mg/ml L-arginine-13C6

15N4) or light (normal) SILAC media. Cells were grown toconfluence and then replated at 10% confluence, still inheavy or light medium, with cell numbers normalizedbetween heavy and light conditions. After treatments, heavyand light cell lysates were prepared as a 1:1 mixture accord-ing to protein concentration, and native afadin was thenimmunoprecipitated.Afadin proteins purified via immunoprecipitation and SDS-

PAGE were subjected to in-gel tryptic digestion. Followingdigestion, total afadin peptides were subjected to titaniumdioxide (TiO2) enrichment to separate phosphopeptide(enriched) and nonphosphopeptide (flow-through) fractionsfor LC-MS/MS analysis.Protein identification and SILAC quantitation were batch-

processed usingMascot Daemon (version 2.2.107, �) andMas-cot Distiller (version 2.3.0.0) from Matrix Science. Data basesearches were conducted using our in-house Mascot Server(version 2.3.0), which has the latest quantitation Toolboxupdate.

RESULTS

Inhibition of GSK-3� Induces Ca2�-independent Depositionof Junction Components—In the MDCK epithelial model sys-tem, low concentrations of extracellular Ca2� disrupt intercel-lular junctions (49), and the restoration of high Ca2� concen-

trations induces the deposition of junction proteins to theplasma membrane. This manipulation is referred to as a “Ca2�

switch” (49). We first sought to determine whether the activityof GSK-3� influences this process.We cultured MDCK cells to confluency in high Ca2�

medium (1.8 mM Ca2�, HCM) and then incubated them in lowCa2� medium (5 �MCa2�, LCM) for 16 h. At 30 min and 1 and2 h after the reintroduction of HCM, cells were lysed in thepresence of phosphatase inhibitors, followed by aWestern blotanalysis. To examine the activity of GSK-3�, we used an anti-body specifically recognizing �-catenin phosphorylated on res-idues Ser33/Ser37/Thr41. These residues were demonstrated tobe phosphorylated by GSK-3� (50). Thus, the extent of theirphosphorylation indirectly reflects the in situ level of GSK-3�activity. We found that, despite a constant level of total�-catenin, the levels of phosphorylated�-cateninwere reducedin cells lysed after the addition of HCMas compared with thosedetected in cells maintained in LCM, suggesting decreasedGSK-3� activity during Ca2�-induced epithelial polarization(Fig. 1A). To determine whether the decreased levels of�-catenin phosphorylation were attributable instead to theactivation of phosphatases during the calcium switch, wetreatedMDCK cells subjected to a calcium switch with okadaicacid, which inhibits a broad spectrum of phosphatases. Wefound that despite an overall increase in �-catenin phosphory-lation, the levels of phosphorylated�-cateninwere still reduced incells during the calcium switch (supplemental Fig. S1A), sug-gesting that the activation of phosphatases did not cause thedecrease in �-catenin phosphorylation. GSK-3� was reportedto be inhibited by Ser9 phosphorylation (51).We did not detectany change in Ser9 phosphorylation during the Ca2� switchusing an antibody recognizingGSK-3�phosphorylated on Ser9,suggesting that Ser9 phosphorylation is not responsible forGSK-3� inhibition during this process (Fig. 1A).To inhibit GSK-3� pharmacologically, we treated MDCK

cells with theGSK-3� inhibitors (52) 10�MSB216763 or 20mM

LiCl for 1 h and found that both treatments decreased�-catenin phosphorylation compared with the treatments withDMSO vehicle control or with 20 mM NaCl, which reproducesthe osmolarity and tonicity effects of LiCl (Fig. 1B). These dataconfirm that SB216763 and LiCl effectively inhibit GSK-3� inthe MDCK cell system. We then investigated the effects ofGSK-3� inhibition on the deposition of tight junction proteinsby examining the time course of the localization of ZO-1 duringCa2� switch in the presence or absence of GSK-3� inhibitors,and we found that ZO-1 deposition is significantly acceleratedin the cells incubated in the presence of SB216763 or LiCl, com-paredwith those incubated inDMSOor inNaCl (supplementalFig. S1,B andC), suggesting that GSK-3� inhibition acceleratesthe deposition of junction proteins during Ca2�-induced junc-tion assembly.Intriguingly, we noticed that in MDCK cells pretreated with

GSK-3� inhibitors, tight junction proteins were relocalized tothe sites of cell-cell contact even before the reintroduction ofCa2�. We found that the deposition of ZO-1 occurs in LCM inthe presence of 10 �M SB216763 or 20 mM LiCl but not in thepresence of DMSO or NaCl (Fig. 1, C and D). We observedfragmentary strands of ZO-1 on plasma membranes as early as



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FIGURE 1. Inhibition of GSK-3� induces Ca2�-independent deposition of junction components. A, confluent MDCK cells were incubated in LCM for 16 h andthen switched back to HCM for the indicated times. Cell lysates were probed with the indicated antibodies in a Western blot analysis. B, confluent MDCK cells weretreated with DMSO, 10 �M SB216763, 20 mM NaCl, or 20 mM LiCl for 2 h. Cell lysates were probed with the indicated antibodies in a Western blot analysis. C, confluentMDCK cells were incubated in LCM for 16 h followed by replacement of the media with fresh LCM supplemented with DMSO, 10 �M SB216763, 20 mM NaCl, or 20 mM

LiCl. Cells were fixed at the indicated time points after the replacement of medium and immunostained for ZO-1. Bar, 30 �m. D, quantification of ZO-1 relocalizationto cell membrane in C. Data are representative of three independent experiments (n � 3). Error bars represent means � S.D. ZO-1 length per cell is within each of thefour randomly picked view fields. The asterisks denote significant difference in the indicated pairs (p � 0.05) by Student’s t test. E, MDCK cells were treated as describedin C, except that they were instead immunostained for occludin. Bar, 30 �m. F, quantification of occludin relocalization to cell membrane in E. Error bars representmeans�S.D. for occludin length per cell within each of the four randomly picked view fields. The asterisks denote significant difference in the indicated pairs (p �0.05)by Student’s t test. G, confluent MDCK cells were incubated in LCM for 16 h followed by replacement of the media with fresh LCM supplemented with the indicatedconcentrations of different test reagents. Cells were fixed 2 and 10 h after the medium change and immunostained for ZO-1. Bar, 30 �m. H, quantification of ZO-1relocalization to cell membrane in G. Data are representative of two independent experiments (n �2). Error bars represent means�S.D. for ZO-1 length per cell withineach of the four randomly picked view fields. The asterisks denote significant differences in the indicated pairs (p � 0.05) by Student’s t test.



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1 h and throughout up to 4 h of SB216763 or LiCl treatment.However, the ZO-1 strands failed to becomemore consolidatedand morphologically organized with further SB216763 or LiClincubation. Instead, most of the ZO-1 strands disappeared at8 h of continued treatment. These results appear to contradictthe widely accepted notion that extracellular Ca2� is requiredto initiate the deposition of junction components (2–4). Thus,we next studied several different proteins associated with thetight junction to assess the generality of this observation. Weexamined the distribution of occludin, a membrane proteinthat is essential for the integrity of tight junctions. Similar towhat was observed with ZO-1, we found that, in cells treatedwith SB216763 or LiCl, fragmentary strands of occludin weredetectable on the cell membrane, primarily at sites of apparentcell-cell contact. Most of these occludin strands disappear afterprolonged incubation for 10 h (Fig. 1, E and F). In addition,following the treatment with SB216763 or LiCl in LCM, weobserved fragmentary strands of PATJ and PAR-3, both ofwhich are components of polarity complexes that are relocal-ized to the plasma membrane during junction assembly. Theseproteins were diffusely organized following DMSO or NaCltreatments (supplemental Fig. S1D). We also investigatedwhether or not these fragmentary junction componentsbecame tightly associated with the actin cytoskeleton. To beginto assess this possibility, wemade use of a TritonX-100 extract-ability assay (53). We separated cell lysates into Triton-solubleand Triton-insoluble fractions. This latter pool is thought toinclude proteins that are strongly associated with the cytoskel-eton. We did not detect any significant transition of the junc-tion components ZO-1, occludin, or claudin from the Triton-soluble pool into the Triton-insoluble pool, suggesting thatthese proteins had not yet become tightly associated with theactin cytoskeleton in response to 2 h ofGSK-3� inhibition (sup-plemental Fig. S1E). We did not detect any significant increasein the transepithelial electrical resistance in cells treated for 2 hwith GSK-3� inhibitors (data not shown), presumably becausethe nascent junction-related structures that we observed wereincomplete and did not produce tight seals.We next sought to determine how different concentrations

of GSK-3� inhibitors would affect the extent and the durationof the Ca2�-independent deposition of junction components.We treated MDCK cells maintained in LCM with the original(10 �M SB216763 or 20 mM LiCl) or quadrupled (40 �M

SB216763 or 80 mM LiCl) concentrations of GSK-3� inhibitorsfor both 2 and 10 h. After 2 h of treatment, we observed asignificant increase in the number and length of ZO-1 strandson plasma membranes with the higher doses of the inhibitors.After 10 h of treatment with either of these high dose GSK-3�inhibitors, substantial numbers of ZO-1 strands were stillretained on the plasma membrane, whereas most of the ZO-1strands disappeared from plasma membrane in cells treatedwith the original concentrations of GSK-3� inhibitors (Fig. 1,Gand H). We found similar results when we stained the cells foroccludin (supplemental Fig. S1, F and G), confirming thathigher concentrations of GSK-3� inhibitors enhance both theextent and the duration of the Ca2�-independent deposition ofjunction components.

Our previous study has shown thatAMPKactivation inducesCa2�-independent deposition of junction components to thesites of cell-cell contact (34). We found that treating cells withhigher concentrations of the AMPK activator, AICAR, alsoenhance both the extent and the duration of the Ca2�-inde-pendent deposition of junction components, as compared withthat detected in cells treated with lower AICAR concentrations(Fig. 1, E–H).Inhibition of GSK-3� Delays the Withdrawal of Junction

Components from the PlasmaMembrane uponCa2�Depletion—Depletion of Ca2� from the medium causes disassembly ofcell-cell junctions in polarized epithelial cells, and the junctioncomponents become diffusely distributed throughout thecytoplasm. We sought to determine whether inhibition ofGSK-3� delays this process.We pretreated confluent polarizedMDCK cells cultured in HCM with GSK-3� inhibitors (10 �M

SB216763 or 20 mM LiCl) for 1 h and then substituted HCMwith LCM, in the continued presence of these inhibitors. Cellswere fixed immediately or 4 or 10 h following the mediumchange and were then immunostained for ZO-1. After both 4and 10 h of Ca2� depletion, we observed significantly moreZO-1 organized on the plasmamembranes of cells treated withSB216763 or LiCl than on those of cells treated with DMSO orNaCl (Fig. 2). We also studied the distribution of occludin dur-

FIGURE 2. Inhibition of GSK-3� delays the withdrawal of junction pro-teins from the plasma membrane upon Ca2� depletion. A, confluentMDCK cells were incubated in HCM with DMSO, 10 �M SB216763, 20 mM NaCl,or 20 mM LiCl for 1 h before they were switched to LCM in the presence of theaforementioned test reagents. Cells were fixed 4 and 10 h after the mediumchange and immunostained for ZO-1. Bar, 30 �m. B, quantification of ZO-1 onthe cell membrane in A. Data are representative of three independent exper-iments (n � 3). Error bars represent means � S.D. for ZO-1 length per cellwithin each of the four randomly picked view fields. The asterisks denotesignificant differences in the indicated pairs (p � 0.05) by Student’s t test.



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ing these treatments, and we found that significantly moreoccludin strands were retained on the plasma membrane after10 h of Ca2� depletion when the cells were treated withSB216763 or LiCl, compared with cells treated with DMSO orNaCl (supplemental Fig. S2). Taken together, these resultsdemonstrate that GSK-3� inhibition delays the withdrawal ofjunction components from the plasma membrane when extra-cellular Ca2� is depleted.Activation of AMPK and Inhibition of GSK-3� Synergistically

Induce Ca2�-independent Deposition of Junction Componentsvia Different Pathways—Because both AMPK activation andGSK-3� inhibition induce similar effects on the deposition ofjunction components, we sought to determine whether thesetwo kinases function in the same pathway. We first examinedwhether AMPK activation leads to GSK-3� inhibition or viceversa.AMPK is activated by phosphorylation on residueThr172,and activated AMPK phosphorylates its substrate acetyl-CoAcarboxylase (35). The phosphorylation status of AMPK andacetyl-CoA carboxylase is widely used as an indicator of AMPKactivity. We treated MDCK cells with 1 mM AICAR for 2 h andfound significantly higher levels of both phosphorylatedAMPKand phosphorylated acetyl-CoA carboxylase, which confirmsthat AMPK is activated. We then examined the levels of phos-phorylated�-catenin and found no significant change, suggest-ing that GSK-3� is not inhibited by AMPK activation (Fig. 3A).We next performed a reciprocal experiment by treatingMDCKcells with 10 �M SB216763 or 20 mM LiCl for 1 h and thenexamining the levels of AMPK activity. We observed signifi-cantly lower levels of �-catenin phosphorylation but no changein the phosphorylation of either AMPK or acetyl-CoA carboxy-lase, suggesting that AMPK is not activated by GSK-3� inhibi-tion (Fig. 3A).We then sought to determine whether the effects of AMPK

activation and GSK-3� inhibition on Ca2�-independent depo-sition of junction components are synergistic. If GSK-3� inhi-bition could further promote the deposition of junction com-ponents when the effects of AMPK activation on this processare maximized, it would constitute a strong argument that theeffects of AMPK activation and GSK-3� inhibition are syner-gistic and that they are unlikely to function in the same path-way. We found that the effects of AMPK activation are satu-rated at 4 mMAICAR (supplemental Fig. S3,A and B). We thencombined GSK-3� inhibitors (10 �M SB216763 or 20 mM LiCl)with 4 mM AICAR in LCM and treated Ca2�-depleted MDCKcells for 2 h.Wewere able to detect significantlymore extensiveZO-1 or occludin strands on the plasma membrane with thecombination of GSK-3� inhibitors and AICAR than wereobserved with the DMSO or NaCl in combination with AICAR

(Fig. 3, B–D) These data indicate that GSK-3� inhibition fur-ther promotes Ca2�-independent deposition of junction com-ponents under conditions in which AMPK effects are maxi-mized, suggesting that these two molecules functionsynergistically via different pathways.If AMPK and GSK-3� employ different pathways to induce

Ca2�-independent deposition of junction components, thenelimination ofAMPKactivity should not influence the effects ofGSK-3� inhibition. To test this hypothesis, we introducedAMPK shRNA into MDCK cells and generated stable cell lines(4-2 and 4-15) in which AMPK expression is reduced by 90%comparedwith those of control cell lines (P7 and LRT) (supple-mental Fig. S3C). We found that AICAR is much less effectivein inducing Ca2�-independent deposition of junction compo-nents in AMPK knockdown cells, as evidenced by many fewerZO-1 strands relocalized to the sites of cell-cell contact thanthose observed in control cell lines. However, when we treatAMPK knockdown cells with GSK-3� inhibitors, we detectedlevels of ZO-1 deposition similar to those found in control celllines (Fig. 3, E and F). Similar results were also observed whenwe stained the cells for occludin (supplemental Fig. S3, D andE). We conclude that the effects of GSK-3� inhibition on thedeposition of junction components are not affected by reducedAMPK activity, further suggesting that AMPK activation andGSK-3� inhibition induce Ca2�-independent deposition ofjunction components via different pathways.Activation of AMPK and Inhibition of GSK-3� Induce Depo-

sition of Junction Components Independent of E-cadherin—Ep-ithelial cells adhere to their neighbors via E-cadherin, whichrequires extracellular Ca2� to stabilize it in its adhesive state.Blockade of adhesion inhibits the formation of the epithelialjunction complex (18). Because AMPK activation and GSK-3�inhibition induce deposition of junction components inde-pendent of extracellular Ca2�, we sought to determine whetherthis process is also independent of E-cadherin.To test the involvement of E-cadherin, we used shRNA con-

structs targeting both E-cadherin and the kidney-specific cad-herin-6, as well as a control construct (kindly provided by Dr.Ian Macara, University of Virginia). The efficacy of these con-structs has been previously demonstrated (20). We transientlytransfected these constructs intoMDCKcells, cultured the cellsfor 48 h in HCM, and found that more than 90% of E-cadherinwas eliminated (supplemental Fig. S4A). These cadherin knock-down cells and control cells were cultured in LCM for 16 hbefore being treated with 1 mMAICAR, 10 �M SB216763, or 20mM LiCl. We found no significant difference in the amount ofZO-1 or occludin that was relocalized to plasma membrane incadherin knockdown cells versus control cells, suggesting that

FIGURE 3. AMPK activation and GSK-3� inhibition synergistically induce Ca2�-independent deposition of junction components via different path-ways. A, MDCK cells were treated with 1 mM AICAR, DMSO, 10 �M SB216763, 20 mM NaCl, or 20 mM LiCl for 2 h. Cell lysates were probed with the indicatedantibodies in a Western blot analysis. ctrl, control. B, confluent MDCK cells were incubated in LCM for 16 h followed by the replacement of the media with freshLCM supplemented with the combination of 4 mM AICAR and DMSO, SB216763, NaCl, or LiCl. Cells were fixed 2 h after the medium change and immunostainedfor ZO-1 and occludin. Bar, 30 �m. C and D, quantification of ZO-1 and occludin relocalization, respectively, to cell membrane in B. Data are representatives oftwo independent experiments (n � 2) for each protein. Error bars represent means � S.D. for strand length per cell within each of the four randomly pickedfields of view. The asterisks denote significant differences in the indicated pairs (p � 0.05) by Student’s t test. E, confluent AMPK knockdown stable MDCK cellsand confluent control cells were incubated in LCM for 16 h followed by the replacement of the media with fresh LCM supplemented with DMSO, SB216763,NaCl, LiCl, or AICAR. Cells were fixed 2 h after the medium change and immunostained for ZO-1. Bar, 30 �m. F, quantification of ZO-1 relocalization to cellmembrane in E. Data are representative of three independent experiments (n � 3). Error bars represent means � S.D. for ZO-1 length per cell within each of thefour randomly picked view fields. p values from Student’s t test are noted in the figure.



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AMPK activation andGSK-3� inhibition induce the depositionof junction components independent of E-cadherin involve-ment (Fig. 4, A and B, and supplemental Fig. S4, B and C). Bydoubly staining cell samples with antibodies directed againstE-cadherin and ZO-1, we also noticed that ZO-1 was relocal-ized to the plasma membrane even in cells where E-cadherinexpression was barely detectable (Fig. 4C).We have demonstrated in a previous report that AMPK is

activated during extracellular Ca2�-induced junction assembly(34). Because E-cadherin is not required in Ca2�-independent

deposition of junction components induced by AMPK activa-tion, we sought to determine whether extracellular Ca2� couldinduce the deposition of junction components in cells whereE-cadherin ismissing.We transfectedMDCKcells with shRNAconstructs targeting cadherins and then subjected these cells toa Ca2� switch. We found that the introduction of extracellularCa2� induced a modest but significant relocalization of junc-tion components to the sites of cell-cell contact in cadherinknockdown cells. We compared the level of the deposition ofjunction components induced by extracellular Ca2� with that

FIGURE 4. Activation of AMPK and inhibition of GSK-3� induce deposition of junction components independent of E-cadherin. A, MDCK cells trans-fected with E-cadherin or control shRNAs were incubated in LCM for 16 h followed by replacement of the media with fresh LCM supplemented with 1 mM

AICAR, 20 mM NaCl, or 20 mM LiCl. Cells were fixed 2 h after the medium change and immunostained for ZO-1. Bar, 30 �m. Luc-ctrl, luciferase control.B, quantification of ZO-1 relocalization to cell membrane in A. Data are representative of three independent experiments (n � 3). Error bars represent means �S.D. for ZO-1 length per cell within each of the four randomly picked view fields. C, MDCK cells transfected with E-cadherin or control shRNAs were incubatedin LCM for 16 h followed by replacement of the media with fresh LCM supplemented with 20 mM LiCl. Cells were fixed 2 h after the medium change andimmunostained for ZO-1 and E-cadherin. Bar, 30 �m. D, MDCK cells transfected with E-cadherin or control shRNAs were incubated in LCM for 16 h. Medium wasreplaced by fresh LCM supplemented with AICAR or HCM. Cells were fixed 2 h after the medium change and immunostained for ZO-1. Bar, 30 �m. E, quanti-fication of ZO-1 relocalization to cell membrane in D. Data are representative of two independent experiments (n � 2). Error bars represent means � S.D. forZO-1 length per cell within each of the four randomly picked view fields. F, MDCK cells transfected with E-cadherin or control shRNAs were incubated in LCMfor 16 h and then switched back to HCM for the indicated times. Cell lysates were probed with the indicated antibodies in a Western blot analysis.



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induced by theAICAR treatment in the absence of extracellularCa2�. We found that in cells where E-cadherin is depleted,these two regimens induced comparable levels of deposition ofjunction components (Fig. 4,D andE). It is interesting to note inthis context that the Ca2� switch led to AMPK activation in theE-cadherin knockdown cells, as revealed by Western blottingfor phosphorylated AMPK (Fig. 4F). Taken together, these dataare consistent with the interpretation that the E-cadherin-in-dependent deposition of junction components stimulated byCa2� switch could be attributable to Ca2� switch-induced acti-vation of AMPK.Afadin Is Required for AMPK Activation, but Not GSK-3�

Inhibition, to Induce the Deposition of Junction Components—The nectin-afadin protein complex mediates cell adhesionindependent of the cadherin-cateninmechanism. The intercel-lular interaction between nectin molecules does not requireextracellular Ca2�. Because the deposition of junction compo-nents induced by eitherAMPK activation orGSK-3� inhibitionis independent of extracellular Ca2�, we sought to investigatewhether these processes are mediated by the nectin-afadinsystem.To assess the involvement of the nectin-afadin system, we

examined the Ca2�-independent deposition of junction com-ponents in cells lacking a functional nectin-afadin complex.Nectins are a family of membrane proteins consisting of multi-ple members, with each member existing in a variety of spliceisoforms (26). The diversity of nectins makes it difficult to effi-ciently eliminate their expression by the RNAi technique. Eachnectin isoform, however, is linked to the actin cytoskeleton viatheir interaction with afadin. Therefore, knocking down afadinfunctionally decreases the availability of the nectin-afadin sys-tem to mediate cell-cell adhesion. We used shRNA constructs

targeting afadin and the aforementioned control construct.Wetransiently transfected these constructs into MDCK cells, cul-tured these cells for 48 h in HCM, and found that about 80% ofafadin was eliminated (supplemental Fig. S5A). These afadinknockdown cells and control cells were cultured in LCM for16 h before they were treated with 1 mM AICAR, 10 �M

SB216763, or 20 mM LiCl. We found that in afadin knockdowncells, there was no significant Ca2�-independent ZO-1 oroccludin relocalization induced by AICAR treatment. In con-trol cells, however, AICAR treatment induced significantlymore relocalization of ZO-1 and occludin to the plasma mem-brane than did the vehicle control (Fig. 5 and supplemental Fig.S5, B and C). This finding suggests that the Ca2�-independentdeposition of junction components induced by AMPK activa-tion requires the nectin-afadin system. To our surprise, how-ever, in afadin knockdown cells, both SB216763 and LiCl treat-ments were still able to relocalize significantly more ZO-1 andoccludin to the sites of cell-cell contact compared with theirrespective DMSO and NaCl controls. There was no significantdifference in the quantity of ZO-1 or occludin relocalized inresponse to GSK-3� inhibition in afadin knockdown cells ver-sus control cells (Fig. 5 and supplemental Fig. S5, B and C),suggesting that GSK-3� inhibition induces deposition of junc-tion components independent of the nectin-afadin system.This observation further supports the conclusion that AMPKactivation and GSK-3� inhibition induce Ca2�-independentdeposition of junction components via different pathways.Afadin Is an AMPK Substrate—Recent studies inDrosophila

(54, 55) provide interesting clues as to howAMPK is associatedwith the polarization events in epithelial cells. (MRLC) wasdemonstrated to be phosphorylated byAMPKboth inDrosoph-ila and in human cells. In Drosophila lacking AMPK expres-

FIGURE 5. Afadin is required to induce the deposition of junction components via AMPK activation but not via GSK-3� inhibition. A, MDCK cellstransfected with afadin or control shRNAs were incubated in LCM for 16 h followed by replacement of the media with fresh LCM supplemented with 1 mM

AICAR, DMSO, 10 �M SB216763, 20 mM NaCl, or 20 mM LiCl. Luc-ctrl, luciferase control. Cells were fixed 2 h after the medium change and immunostained forZO-1. Bar, 30 �m. B, quantification of ZO-1 relocalization to cell membrane in A. Data are representative of three independent experiments (n � 3). Error barsrepresent means � S.D. for ZO-1 length per cell within each of the four randomly picked view fields. The asterisk denotes significant differences in the indicatedpairs (p � 0.05) by Student’s t test.



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sion, phosphorylation of Sqh (the Drosophila homolog ofMRLC) was reduced. Transgenic expression of an activatedphosphomimetic version of Sqh could rescue many aspects ofthe epithelial polarity defects found in Ampk� mutants, sug-gesting that AMPK regulates epithelial cell polarization pri-marily through its phosphorylation of MRLC. We thereforeexamined MRLC phosphorylation with an antibody recogniz-ing phosphorylated human MRLC in Ca2�-depleted MDCKcells treated with AICAR. We did not observe any change inMRLC phosphorylation in response to the treatment, suggest-ing that MRLC is not likely to be a downstream target forAMPK in inducing Ca2�-independent deposition of junctioncomponents. We also examined MRLC phosphorylation inMDCK cells subjected to a Ca2� switch. Although AMPK isactivated in this process, we were not able to detect anyconcomitant change in the levels of MRLC phosphorylation(Fig. 6A).The observation that the knockdown of afadin inhibits the

deposition of junction components induced by AMPK activa-tion suggests thatAMPK influences afadin function, possibly bydirect phosphorylation. To examine whether or not afadin is anAMPK substrate, we used an in vitro phosphorylation assay.We immunoprecipitated endogenous afadin from MDCK celllysates, and the immunoprecipitated proteins were then mixedwith recombinant AMPK protein and radioactively labeled

[�-32P]ATP, in the presence or absence of an AMPK inhibitorcompound C. After the kinase reactions were terminated, theproteins eluted from the beads were separated by SDS-PAGE.The phosphorylation profile was revealed by autoradiography.We also used an AMPK substrate, SAMS peptide, as a positivecontrol.We found that recombinant AMPKprotein was able toincorporate radioactive phosphate groups into the SAMS pep-tide, and this incorporation was dramatically reduced by com-pound C (Fig. 6B). The autoradiography profile of the in vitrophosphorylation assay revealed a band corresponding to themolecular weight of afadin in samples from the reaction inwhich compound C was not included, indicating that afadinwas phosphorylated. In samples in which compound C wasincluded in the reaction, the intensity of the afadin band wassignificantly reduced to a level comparable with that found insamples from the reactions missing the AMPK recombinantprotein. These data demonstrate that AMPK activity catalyzesthe incorporation of radioactive phosphate groups into afadin(Fig. 6C) and confirm that afadin is an AMPK substrate in vitro.We also performed phosphoproteomic studies on afadinimmunoprecipitates prepared from intact MDCK cells to sur-vey changes in the phosphorylation status of endogenouslyexpressed afadin. We were able to identify afadin phosphopep-tides that bear the consensus sequence for AMPK recognitionand phosphorylation (supplemental Table S1). We also foundthat the phosphorylation on at least one of these peptides wassubstantially increased in cells treatedwith theAMPKactivatorAICAR. These data therefore suggest that afadin is also anAMPK substrate in vivo (supplemental Fig. S6).Wenext sought to investigate the functional consequences of

afadin phosphorylation induced by AMPK activation. Becauseafadin directly interacts with ZO-1 (32), we hypothesized thatAMPK activation induced by either AICAR treatment or a cal-cium switch would increase the extent of the interactionbetween afadin and ZO-1, thereby facilitating the assembly oftight junctions. We immunoprecipitated afadin from cellstreated with AICAR, as well as from cells lysed at different timepoints following a calcium switch, and then blotted the result-ing immunoprecipitates to detect ZO-1 that co-precipitatedwith afadin. We found that in cells treated with AICAR, moreZO-1 co-precipitated with afadin compared with that detectedin control cells (Fig. 6D). We also detected increased amountsof ZO-1 interacting with afadin in cells when the culture con-ditions were switched from LCM to HCM (Fig. 6E). Theseresults suggested that AMPK activation might facilitate tightjunction assembly by phosphorylating afadin and inducing orstabilizing its association with tight junction components suchas ZO-1.

DISCUSSION

We have demonstrated that GSK-3� is involved in the regu-lation of an important step in epithelial cell polarization. Phar-macological inhibition of GSK-3� activity leads to the deposi-tion of junction components to the sites of cell-cell contact incells maintained in LCM.We found that AMPK activation andGSK-3� inhibition independently induce Ca2�-independentdeposition of junction components. Furthermore, the deposi-tion of junction components in response to both treatments is

FIGURE 6. Afadin is an AMPK substrate. A, confluent MDCK cells were incu-bated in LCM for 16 h and then switched back to HCM for the indicated times.In addition, a separate set of MDCK cells maintained in LCM was treated with1 mM AICAR for 2 h. Cells lysates were probed with the indicated antibodies ina Western blot analysis. B, AMPK was incubated with SAMS peptide alongwith [�-32P]ATP in the presence or absence of compound C (Comp. C). SAMSpeptide incubated in the indicated conditions was subjected to scintillationcounting. C, afadin in MDCK cell lysates was immunoprecipitated (IP), andAMPK was then added to the immunoprecipitate along with [�-32P]ATP in thepresence or absence of compound C. After incubation, proteins were sepa-rated by SDS-PAGE, and the radioactive signal was detected by autoradio-graphy. D and E, confluent MDCK cells were incubated in LCM for 16 h thenswitched back to HCM for the indicated times (D) or exposed to 1 mM AICARfor 2 h (E). Cell lysates were immunoprecipitated using an anti-afadin anti-body. Equal amounts of immunoprecipitates were then separated on SDS-PAGE and probed for ZO-1. Total cell lysates were simultaneously subjectedto immunoblotting using the anti-afadin and anti-ZO-1 antibodies.



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independent of E-cadherin. We also showed that the nectin-afadin cell adhesion system is required for the deposition ofjunction components induced byAMPK activation, but it is notrequired for that induced by GSK-3� inhibition.We have iden-tified afadin as a substrate for AMPK using an in vitro phosphor-ylation assay and have shown that it is also phosphorylated invivo using phosphoproteomic analysis of intact cells.We have focused primarily on the Ca2�-independent depo-

sition of junction components in this study. To understand howthe kinase activities of GSK-3� and AMPKmight contribute tothis process, it is worth reviewing the biological programof tight junction assembly, much of which is poorly understood.The early stages of tight junction assembly are believed toinvolve several critical events. First, neighboring cellsmust con-tact one another, permitting the engagement of cell adhesionmolecules in a trans-interacting manner to form nascent celladhesions (56). At this point, signal transduction pathways areactivated that induce cytoskeleton reorganization and largescale delivery of both tight and adherens junction proteins intocell membranes (57, 58). The formation of a supporting cyto-skeleton framework and the clustering of junction proteinsboth stabilize and strengthen the nascent cell adhesions. Sub-sequently, tight junction protein complexes separate fromadherens junctions and move apically to form mature tightjunctions (33). Under normal physiological circumstances, thisprocess is Ca2�-dependent because E-cadherin, themajor arbi-ter of epithelial cell adhesion, requires Ca2� to actively interactwith its counterpart on a neighboring cell (18).Our data suggest that, in the processes through which

AMPK activation and GSK-3� inhibition induce the recruit-ment of tight junction components to the cell surface inde-pendent of Ca2�, the prerequisite for interactions betweenE-cadherin molecules on the surfaces of adjacent cells can bebypassed. This may be achieved by strengthening the trans-interactions of other cell adhesion molecules, such as thoseof the nectin-afadin system, and/or by modulating cytoskel-eton dynamics near the cell membrane so that nascent cell-cell contacts are more stable even without the participationof E-cadherin.MRLC has emerged as an AMPK effector in regulating

epithelial polarity in recent Drosophila studies (54, 55).MRLC was reported to be phosphorylated by AMPK in vitro,and expression of an activated phosphomimetic version ofMRLC could reverse epithelial polarity defects caused by theloss of AMPK. MRLC has also been reported in many studiesto be implicated in the reorganization of actin cytoskeleton.Therefore, AMPK could potentially modulate cytoskeletondynamics by phosphorylating MRLC and thus regulating itsactivity. In our study, however, neither AICAR treatmentnor Ca2� switch induced any change in the level of MRLCphosphorylation, suggesting that MRLC is not likely anAMPK target in inducing Ca2�-independent deposition ofjunction components, at least in the mammalian MDCK cellsystem.We demonstrated that afadin can be phosphorylated by

AMPK, and these findings suggest that afadin is a promisingcandidate to serve as the effector of AMPK in inducing junctionassembly. It appears that, when AMPK is activated by AICAR,

afadin plays an essential role in relocalizing tight junction com-ponents to the sites of cell-cell contact in a Ca2�-independentmanner. This conclusion receives support from the fact thatknocking down afadin expression prevented AMPK activationfrom inducing Ca2�-independent junction assembly. We alsodemonstrated that AMPK activation by either AICAR treat-ment or calcium switch increases the extent of the interactionbetween afadin and ZO-1, resulting in a connection betweenthe nectin-afadin system and the tight junction complex. It istherefore possible that activation of the nectin-afadin systemmight be sufficient on its own to initiate tight junction assem-bly. In this context, it is interesting to note that nectin junctionsare formed very early in the process of epithelialmorphogenesisin the developing kidney and that nectin expression occursprior to that of the transmembrane tight junction proteinoccludin. Furthermore, expression of a dominant negative nec-tin protein disrupts epithelial cyst formation whenMDCK cellsare grown in three-dimensional collagen gels (30). Takentogether with our findings, these data suggest the interestingpossibility that AMPK may act to initiate or reinforce nectin-afadin-mediated junction formation at an early stage of celljunction assembly, after which the formation of fully maturejunctions is carried forward by Ca2� and E-cadherin-depen-dent processes.Although a number of recent advances link AMPK with epi-

thelial cell polarity, the role of GSK-3� in the regulation ofcytoskeleton dynamics and cell polarity has not been as exten-sively studied. In neurons, GSK-3� inhibition ensures theextension of axons by preventing axonal microtubules frombeing disrupted by the GSK-3� substrate CRMP-2. The PI3Kpathway and Akt have been shown to be required for GSK-3�inhibition in this process (43). In migrating astrocytes, Cdc42and PAR6 promote the activation of atypical PKC� at the lead-ing edge, which consequently phosphorylates and inhibitsGSK-3�, leading to the reorganization of cytoskeleton via ade-nomatous polyposis coli (47, 59).Although the upstream and downstreammolecules involved

in the deposition of junction components induced by GSK-3�inhibition are still under investigation, our study has providedsome clues in this regard. GSK-3� is known to be inhibitedwhen its Ser9 residue is phosphorylated by several upstreamkinases such as Akt and atypical PKC�. We determined, how-ever, that although the activity of GSK-3� is inhibited, the levelof GSK-3� Ser9 phosphorylation remained constant duringjunction assembly, suggesting that in this process GSK-3� isinhibited through a mechanism independent of Ser9 phosphor-ylation and thus independent of its regulation by Akt and atyp-ical PKC�.

In summary, we have identified AMPK andGSK-3� as effec-tors in novel kinase pathways that lead to the deposition ofjunction components without the participation of extracellularCa2� or of the Ca2�-dependent epithelial cell adhesionmachinery. We also identified afadin as a substrate for AMPKin this process. Future investigation will be required to identifythe downstream targets of GSK-3�, as well as to fully under-stand the roles that each of these pathways play in junctionformation in vivo.



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Acknowledgments—We thank Dr. D. Grahame Hardie for AMPK �1antibody; Dr. IanMacara for RNAi constructs; Dr. Agnes Kim for helpwith the AMPK activity assay; OrLando Yarborough for help with theSILAC labeling; and allmembers of theCaplan laboratory for insight-ful advice and discussions.

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