Multi-PDZ domain protein MUPP1 is a cellular target for both adenovirus E4-ORF1 and high-risk papillomavirus type 18 E6 oncoproteins - PubMed (original) (raw)

Multi-PDZ domain protein MUPP1 is a cellular target for both adenovirus E4-ORF1 and high-risk papillomavirus type 18 E6 oncoproteins

S S Lee et al. J Virol. 2000 Oct.

Abstract

A general theme that has emerged from studies of DNA tumor viruses is that otherwise unrelated oncoproteins encoded by these viruses often target the same important cellular factors. Major oncogenic determinants for human adenovirus type 9 (Ad9) and high-risk human papillomaviruses (HPV) are the E4-ORF1 and E6 oncoproteins, respectively, and although otherwise unrelated, both of these viral proteins possess a functional PDZ domain-binding motif that is essential for their transforming activity and for binding to the PDZ domain-containing and putative tumor suppressor protein DLG. We report here that the PDZ domain-binding motifs of Ad9 E4-ORF1 and high-risk HPV-18 E6 also mediate binding to the widely expressed cellular factor MUPP1, a large multi-PDZ domain protein predicted to function as an adapter in signal transduction. With regard to the consequences of these interactions in cells, we showed that Ad9 E4-ORF1 aberrantly sequesters MUPP1 within the cytoplasm of cells whereas HPV-18 E6 targets this cellular protein for degradation. These effects were specific because mutant viral proteins unable to bind MUPP1 lack these activities. From these results, we propose that the multi-PDZ domain protein MUPP1 is involved in negatively regulating cellular proliferation and that the transforming activities of two different viral oncoproteins depend, in part, on their ability to inactivate this cellular factor.

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Figures

FIG. 1

The partial mouse protein 9BP-1 represents the carboxyl terminus of the mouse multi-PDZ protein MUPP1. (A) PDZ domain organizations of the partial mouse protein 9BP-1 (688 aa) and the mouse multi-PDZ domain protein MUPP1 (2,055 aa). Note that the domain organization of 9BP-1 is identical to that of the carboxyl-terminal region of MUPP1. The unique protein region used to generate MUPP1 antisera is indicated. (B) The partial mouse protein 9BP-1 exhibits 99% amino acid sequence identity to the carboxyl-terminal region of the mouse MUPP1 protein (aa 1368 to 2055). Highlighted sequences denote PDZ homology domains. Sequence alignment was performed using the Align Global Sequence Alignment algorithm from the Baylor College of Medicine Search Launcher Web site.

FIG. 2

9ORF1-associated protein p220 displays similar properties to both 9BP-1 and MUPP1. (A) 9BP-1 and 9ORF1-associated protein p220 comigrate and exhibit identical species-specific gel mobilities. Proteins from RIPA buffer-lysed mouse NIH 3T3, rat CREF, human 293, and human TE85 cell lines were either immunoblotted with 9BP-1 antiserum (left) or first subjected to a GST pulldown assay with the indicated fusion protein and then blotted with a radiolabeled 9ORF1 protein probe (right). For the experiment shown in the left or right panel, 100 or 2.5 mg of cell proteins was used, respectively, and the protein gels were run in parallel. Asterisks indicate 9ORF1-associated protein p220. (B) MUPP1 antiserum cross-reacts with 9BP-1 protein derived from several different species. Cell proteins (2.5 mg) in RIPA buffer from the indicated cell lines were first immunoprecipitated (IP) with either 9BP-1 antiserum (α-9BP-1) or the matched preimmune serum (pre) and then immunoblotted with MUPP1 antiserum. Also note that the 9BP-1 protein detected in panel A (left) and the MUPP1 protein detected here exhibited identical species-specific gel mobilities.

FIG. 3

9ORF1 binds MUPP1 in vitro. GST-9ORF1 binds HA epitope-tagged rat MUPP1 protein (HAMUPP1) expressed in COS7 cells. Cells were lipofected with 4 μg of either empty GW1 plasmid (vector) or GW1-HAMUPP1 plasmid, and cell proteins in RIPA buffer were either immunoblotted with HA antibodies (left) or first subjected to a GST pulldown assay with the indicated wild-type or mutant E4-ORF1 fusion protein (Table 1) and then immunoblotted with HA antibodies (right). COS7 cell proteins at 100 μg or 1 mg were used in the experiment shown in the left and right panels, respectively.

FIG. 4

9ORF1 complexes with MUPP1 in cells. (A) 9ORF1 complexes with HA epitope-tagged rat MUPP1 protein (HAMUPP1) expressed in COS7 cells. Cells were lipofected with 6 μg of GW1-HAMUPP1 plasmid and 2 μg of either empty GW1 plasmid (vector) or a GW1 plasmid expressing wild-type or the indicated mutant 9ORF1 protein. Cell proteins in RIPA buffer were either immunoblotted with HA antibodies or 9ORF1 antiserum (left) or first immunoprecipitated (IP) with 9ORF1 antiserum (α-9ORF1) and then immunoblotted with HA antibodies or 9ORF1 antiserum (right). Cell proteins at 100 or 800 μg were used in the experiment shown in the left and right panels, respectively. (B) 9ORF1 complexes with endogenous MUPP1 of CREF cells. Cell proteins in RIPA buffer were either immunoblotted with MUPP1 or 9ORF1 antiserum (top) or first immunoprecipitated (IP) with 9ORF1 antiserum or the matched preimmune serum (pre) (bottom left) or, alternatively, with either MUPP1 antiserum (α-MUPP1) or the matched preimmune serum (pre) (bottom right) and then immunoblotted with either MUPP1 or 9ORF1 antiserum. CREF cell proteins at 100 μg, 3 mg, and 3 mg were used in the experiments in the top, bottom left, and bottom right panels, respectively. (C) 9ORF1 complexes with MUPP1 in the Ad9-induced rat mammary tumor cell line 20-8. This tumor cell line contains a single integrated copy of the entire Ad9 genome (unpublished results). Cell proteins (3 mg) in RIPA buffer were first immunoprecipitated (IP) with either 9BP-1 antiserum (α-9BP-1) or the matched preimmune serum and then immunoblotted with either 9ORF1 or 9BP-1 antiserum.

FIG. 4

FIG. 5

MUPP1 PDZ7 and PDZ10 mediate binding to 9ORF1. (A) Illustration of the full-length MUPP1 polypeptide and 10 different MUPP1 GST fusion protein constructs used in protein blotting assays. (B) 9ORF1 binds MUPP1 PDZ7 and PDZ10 in vitro. Approximately 1 μg of each indicated MUPP1 GST fusion protein was immobilized on a membrane and protein blotted with a radiolabeled 9ORF1 protein probe. As a control, the membrane was stained with Coomassie brilliant blue dye to verify that an equivalent amount of each fusion protein was used in the experiment (data not shown). (C) A MUPP1 deletion mutant lacking both PDZ7 and PDZ10 fails to complex with 9ORF1 in COS7 cells. Cells were lipofected with 6 μg of a GW1 plasmid expressing wild-type or the indicated deletion mutant MUPP1 protein together with 2 μg of either empty GW1 plasmid (vector) or the GW1-9ORF1wt plasmid. Cell proteins in RIPA buffer were either immunoblotted with HA antibodies (top) or first immunoprecipitated (IP) with 9ORF1 antiserum (α-9ORF1) and then immunoblotted with HA antibodies or 9ORF1 antiserum (bottom). Cell proteins at 50 and 750 μg were used in the experiments in the top and bottom panels, respectively.

FIG. 6

9ORF1 aberrantly sequesters MUPP1 within punctate bodies in the cytoplasm of cells. (A) Determination of the subcellular localization of MUPP1 in normal CREF cells (CREF) or CREF cell lines stably expressing wild-type (CREF-9ORF1) or the indicated mutant 9ORF1 protein (CREF-IIIA, CREF-IIIC, and CREF-IIID). IF microscopy assays were performed with either MUPP1 antiserum (α-MUPP1) or the matched preimmune serum (pre). Although all of the CREF cell lines expressed similar amounts of MUPP1 protein (see Fig. 7A), the MUPP1 staining for CREF-9ORF1 cells appeared brighter than that for the other CREF lines. This effect probably resulted from the large amounts of MUPP1 protein concentrated within the cytoplasmic punctate bodies. Discontinuous cell-cell contact staining for MUPP1 was most evident in normal CREF cells, and CREF-IIIA and CREF-IIIC lines, all of which exhibited similar MUPP1 staining patterns. As an example of this cell-cell contact staining, two adjacent CREF-IIIC cells within the delimited rectangular region are shown offset at higher magnification. (B) 9ORF1 and MUPP1 colocalize within punctate bodies in the cytoplasm of CREF cells. Double-labeling IF microscopy assays using both MUPP1 antiserum and HA antibodies (α-HA) were performed with CREF cells stably expressing HA epitope-tagged 9ORF1 protein (CREF-HA9ORF1). Each of the three panels shows the identical field containing the same three cells. The top left and top right panels show the MUPP1 and 9ORF1 staining patterns, respectively, whereas the bottom panel shows the merged images.

FIG. 6

FIG. 7

9ORF1 aberrantly redistributes MUPP1 into the RIPA buffer-insoluble fraction of cells. (A) Similar amounts of MUPP1 and 9ORF1 proteins within CREF cell lines stably expressing wild-type and mutant 9ORF1 proteins. Cell proteins (100 μg) extracted with sample buffer were immunoblotted with MUPP1 antiserum or 9ORF1 antiserum. (B) Wild-type 9ORF1 specifically redistributes MUPP1 into the RIPA buffer-insoluble fraction of CREF cells. Cells from the indicated CREF lines were lysed in RIPA buffer and centrifuged to yield a RIPA buffer-soluble supernatant fraction and a RIPA buffer-insoluble pellet fraction (see Materials and Methods). Cell proteins from an equivalent volume of either the soluble or insoluble fraction were immunoblotted with MUPP1 or 9ORF1 antiserum. (C) Most MUPP1 protein is complexed with 9ORF1 in 9ORF1-expressing CREF cells. Cell proteins (3 mg) in the RIPA buffer-soluble fraction of normal CREF cells or wild-type 9ORF1-expressing CREF cells were subjected to five serial immunoprecipitations with 9ORF1 antiserum. Relative amounts of MUPP1 and 9ORF1 protein remaining in this fraction (100 μg of protein) “before” and “after” performing the serial immunoprecipitations were determined by immunoblot analysis. (D) Wild-type 9ORF1 also specifically redistributes HA epitope-tagged rat MUPP1 (HAMUPP1) into the RIPA buffer-insoluble fraction of 293 cells. Cells were lipofected with 1 μg of GW1-HAMUPP1 plasmid and 3 μg of either empty GW1 plasmid (vector) or a GW1 plasmid expressing wild-type or the indicated mutant 9ORF1 protein. Cell fractionation assays were performed as described for panel B, except that cell proteins were immunoblotted with HA antibodies or 9ORF1 antiserum.

FIG. 8

18E6 binds MUPP1 in vitro. GST-18E6 binds HA epitope-tagged rat MUPP1 (HAMUPP1) exogenously expressed in COS7 cells. Cells were lipofected with 8 μg of GW1-HAMUPP1 plasmid, and cell proteins (250 μg) in RIPA buffer were first subjected to a GST pulldown assay with the indicated fusion protein and then immunoblotted with HA antibodies.

FIG. 9

18E6 promotes the degradation of the MUPP1 protein in vitro. In vitro-translated MUPP1, DLG, or p53 protein was incubated for the indicated times with a 5- to 10-fold molar excess of in vitro-translated 18E6 protein (+) or with an equivalent volume of a water-primed in vitro translation reaction mixture (−). Proteins from each reaction were subjected to immunoprecipitation with MUPP1, DLG, or p53 antibodies, respectively, and detected by autoradiography.

FIG. 10

18E6 reduces the steady-state levels of MUPP1 protein in cells. (A) 18E6 reduces the steady-state levels of HA epitope-tagged rat MUPP1 (HAMUPP1) protein expressed in COS7 cells. Cells were lipofected with 1 μg of GW1-HAMUPP1 plasmid and 4 μg of either empty GW1 plasmid (vector) or a GW1 plasmid expressing HA epitope-tagged wild-type or the indicated mutant 18E6 protein or expressing HA epitope-tagged wild-type 11E6 protein. Cell proteins (30 μg) in RIPA buffer were immunoblotted with HA antibodies. (B) 18E6 does not bind HA epitope-tagged PDZ-protein ZO-1 (HAZO-1). Cells were lipofected with 3 μg of either empty GW1 plasmid (vector), GW1-HADLG plasmid, or GW1-HAZO-1 plasmid, and cell proteins in RIPA buffer were either immunoblotted with HA antibodies (left) or first subjected to a GST pulldown assay with the indicated fusion protein and then immunoblotted with HA antibodies (right). COS7 cell proteins at 10 and 75 μg were used in the experiments in the left and right panels, respectively. HADLG was included as a positive control in these binding assays (28). (C) 18E6 does not reduce HAZO-1 protein levels in COS7 cells. COS7 cells were lipofected with 0.01 μg of GW1-HAZO-1 plasmid and 4 μg of either empty GW1 plasmid (vector) or a GW1 plasmid expressing HA epitope-tagged wild-type or the indicated mutant 18E6 protein. Cell proteins (30 μg) in RIPA buffer were immunoblotted with HA antibodies.

FIG. 11

18E6 decreases the half-life of the MUPP1 protein in cells. A total of 5.5 × 105 COS7 cells were lipofected with 5 μg of empty GW1 plasmid (vector) or 1 μg of GW1-HAMUPP1 plasmid and 4 μg of either empty GW1 plasmid (HAMUPP1) or the GW1-18E6 plasmid (HAMUPP1 + 18E6). At 24 h posttransfection, cells were pulse-labeled and then chased for the indicated times (see Materials and Methods). Cell proteins (200 μg) were immunoprecipitated with HA antibodies (α-HA), and HAMUPP1 protein was detected by autoradiography and quantified with a PhosphorImager.

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Multi-PDZ domain protein MUPP1 is a cellular target for both adenovirus E4-ORF1 and high-risk papillomavirus type 18 E6 oncoproteins - PubMed (original) (raw)