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First published online 19 February 2008
doi: 10.1242/jcs.014878

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G12 regulates protein interactions within the MDCK cell tight junction and inhibits tight-junction assembly

Ernesto Sabath^1,*, Hideyuki Negoro^1,*, Sarah Beaudry¹, Manuel Paniagua¹, Susanne Angelow², Jagesh Shah¹, Nicholas Grammatikakis³, Alan S. L. Yu² and Bradley M. Denker^1,

¹ Renal Division, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA
² Nephrology Division, University of Southern California, Los Angeles, CA 90089, USA
³ Institute of Biology, National Centre for Scientific Research `Demokritos', Athens, Greece

View larger version (84K): [in this window] [in a new window]   Fig. 1. Src is a component of the TJ complex. (A) Western blots for ZO-1 and total Src after ZO-1 immunoprecipitation from QL12-MDCK cells in +/–dox conditions. ZO-1 hybridoma was added undiluted to an equivalent amount of cell lysates prepared as described in the Materials and Methods. Negative controls were rat ascites using IgG beads. Arrows indicate the ZO-1 and Src bands. (B) Lysates from G12- and QL12-MDCK cells (+/–dox) were immunoprecipitated with ZO-1 and probed with antibodies against active Src (pTyr419) and ZO-1. (C) Confocal microscopy of G12-MDCK cells +/–dox double stained for ZO-1 and Src as described in the Materials and Methods. (Ca,Ch) White box indicates the cell that was examined at higher magnification in the panels to the right. In x-y and lat-z panels, a region of the membrane was identified (white enclosure) for additional analysis. The lat-z images represent the apical 5 µm region of the cell (total height approximately 8 µm). (Ca-Cg) +dox; (Ch-Cn) –dox. FITC-ZO-1, Texas-red c-Src and merged images are shown for both conditions in x-y and lat-z orientation. Scale bars: 10 µm (Ca,Ch), 5 µm (Cb-Cg,Ci-Cn).

View larger version (47K): [in this window] [in a new window]   Fig. 2. ZO-1 and ZO-2 are tyrosine phosphorylated in QL12-MDCK cells by Src. Immunoprecipitations of the indicated TJ proteins were performed as described in the Materials and Methods followed by western blotting with 4G10 antibody. Identical amounts of lysate from QL12-MDCK cells +/–dox for 72 hours were used for each precipitation. Immunoprecipitations were repeated from cells cultured in PP2 (10 µM) or genistein (2 µM), which were added to the medium at the time of medium change to –dox. The intensity of the phosphotyrosine (pTyr) band (arrows) in each condition is normalized to the amount of protein in the precipitate. This fraction was divided by the fraction of pTyr detected in +dox cells with no inhibitor condition. Bar graphs on the right summarize the results from three experiments. (A) ZO-1; (B) ZO-2; (C) occludin; and (D) ZO-1 in G12-MDCK +/–dox. *P<0.05.

View larger version (36K): [in this window] [in a new window]   Fig. 3. G12/Src activation disrupts interactions of claudin 1 and occludin with ZO-1. (A) Specificity of ZO-1 immunoprecipitations. G12-MDCK cells were cultured +/–dox +/–PP2 and immunoprecipitated with rat monoclonal R46 antibody or the equivalent amount of ascites control. Western blot for ZO-1 in cell lysates and immunoprecipitations using the identical antibody is shown. (B) G12- and QL-12 MDCK cells +/–dox were immunoprecipitated with ZO-1 antibody as described in the Materials and Methods. Immunoprecipitates were analyzed by western blotting to ZO-1 followed by reprobing with antibodies to other TJ proteins (ZO-2, occludin and claudin 1). A parallel analysis was performed in QL12-MDCK cells pre-treated with the Src inhibitor PP2 (10 µM) added at the time of switch to –dox medium. (C) Bar graphs summarizing the fraction of TJ protein remaining associated with ZO-1. The results are the mean ± s.e.m. of three experiments normalized to the ZO-1 immunoprecipitates (+dox). *P<0.05.

View larger version (51K): [in this window] [in a new window]   Fig. 4. Hsp90 is necessary for G12/Src regulation of permeability and TJ structure. (A) QL12-MDCK cells in +/–dox and +/–GA (2 µM) for 72 hours were analyzed for TER as described in the Materials and Methods. (B) Sodium and chloride permeability were determined in QL12-MDCK cells +/–dox and +/–GA as described in Materials and Methods. (C) Effect of GA in QL12-MDCK cells +/–dox on size selectivity for cation permeability relative to sodium [lithium (Li; 1.2 Å), potassium (K; 2.66 Å), rubidium (Rb; 2.96 Å), cesium (Cs; 3.38 Å) and arginine hydrocholoride (6.96 Å)]. (D) Immunofluorescence microscopy of ZO-1, occludin and claudin 1 in QL12-MDCK cells +/–dox and –dox +GA. Arrow highlights area of membrane thickening. *P<0.05. Scale bar: 5 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 5. Hsp90 is a component of the activated G12 complex and is necessary for Src activation and ZO-2 phosphorylation. (A) Src activation in QL12-MDCK cells +/–dox and +/–GA. Identical amounts of cell lysate were analyzed by western blotting with antibodies specific for pTyr-419 as described in the Materials and Methods. Membranes were stripped and reanalyzed for total Src and occludin. (B) Band intensity was quantified as described above and the pTyr-419 immunoreactivity normalized to total Src. The relative amount of pTyr419 for –dox and –dox +GA is shown in comparison to the +dox condition. (C) Phosphotyrosine content of immunoprecipitated ZO-2 from QL12-MDCK cells +/–dox and +/–GA. ZO-2 was immunoprecipitated and analyzed with 4G10 anti-phosphotyrosine as described above. (D) Interaction of G12 with ZO-1 in QL12-MDCK cells +/–dox and +/–GA. ZO-1 was immunoprecipitated under the specified conditions and precipitates were analyzed for G12 by western blotting. Control is lysate with beads alone. (E) Steady-state protein levels of TJ proteins and G12 in QL12-MDCK +/–dox and +/–GA (2 µM). (F) Validation of GST-TPR pull-down of activated G12. QL12- and G12-MDCK cells were cultured in –dox, and lysates (800 µg) were pulled-down with 1 µg GST or GST-TPR, as described in the Materials and Methods, and analyzed by western blotting for G12. 10% of the input of wild-type G12 is shown in the lysate lane. (G) Hsp90 western blot of GST-TPR pull-downs from G12- and QL12-MDCK cells in –dox.

View larger version (38K): [in this window] [in a new window]   Fig. 6. Endogenous G12 is activated during TJ assembly in the calcium switch and thrombin delays TJ assembly. (A) Calcium switch in control G12-MDCK cells +dox. Cells were cultured overnight in low calcium (LC; t=0) medium and then switched back to normal calcium (NC) medium. TER measurements were obtained at the specified intervals. Thrombin (2 U/ml) was added at t=0. Results are the mean ± s.e.m. for n=4 at each time point. The experiment was repeated three times with nearly identical results. (B) Western blot of G12 +dox cells +/–thrombin analyzed by GST-TPR pull-down at the specified times after the calcium switch. (C) Calcium switch in G12-MDCK cells with induced G12 expression (–dox). (D) Western blot of G12 –dox cells +/–thrombin analyzed by GST-TPR pull-down at the specified times after the calcium switch. The first lane of the top panel shows the induced G12 and is 10% of the protein used for the pull-downs. (E) Western blots for G13 on G12-MDCK cells +/–dox and +/–thrombin and pulled-down with GST-TPR as described in C. For each panel, the +/–thrombin results were obtained from a single gel.

View larger version (21K): [in this window] [in a new window]   Fig. 7. Bradykinin activates G13 without affecting TJ assembly. (A,B) Calcium-switch experiments in G12-MDCK cells (A, +dox; B, –dox) +/–bradykinin (100 µM) at t=0 as described in Fig. 6. (C) GST-TPR pull-downs from bradykinin-stimulated cells at the specified times after the calcium switch in G12-MDCK cells +/–dox. Western blots were probed for G12 and were reprobed for G13 (designated by arrows). The bradykinin-stimulated cells (+/–dox) were analyzed on a single blot.

View larger version (25K): [in this window] [in a new window]   Fig. 8. Inhibiting Src with PP2 and Hsp90 with GA blocks thrombin-stimulated delay in TJ assembly. (A) Calcium switch in +dox G12-MDCK cells +/–thrombin and +/–PP2 (10 µM) added at t=0. (B) Calcium switch in +dox G12-MDCK cells +/–thrombin and +/–GA (2 µM) added at t=0.

View larger version (28K): [in this window] [in a new window]   Fig. 9. Thrombin stimulates the loss of barrier function in intact monolayers through G12, Src and Hsp90. (A) Confluent monolayers of G12-MDCK cells in +dox on Transwell filters were stimulated with thrombin (2 U/ml) at t=0 and TER was measured over 12 hours. Results are the mean ± s.e.m. of n=4 and were normalized to the untreated control. (B) Monolayers were stimulated at t=0 with thrombin (2 U/ml), PP2 (10 µM), GA (0.2 µM), thrombin plus PP2 or GA, bradykinin (100 µM), or bradykinin plus PP2 or GA and compared to vehicle (control). TER was measured at numerous time points. The results (normalized to the untreated control) at 40 minutes are shown (n=4 ± s.e.m.). *P<0.05. (C) Confluent G12-MDCK cells in +dox were stimulated at t=0 with thrombin and lysates were prepared at t=0 (no thrombin), 15 and 30 minutes of stimulation for analysis by GST-TPR pull-down. Western blots to G12 and Hsp90 were performed as described above.

View larger version (21K): [in this window] [in a new window]   Fig. 10. G12 as a check-point in TJ regulation. At baseline (A), claudins from neighboring cells interact with each other in the paracellular space and with ZO-1 in the TJ. The TJ protein complex contains numerous proteins. In this figure, ZO-1, ZO-2 and ZO-3 are designated ZO. Src might interact directly or indirectly with the ZO complex. G12 directly interacts with ZO-1 and ZO-2, and Hsp90 is probably not constitutively localized in this domain but is recruited to the activated G12 complex (denoted with * in B). Activated G12 (QL mutation or thrombin) activates Src (denoted with * in B) leading to increased tyrosine phosphorylation of ZO-1 and ZO-2 (P-Y). (C) Phosphorylation of ZO-1 and ZO-2 leads to disruption of the ZO-1 protein complex, intracellular localization of claudin 1 and loss of barrier function. In addition to the loss of barrier function, under some conditions of persistent activation, this could lead to persistent disassembly of the TJ. Once TJs are disrupted and stimulated to reassemble (as in the calcium switch), activation of G12 (using Hsp90 and Src) leads to a brake on TJ assembly.

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