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Protein Kinase C Regulates the Phosphorylation and Cellular Localization of Occludin

Protein Kinase C Regulates the Phosphorylation and Cellular Localization of Occludin THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 42, Issue of October 19, pp. 38480 –38486, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Protein Kinase C Regulates the Phosphorylation and Cellular Localization of Occludin* Received for publication, May 30, 2001, and in revised form, August 6, 2001 Published, JBC Papers in Press, August 13, 2001, DOI 10.1074/jbc.M104923200 Anna Y. Andreeva‡, Eberhard Krause‡, Eva-Christina Mu ¨ ller§, Ingolf E. Blasig‡¶, and Darkhan I. Utepbergenov‡ From the ‡Forschungsinstitut fu ¨ r Molekulare Pharmakologie, 13125 Berlin-Buch and §Charite ´, Humboldt Universita ¨t Berlin, 13092 Berlin, Germany Occludin is an integral membrane phosphoprotein spe- critical step in various cellular processes, including the estab- cifically associated with tight junctions, contributing to lishment of epithelial cell polarity and developmental pattern- the structure and function of this intercellular seal. Oc- ing (3). Previous data suggest that TJ function may be regu- cludin function is thought to be regulated by phosphoryl- lated by the phosphorylation of certain proteins (4 – 6). ation, but no information is available on the molecular Diacylglycerols have been shown to trigger the formation of TJ, pathways involved. In the present study, the involvement suggesting the involvement of protein kinase C; the identities of the protein kinase C pathway in the regulation of the of the molecular pathways involved remain, however, elusive phosphorylation and cellular distribution of occludin has (4). been investigated. Phorbol 12-myristate 13-acetate and The integral membrane protein occludin was recently iden- 1,2-dioctanoylglycerol induced the rapid phosphorylation tified as a component of TJ of epithelial and endothelial cells (7, of occludin in Madin-Darby canine kidney cells cultured 8). Occludin comprises four transmembrane domains, two ex- in low extracellular calcium medium with a concomitant tracellular loops, and three cytoplasmic domains (one intracel- translocation of occludin to the regions of cell-cell con- lular short turn, a short NH -terminal domain, and a long tact. The extent of occludin phosphorylation as well as its COOH-terminal domain). Accumulating evidence suggests incorporation into tight junctions induced by protein ki- that occludin plays an important role in tight junctions, al- nase C activators or calcium switch were markedly de- creased by the protein kinase C inhibitor GF-109203X. In though embryonic stem cells lacking occludin are able to form addition, in vitro experiments showed that the recombi- well developed TJs (9). The overexpression of mutant forms of nant COOH-terminal domain of murine occludin could be occludin in cultured epithelial cells leads to changes in the phosphorylated by purified protein kinase C. Ser of barrier and fence function (10, 11). The addition of synthetic occludin was identified as an in vitro protein kinase C peptides corresponding to the extracellular loops of occludin to phosphorylation site using peptide mass fingerprint anal- epithelial cells results in the disappearance of TJ and inhibi- ysis and electrospray ionization tandem mass spectros- tion of cell adhesion (12–14). Finally, occludin knock-out mice copy. These findings indicate that protein kinase C is show a complex phenotype, including retarded growth and involved in the regulation of occludin function at tight various histological abnormalities, suggesting that the func- junctions. tions of both TJ and occludin are more complex than was supposed previously (15). 1 Occludin has been shown to be highly phosphorylated (16, Tight junctions (TJs), the most apical component of the 17). Tight junction assembly induced by calcium switch is par- junctional complex of epithelial and endothelial cells, form a alleled by occludin phosphorylation and incorporation into the diffusion barrier limiting the flux of hydrophilic molecules TJ of MDCK cells (16, 17). It has also been shown that highly through the paracellular pathway and maintain cell polarity by phosphorylated occludin molecules are selectively concentrated acting as a boundary between the apical and basolateral at TJs, whereas non- or less phosphorylated occludin is local- plasma membrane domains (reviewed in Refs. 1 and 2). The ized in the cytoplasm (16). Occludin becomes phosphorylated dynamic rearrangement of cell-cell junctions including TJs is a and associates with ZO-1 during certain stages of mouse em- This is an open access article under the CC BY license. bryo development, and these processes are proposed to regulate * This work was supported in part by Deutsche Forschungsgemein- TJ biogenesis and the timing of blastocyst formation (18). In shaft Grants SFB 507 TP A2, DFG GK 238-2, DFG BL 308/6-1, and addition, recent results suggest that the phosphorylation of BMBF BEO 0311466C. The costs of publication of this article were occludin may regulate tight junction permeability in response defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 to histamine and lysophosphatidic acid (19). The findings above U.S.C. Section 1734 solely to indicate this fact. led to the conclusion that occludin function at TJ is regulated ¶ To whom correspondence should be addressed: Forschungsinstitut by phosphorylation. The molecular pathways regulating occlu- fu ¨ r Molekulare Pharmakologie, Bldg. 81, Rm. A2.06, Robert-Ro ¨ ssle-Str. 10, 13125 Berlin-Buch, Germany. Tel.: 49-30-94793321; Fax: 49-30- din phosphorylation remain unclear. 94793243; E-mail: [email protected]. In the present study, we analyzed the effect of phorbol 12- The abbreviations used are: TJ, tight junction; diC8, 1,2-dioctanoyl- myristate 13-acetate (PMA) and 1,2-dioctanoylglycerol (diC8), glycerol; ESI, electrospray ionization; FCS, fetal calf serum; LC, low activators of protein kinase C, on the phosphorylation and calcium medium; NC, normal calcium medium; MALDI, matrix- assisted laser desorption/ionization; MBP, maltose-binding protein; cellular localization of occludin in monolayers of MDCK cells MDCK, Madine-Darby canine kidney; PAGE, polyacrylamide gel elec- incubated in low extracellular Ca medium. PMA and diC8 trophoresis; PBS, phosphate-buffered saline; PKC, protein kinase C; induced a rapid phosphorylation of occludin and its redistribu- TOF, time of flight; MS, mass spectrometry; MS/MS, tandem mass tion to the regions of cell-cell contact. The phosphorylation and spectrometry; MEM, minimal essential medium; PMA, phorbol 12- myristate 13-acetate. incorporation of occludin into tight junctions induced by PMA, 38480 This paper is available on line at http://www.jbc.org Occludin Phosphorylation by PKC 38481 X-100-soluble fraction). The remaining material was scraped into lysis diC8, or calcium switch were inhibited by a PKC inhibitor. buffer (25 mM Hepes/NaOH, pH 7.4, 150 mM NaCl, 4 mM EDTA, 25 mM Furthermore, Ser of the recombinant COOH-terminal do- NaF, 1% SDS), boiled for 5 min, and centrifuged for 10 min at 14,000 main of murine occludin was found to be phosphorylated in g (the resulting extract is referred to as the Triton X-100-insoluble vitro by purified protein kinase C. These findings suggest that fraction). One-dimensional SDS-PAGE (8% gel) was performed accord- the regulation of phosphorylation and cellular distribution of ing to Laemmli (20). For immunoblotting, proteins separated by SDS- occludin are mediated by protein kinase C. PAGE were electrophoretically transferred to nitrocellulose sheets, which were then incubated with the antibodies. The antibodies were MATERIALS AND METHODS detected with a blotting detection kit. To detect protein phosphoryla- Reagents, Antibodies, and Cells—Protease inhibitors and protein tion, samples were blotted as above and the P signal was detected by A-Sepharose were from Sigma (Taufkirchen, Germany). Protein kinase autoradiography. Proteins transferred on the nitrocellulose membrane C, PMA, 1,2-dioctanoylglycerol, and GF-109203X were from Alexis were visualized by silver staining. Deutschland GmbH (Gruenberg, Germany). TRIzol reagent and flasks Occludin Immunoprecipitation and Alkaline Phosphatase Treat- on a slide were from Life Technologies GmbH (Technologiepark ment—MDCK cells cultured on 8.8-cm Petri dishes were washed twice Karlsruhe, Germany). Cy3- and horseradish peroxidase-conjugated with ice-cold PBS and extracted with 1 ml of ice-cold immunoprecipi- goat anti-rabbit monoclonal antibodies and rabbit polyclonal anti-occlu- tation buffer (25 mM Hepes/NaOH, pH 7.4, 150 mM NaCl, 4 mM EDTA, din antibodies were purchased from Zymed Laboratories Inc. (San 25 mM NaF, 1% Triton X-100, 1 M leupeptin, 0.3 M aprotinin, 0.1 mM Francisco, CA). MDCK cells were obtained from Dr. Swaroop (MDC, phenylmethylsulfonyl fluoride, 1 M pepstatin) for 30 min and collected Berlin, Germany). The pMAL fusion and purification system, BamHI, in 1.5-ml tubes. After centrifugation (13,000  g, 15 min, 4 °C), the and SalI were purchased from New England Biolabs GmbH (Frankfurt pellet was resuspended in 100 l of lysis buffer, heated 5 min at 95 °C, am Main, Germany). Taq polymerase, alkaline phosphatase, and Molo- and combined with 900 l of immunoprecipitation buffer. After centrif- ney murine leukemia virus reverse transcriptase were from Promega ugation (13,000  g for 15 min), the supernatant was pretreated with a GmbH (Mannheim, Germany). [- P]ATP and the blotting detection 15-l bed volume of protein A-Sepharose. For immunoprecipitation 1 g kit were from Amersham Pharmacia Biotech Europe GmbH (Freiburg, of anti-occludin antibodies and a 10-l bed volume of protein A-Sepha- Germany). rose were added to the supernatant and rotated for1hat4 °C. Beads Occludin Expression Construct and in Vitro Phosphorylation of Re- were washed five times with 1 ml of immunoprecipitation buffer and combinant Occludin—Total RNA was isolated from 50 mg of murine then resuspended in 100 l of phosphatase buffer (50 mM Tris/HCl, pH kidney using TRIzol reagent and reverse transcription was performed 9.3, 1 mM MgCl , 0.1 mM ZnCl ,1mM spermidine) with or without 20 2 2 using Moloney murine leukemia virus reverse transcriptase and ran- units of calf intestinal alkaline phosphatase. After1hof incubation at dom hexamer primers. A 777-base pair fragment encoding amino acids 37 °C, SDS-PAGE buffer was added and beads were boiled to elute the 264 –521 of murine occludin was amplified using the primers 5-AG- immunoprecipitates for subsequent PAGE. GATCCAAAACCCGAAGAAAGATGGATCGG-3, and 5-TTGTCGAC- Identification of Phosphorylation Sites by Mass Spectrometry—For TAAGGTTTCCGTCTGTCATAGTC-3 (BamHI and SalI sites are un- MALDI analysis, 15 l of occludin solution taken from the phosphoryl- derlined). The amplified product was cloned using the TOPO-TA ation reaction were resolved by SDS-PAGE. The proteins were detected cloning kit (Invitrogen, Carlsbad, CA). Several clones were sequenced by staining with a Colloidal Blue staining kit (Novex, San Diego, CA). (Taq DyeDeoxy-Terminator cycle sequencing kit, Applied Biosystems, Phosphorylated and non-phosphorylated occludin bands were excised Weiterstadt, Germany); a clone containing no mutations was selected, from the stained gels, washed with 50% (v/v) acetonitrile in 25 mM and its BamHI-SalI fragment was subcloned into the pMAL-c2x plas- ammonium bicarbonate, dehydrated in acetonitrile, and dried in a mid to produce a plasmid coding for the COOH-terminal domain of vacuum centrifuge. Disulfide bonds were reduced by incubation in 30 l occludin fused with maltose-binding protein. The fusion protein was of 10 mM dithiothreitol in 100 mM ammonium bicarbonate for 45 min at overexpressed in Escherichia coli and purified over an amylose column 55 °C. Alkylation was performed by replacing the dithiothreitol solution according to the manufacturer’s instructions. with 55 mM iodoacetamide in 100 mM ammonium bicarbonate. After a The in vitro phosphorylation of occludin was performed as follows. 20-min incubation at 25 °C in the dark, the gel pieces were washed with Purified recombinant occludin fragment (5 g) was mixed with 16 ng of 50 –100 l of 50% (v/v) acetonitrile in 25 mM ammonium bicarbonate, protein kinase C in 50 mM Tris/HCl buffer, pH 7.5, containing 10 mM dehydrated in acetonitrile, and dried in a vacuum centrifuge. The gel MgCl ,2mM CaCl ,1mM dithiothreitol, 0.2 mM ATP, 5 nM PMA, and 5 pieces were re-swollen in 10 lof5mM ammonium bicarbonate, con- 2 2 Ci of [- P]ATP in a reaction volume of 40 l. In other experiments taining 300 ng of endoproteinase Lys-C (sequencing grade, Roche Di- GF-109203X at a concentration of 5 M was used to inhibit PKC. agnostics, Mannheim, Germany). After 15 min, 5 lof5mM ammonium Phosphorylation of MBP--galactosidase (the expression product of the bicarbonate was added to keep the gel pieces moist during Lys-C cleav- pMal-c2x plasmid) was performed as for occludin, but double the age (37 °C, overnight). To extract the peptides, 15 l of 0.5% (v/v) amount of PKC was used. trifluoroacetic acid in acetonitrile was added, and the samples were Cell Culture, Low Calcium Medium Culture, and Calcium Switch— sonicated for 5 min. The separated liquid was dried under vacuum and MDCK cells were cultured in MEM with 10% FCS. Low Ca medium redissolved in 10 l of 0.1% (v/v) trifluoroacetic acid in water. The (LC) was prepared from S-MEM (calcium-free MEM) and 5% FCS peptides were purified over a C18 reversed-phase minicolumn filled in pretreated with Chelex resin (Bio-Rad Laboratories GmbH, Mu ¨ nchen, a micropipette tip (ZipTip C18, Millipore, Bedford, MA) for mass spec- Germany) as described in Ref. 19. Confluent monolayers of MDCK cells trometry analysis. Purification was performed according to the manu- were grown in normal calcium (NC) medium (MEM with 1.8 mM CaCl facturer’s manual, except that peptides were eluted with 3 lof60% and 5% FCS), washed twice with PBS, and then transferred to low Ca (v/v) acetonitrile, 0.3% (v/v) trifluoroacetic acid or with 5 l of 60% (v/v) medium for 20 h prior to the addition of normal calcium medium, PMA, acetonitrile, 0.2% (v/v) formic acid for matrix-assisted laser desorption/ or diC8. In other experiments GF-109203X at a concentration of 5 M ionization mass spectrometry (MALDI-MS) and nanoelectrospray tan- was added 30 min before the addition of normal calcium medium (or low dem mass spectrometry (nanoESI-MS/MS), respectively. calcium medium with PKC activators) containing 5 M GF-109203X. MALDI-MS measurements were performed on a Voyager-DE STR Immunofluorescence Microscopy—Cells were grown in the flasks on BioSpectrometry work station MALDI-TOF mass spectrometer (Per- slides, washed with PBS and fixed in 1% paraformaldehyde in PBS for septive Biosystems, Inc., Framingham, MA). One l of the analyte 15 min. After three washes with PBS, cells were permeabilized with solution was mixed with 1 lof -cyano-4-hydroxycinnamic acid matrix 0.2% Triton X-100 in PBS, soaked in blocking solution (1% bovine solution consisting of 10 mg of matrix dissolved in 1 ml of 0.3% triflu- serum albumin in PBS) for 60 min, and then incubated with 300 lof oroacetic acid in acetonitrile-water (1:1, v/v). One l of the resulting anti-occludin antibodies (diluted 100 times). Samples were washed mixture was applied to the sample plate. Samples were air-dried at three times with 0.2% bovine serum albumin and then incubated for 60 ambient temperature (24 °C). Measurements were performed in the min with Cy3-conjugated goat anti-rabbit antibodies. After four washes reflection mode at an acceleration voltage of 20 kV, 70% grid voltage, coverslips were mounted over the cells with the aid of mounting me- and a delay of 200 ns. Each spectrum obtained was the mean of 256 dium (Shandon, Pittsburgh, PA). Samples were examined using a flu- laser shots. orescence microscope equipped with the AxioVision image acquisition For nanoESI-MS/MS, 5 l of the peptide mixture were lyophilized and analysis system (Carl Zeiss, Jena, Germany). and dissolved in 5 l of methanol, 1% formic acid (1:1, v/v). The MS/MS Protein Blotting and Analysis—Cells were washed twice with PBS measurements were performed with a nanoelectrospray hybrid quadru- and extracted for 30 min with PBS containing 1% Triton X-100, 1 M pole mass spectrometer (Q-Tof, Micromass, Manchester, United King- leupeptin, 0.3 M aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, and dom). The collision gas was argon at a pressure of 6.0  10 millibars 1mM pepstatin at 4 °C (the resulting extract is referred to as the Triton in the collision cell. 38482 Occludin Phosphorylation by PKC FIG.2. PMA and diC8 induce the phosphorylation of occludin in MDCK cells incubated in low Ca medium. Panel A, immuno- blot analysis of occludin in the Triton X-100-soluble (S) and -insoluble (I) fractions of MDCK cells incubated in normal Ca medium (lane 1) or in low Ca medium (lane 2). MDCK cells cultivated in low calcium medium were subjected to a calcium switch (lane 3) or treated with 2 nM (lane 4), 25 nM PMA (lane 5), or 0.5 mM diC8 for4h(lane 6). Treatment of MDCK cells cultivated in low calcium medium with PMA or diC8 induced an upward band shift. B, immunoblot analysis of the Triton X-100-insoluble fraction of immunoprecipitated occludin from MDCK cells cultivated in low calcium medium and stimulated with 10 nM PMA for 20 min. Samples were either left untreated (lane 1) or subjected to treatment with alkaline phosphatase (lane 2), resolved by electrophore- sis, and then analyzed by immunoblotting. Alkaline phosphatase treat- ment completely abolished the upward band shift caused by PMA treatment. The arrow shows the position of non-phosphorylated occlu- din, the dash shows the position of antibody heavy chain, and a bracket denotes the position of multiple phosphorylated forms of occludin. the occludin was translocated to the plasma membrane at regions of cell-cell contact (Fig. 1C, arrows). Staining of occlu- din at cell-cell contacts was not as continuous or intensive as in the cells grown in NC (Fig. 1A). Prolongation of the incubation period up to 4 h further increased the amount of occludin translocated to the regions of cell-cell contact (Fig. 1D). When 25 nM PMA was used to stimulate cells, occludin translocated to the periphery showed a more discontinuous and intensive staining as compared with samples treated with 2 nM PMA (Fig. 1E). Most of the occludin was still localized in the cyto- FIG.1. PMA and diC8 induce the translocation of occludin plasm, a significant portion in large granular structures (Fig. from the cytoplasm to the lateral cell membrane in MDCK cells 2 1E, arrowheads). Prolongation of the incubation time up to 4 h incubated in low Ca medium. MDCK cells were incubated in low did not change the distribution pattern significantly (Fig. 1F), calcium medium for 20 h after which PMA, diC8, or Ca was added. Cells cultivated in normal calcium medium show continuous staining of although the decrease in the amount of occludin localized at occludin at cell-cell contacts (A). After cultivation of cells in low calcium cell-cell contacts was notable. The treatment of MDCK cells in medium, the occludin signal was detected mainly in small granular LC with the another protein kinase C activator diC8 resulted in structures in the cytoplasm (B). Two hours after the addition of 2 nM the redistribution of occludin, which was similar to the effect of PMA, staining of occludin at cell borders (arrows) was evident (C). Prolongation of the incubation period to 4 h further increased the 2nM PMA. Fragmentary staining of occludin at cell-cell con- amount of occludin migrating to the cell borders (D). When 25 nM PMA tacts was evident 2 h after the addition of diC8 (Fig. 1G). The was used to stimulate the cells for 2 h, large granular structures were amount of occludin translocated to cell-cell contacts further found in the cytoplasm (arrowheads) and occludin staining at the cell increased when the incubation time was prolonged up to 4 h borders became discontinuous (E). Prolongation of the incubation pe- riod (25 nM PMA) up to 4 h led to some decrease in the amount of (Fig. 1H). occludin found at cell-cell contacts (F). Two hours after the addition of Effect of PKC Activators on the Triton X-100 Solubility and 0.5 mM diC8, staining of occludin at cell borders was evident (G). Phosphorylation of Occludin in MDCK Cells in LC—Previous Prolongation of the incubation period to 4 h further increased the studies have shown that occludin localized in TJ cannot be amount of occludin at cell borders (H). Bar,10 m. extracted with non-ionic detergents from confluent epithelial cells and that it shows a decreased electrophoretic mobility due RESULTS to extensive phosphorylation (16, 17, 21). In accordance with Effect of PKC Activators on the Localization of Occludin in literature data (17), occludin extracted with Triton X-100 from MDCK Cells in Low Calcium Medium—In view of the fact that MDCK cells grown in NC migrated as a single band with an diacylglycerols are known to induce the formation of tight apparent molecular mass of 60 kDa, whereas that from the junction strands in MDCK cells cultivated in low calcium me- Triton X-100-insoluble fraction migrated as multiple unre- dium (4), we assumed that occludin phosphorylation and re- solved bands with apparent molecular masses ranging between cruitment into tight junctions are mediated by PKC. To verify 60 and 82 kDa (Fig. 2A, lane 1). When MDCK cells were this hypothesis, we investigated the effect of PKC activators on incubated in LC in order to down-regulate TJs, the high mo- the phosphorylation and cellular distribution of occludin in lecular mass bands disappeared from the Triton X-100-insolu- MDCK cells cultured in low calcium medium, which disrupts ble fraction and the amount of Triton X-100-insoluble occludin tight junctions. In accordance with literature data (7, 10), oc- decreased significantly (Fig. 2A, lane 2). When TJ re-assembly cludin was intensively stained at cell-cell contacts in confluent was induced by calcium switch, the banding pattern of Triton MDCK cells in normal Ca medium, indicating that continu- X-100-insoluble occludin was restored (Fig. 2A, lane 3). Four ous TJs were formed (Fig. 1A). After incubation of the cells in hours after the addition of 2 nM PMA to the cells in LC, the LC, occludin became localized almost exclusively in the cyto- amount of Triton X-100-insoluble occludin increased signifi- plasm, indicating a down-regulation of TJs (Fig. 1B). When cantly and a portion of occludin underwent an upward band PMA at 2 nM was added for2htothe cells in LC, a portion of shift (Fig. 2A, lane 4). Treatment of cells in LC with 25 nM PMA Occludin Phosphorylation by PKC 38483 FIG.3. Time and concentration dependence of occludin phos- phorylation in MDCK cells upon treatment with PMA. Figure shows immunoblot analysis of occludin in the Triton X-100-insoluble fraction of MDCK cells. After incubation of cell monolayers in low calcium medium for 20 h, PMA at the indicated concentrations was added for1h(A)or25nM PMA was added for the indicated periods of time (B). for 4 h resulted in an additional increase in the amounts of FIG.4. Effects of the PKC inhibitor GF-109203X on the redis- Triton X-100-insoluble occludin and in the degree of upward tribution of occludin during tight junction assembly induced by band shift, with a concomitant decrease in the amount of Triton Ca switch or PKC activators. Figure shows immunofluorescence X-100-soluble occludin (Fig. 2A, lane 5). The decrease is prob- localization of occludin 4 h after addition of calcium (A and B), PMA (C and D), or diC8 (E and F) to MDCK cells placed in low calcium medium. ably due to increased proteolysis, since high concentrations of 5 M GF-109203X was added (B, D, and F) to evaluate the involvement PMA are known to cause degradation of occludin (21, 22). An of PKC in the redistribution of occludin. Bar,10 m. increase in Triton X-100-insoluble occludin and an upward band shift were also evident in cells treated with 0.5 mM diC8 for 4 h (Fig. 2A, lane 6). switching to NC, continuous occludin staining was evident at The multiple bands revealed by immunoblotting analysis intercellular contacts, indicating the recruitment of occludin probably represent differently phosphorylated occludin mole- into tight junctions (Fig. 4A). Inhibition of PKC with 5 M cules. In order to show that the upward band shift induced by GF-109203X resulted in very weak staining of occludin at the PMA was due to phosphorylation, MDCK cells were treated intercellular contacts but a prominent staining of occludin in with PMA and occludin immunoprecipitated from the Triton cytoplasm (Fig. 4B). It can be concluded from these data that X-100-insoluble fraction was subjected to in vitro phosphatase PKC participates in the targeting of occludin into TJs. Further- treatment. Fig. 2B shows that the additional high molecular more, GF-109203X inhibited the redistribution of occludin to weight occludin bands disappeared on alkaline phosphatase the regions of cell-cell contacts induced by PMA (Fig. 4, C and treatment. Therefore, the upward band shift of occludin in- D) and diC8 (Fig. 4, E and F). duced by PMA is due to phosphorylation of occludin, and In order to study the involvement of PKC in the phosphoryl- changes in the cellular distribution of occludin induced by the ation of occludin, the effect of GF-109203X on calcium switch- treatment of MDCK cells in LC by PMA or diC8 are paralleled induced occludin phosphorylation was investigated. The char- by its phosphorylation. acteristic multiple-band pattern of occludin is seen on the Fig. 3 shows the time and concentration dependence of PMA- immunoblot in Triton X-100-insoluble fractions of MDCK cells induced phosphorylation of occludin. As high molecular weight incubated in NC medium (Fig. 5, NC). High molecular weight bands corresponding to phosphorylated occludin were invari- bands disappeared when cells were incubated in LC medium, ably found to be Triton X-100-insoluble, only Triton X-100- indicating the dephosphorylation of occludin (Fig. 5, LC). Four insoluble fractions were analyzed. Addition of PMA to MDCK hours after switching to NC, a profound upward band shift of cells incubated in LC medium resulted in a dose-dependent occludin was visible on the immunoblot, reflecting the in- phosphorylation of occludin (Fig. 3A). When PMA was added creased phosphorylation of occludin (Fig. 5, Ca ). When the for 1 h, the phosphorylation was evident at 2.5 nM PMA and calcium switch was performed in the presence of 5 M GF- reached a maximum at 40 nM PMA. The time dependence of 109203X, the extent of occludin phosphorylation was markedly occludin phosphorylation in MDCK cells in LC induced by 25 decreased, indicating the involvement of PKC. Similarly, the nM PMA is shown on Fig. 3B. The upward band shift became increase in phosphorylation of occludin induced by PMA and evident after 5 min and reached a maximum 40 min after PMA diC8 was significantly decreased by GF-109203X, again indi- addition. cating the involvement of PKC (Fig. 5, PMA and diC8). Phosphorylation and Redistribution of Occludin Induced by Occludin as a Substrate of PKC—To investigate whether Ca Switch PMA and diC8 Is PKC-dependent—The striking occludin may serve as a substrate for protein kinase C, recom- effects of the PKC activators PMA and diC8 on the phospho- binant occludin (MBP fusion protein of the soluble cytoplasmic rylation and distribution of occludin suggest that PKC may be COOH-terminal domain of murine occludin or MBP-occludin) a physiological regulator of occludin function. To investigate was incubated in vitro with purified PKC (a mixture of , I, the involvement of PKC in the regulation of occludin incorpo- II, and  isoforms of PKC) in the presence of [- P]ATP. As ration into TJs, the assembly of tight junctions was induced by shown on Fig. 6 (lane 1), an autoradiography signal was de- the calcium switch procedure in the presence or absence of the tected corresponding to a protein of 74 kDa (the predicted PKC inhibitor GF-109203X and occludin localization was as- molecular mass of MBP-occludin is 73.6 kDa), implying that sessed by immunofluorescence microscopy. Four hours after MBP-occludin was phosphorylated by protein kinase C. No 38484 Occludin Phosphorylation by PKC DISCUSSION Occludin phosphorylation has been implicated in the regula- tion of tight junction function (16, 19, 21), but the molecular pathways involved remain unclear. The present study estab- lishes the importance of the protein kinase C pathway in the phosphorylation and cellular localization of occludin. FIG.5. PKC inhibitor GF-109203X decreases the phosphoryla- 2 The redistribution of occludin from the cytoplasm to the tion of occludin during tight junction assembly induced by Ca lateral plasma membrane has been observed after the treat- switch or by PKC activators. Figure shows immunoblot analysis of occludin in Triton X-100-insoluble fractions of MDCK cells. Cells were ment of MDCK cells in LC with 2 nM PMA or 0.5 mM of more harvested after cultivation in NC or in LC. Normal calcium medium specific PKC activator diC8. This probably reflects the inser- (Ca ), 2.5 nM PMA (PMA), or 0.5 mM diC8 (diC8) were added to tion of occludin into TJs, since the formation of tight junction the cells in LC either with or without 5 M GF109203X (inh.). fibrils was reported under similar conditions (4). The amount of occludin translocated to the lateral plasma membrane in- creases with PMA concentration, but the staining of occludin becomes discontinuous. This observation is in agreement with studies reporting the disruption of TJs and increase in occludin proteolysis in response to the treatment of MDCK cells with very high (0.1–1 M) concentrations of PMA (21, 22). The results presented here clearly indicate that a Triton X-100-insoluble pool of occludin is phosphorylated upon the addition of the protein kinase C activators PMA or diC8 to FIG.6. Protein kinase C phosphorylates the COOH-terminal MDCK cells incubated in low calcium medium. Previously, the domain of recombinant occludin. MBP-occludin was incubated with 32 time course of occludin phosphorylation induced by a calcium PKC in a buffer containing [- P]ATP followed by SDS-PAGE, transfer onto nitrocellulose membrane, silver staining, and autoradiography. An switch was shown to be paralleled by TJ formation (16). In this autoradiography signal was visible at 74 kDa, indicating that MBP- study, a low but easily distinguishable level of upward band occludin is phosphorylated by PKC (lane 1). No signal was detected in shift of occludin caused by the 4-h incubation of MDCK cells control samples from which occludin (lane 2) or protein kinase C (lane with 2 nM PMA correlates well with the low amounts of occlu- 3) were omitted. The phosphorylation was strongly inhibited by 5 M din translocated to the lateral cell membrane. Correspond- GF-109203X (lane 4). The control protein MBP--galactosidase was only weakly phosphorylated, although the amount of PKC was doubled ingly, the increased phosphorylation caused by 25 nM PMA is (lane 5). paralleled by the increased amounts of occludin translocated to TJs. These observations support the assumption that the phos- phorylation of occludin in response to PMA or diC8 is involved autoradiography signal was detected when MBP-occludin or in the regulation of TJ assembly. The degree and rate of occlu- PKC was omitted (Fig. 6, lanes 2 and 3), excluding the possi- din phosphorylation induced by PMA are the same or even bilities that the autoradiography signal was due to autophos- higher compared with those induced by the calcium switch; phorylation of PKC or to any kinase activity that might have phosphorylation is induced by rather low concentrations of co-purified with MBP-occludin. The phosphorylation was PMA, indicating that diacylglycerols, along with calcium ions, strongly inhibited by 5 M GF-109203X (Fig. 6, lane 4). Another are physiological regulators of occludin phosphorylation. High MBP fusion protein, MBP--galactosidase, with a predicted molecular weight forms of occludin that appeared after the molecular mass of 53.5 kDa was phosphorylated to a much treatment of the cells in LC by PMA or diC8 were invariably lower extent, indicating the relative specificity of occludin not extractable with Triton X-100. It can be concluded that the phosphorylation by PKC (Fig. 6, lane 5). phosphorylation regulates the association of occludin with the To determine which sites of occludin are phosphorylated by cytoskeleton and/or detergent-resistant membrane microdo- PKC, occludin was analyzed by in-gel digestion using chymo- mains (24). Future investigations should clarify which deter- trypsin, trypsin, Asp-N, or Lys-C, followed by peptide mass gent-insoluble structures interact with occludin and how the fingerprinting by matrix-assisted laser desorption ionization phosphorylation of occludin regulates these interactions. time-of-flight (MALDI-TOF) mass spectrometry. Comparison of Both the phosphorylation of occludin and its incorporation the peptide mass fingerprints of phosphorylated and non-phos- into tight junctions induced by calcium switch were markedly inhibited by the PKC inhibitor GF-109203X. These observa- phorylated occludin revealed a distinct mass peak at m/z tions indicate that PKC participates in the regulation of occlu- 1256.532 (theoretical m/z 1256.535) corresponding to the phos- 333 341 din function during tight junction assembly induced by calcium phorylated fragment RSYPESFYK after Lys-C digestion switch. Previously, the application of the PKC inhibitor has of occludin treated with PKC (Fig. 7a). This peak was not been shown to inhibit the development of transepithelial elec- present when PKC was omitted (Fig. 7b). The site of phospho- trical resistance, a functional measure of tight junction biogen- rylation within the phosphorylated occludin fragment was de- esis (25). Occludin, the only known transmembrane protein of termined by nanoelectrospray ionization tandem mass spec- TJ whose localization in tight junctions is dependent on its trometry (ESI-MS/MS). Based on the double charge ion with phosphorylation state, may be a key target in the PKC-medi- m/z 628.78, the spectrum (Fig. 8) shows a peak corresponding ated regulation of tight junctions. Other authors have noted to the neutral loss of H PO (98 Da). The evidence for the 3 4 similarities between occludin and connexins in terms of phos- phosphorylation of Ser comes from the mass of the b and b 2 3 phorylation and structure (16). The data presented here extend ions corresponding to the non-phosphorylated NH -terminal the similarities further; connexin43 is phosphorylated by PKC part, whereas the ions with a mass greater than 700 Da appear in response to PMA, and the phosphorylation dramatically as b -98, b -98, b -98, and y -98. No evidence of a concurrent 6 7 8 6 changes gap junction channel conductivity (26). Possibly, the phosphorylation was found at any other position. From mass assembly of the junctional complex is regulated by PKC via spectrometry data, it can be concluded that Ser of mouse the phosphorylation of connexin43 and occludin to coordinate occludin is phosphorylated by protein kinase C in vitro. the formation of a functionally active barrier and the function Occludin Phosphorylation by PKC 38485 FIG.7. Peptide mass fingerprint analysis of in vitro phosphorylated and non-phosphorylated occludin. Proteins were in-gel digested with Lys-C; the peptides were extracted, purified over a C18 reversed-phase minicolumn, mixed with an -cyano-4-hydroxycinnamic acid matrix, and analyzed by MALDI-TOF-MS. Upper and lower panels show portions of spectra of the in vitro phosphorylated (a) and non-phosphorylated occludin (b). The peaks with m/z 1256.532 (theoretical m/z 1256.535) and 1176.575 (theoretical m/z 1176.569) correspond, respectively, to the phosphorylated and non-phosphorylated sequence RSYPESFYK. FIG.8. Identification of Ser as an in vitro phosphorylation site. MS/MS spectrum of the peptide RSYPEpSFYK (pS, phosphoserine residue) with the dou- ble charged ion m/z  628.78 Da. The base peak with m/z  579.73 Da results from the neutral loss of H PO (98 Da). 3 4 Parts of the spectrum are magnified by 2 to aid the observation of lower abundance product ions. Relevant ions are labeled according to the accepted nomenclature (23). Both the b ions and the y ions in the spectrum that contain the phosphoserine are produced by consecutive fragmenta- tion reactions, whereby the amide bond is broken and H PO is lost, or vice versa. 3 4 The masses of the b and b ions corre- 2 3 spond to the unphosphorylated NH -ter- minal part, whereas the ions with a mass greater than 700 Da confirm the phospho- rylation of Ser . of intercellular channels. The phosphorylation and redistribu- terminal domain of occludin in vitro, whereas a number of tion of occludin induced by PKC activators were strongly in- tested kinases did not phosphorylate occludin (31). However, hibited by the application of GF-109203X. These observations there are no indications of the involvement of casein kinase 2 in further substantiate the specificity of the regulation of occludin the regulation of TJ functions. functions by PKC. Despite the use of four different proteases to produce various The simplest explanation for the observed occludin phospho- peptides of occludin, Ser of occludin was the only phospho- rylation induced by PMA or diC8 is that PKC directly phospho- rylation site for classical PKCs that was positively identified by rylates occludin when stimulated by PMA, although the possi- MS. On the other hand, the presence of multiple phosphoryl- bility that PKC activates an intermediate kinase(s) cannot be ated forms of occludin in vivo was demonstrated by two-dimen- ruled out. Therefore, the in vitro phosphorylation of the recom- sional gel electrophoresis (19). The appearance of multiple binant occludin COOH-terminal domain by PKC was investi- phosphatase-sensitive occludin bands resulting from treatment gated. The carboxyl-terminal domain of occludin has been re- of MDCK cells with PMA indicates the multiple phosphoryla- cently shown to be responsible for the targeting of occludin to tion of occludin. Whereas it is possible that some phosphoryl- the TJs (27) and for its interaction with other proteins (28, 29). ation sites might not have been identified by MS, the absence of The results suggest that the COOH-terminal domain of occlu- additional phosphorylation sites suggests that other kinase(s) din may be phosphorylated by classical isoforms of PKC, which must phosphorylate occludin in vivo. These may include non- are known to be expressed in MDCK cells (30). To date, only classical PKC isoforms, some of which are known to be associ- casein kinase 2 has been reported to phosphorylate the COOH- ated with TJs (32, 33) and which have different substrate 38486 Occludin Phosphorylation by PKC 10. Balda, M. S., Whitney, J. A., Flores, C., Gonzalez, S., Cereijido, M., and Matter, sequence motifs from those of classical PKCs (34), or an inter- K. (1996) J. Cell Biol. 134, 1031–1049 mediate kinase(s) which is activated by PKC-dependent phos- 11. Bamforth, S. D., Kniesel, U., Wolburg, H., Engelhardt, B., and Risau, W. (1999) J. Cell Sci. 112, 1879 –1888 phorylation and subsequently phosphorylates occludin at dif- 338 12. Wong, V., and Gumbiner, B. M. (1997) J. Cell Biol. 136, 399 – 409 ferent positions. Ser is located within a 100-amino acid 13. Lacaz-Vieira, F., Jaeger, M. M., Farshori, P., and Kachar, B. (1999) J. Membr. stretch of occludin, which was shown to be dispensable for the Biol. 168, 289 –297 14. Van Itallie, C. M., and Anderson, J. M. (1997) J. Cell Sci. 110, 1113–1121 binding to ZO-1 (29) and for the targeting of occludin to TJs 15. Saitou, M., Furuse, M., Sasaki, H., Schulzke, J. D., Fromm, M., Takano, H., (27). Therefore, the significance of the phosphorylation on Noda, T., and Tsukita, S. (2000) Mol. Biol. Cell 11, 4131– 4142 Ser 16. Sakakibara, A., Furuse, M., Saitou, M., Ando-Akatsuka, Y., and Tsukita, S. is not clear. (1997) J. Cell Biol. 137, 1393–1401 Taken together, our data outline the importance of the pro- 17. Wong, V. (1998) Am. J. Physiol. 273, C1859 –C1867 tein kinase C pathway in the regulation of occludin function at 18. Sheth, B., Moran, B., Anderson, J. M., and Fleming, T. P. (2000) Development 127, 831– 840 tight junctions. Further analysis of the PKC pathway will lead 19. Hirase, T., Kawashima, S., Wong, E. Y., Ueyama, T., Rikitake, Y., Tsukita, S., to a better understanding of the functional biochemistry of Yokoyama, M., and Staddon, J. M. (2001) J. Biol. Chem. 276, 10423–10431 occludin and tight junctions. 20. Laemmli, U. K. (1970) Nature 227, 680 – 685 21. Farshori, P., and Kachar, B. (1999) J. Membr. Biol. 170, 147–156 22. Clarke, H., Soler, A. P., and Mullin, J. M. (2000) J. Cell Sci. 113, 3187–3196 Acknowledgments—We thank Gislinde Hartmann and Heidemarine 23. Biemann, K., and Scoble, H. A. (1987) Science 237, 992–998 Lerch for technical assistance. We appreciate the assistance of John 24. Nusrat, A., Parkos, C. A., Verkade, P., Foley, C. S., Liang, T. W., Innis- Dickson in preparation of this manuscript. We are grateful to Dr. Whitehouse, W., Eastburn, K. K., and Madara, J. L. (2000) J. Cell Sci. 113, Reiner Haseloff for help in the radioactivity experiments. 1771–1781 25. Stuart, R. O., and Nigam, S. K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, REFERENCES 6072– 6076 26. Lampe, P. D., TenBroek, E. M., Burt, J. M., Kurata, W. E., Johnson, R. G., and 1. Matter, K., and Balda, M. S. (2000) Semin. Cell Dev. Biol. 11, 281–289 Lau, A. F. (2000) J. Cell Biol. 149, 1503–1512 2. Gonzalez-Mariscal, L., Betanzos, A., and Avila-Flores, A. (2000) Semin. Cell Dev. Biol. 11, 315–324 27. Mitic, L. L., Schneeberger, E. E., Fanning, A. S., and Anderson, J. M. (1999) J. 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J. Biochem. 264, 374 –384 Tsukita, S. (1993) J. Cell Biol. 123, 1777–1788 8. Hirase, T., Staddon, J. M., Saitou, M., Ando-Akatsuka, Y., Itoh, M., Furuse, 32. Dodane, V., and Kachar, B. (1996) J. Membr. Biol. 149, 199 –209 33. Izumi, Y., Hirose, H., Tamai, Y., Hirai, S.-I., Nagashima, Y., Fujimoto, T., M., Fujimoto, K., Tsukita, S., and Rubin, L. L. (1997) J. Cell Sci. 110, 1603–1613 Tabuse, Y., Kemphues, K. J., and Ohno, S. (1998) J. Cell Biol. 143, 95–106 34. Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., and Cantley, L. C. 9. Saitou, M., Fujimoto, K., Doi, Y., Itoh, M., Fujimoto, T., Furuse, M., Takano, H., Noda, T., and Tsukita, S. (1998) J. Cell Biol. 141, 397– 408 (1997) J. Biol. Chem. 272, 952–960 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Protein Kinase C Regulates the Phosphorylation and Cellular Localization of Occludin

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 42, Issue of October 19, pp. 38480 –38486, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Protein Kinase C Regulates the Phosphorylation and Cellular Localization of Occludin* Received for publication, May 30, 2001, and in revised form, August 6, 2001 Published, JBC Papers in Press, August 13, 2001, DOI 10.1074/jbc.M104923200 Anna Y. Andreeva‡, Eberhard Krause‡, Eva-Christina Mu ¨ ller§, Ingolf E. Blasig‡¶, and Darkhan I. Utepbergenov‡ From the ‡Forschungsinstitut fu ¨ r Molekulare Pharmakologie, 13125 Berlin-Buch and §Charite ´, Humboldt Universita ¨t Berlin, 13092 Berlin, Germany Occludin is an integral membrane phosphoprotein spe- critical step in various cellular processes, including the estab- cifically associated with tight junctions, contributing to lishment of epithelial cell polarity and developmental pattern- the structure and function of this intercellular seal. Oc- ing (3). Previous data suggest that TJ function may be regu- cludin function is thought to be regulated by phosphoryl- lated by the phosphorylation of certain proteins (4 – 6). ation, but no information is available on the molecular Diacylglycerols have been shown to trigger the formation of TJ, pathways involved. In the present study, the involvement suggesting the involvement of protein kinase C; the identities of the protein kinase C pathway in the regulation of the of the molecular pathways involved remain, however, elusive phosphorylation and cellular distribution of occludin has (4). been investigated. Phorbol 12-myristate 13-acetate and The integral membrane protein occludin was recently iden- 1,2-dioctanoylglycerol induced the rapid phosphorylation tified as a component of TJ of epithelial and endothelial cells (7, of occludin in Madin-Darby canine kidney cells cultured 8). Occludin comprises four transmembrane domains, two ex- in low extracellular calcium medium with a concomitant tracellular loops, and three cytoplasmic domains (one intracel- translocation of occludin to the regions of cell-cell con- lular short turn, a short NH -terminal domain, and a long tact. The extent of occludin phosphorylation as well as its COOH-terminal domain). Accumulating evidence suggests incorporation into tight junctions induced by protein ki- that occludin plays an important role in tight junctions, al- nase C activators or calcium switch were markedly de- creased by the protein kinase C inhibitor GF-109203X. In though embryonic stem cells lacking occludin are able to form addition, in vitro experiments showed that the recombi- well developed TJs (9). The overexpression of mutant forms of nant COOH-terminal domain of murine occludin could be occludin in cultured epithelial cells leads to changes in the phosphorylated by purified protein kinase C. Ser of barrier and fence function (10, 11). The addition of synthetic occludin was identified as an in vitro protein kinase C peptides corresponding to the extracellular loops of occludin to phosphorylation site using peptide mass fingerprint anal- epithelial cells results in the disappearance of TJ and inhibi- ysis and electrospray ionization tandem mass spectros- tion of cell adhesion (12–14). Finally, occludin knock-out mice copy. These findings indicate that protein kinase C is show a complex phenotype, including retarded growth and involved in the regulation of occludin function at tight various histological abnormalities, suggesting that the func- junctions. tions of both TJ and occludin are more complex than was supposed previously (15). 1 Occludin has been shown to be highly phosphorylated (16, Tight junctions (TJs), the most apical component of the 17). Tight junction assembly induced by calcium switch is par- junctional complex of epithelial and endothelial cells, form a alleled by occludin phosphorylation and incorporation into the diffusion barrier limiting the flux of hydrophilic molecules TJ of MDCK cells (16, 17). It has also been shown that highly through the paracellular pathway and maintain cell polarity by phosphorylated occludin molecules are selectively concentrated acting as a boundary between the apical and basolateral at TJs, whereas non- or less phosphorylated occludin is local- plasma membrane domains (reviewed in Refs. 1 and 2). The ized in the cytoplasm (16). Occludin becomes phosphorylated dynamic rearrangement of cell-cell junctions including TJs is a and associates with ZO-1 during certain stages of mouse em- This is an open access article under the CC BY license. bryo development, and these processes are proposed to regulate * This work was supported in part by Deutsche Forschungsgemein- TJ biogenesis and the timing of blastocyst formation (18). In shaft Grants SFB 507 TP A2, DFG GK 238-2, DFG BL 308/6-1, and addition, recent results suggest that the phosphorylation of BMBF BEO 0311466C. The costs of publication of this article were occludin may regulate tight junction permeability in response defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 to histamine and lysophosphatidic acid (19). The findings above U.S.C. Section 1734 solely to indicate this fact. led to the conclusion that occludin function at TJ is regulated ¶ To whom correspondence should be addressed: Forschungsinstitut by phosphorylation. The molecular pathways regulating occlu- fu ¨ r Molekulare Pharmakologie, Bldg. 81, Rm. A2.06, Robert-Ro ¨ ssle-Str. 10, 13125 Berlin-Buch, Germany. Tel.: 49-30-94793321; Fax: 49-30- din phosphorylation remain unclear. 94793243; E-mail: [email protected]. In the present study, we analyzed the effect of phorbol 12- The abbreviations used are: TJ, tight junction; diC8, 1,2-dioctanoyl- myristate 13-acetate (PMA) and 1,2-dioctanoylglycerol (diC8), glycerol; ESI, electrospray ionization; FCS, fetal calf serum; LC, low activators of protein kinase C, on the phosphorylation and calcium medium; NC, normal calcium medium; MALDI, matrix- assisted laser desorption/ionization; MBP, maltose-binding protein; cellular localization of occludin in monolayers of MDCK cells MDCK, Madine-Darby canine kidney; PAGE, polyacrylamide gel elec- incubated in low extracellular Ca medium. PMA and diC8 trophoresis; PBS, phosphate-buffered saline; PKC, protein kinase C; induced a rapid phosphorylation of occludin and its redistribu- TOF, time of flight; MS, mass spectrometry; MS/MS, tandem mass tion to the regions of cell-cell contact. The phosphorylation and spectrometry; MEM, minimal essential medium; PMA, phorbol 12- myristate 13-acetate. incorporation of occludin into tight junctions induced by PMA, 38480 This paper is available on line at http://www.jbc.org Occludin Phosphorylation by PKC 38481 X-100-soluble fraction). The remaining material was scraped into lysis diC8, or calcium switch were inhibited by a PKC inhibitor. buffer (25 mM Hepes/NaOH, pH 7.4, 150 mM NaCl, 4 mM EDTA, 25 mM Furthermore, Ser of the recombinant COOH-terminal do- NaF, 1% SDS), boiled for 5 min, and centrifuged for 10 min at 14,000 main of murine occludin was found to be phosphorylated in g (the resulting extract is referred to as the Triton X-100-insoluble vitro by purified protein kinase C. These findings suggest that fraction). One-dimensional SDS-PAGE (8% gel) was performed accord- the regulation of phosphorylation and cellular distribution of ing to Laemmli (20). For immunoblotting, proteins separated by SDS- occludin are mediated by protein kinase C. PAGE were electrophoretically transferred to nitrocellulose sheets, which were then incubated with the antibodies. The antibodies were MATERIALS AND METHODS detected with a blotting detection kit. To detect protein phosphoryla- Reagents, Antibodies, and Cells—Protease inhibitors and protein tion, samples were blotted as above and the P signal was detected by A-Sepharose were from Sigma (Taufkirchen, Germany). Protein kinase autoradiography. Proteins transferred on the nitrocellulose membrane C, PMA, 1,2-dioctanoylglycerol, and GF-109203X were from Alexis were visualized by silver staining. Deutschland GmbH (Gruenberg, Germany). TRIzol reagent and flasks Occludin Immunoprecipitation and Alkaline Phosphatase Treat- on a slide were from Life Technologies GmbH (Technologiepark ment—MDCK cells cultured on 8.8-cm Petri dishes were washed twice Karlsruhe, Germany). Cy3- and horseradish peroxidase-conjugated with ice-cold PBS and extracted with 1 ml of ice-cold immunoprecipi- goat anti-rabbit monoclonal antibodies and rabbit polyclonal anti-occlu- tation buffer (25 mM Hepes/NaOH, pH 7.4, 150 mM NaCl, 4 mM EDTA, din antibodies were purchased from Zymed Laboratories Inc. (San 25 mM NaF, 1% Triton X-100, 1 M leupeptin, 0.3 M aprotinin, 0.1 mM Francisco, CA). MDCK cells were obtained from Dr. Swaroop (MDC, phenylmethylsulfonyl fluoride, 1 M pepstatin) for 30 min and collected Berlin, Germany). The pMAL fusion and purification system, BamHI, in 1.5-ml tubes. After centrifugation (13,000  g, 15 min, 4 °C), the and SalI were purchased from New England Biolabs GmbH (Frankfurt pellet was resuspended in 100 l of lysis buffer, heated 5 min at 95 °C, am Main, Germany). Taq polymerase, alkaline phosphatase, and Molo- and combined with 900 l of immunoprecipitation buffer. After centrif- ney murine leukemia virus reverse transcriptase were from Promega ugation (13,000  g for 15 min), the supernatant was pretreated with a GmbH (Mannheim, Germany). [- P]ATP and the blotting detection 15-l bed volume of protein A-Sepharose. For immunoprecipitation 1 g kit were from Amersham Pharmacia Biotech Europe GmbH (Freiburg, of anti-occludin antibodies and a 10-l bed volume of protein A-Sepha- Germany). rose were added to the supernatant and rotated for1hat4 °C. Beads Occludin Expression Construct and in Vitro Phosphorylation of Re- were washed five times with 1 ml of immunoprecipitation buffer and combinant Occludin—Total RNA was isolated from 50 mg of murine then resuspended in 100 l of phosphatase buffer (50 mM Tris/HCl, pH kidney using TRIzol reagent and reverse transcription was performed 9.3, 1 mM MgCl , 0.1 mM ZnCl ,1mM spermidine) with or without 20 2 2 using Moloney murine leukemia virus reverse transcriptase and ran- units of calf intestinal alkaline phosphatase. After1hof incubation at dom hexamer primers. A 777-base pair fragment encoding amino acids 37 °C, SDS-PAGE buffer was added and beads were boiled to elute the 264 –521 of murine occludin was amplified using the primers 5-AG- immunoprecipitates for subsequent PAGE. GATCCAAAACCCGAAGAAAGATGGATCGG-3, and 5-TTGTCGAC- Identification of Phosphorylation Sites by Mass Spectrometry—For TAAGGTTTCCGTCTGTCATAGTC-3 (BamHI and SalI sites are un- MALDI analysis, 15 l of occludin solution taken from the phosphoryl- derlined). The amplified product was cloned using the TOPO-TA ation reaction were resolved by SDS-PAGE. The proteins were detected cloning kit (Invitrogen, Carlsbad, CA). Several clones were sequenced by staining with a Colloidal Blue staining kit (Novex, San Diego, CA). (Taq DyeDeoxy-Terminator cycle sequencing kit, Applied Biosystems, Phosphorylated and non-phosphorylated occludin bands were excised Weiterstadt, Germany); a clone containing no mutations was selected, from the stained gels, washed with 50% (v/v) acetonitrile in 25 mM and its BamHI-SalI fragment was subcloned into the pMAL-c2x plas- ammonium bicarbonate, dehydrated in acetonitrile, and dried in a mid to produce a plasmid coding for the COOH-terminal domain of vacuum centrifuge. Disulfide bonds were reduced by incubation in 30 l occludin fused with maltose-binding protein. The fusion protein was of 10 mM dithiothreitol in 100 mM ammonium bicarbonate for 45 min at overexpressed in Escherichia coli and purified over an amylose column 55 °C. Alkylation was performed by replacing the dithiothreitol solution according to the manufacturer’s instructions. with 55 mM iodoacetamide in 100 mM ammonium bicarbonate. After a The in vitro phosphorylation of occludin was performed as follows. 20-min incubation at 25 °C in the dark, the gel pieces were washed with Purified recombinant occludin fragment (5 g) was mixed with 16 ng of 50 –100 l of 50% (v/v) acetonitrile in 25 mM ammonium bicarbonate, protein kinase C in 50 mM Tris/HCl buffer, pH 7.5, containing 10 mM dehydrated in acetonitrile, and dried in a vacuum centrifuge. The gel MgCl ,2mM CaCl ,1mM dithiothreitol, 0.2 mM ATP, 5 nM PMA, and 5 pieces were re-swollen in 10 lof5mM ammonium bicarbonate, con- 2 2 Ci of [- P]ATP in a reaction volume of 40 l. In other experiments taining 300 ng of endoproteinase Lys-C (sequencing grade, Roche Di- GF-109203X at a concentration of 5 M was used to inhibit PKC. agnostics, Mannheim, Germany). After 15 min, 5 lof5mM ammonium Phosphorylation of MBP--galactosidase (the expression product of the bicarbonate was added to keep the gel pieces moist during Lys-C cleav- pMal-c2x plasmid) was performed as for occludin, but double the age (37 °C, overnight). To extract the peptides, 15 l of 0.5% (v/v) amount of PKC was used. trifluoroacetic acid in acetonitrile was added, and the samples were Cell Culture, Low Calcium Medium Culture, and Calcium Switch— sonicated for 5 min. The separated liquid was dried under vacuum and MDCK cells were cultured in MEM with 10% FCS. Low Ca medium redissolved in 10 l of 0.1% (v/v) trifluoroacetic acid in water. The (LC) was prepared from S-MEM (calcium-free MEM) and 5% FCS peptides were purified over a C18 reversed-phase minicolumn filled in pretreated with Chelex resin (Bio-Rad Laboratories GmbH, Mu ¨ nchen, a micropipette tip (ZipTip C18, Millipore, Bedford, MA) for mass spec- Germany) as described in Ref. 19. Confluent monolayers of MDCK cells trometry analysis. Purification was performed according to the manu- were grown in normal calcium (NC) medium (MEM with 1.8 mM CaCl facturer’s manual, except that peptides were eluted with 3 lof60% and 5% FCS), washed twice with PBS, and then transferred to low Ca (v/v) acetonitrile, 0.3% (v/v) trifluoroacetic acid or with 5 l of 60% (v/v) medium for 20 h prior to the addition of normal calcium medium, PMA, acetonitrile, 0.2% (v/v) formic acid for matrix-assisted laser desorption/ or diC8. In other experiments GF-109203X at a concentration of 5 M ionization mass spectrometry (MALDI-MS) and nanoelectrospray tan- was added 30 min before the addition of normal calcium medium (or low dem mass spectrometry (nanoESI-MS/MS), respectively. calcium medium with PKC activators) containing 5 M GF-109203X. MALDI-MS measurements were performed on a Voyager-DE STR Immunofluorescence Microscopy—Cells were grown in the flasks on BioSpectrometry work station MALDI-TOF mass spectrometer (Per- slides, washed with PBS and fixed in 1% paraformaldehyde in PBS for septive Biosystems, Inc., Framingham, MA). One l of the analyte 15 min. After three washes with PBS, cells were permeabilized with solution was mixed with 1 lof -cyano-4-hydroxycinnamic acid matrix 0.2% Triton X-100 in PBS, soaked in blocking solution (1% bovine solution consisting of 10 mg of matrix dissolved in 1 ml of 0.3% triflu- serum albumin in PBS) for 60 min, and then incubated with 300 lof oroacetic acid in acetonitrile-water (1:1, v/v). One l of the resulting anti-occludin antibodies (diluted 100 times). Samples were washed mixture was applied to the sample plate. Samples were air-dried at three times with 0.2% bovine serum albumin and then incubated for 60 ambient temperature (24 °C). Measurements were performed in the min with Cy3-conjugated goat anti-rabbit antibodies. After four washes reflection mode at an acceleration voltage of 20 kV, 70% grid voltage, coverslips were mounted over the cells with the aid of mounting me- and a delay of 200 ns. Each spectrum obtained was the mean of 256 dium (Shandon, Pittsburgh, PA). Samples were examined using a flu- laser shots. orescence microscope equipped with the AxioVision image acquisition For nanoESI-MS/MS, 5 l of the peptide mixture were lyophilized and analysis system (Carl Zeiss, Jena, Germany). and dissolved in 5 l of methanol, 1% formic acid (1:1, v/v). The MS/MS Protein Blotting and Analysis—Cells were washed twice with PBS measurements were performed with a nanoelectrospray hybrid quadru- and extracted for 30 min with PBS containing 1% Triton X-100, 1 M pole mass spectrometer (Q-Tof, Micromass, Manchester, United King- leupeptin, 0.3 M aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, and dom). The collision gas was argon at a pressure of 6.0  10 millibars 1mM pepstatin at 4 °C (the resulting extract is referred to as the Triton in the collision cell. 38482 Occludin Phosphorylation by PKC FIG.2. PMA and diC8 induce the phosphorylation of occludin in MDCK cells incubated in low Ca medium. Panel A, immuno- blot analysis of occludin in the Triton X-100-soluble (S) and -insoluble (I) fractions of MDCK cells incubated in normal Ca medium (lane 1) or in low Ca medium (lane 2). MDCK cells cultivated in low calcium medium were subjected to a calcium switch (lane 3) or treated with 2 nM (lane 4), 25 nM PMA (lane 5), or 0.5 mM diC8 for4h(lane 6). Treatment of MDCK cells cultivated in low calcium medium with PMA or diC8 induced an upward band shift. B, immunoblot analysis of the Triton X-100-insoluble fraction of immunoprecipitated occludin from MDCK cells cultivated in low calcium medium and stimulated with 10 nM PMA for 20 min. Samples were either left untreated (lane 1) or subjected to treatment with alkaline phosphatase (lane 2), resolved by electrophore- sis, and then analyzed by immunoblotting. Alkaline phosphatase treat- ment completely abolished the upward band shift caused by PMA treatment. The arrow shows the position of non-phosphorylated occlu- din, the dash shows the position of antibody heavy chain, and a bracket denotes the position of multiple phosphorylated forms of occludin. the occludin was translocated to the plasma membrane at regions of cell-cell contact (Fig. 1C, arrows). Staining of occlu- din at cell-cell contacts was not as continuous or intensive as in the cells grown in NC (Fig. 1A). Prolongation of the incubation period up to 4 h further increased the amount of occludin translocated to the regions of cell-cell contact (Fig. 1D). When 25 nM PMA was used to stimulate cells, occludin translocated to the periphery showed a more discontinuous and intensive staining as compared with samples treated with 2 nM PMA (Fig. 1E). Most of the occludin was still localized in the cyto- FIG.1. PMA and diC8 induce the translocation of occludin plasm, a significant portion in large granular structures (Fig. from the cytoplasm to the lateral cell membrane in MDCK cells 2 1E, arrowheads). Prolongation of the incubation time up to 4 h incubated in low Ca medium. MDCK cells were incubated in low did not change the distribution pattern significantly (Fig. 1F), calcium medium for 20 h after which PMA, diC8, or Ca was added. Cells cultivated in normal calcium medium show continuous staining of although the decrease in the amount of occludin localized at occludin at cell-cell contacts (A). After cultivation of cells in low calcium cell-cell contacts was notable. The treatment of MDCK cells in medium, the occludin signal was detected mainly in small granular LC with the another protein kinase C activator diC8 resulted in structures in the cytoplasm (B). Two hours after the addition of 2 nM the redistribution of occludin, which was similar to the effect of PMA, staining of occludin at cell borders (arrows) was evident (C). Prolongation of the incubation period to 4 h further increased the 2nM PMA. Fragmentary staining of occludin at cell-cell con- amount of occludin migrating to the cell borders (D). When 25 nM PMA tacts was evident 2 h after the addition of diC8 (Fig. 1G). The was used to stimulate the cells for 2 h, large granular structures were amount of occludin translocated to cell-cell contacts further found in the cytoplasm (arrowheads) and occludin staining at the cell increased when the incubation time was prolonged up to 4 h borders became discontinuous (E). Prolongation of the incubation pe- riod (25 nM PMA) up to 4 h led to some decrease in the amount of (Fig. 1H). occludin found at cell-cell contacts (F). Two hours after the addition of Effect of PKC Activators on the Triton X-100 Solubility and 0.5 mM diC8, staining of occludin at cell borders was evident (G). Phosphorylation of Occludin in MDCK Cells in LC—Previous Prolongation of the incubation period to 4 h further increased the studies have shown that occludin localized in TJ cannot be amount of occludin at cell borders (H). Bar,10 m. extracted with non-ionic detergents from confluent epithelial cells and that it shows a decreased electrophoretic mobility due RESULTS to extensive phosphorylation (16, 17, 21). In accordance with Effect of PKC Activators on the Localization of Occludin in literature data (17), occludin extracted with Triton X-100 from MDCK Cells in Low Calcium Medium—In view of the fact that MDCK cells grown in NC migrated as a single band with an diacylglycerols are known to induce the formation of tight apparent molecular mass of 60 kDa, whereas that from the junction strands in MDCK cells cultivated in low calcium me- Triton X-100-insoluble fraction migrated as multiple unre- dium (4), we assumed that occludin phosphorylation and re- solved bands with apparent molecular masses ranging between cruitment into tight junctions are mediated by PKC. To verify 60 and 82 kDa (Fig. 2A, lane 1). When MDCK cells were this hypothesis, we investigated the effect of PKC activators on incubated in LC in order to down-regulate TJs, the high mo- the phosphorylation and cellular distribution of occludin in lecular mass bands disappeared from the Triton X-100-insolu- MDCK cells cultured in low calcium medium, which disrupts ble fraction and the amount of Triton X-100-insoluble occludin tight junctions. In accordance with literature data (7, 10), oc- decreased significantly (Fig. 2A, lane 2). When TJ re-assembly cludin was intensively stained at cell-cell contacts in confluent was induced by calcium switch, the banding pattern of Triton MDCK cells in normal Ca medium, indicating that continu- X-100-insoluble occludin was restored (Fig. 2A, lane 3). Four ous TJs were formed (Fig. 1A). After incubation of the cells in hours after the addition of 2 nM PMA to the cells in LC, the LC, occludin became localized almost exclusively in the cyto- amount of Triton X-100-insoluble occludin increased signifi- plasm, indicating a down-regulation of TJs (Fig. 1B). When cantly and a portion of occludin underwent an upward band PMA at 2 nM was added for2htothe cells in LC, a portion of shift (Fig. 2A, lane 4). Treatment of cells in LC with 25 nM PMA Occludin Phosphorylation by PKC 38483 FIG.3. Time and concentration dependence of occludin phos- phorylation in MDCK cells upon treatment with PMA. Figure shows immunoblot analysis of occludin in the Triton X-100-insoluble fraction of MDCK cells. After incubation of cell monolayers in low calcium medium for 20 h, PMA at the indicated concentrations was added for1h(A)or25nM PMA was added for the indicated periods of time (B). for 4 h resulted in an additional increase in the amounts of FIG.4. Effects of the PKC inhibitor GF-109203X on the redis- Triton X-100-insoluble occludin and in the degree of upward tribution of occludin during tight junction assembly induced by band shift, with a concomitant decrease in the amount of Triton Ca switch or PKC activators. Figure shows immunofluorescence X-100-soluble occludin (Fig. 2A, lane 5). The decrease is prob- localization of occludin 4 h after addition of calcium (A and B), PMA (C and D), or diC8 (E and F) to MDCK cells placed in low calcium medium. ably due to increased proteolysis, since high concentrations of 5 M GF-109203X was added (B, D, and F) to evaluate the involvement PMA are known to cause degradation of occludin (21, 22). An of PKC in the redistribution of occludin. Bar,10 m. increase in Triton X-100-insoluble occludin and an upward band shift were also evident in cells treated with 0.5 mM diC8 for 4 h (Fig. 2A, lane 6). switching to NC, continuous occludin staining was evident at The multiple bands revealed by immunoblotting analysis intercellular contacts, indicating the recruitment of occludin probably represent differently phosphorylated occludin mole- into tight junctions (Fig. 4A). Inhibition of PKC with 5 M cules. In order to show that the upward band shift induced by GF-109203X resulted in very weak staining of occludin at the PMA was due to phosphorylation, MDCK cells were treated intercellular contacts but a prominent staining of occludin in with PMA and occludin immunoprecipitated from the Triton cytoplasm (Fig. 4B). It can be concluded from these data that X-100-insoluble fraction was subjected to in vitro phosphatase PKC participates in the targeting of occludin into TJs. Further- treatment. Fig. 2B shows that the additional high molecular more, GF-109203X inhibited the redistribution of occludin to weight occludin bands disappeared on alkaline phosphatase the regions of cell-cell contacts induced by PMA (Fig. 4, C and treatment. Therefore, the upward band shift of occludin in- D) and diC8 (Fig. 4, E and F). duced by PMA is due to phosphorylation of occludin, and In order to study the involvement of PKC in the phosphoryl- changes in the cellular distribution of occludin induced by the ation of occludin, the effect of GF-109203X on calcium switch- treatment of MDCK cells in LC by PMA or diC8 are paralleled induced occludin phosphorylation was investigated. The char- by its phosphorylation. acteristic multiple-band pattern of occludin is seen on the Fig. 3 shows the time and concentration dependence of PMA- immunoblot in Triton X-100-insoluble fractions of MDCK cells induced phosphorylation of occludin. As high molecular weight incubated in NC medium (Fig. 5, NC). High molecular weight bands corresponding to phosphorylated occludin were invari- bands disappeared when cells were incubated in LC medium, ably found to be Triton X-100-insoluble, only Triton X-100- indicating the dephosphorylation of occludin (Fig. 5, LC). Four insoluble fractions were analyzed. Addition of PMA to MDCK hours after switching to NC, a profound upward band shift of cells incubated in LC medium resulted in a dose-dependent occludin was visible on the immunoblot, reflecting the in- phosphorylation of occludin (Fig. 3A). When PMA was added creased phosphorylation of occludin (Fig. 5, Ca ). When the for 1 h, the phosphorylation was evident at 2.5 nM PMA and calcium switch was performed in the presence of 5 M GF- reached a maximum at 40 nM PMA. The time dependence of 109203X, the extent of occludin phosphorylation was markedly occludin phosphorylation in MDCK cells in LC induced by 25 decreased, indicating the involvement of PKC. Similarly, the nM PMA is shown on Fig. 3B. The upward band shift became increase in phosphorylation of occludin induced by PMA and evident after 5 min and reached a maximum 40 min after PMA diC8 was significantly decreased by GF-109203X, again indi- addition. cating the involvement of PKC (Fig. 5, PMA and diC8). Phosphorylation and Redistribution of Occludin Induced by Occludin as a Substrate of PKC—To investigate whether Ca Switch PMA and diC8 Is PKC-dependent—The striking occludin may serve as a substrate for protein kinase C, recom- effects of the PKC activators PMA and diC8 on the phospho- binant occludin (MBP fusion protein of the soluble cytoplasmic rylation and distribution of occludin suggest that PKC may be COOH-terminal domain of murine occludin or MBP-occludin) a physiological regulator of occludin function. To investigate was incubated in vitro with purified PKC (a mixture of , I, the involvement of PKC in the regulation of occludin incorpo- II, and  isoforms of PKC) in the presence of [- P]ATP. As ration into TJs, the assembly of tight junctions was induced by shown on Fig. 6 (lane 1), an autoradiography signal was de- the calcium switch procedure in the presence or absence of the tected corresponding to a protein of 74 kDa (the predicted PKC inhibitor GF-109203X and occludin localization was as- molecular mass of MBP-occludin is 73.6 kDa), implying that sessed by immunofluorescence microscopy. Four hours after MBP-occludin was phosphorylated by protein kinase C. No 38484 Occludin Phosphorylation by PKC DISCUSSION Occludin phosphorylation has been implicated in the regula- tion of tight junction function (16, 19, 21), but the molecular pathways involved remain unclear. The present study estab- lishes the importance of the protein kinase C pathway in the phosphorylation and cellular localization of occludin. FIG.5. PKC inhibitor GF-109203X decreases the phosphoryla- 2 The redistribution of occludin from the cytoplasm to the tion of occludin during tight junction assembly induced by Ca lateral plasma membrane has been observed after the treat- switch or by PKC activators. Figure shows immunoblot analysis of occludin in Triton X-100-insoluble fractions of MDCK cells. Cells were ment of MDCK cells in LC with 2 nM PMA or 0.5 mM of more harvested after cultivation in NC or in LC. Normal calcium medium specific PKC activator diC8. This probably reflects the inser- (Ca ), 2.5 nM PMA (PMA), or 0.5 mM diC8 (diC8) were added to tion of occludin into TJs, since the formation of tight junction the cells in LC either with or without 5 M GF109203X (inh.). fibrils was reported under similar conditions (4). The amount of occludin translocated to the lateral plasma membrane in- creases with PMA concentration, but the staining of occludin becomes discontinuous. This observation is in agreement with studies reporting the disruption of TJs and increase in occludin proteolysis in response to the treatment of MDCK cells with very high (0.1–1 M) concentrations of PMA (21, 22). The results presented here clearly indicate that a Triton X-100-insoluble pool of occludin is phosphorylated upon the addition of the protein kinase C activators PMA or diC8 to FIG.6. Protein kinase C phosphorylates the COOH-terminal MDCK cells incubated in low calcium medium. Previously, the domain of recombinant occludin. MBP-occludin was incubated with 32 time course of occludin phosphorylation induced by a calcium PKC in a buffer containing [- P]ATP followed by SDS-PAGE, transfer onto nitrocellulose membrane, silver staining, and autoradiography. An switch was shown to be paralleled by TJ formation (16). In this autoradiography signal was visible at 74 kDa, indicating that MBP- study, a low but easily distinguishable level of upward band occludin is phosphorylated by PKC (lane 1). No signal was detected in shift of occludin caused by the 4-h incubation of MDCK cells control samples from which occludin (lane 2) or protein kinase C (lane with 2 nM PMA correlates well with the low amounts of occlu- 3) were omitted. The phosphorylation was strongly inhibited by 5 M din translocated to the lateral cell membrane. Correspond- GF-109203X (lane 4). The control protein MBP--galactosidase was only weakly phosphorylated, although the amount of PKC was doubled ingly, the increased phosphorylation caused by 25 nM PMA is (lane 5). paralleled by the increased amounts of occludin translocated to TJs. These observations support the assumption that the phos- phorylation of occludin in response to PMA or diC8 is involved autoradiography signal was detected when MBP-occludin or in the regulation of TJ assembly. The degree and rate of occlu- PKC was omitted (Fig. 6, lanes 2 and 3), excluding the possi- din phosphorylation induced by PMA are the same or even bilities that the autoradiography signal was due to autophos- higher compared with those induced by the calcium switch; phorylation of PKC or to any kinase activity that might have phosphorylation is induced by rather low concentrations of co-purified with MBP-occludin. The phosphorylation was PMA, indicating that diacylglycerols, along with calcium ions, strongly inhibited by 5 M GF-109203X (Fig. 6, lane 4). Another are physiological regulators of occludin phosphorylation. High MBP fusion protein, MBP--galactosidase, with a predicted molecular weight forms of occludin that appeared after the molecular mass of 53.5 kDa was phosphorylated to a much treatment of the cells in LC by PMA or diC8 were invariably lower extent, indicating the relative specificity of occludin not extractable with Triton X-100. It can be concluded that the phosphorylation by PKC (Fig. 6, lane 5). phosphorylation regulates the association of occludin with the To determine which sites of occludin are phosphorylated by cytoskeleton and/or detergent-resistant membrane microdo- PKC, occludin was analyzed by in-gel digestion using chymo- mains (24). Future investigations should clarify which deter- trypsin, trypsin, Asp-N, or Lys-C, followed by peptide mass gent-insoluble structures interact with occludin and how the fingerprinting by matrix-assisted laser desorption ionization phosphorylation of occludin regulates these interactions. time-of-flight (MALDI-TOF) mass spectrometry. Comparison of Both the phosphorylation of occludin and its incorporation the peptide mass fingerprints of phosphorylated and non-phos- into tight junctions induced by calcium switch were markedly inhibited by the PKC inhibitor GF-109203X. These observa- phorylated occludin revealed a distinct mass peak at m/z tions indicate that PKC participates in the regulation of occlu- 1256.532 (theoretical m/z 1256.535) corresponding to the phos- 333 341 din function during tight junction assembly induced by calcium phorylated fragment RSYPESFYK after Lys-C digestion switch. Previously, the application of the PKC inhibitor has of occludin treated with PKC (Fig. 7a). This peak was not been shown to inhibit the development of transepithelial elec- present when PKC was omitted (Fig. 7b). The site of phospho- trical resistance, a functional measure of tight junction biogen- rylation within the phosphorylated occludin fragment was de- esis (25). Occludin, the only known transmembrane protein of termined by nanoelectrospray ionization tandem mass spec- TJ whose localization in tight junctions is dependent on its trometry (ESI-MS/MS). Based on the double charge ion with phosphorylation state, may be a key target in the PKC-medi- m/z 628.78, the spectrum (Fig. 8) shows a peak corresponding ated regulation of tight junctions. Other authors have noted to the neutral loss of H PO (98 Da). The evidence for the 3 4 similarities between occludin and connexins in terms of phos- phosphorylation of Ser comes from the mass of the b and b 2 3 phorylation and structure (16). The data presented here extend ions corresponding to the non-phosphorylated NH -terminal the similarities further; connexin43 is phosphorylated by PKC part, whereas the ions with a mass greater than 700 Da appear in response to PMA, and the phosphorylation dramatically as b -98, b -98, b -98, and y -98. No evidence of a concurrent 6 7 8 6 changes gap junction channel conductivity (26). Possibly, the phosphorylation was found at any other position. From mass assembly of the junctional complex is regulated by PKC via spectrometry data, it can be concluded that Ser of mouse the phosphorylation of connexin43 and occludin to coordinate occludin is phosphorylated by protein kinase C in vitro. the formation of a functionally active barrier and the function Occludin Phosphorylation by PKC 38485 FIG.7. Peptide mass fingerprint analysis of in vitro phosphorylated and non-phosphorylated occludin. Proteins were in-gel digested with Lys-C; the peptides were extracted, purified over a C18 reversed-phase minicolumn, mixed with an -cyano-4-hydroxycinnamic acid matrix, and analyzed by MALDI-TOF-MS. Upper and lower panels show portions of spectra of the in vitro phosphorylated (a) and non-phosphorylated occludin (b). The peaks with m/z 1256.532 (theoretical m/z 1256.535) and 1176.575 (theoretical m/z 1176.569) correspond, respectively, to the phosphorylated and non-phosphorylated sequence RSYPESFYK. FIG.8. Identification of Ser as an in vitro phosphorylation site. MS/MS spectrum of the peptide RSYPEpSFYK (pS, phosphoserine residue) with the dou- ble charged ion m/z  628.78 Da. The base peak with m/z  579.73 Da results from the neutral loss of H PO (98 Da). 3 4 Parts of the spectrum are magnified by 2 to aid the observation of lower abundance product ions. Relevant ions are labeled according to the accepted nomenclature (23). Both the b ions and the y ions in the spectrum that contain the phosphoserine are produced by consecutive fragmenta- tion reactions, whereby the amide bond is broken and H PO is lost, or vice versa. 3 4 The masses of the b and b ions corre- 2 3 spond to the unphosphorylated NH -ter- minal part, whereas the ions with a mass greater than 700 Da confirm the phospho- rylation of Ser . of intercellular channels. The phosphorylation and redistribu- terminal domain of occludin in vitro, whereas a number of tion of occludin induced by PKC activators were strongly in- tested kinases did not phosphorylate occludin (31). However, hibited by the application of GF-109203X. These observations there are no indications of the involvement of casein kinase 2 in further substantiate the specificity of the regulation of occludin the regulation of TJ functions. functions by PKC. Despite the use of four different proteases to produce various The simplest explanation for the observed occludin phospho- peptides of occludin, Ser of occludin was the only phospho- rylation induced by PMA or diC8 is that PKC directly phospho- rylation site for classical PKCs that was positively identified by rylates occludin when stimulated by PMA, although the possi- MS. On the other hand, the presence of multiple phosphoryl- bility that PKC activates an intermediate kinase(s) cannot be ated forms of occludin in vivo was demonstrated by two-dimen- ruled out. Therefore, the in vitro phosphorylation of the recom- sional gel electrophoresis (19). The appearance of multiple binant occludin COOH-terminal domain by PKC was investi- phosphatase-sensitive occludin bands resulting from treatment gated. The carboxyl-terminal domain of occludin has been re- of MDCK cells with PMA indicates the multiple phosphoryla- cently shown to be responsible for the targeting of occludin to tion of occludin. Whereas it is possible that some phosphoryl- the TJs (27) and for its interaction with other proteins (28, 29). ation sites might not have been identified by MS, the absence of The results suggest that the COOH-terminal domain of occlu- additional phosphorylation sites suggests that other kinase(s) din may be phosphorylated by classical isoforms of PKC, which must phosphorylate occludin in vivo. These may include non- are known to be expressed in MDCK cells (30). To date, only classical PKC isoforms, some of which are known to be associ- casein kinase 2 has been reported to phosphorylate the COOH- ated with TJs (32, 33) and which have different substrate 38486 Occludin Phosphorylation by PKC 10. Balda, M. S., Whitney, J. A., Flores, C., Gonzalez, S., Cereijido, M., and Matter, sequence motifs from those of classical PKCs (34), or an inter- K. (1996) J. Cell Biol. 134, 1031–1049 mediate kinase(s) which is activated by PKC-dependent phos- 11. Bamforth, S. D., Kniesel, U., Wolburg, H., Engelhardt, B., and Risau, W. (1999) J. Cell Sci. 112, 1879 –1888 phorylation and subsequently phosphorylates occludin at dif- 338 12. Wong, V., and Gumbiner, B. M. (1997) J. Cell Biol. 136, 399 – 409 ferent positions. Ser is located within a 100-amino acid 13. Lacaz-Vieira, F., Jaeger, M. M., Farshori, P., and Kachar, B. (1999) J. Membr. stretch of occludin, which was shown to be dispensable for the Biol. 168, 289 –297 14. Van Itallie, C. M., and Anderson, J. M. (1997) J. Cell Sci. 110, 1113–1121 binding to ZO-1 (29) and for the targeting of occludin to TJs 15. Saitou, M., Furuse, M., Sasaki, H., Schulzke, J. D., Fromm, M., Takano, H., (27). Therefore, the significance of the phosphorylation on Noda, T., and Tsukita, S. 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Published: Oct 1, 2001

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