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First published online 1 September 2005
doi: 10.1242/jcs.02560

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Viral oncoprotein-induced mislocalization of select PDZ proteins disrupts tight junctions and causes polarity defects in epithelial cells

Isabel J. Latorre^1,*, Michael H. Roh^2,*, Kristopher K. Frese¹, Robert S. Weiss^1,, Ben Margolis² and Ronald T. Javier^1,

¹ Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA
² Department of Biological Chemistry, Howard Hughes Medical Institute, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109, USA

View larger version (48K): [in a new window]   Fig. 1. E4-ORF1 directly binds to PDZ proteins expressed in MDCK cells. (A) E4-ORF1 directly binds to MDCK cell-derived PDZ proteins having gel mobilities similar to the known E4-ORF1-interacting PDZ proteins MAGI-1, ZO-2 and SAP97, but not MUPP1, expressed in TE85 cells. The detection of an unknown 220 kDa protein (p220) is indicated. Extracts (1.2 mg protein) of RIPA buffer-lysed TE85 and MDCK cells were subjected to pulldown reactions with the indicated GST fusion proteins, and recovered proteins, immobilized on a membrane, were blotted with a radiolabeled GST-E4-ORF1 protein probe. Proteins were detected by autoradiography. A vertical line indicates boundary between spliced lanes from the same gel. (B) MDCK cells lack detectable MUPP1 expression. Extracts (100 µg protein) of RIPA buffer-lysed TE85, CREF and MDCK cells were immunoblotted with MUPP1 antiserum. TE85 and CREF cells represented positive controls for MUPP1 expression in this experiment.

View larger version (17K): [in a new window]   Fig. 2. PATJ is a common cellular target for the adenovirus E4-ORF1 and high-risk HPV type 18 E6 oncoproteins. (A) PATJ PDZ8 mediates binding to E4-ORF1. COS7 cells on 6 cm dishes were lipofected with pRK5-myc-PATJ-wt, -PDZ6, or -PDZ8 (3 µg). Extracts (200 µg protein) of RIPA buffer-lysed cells were subjected to pulldown reactions with the indicated GST-E4-ORF1 fusion protein, and recovered proteins were immunoblotted with Myc antibody (left panel). An immunoblot assay of cell extracts with Myc antibody shows 3/40th of the input protein for each reaction (right panel). The control lane represents extracts from cells lipofected with pRK5-myc plasmid (3 µg). Vertical lines indicate boundary between spliced lanes from the same gel. (B) PATJ binds E4-ORF1 and the high-risk HPV-18 E6 oncoprotein. COS7 cells on 6 cm dishes were lipofected with pRK5-myc-PATJ (3 µg). Extracts (160 µg protein) of RIPA buffer-lysed cells were subjected to pulldown reactions with the indicated GST fusion proteins, and recovered proteins were immunoblotted with Myc antibody. An immunoblot assay of cell extracts with the Myc antibody shows 1/16 of the input protein for each reaction (rightmost lane). 18E6, HPV-18 E6; 11E6, HPV-11 E6.

View larger version (34K): [in a new window]   Fig. 3. E4-ORF1 binds endogenous PATJ and redistributes it into detergent-insoluble complexes in MDCK cells. (A) Endogenous PATJ co-immunoprecipitates (IP) with E4-ORF1 in MDCK cells. Extracts (900 µg protein) of RIPA buffer-lysed MDCK cells, MDCK-E4-ORF1 cells and MDCK-IIIA cells were immunoprecipitated with E4-ORF1 antibodies, and recovered proteins were immunoblotted with PATJ and E4-ORF1 antibodies. (B) E4-ORF1 causes PATJ to redistribute into detergent-insoluble complexes in MDCK cells. Detergent-soluble or detergent-insoluble fractions of MDCK cells, MDCK-E4-ORF1 cells and MDCK-IIIA cells were prepared as described in the Materials and Methods, and an equivalent amount of each fraction was immunoblotted with PATJ or E4-ORF1 antibodies. An immunoblot assay of total cell extracts with PATJ antibody shows equivalent amounts of PATJ in the three lines (bottom panel). Vertical lines indicate the boundary between spliced lanes from the same gel.

View larger version (51K): [in a new window]   Fig. 4. E4-ORF1 blocks proper TJ localization of PATJ, PALS1 and Par3 in MDCK cells. (A) YFP-PATJ localizes at TJs of polarized MDCK-YFP-PATJ cells. Cells were fixed, permeabilized, and mounted on slides as described in the Materials and Methods. YFP-PATJ was directly visualized by fluorescence microscopy. (B) Transient expression of wt E4-ORF1 ablates proper TJ localization of YFP-PATJ, and the two proteins co-localize in punctate bodies. MDCK-YFP-PATJ cells were transfected with pCMVBam3-Neo-E4-ORF1 (5 µg). Each of the three panels represents the same field showing either YFP-PATJ staining, E4-ORF1 immunostaining, or their combined images (merge). Arrowheads indicate punctate bodies where YFP-PATJ and E4-ORF1 co-localize. (C) Endogenous PATJ, PALS1 and Par3 lack proper TJ localization in MDCK-E4-ORF1 cells. MDCK-E4-ORF1 cells and MDCK-IIIA cells were immunostained with PATJ, PALS1 or Par3 antibodies. Scale bars: 20 µm.

View larger version (71K): [in a new window]   Fig. 5. E4-ORF1 disrupts proper TJ localization of ZO-1 and ZO-2 in MDCK cells. (A) ZO-2 localization to TJs is impaired in MDCK-E4-ORF1 cells. Control MDCK-IIIA cells and MDCK-E4-ORF1 cells were immunostained with ZO-2 or SAP97 antibodies. Arrowheads indicate representative punctate bodies where ZO-2 becomes aberrantly sequestered in MDCK-E4-ORF1 cells. (B) Endogenous ZO-1 fails to associate with TJs in MDCK-E4-ORF1 cells. Control MDCK-IIIA cells and MDCK-E4-ORF1 cells were immunostained with ZO-1 (middle panels) and E4-ORF1 (left panels) antibodies. The combined images are shown in the right panels (merge). Scale bars: 20 µm (A); 40 µm (B).

View larger version (21K): [in a new window]   Fig. 6. Wt E4-ORF1, but not mutant IIB or IIIA E4-ORF1, activates the PI 3-kinase effector PKB in MDCK cells. (A) PKB activation by E4-ORF1 in MDCK cells. MDCK cells on 6 cm dishes were lipofected with pGW1-HA-PKB (1.0 µg) in combination with pGW1-wt-E4-ORF1, -IIB-E4-ORF1 or -IIIA-E4-ORF1 (50 ng). Extracts (120 µg protein) of RIPA buffer-lysed, serum-starved cells were immunoblotted with (P)Ser473PKB or HA antibodies. (B) Wt and mutant E4-ORF1 proteins are expressed at comparable levels in MDCK cells. MDCK cells on 6 cm dishes were lipofected with pGW1-HA-PKB (0.5 µg) in combination with pGW1-wt-E4-ORF1, -IIB-E4-ORF1 or -IIIA-E4-ORF1 (2.5 µg). Extracts (100 µg protein) of sample buffer-lysed, serum-starved cells were immunoblotted with E4-ORF1, or HA antibodies.

View larger version (97K): [in a new window]   Fig. 7. PI 3-kinase activation by E4-ORF1 is not required to block TJ localization of PDZ proteins in MDCK cells. (A) Endogenous ZO-1 fails to associate with TJs in MDCK cells expressing mutant IIB E4-ORF1. Cells of the MDCK-IIB pool were immunostained with ZO-1 (middle panel) or E4-ORF1 (left panel) antibodies. The combined images are shown in the right panel (merge). Scale bars: 40 µm. (B) Endogenous PATJ and ZO-2 also fail to associate with TJs in MDCK cells expressing mutant IIB E4-ORF1. Cells of the MDCK-IIB pool were immunostained with ZO-1, PATJ, and ZO-2 antibodies. The top three panels represent the same field of cells showing immunostaining of ZO-1, PATJ, or the merge of the latter two images (merge). The bottom three panels represent the same field of cells showing immunostaining of ZO-1, ZO-2, and the merged images (merge). Dashed lines outline groups of cells inferred to express E4-ORF1 based on the results shown in (A).

View larger version (58K): [in a new window]   Fig. 8. Threshold levels of E4-ORF1 expression are required to block TJ localization of PDZ proteins in MDCK cells. (A) High or low levels of E4-ORF1 protein expression by two MDCK lines. Extracts of sample buffer-lysed MDCK cells, MDCK-E4-ORF1-low cells and MDCK-E4-ORF1 cells (180 µg protein) were immunoblotted with E4-ORF1 or Erk1/2 antibodies. Erk1/2 served as a loading control. (B) The localization pattern of E4-ORF1 depends on its expression level in MDCK cells. MDCK-E4-ORF1-low cells or MDCK-E4-ORF1 cells were immunostained with E4-ORF1 antibodies. Scale bar: 40 µm. (C) E4-ORF1 must be expressed at a minimal threshold level to block TJ localization of PDZ proteins in MDCK cells. MDCK-E4-ORF1-low cells and MDCK-E4-ORF1 cells were immunostained with PATJ, ZO-2, SAP97 or ß-catenin antibodies. (D) E4-ORF1 protein levels progressively increase during an Ad9 infection of epithelial cells. A549 cells were infected with Ad9 virus for the indicated times, and extracts (80 µg protein) of sample buffer-lysed cells were immunoblotted with E4-ORF1 antibodies.

View larger version (19K): [in a new window]   Fig. 9. E4-ORF1 functionally disrupts TJs in MDCK cells. (A) Prevention or delay of TER establishment in MDCK-E4-ORF1 and MDCK-E4-ORF1-low monolayers, respectively. The arrow indicates the time when TER values became stabilized (72 hours post-plating). CREF fibroblasts were used as a negative control in the assay. (B) Comparable E4-ORF1 expression by MDCK-E4-ORF1 cells and MDCK-IIB cells. Extracts (150 µg protein) of the indicated sample buffer-lysed cells were immunoblotted with E4-ORF1 or Erk1/2 antibodies. Erk1/2 served as a loading control. (C) Prevention of TER establishment in MDCK-IIB monolayers. TER values were determined for fully confluent cells on Transwell filters at the indicated times post-plating.

View larger version (36K): [in a new window]   Fig. 10. E4-ORF1 causes cell polarity defects in MDCK cells. Shown are cysts derived from MDCK-IIIA cells or MDCK-E4-ORF1 cells co-immunostained with gp135 and E-cadherin antibodies. Representative cysts from independent experiments 1 and 2 were photographed at 7 days (MDCK-IIIA cells) or 10 days and 14 days (MDCK-E4-ORF1 cells) post-suspension. Scale bars are 10 µm (experiment 1) and 20 µm (experiment 2).

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