Title: Oncogenic function for the Dlg1 mammalian homolog of the Drosophila discs-large tumor suppressor
Abstract: Article2 March 2006free access Oncogenic function for the Dlg1 mammalian homolog of the Drosophila discs-large tumor suppressor Kristopher K Frese Kristopher K Frese Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USAPresent address: Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Search for more papers by this author Isabel J Latorre Isabel J Latorre Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USAPresent address: Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Search for more papers by this author Sang-Hyuk Chung Sang-Hyuk Chung Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Georgina Caruana Georgina Caruana Department of Anatomy and Cell Biology, Monash University, Clayton, Victoria, Australia Search for more papers by this author Alan Bernstein Alan Bernstein Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada Search for more papers by this author Stephen N Jones Stephen N Jones Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Lawrence A Donehower Lawrence A Donehower Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Monica J Justice Monica J Justice Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Craig C Garner Craig C Garner Department of Psychiatry and Behavioral Science, Nancy Pritzker Laboratory, Stanford University, Palo Alto, CA, USA Search for more papers by this author Ronald T Javier Corresponding Author Ronald T Javier Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Kristopher K Frese Kristopher K Frese Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USAPresent address: Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Search for more papers by this author Isabel J Latorre Isabel J Latorre Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USAPresent address: Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Search for more papers by this author Sang-Hyuk Chung Sang-Hyuk Chung Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Georgina Caruana Georgina Caruana Department of Anatomy and Cell Biology, Monash University, Clayton, Victoria, Australia Search for more papers by this author Alan Bernstein Alan Bernstein Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada Search for more papers by this author Stephen N Jones Stephen N Jones Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Lawrence A Donehower Lawrence A Donehower Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Monica J Justice Monica J Justice Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Craig C Garner Craig C Garner Department of Psychiatry and Behavioral Science, Nancy Pritzker Laboratory, Stanford University, Palo Alto, CA, USA Search for more papers by this author Ronald T Javier Corresponding Author Ronald T Javier Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Author Information Kristopher K Frese1,‡, Isabel J Latorre1,‡, Sang-Hyuk Chung1, Georgina Caruana2, Alan Bernstein3, Stephen N Jones4, Lawrence A Donehower1, Monica J Justice5, Craig C Garner6 and Ronald T Javier 1 1Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA 2Department of Anatomy and Cell Biology, Monash University, Clayton, Victoria, Australia 3Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada 4Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA, USA 5Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA 6Department of Psychiatry and Behavioral Science, Nancy Pritzker Laboratory, Stanford University, Palo Alto, CA, USA ‡These authors contributed equally to this work *Corresponding author. Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA. Tel.: +1 713 798 3898; Fax: +1 713 798 3586; E-mail: [email protected] The EMBO Journal (2006)25:1406-1417https://doi.org/10.1038/sj.emboj.7601030 Present address: Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The fact that several different human virus oncoproteins, including adenovirus type 9 E4-ORF1, evolved to target the Dlg1 mammalian homolog of the membrane-associated Drosophila discs-large tumor suppressor has implicated this cellular factor in human cancer. Despite a general belief that such interactions function solely to inactivate this suspected human tumor suppressor protein, we demonstrate here that E4-ORF1 specifically requires endogenous Dlg1 to provoke oncogenic activation of phosphatidylinositol 3-kinase (PI3K) in cells. Based on our results, we propose a model wherein E4-ORF1 binding to Dlg1 triggers the resulting complex to translocate to the plasma membrane and, at this site, to promote Ras-mediated PI3K activation. These findings establish the first known function for Dlg1 in virus-mediated cellular transformation and also surprisingly expose a previously unrecognized oncogenic activity encoded by this suspected cellular tumor suppressor gene. Introduction Dlg1, also known as hDlg or SAP97, is a mammalian homolog of the Drosophila discs-large (Dlg) tumor suppressor protein (Lue et al, 1994; Muller et al, 1995). These proteins localize to specialized cell–cell contact membrane sites (i.e., septate, adherens, and synaptic junctions) and are required for both junction formation and proper polarity establishment in cells (Humbert et al, 2003; Nguyen et al, 2003; Laprise et al, 2004). As MAGUK family proteins, they lack catalytic activity and instead consist of multiple protein interaction modules, including three PDZ domains, an SH3 domain, and a guanylate kinase-homology (GK) domain (Gonzalez-Mariscal et al, 2000). Accordingly, these polypeptides function as molecular scaffolds to organize their targets into supramolecular signaling complexes and to translocate them to the plasma membrane (Kim and Sheng, 2004). Evidence supports the assertion that Drosophila Dlg and mammalian Dlg1 encode evolutionarily conserved tumor suppressor proteins. Drosophila Dlg mutations cause cellular overgrowth in the larval brain and imaginal discs, where cells exhibit a neoplastic morphology (Woods et al, 1996). Upon transplantation into wild-type (wt) flies, Dlg-mutant imaginal discs produce cancerous outgrowths resembling metastatic tumors (Woodhouse et al, 1998), perhaps reflecting the capacity of Dlg to prevent abnormal cellular invasion into adjacent tissues (Goode and Perrimon, 1997). Furthermore, transgenic expression of Dlg1 by Drosophila Dlg mutants rescues their cellular overgrowth phenotype (Thomas et al, 1997), demonstrating conservation of the tumor suppressor function. Dlg1 likewise prevents unscheduled cellular proliferation in ocular lenses of mice (Nguyen et al, 2003), sustains mutations accompanied by a loss of heterozygosity in human breast carcinomas (Fuja et al, 2004), and exhibits reduced expression in invasive human cervical carcinomas (Cavatorta et al, 2004; Lin et al, 2004). Reports showing that three different human virus oncoproteins independently evolved to target Dlg1 in cells provide the most compelling evidence implicating this cellular factor in human cancer. Human adenovirus type 9 (Ad9) elicits mammary tumors in experimental animals (Javier et al, 1991), and high-risk human papillomavirus (HPV) and human T-cell leukemia virus type 1 (HTLV-1) are etiological agents for cervical carcinoma and adult T-cell leukemia, respectively (Barmak et al, 2003; Heise, 2003). Strikingly, the otherwise unrelated oncogenic determinants of these tumor viruses (Ad9 E4-ORF1, HPV E6, and HTLV-1 Tax) similarly possess a carboxyl-terminal PDZ domain-binding motif (PBM) that mediates binding to several different cellular PDZ proteins (Lee et al, 1997; Rousset et al, 1998; Mantovani and Banks, 2001), including Dlg1, which is the only PDZ-protein target common to all three viral proteins. Additionally, the PBM plays a key role in cellular transformation induced by these oncoproteins (Lee et al, 1997; Watson et al, 2003; Hirata et al, 2004). Evidence specifically implicating endogenous Dlg1 in this process is currently lacking, but it has been reasonably hypothesized that the tumorigenic potentials of these viral proteins stem partly from an ability to inactivate this suspected tumor suppressor protein. Consistent with this idea, HPV E6 promotes degradation of overexpressed Dlg1 (Gardiol et al, 1999) and HTLV-1 Tax overcomes cell-cycle arrest caused by Dlg1 overexpression (Suzuki et al, 1999), although one caveat is the uncertain physiological relevance of these findings. The oncogenic potential of Ad9 E4-ORF1 was recently demonstrated to depend on its ability to promote constitutive, growth factor-independent stimulation of cellular phosphatidylinositol 3-kinase (PI3K) (Frese et al, 2003). This critical activity of E4-ORF1 requires the PBM, which mediates interactions with Dlg1 (Lee et al, 1997) and three other cellular PDZ proteins (MUPP1, MAGI-1, and ZO-2) (Glaunsinger et al, 2000, 2001; Lee et al, 2000). We report here that specific binding of E4-ORF1 to endogenous Dlg1 promotes Ras-mediated PI3K activation in cells, revealing the first established function for Dlg1 in viral oncoprotein-mediated cellular transformation. The additional finding that functional as opposed to inactivated Dlg1 mediates E4-ORF1-induced PI3K activation also exposed a surprising oncogenic activity for this suspected tumor suppressor protein. Results Mutation of MUPP1, MAGI-1, or Dlg1 fails to reproduce PI3K activation induced by E4-ORF1 Given that Dlg1 as well as ZO-2 (Chlenski et al, 1999a, 1999b, 2000) are suspected tumor suppressors and that E4-ORF1 sequesters MUPP1, MAGI-1, and ZO-2 in the cytoplasm of cells (Glaunsinger et al, 2000, 2001; Lee et al, 2000), we postulated that PI3K activation provoked by Ad9 E4-ORF1 may stem from inactivation of one of these cellular targets. If so, disruption of the corresponding cellular gene should recapitulate E4-ORF1-induced PI3K activation in cells. To explore this idea, we isolated mouse embryo fibroblasts (MEF) from wt mice and matched littermates carrying either a large deletion mutation encompassing the entire MUPP1 gene (Bell et al, 1995) or a targeted mutation interrupting either the MAGI-1 gene (in preparation) or the Dlg1 gene (Caruana and Bernstein, 2001). The lack of ZO-2 mutant mice precluded inclusion of the respective mutant MEF in this study; however, ZO-2 seems unlikely to play a role in E4-ORF1-induced PI3K activation given that E4-ORF1 proteins encoded by human Ad3, Ad5, and Ad12 activate PI3K (Frese et al, 2003), yet do not bind this single, unique PDZ-protein target of Ad9 E4-ORF1 (Glaunsinger et al, 2001). MUPP1−/− or MAGI-1−/− MEF failed to express the respective wt gene product, as did Dlg1gt/gt MEF, which instead expressed the Dlg/β-geo fusion protein containing the amino-terminal 549 residues of the ∼900-residue wt Dlg1 polypeptide (Supplementary Figure 1A and B). Following serum deprivation and subsequent stimulation by platelet-derived growth factor (PDGF), all mutant MEF and matched wt MEF displayed comparable basal and PDGF-induced levels of activated, phosphorylated protein kinase B (PKB) (Supplementary Figure 1A), a key downstream effector of PI3K. Throughout this study, Thr308- and Ser473-phosphospecific PKB antibodies yielded identical results, so each was used interchangeably. As Dlg/β-geo could feasibly retain some function, including the postulated capacity to suppress PI3K activation, we stably downregulated this protein in Dlg1gt/gt MEF using a Dlg1-specific short hairpin RNA (shRNA). Despite substantial, specific downregulation of Dlg/β-geo, Dlg1 shRNA-expressing Dlg1gt/gt MEF displayed basal and PDGF-induced levels of activated PKB identical to those of Dlg1gt/gt MEF (Supplementary Figure 1C). These observations did not support our hypothesis that E4-ORF1-induced PI3K activation simply results from functional inactivation of MUPP1, MAGI-1, or Dlg1 in cells. PI3K activation and anchorage-independent growth induced by E4-ORF1 depend on Dlg1 Our negative results prompted consideration of the opposite hypothesis, wherein PI3K activation induced by E4-ORF1 requires one of its cellular PDZ-protein targets. This model predicted instead that MUPP1−/−, MAGI-1−/−, or Dlg1gt/gt MEF might fail to support E4-ORF1-induced PKB activation. Significantly, during transient E4-ORF1 expression, Dlg1gt/gt MEF, but not MUPP1−/− or MAGI-1−/− MEF, displayed a substantial defect in supporting this activity compared to matched wt or heterozygous mutant MEF (Figure 1A). Though prolonged exposures revealed that Dlg1gt/gt MEF retained a weak capacity to support E4-ORF1-induced PKB activation, this remnant activity was eliminated by Dlg1 shRNA expression (Figure 1B). Additionally, whereas Dlg1+/+ MEF stably expressing E4-ORF1 showed constitutive activation of endogenous PKB and grew in soft agar, Dlg1gt/gt MEF stably expressing E4-ORF1 lacked these phenotypes (Figure 2A and B), similar to the former MEF treated with the PI3K inhibitor drug LY294002 (LY) (Figure 2C and D). Also notable was that Dlggt/gt MEF and Dlggt/gt MEF expressing the Dlg1 shRNA failed to grow in soft agar (Figure 2B). These findings demonstrated a requirement for functional rather than inactivated Dlg1 in both PI3K activation and oncogenic transformation induced by E4-ORF1 in MEF. Figure 1.E4-ORF1-induced PKB activation depends on Dlg1. (A) E4-ORF1-induced PKB activation is deficient in Dlg1gt/gt MEF. Cells on 6-cm dishes were lipofected with pGW1-HA-PKB (0.5, 1, or 1 μg for MUPP1, MAGI-1, or Dlg1 MEF, respectively) and either empty pGW1 (−) or pGW1-E4-ORF1 (+) (100, 20, or 50 ng for MUPP1, MAGI-1, or Dlg1 MEF, respectively). Cells were serum starved, and equal amounts of cell extracts were blotted with antibody to (P)Thr308 PKB, (P)Ser473 PKB, HA, MUPP1, MAGI-1, or Dlg1. For this type of experiment, the small quantity of E4-ORF1 plasmid needed to achieve optimal HA-PKB activation did not yield detectable levels of E4-ORF1 expression. (B) Dlg1 shRNA expression abrogates weak PKB activation induced by E4-ORF1 in Dlg1gt/gt MEF. On 6-cm dishes, Dlg1gt/gt MEF stably transfected with either empty pSUPER (−) or pSUPER-Dlg1 shRNA (+) were lipofected with pGW1-HA-PKB (0.5 μg) and either empty pGW1 (−) or pGW1-E4-ORF1 (10, 20, or 50 ng). Cells were serum starved, and equal amounts of cell extracts were blotted with antibody to (P)Thr308 PKB, HA, or Dlg1. Download figure Download PowerPoint Figure 2.Dlg1 is also required for E4-ORF1 to induce anchorage-independent growth in MEF. (A) Dlg1gt/gt MEF stably expressing E4-ORF1 fail to accumulate activated, endogenous PKB. Dlg1+/+ or Dlg1gt/gt MEF stably transfected with either empty pBABE (vector) or pBABE-E4-ORF1 (E4-ORF1) were serum starved, and equal amounts of cell extracts were blotted with antibody to (P)Thr308 PKB, PKB, or E4-ORF1. (B) Dlg1gt/gt MEF stably expressing E4-ORF1 fail to grow in soft agar. 105 viable MEF stably transfected with empty pBABE (vector), pBABE-E4-ORF1 (E4-ORF1), or pSUPER-Dlg1 shRNA (Dlg1 shRNA) were suspended in agar and photographed 21 days later. (C) LY294002 (LY) treatment blocks endogenous PKB activation in Dlg1+/+ MEF stably expressing E4-ORF1. Dlg1+/+ MEF stably transfected with pBABE (vector) or pBABE-E4-ORF1 (E4-ORF1) were serum starved, and were treated with DMSO vehicle or 10 μM LY for 15 min. Equal amounts of cell extracts were analyzed as described above in A. (D) LY treatment inhibits anchorage-independent growth of Dlg1+/+ MEF stably expressing E4-ORF1. Soft agar assays were conducted as described above in (B), except that the culture medium contained DMSO vehicle (−LY) or 10 μM LY (+LY). Download figure Download PowerPoint I3-containing Dlg1 isoforms support E4-ORF1-induced PI3K activation Due to several short insertion elements (e.g., I1, I2, I3) that arise by alternative splicing (see Supplementary Figure 1B), multiple Dlg1 isoforms can be produced in cells (Lue et al, 1994; McLaughlin et al, 2002). Despite comparable binding of E4-ORF1 to the Dlg1-I2, -I3, -I1I2, and -I1I3 isoforms (Figure 3A), transient expression of green fluorescent protein (GFP)-tagged Dlg1-I3, but not GFP-Dlg1-I2, restored robust E4-ORF1-induced PKB activation in Dlg1 shRNA-expressing Dlg1gt/gt MEF (Figure 3B). A nucleotide mismatch between the mouse Dlg1 shRNA and the rat Dlg1 cDNA used to construct Dlg1 plasmids permitted GFP-Dlg1 expression in these cells. Figure 3.I3-containing Dlg1 isoforms mediate E4-ORF1-induced PKB activation. (A) E4-ORF1 binds comparably to four Dlg1 isoforms. COS7 cells on 6-cm dishes were lipofected with pGW1-E4-ORF1 (1.5 μg) and either empty pGW1 (−) or the indicated pGW1-HA-Dlg1 isoform plasmid (1.5 μg). Equal amounts of cell extracts were precipitated with HA antibody. Recovered proteins (left) or cell extracts (right) were blotted with antibody to HA or E4-ORF1. (B) Dlg1-I3, but not Dlg1-I2, rescues the defect of Dlg1 shRNA-expressing Dlg1gt/gt MEF in supporting E4-ORF1-induced PKB activation. The latter cells on 6-cm dishes were lipofected with pGW1-HA-PKB (0.5 μg), either empty pGW1 (−) or pGW1-E4-ORF1 (+) (20 ng), and either empty peGFP (−) or the indicated peGFP-Dlg1 isoform plasmid (2.5 μg). Cells were serum starved, and equal amounts of cell extracts were blotted with antibody to (P)Thr308 PKB, HA, or GFP. (C) Low, undetectable levels of Dlg1-I3 expression are sufficient to enhance E4-ORF1-induced PKB activation in Dlg1gt/gt MEF. The latter cells on 6-cm dishes were lipofected with pGW1-HA-PKB (0.8 μg), either empty pGW1 (−) or pGW1-E4-ORF1 (+) (20 ng), and either empty pGW1 (−) or pGW1-HA-Dlg1-I3 (right: 11 ng, 110 ng, 1.1 μg, or 2.2 μg; left: 0.5 μg). Cells were serum starved, and equal amounts of cell extracts were blotted with antibody to (P)Thr308 PKB or HA. (D) Other PDZ proteins fail to augment E4-ORF1-induced PKB activation in Dlg1gt/gt MEF. The latter cells on 6-cm dishes were lipofected with either empty pGW1 (−) or pGW1-HA-PKB (+) (0.4 μg), either empty pCMVBamNeo (−) or pCMVBamNeo-E4-ORF1 (+) (0.1 μg), and either empty peYFP (−) or peYFP-Dlg1-I3 (0.3 μg), -Dlg1-I2 (0.3 μg), -MUPP1 (0.3 μg), -MAGI-1c (0.3 μg), or -ZO-2 (0.1 μg), or peGFP-SAP102 (0.3 μg). Cells were serum starved, and equal amounts of cell extracts were blotted with antibody to (P)Thr308 PKB, HA, or GFP. (E) Dlg1-I3 likewise enhances PKB activation induced by E4-ORF1 encoded by other human adenoviruses. Dlg1gt/gt MEF on 6-cm dishes were lipofected with pGW1-HA-PKB (0.4 μg), either empty pCMVBamNeo (−) or the indicated pCMVBamNeo-E4-ORF1 plasmid (Ad9, 0.1 μg; Ad3, 0.6 μg; Ad5, 0.6 μg), and either empty peGFP (−) or peGFP-Dlg1-I3 (+) (0.4 μg). Cells were serum starved, and equal amounts of cell extracts were blotted with antibody to (P)Thr308 PKB, HA, or SAP97. (F) Dlg1-I3 fails to augment PKB activation by other cellular and viral oncoproteins in Dlg1gt/gt MEF. The latter cells on 6-cm dishes were lipofected with pGW1-HA-PKB (0.4 μg), either empty pGW1 (−) or pCMVBamNeo-E4-ORF1 (0.1 μg), pGW1-RasV12 (0.2 μg), or pPyMT (0.2 μg), and either empty peGFP (−) or peGFP-Dlg1-I3 (+) (0.2 μg). Cells were serum starved, and equal amounts of cell extracts were blotted with antibody to (P)Thr308 PKB, HA, or SAP97. Download figure Download PowerPoint In Dlg1gt/gt MEF, E4-ORF1-induced PKB activation increased markedly even at low undetectable levels of HA epitope-tagged (HA)-Dlg1-I3 expression, peaked at moderate levels, and did not increase appreciably at higher levels (Figure 3C). By contrast, GFP-tagged Dlg1-I2, MUPP1, MAGI-1, ZO-2, or Dlg1-related SAP102 (Thomas et al, 1997) lacked any such activity (Figure 3D). GFP-Dlg1-I3 likewise enhanced PKB activation induced by related human Ad3 and Ad5 E4-ORF1 proteins (Frese et al, 2003) (Figure 3E), but not by cellular H-RasV12 or polyomavirus middle T (PyMT) (Figure 3F). Additionally, Dlg1-I1I2 and Dlg1-I1I3 mirrored Dlg1-I2 and Dlg1-I3, respectively, in their capacities to support E4-ORF1-induced PKB activation (e.g., see Figure 4B) (data not shown). Thus, wt Dlg1 mediates E4-ORF1-induced PI3K activation in cells, and I3-containing Dlg1 isoforms mainly provide this function. The lack of an I3 element in Dlg/β-geo explains, at least in part, its defect in supporting E4-ORF1-induced PI3K activation. Figure 4.The I3, PDZ1+2, and U3 or SH3 elements of Dlg1 are required for E4-ORF1-induced PKB activation. (A) Illustration of Dlg1 isoforms and deletion mutants. Mutants below Dlg1-I3 are derived from this isoform. (B) The PDZ1+2 conformational unit is required for Dlg1-I3 to augment E4-ORF1-induced PKB activation in Dlg1gt/gt MEF. The latter cells on 6-cm dishes were lipofected with pGW1-HA-PKB (0.4 μg), either empty pGW1 (−) or pGW1-E4-ORF1 (+) (20 ng), and either empty peGFP (−) or the indicated peGFP-Dlg1 plasmid (0.4 μg). Cells were serum starved, and equal amounts of cell extracts were blotted with antibody to (P)Thr308 PKB, HA, or GFP. (C) The PDZ1+2 conformational unit is necessary and sufficient to mediate binding of Dlg1 to E4-ORF1. For the co-precipitation assay (left), 293 T cells on 6-cm dishes were lipofected with pGW1-E4-ORF1 (1 μg) and either pGW1-Dlg1-I2 or pGW1-Dlg1-I2ΔPDZ1+2 (1 μg). Equal amounts of cell extracts were precipitated with Dlg1 antibody. Recovered proteins (left) and cell extracts (right) were blotted with antibody to SAP97 or E4-ORF1. For the GST pulldown assay (right), 293 T cells on 6-cm dishes were lipofected with the indicated pGW1-HA-Dlg1-PDZ domain plasmid (1 μg). An equal amount of cell extract was incubated with GST-E4-ORF1 fusion protein (5 μg). Recovered proteins (left) and cell extracts (right) were blotted with HA antibody. (D) Either U3 or SH3 is required for Dlg1 to augment E4-ORF1-induced PKB activation in Dlg1gt/gt MEF. The latter cells on 6-cm dishes were lipofected with pGW1-HA-PKB (0.4 μg), either empty pGW1 (−) or pGW1-E4-ORF1 (+) (20 ng), and either empty peGFP (−) or the indicated peGFP-Dlg1 plasmid (left, 0.1 μg; right, 0.4 μg). Cells were serum starved, and equal amounts of cell extracts were blotted with antibody to (P)Thr308 PKB, HA, or GFP. Download figure Download PowerPoint Multiple Dlg1 domains contribute to E4-ORF1-induced PI3K activation We next examined the capacity of GFP-Dlg1-I3 deletion mutants (Figure 4A; group 1) to rescue E4-ORF1-induced PI3K activation in Dlg1gt/gt MEF. Like wt Dlg1-I3, Dlg1-I3 mutants missing the amino-terminal region (NT), PDZ3, SH3, or GK restored robust E4-ORF1-induced PKB activation (Figure 4B). The modest defect of Dlg1-I3ΔGK probably arises indirectly from its unique, partial nuclear accumulation (Kohu et al, 2002) (data not shown). By contrast, Dlg1-I3 mutants missing PDZ1 or PDZ2 exhibited modest or severe activity loss, respectively, whereas Dlg1-I3 mutants missing either all three PDZ domains or PDZ1+2, which forms a single conformational unit (Lue et al, 1996), lacked any activity (Figure 4B). Given that PDZ1+2 was both necessary and sufficient to mediate Dlg1 binding to E4-ORF1 (Figure 4C), the defects of the latter Dlg1 mutants likely reflect nearly complete or complete disruption of this interaction. To assess possible roles for other Dlg1 domains in E4-ORF1-induced PI3K activation, we examined additional Dlg1 mutants that retain PDZ1+2 and I3, but lack certain other elements (Figure 4A; group 2). We excluded mutants with the ΔGK mutation due to their confounding partial or complete nuclear localization. Whereas Dlg1 mutants missing either the unique region between PDZ2 and PDZ3 (U3) or the SH3 domain restored E4-ORF1-induced PKB activation, Dlg1 mutants lacking both elements did not (Figure 4D), suggesting that U3 and SH3 have redundant functions for this activity. In summary, Dlg1 requires PDZ1+2, I3, and either U3 or SH3 to support E4-ORF1-induced PI3K activation. E4-ORF1 causes endogenous Dlg1 to accumulate at the plasma membrane E4-ORF1 sequesters endogenous MUPP1, MAGI-1, and ZO-2 aberrantly within detergent-insoluble complexes in the cytoplasm of cells, suggesting functional inactivation of these cellular targets. We postulated that E4-ORF1 differentially affects Dlg1 in cells because our results implicated functional rather than inactivated Dlg1 in E4-ORF1-induced PI3K activation. In rat CREF fibroblasts stably expressing E4-ORF1, endogenous ZO-2, but not endogenous Dlg1, was sequestered within detergent-insoluble complexes (Figure 5A) that appeared as cytoplasmic punctae in indirect immunofluorescence (IF) assays (Figure 5B). Close inspection of these IF assays revealed that, in ∼50% of the E4-ORF1-expressing CREF cells, but in none of the control CREF cells, some Dlg1 accumulated at the plasma membrane (Figure 5B), the known location for E4-ORF1-induced PI3K activation (Frese et al, 2003). Moreover, treatment with chlorpromazine, an inhibitor of receptor-mediated endocytosis (Sieczkarski and Whittaker, 2002), increased detection of Dlg1 plasma membrane staining to 95% of E4-ORF1-expressing CREF cells, but to less than 10% of control CREF cells (Figure 5C), implying that E4-ORF1-induced Dlg1 membrane accumulation is a dynamic process continually counterbalanced by endocytosis. Figure 5.Binding of E4-ORF1 to Dlg1-I3 promotes their translocation to the plasma membrane. (A) E4-ORF1 fails to sequester endogenous Dlg1 within detergent-insoluble complexes. Confluent CREF cells stably transfected with empty pBABE (vector) or pBABE-E4-ORF1 (E4-ORF1) were lysed either in sample buffer to isolate total cell extracts (T) or in RIPA buffer to isolate detergent-soluble (S) and detergent-insoluble (I) fractions. Equal amounts of cell extracts were blotted with antibody to SAP97, ZO-2, or E4-ORF1. (B) E4-ORF1 promotes endogenous Dlg1 to accumulate at the plasma membrane. Confluent cells described in (A) were subjected to indirect IF assays with antibody to SAP97 or ZO-2 and visualized by fluorescence microscopy. Dlg1 and ZO-2 signals are green, whereas DAPI-stained nuclei are red. The top center and right panels show Dlg1 staining for two independent CREF-E4-ORF1 lines. White arrows denote examples of Dlg1 plasma membrane staining absent in control CREF cells. (C) Chlorpromazine (cpz) substantially increases Dlg1 plasma membrane staining in E4-ORF1-expressing cells. Confluent cells described in (A) were untreated or treated with cpz for 30 min and subjected to indirect IF assays with SAP97 antibody. Results represent the average of two independent experiments, where >100 cells were analyzed. (D) E4-ORF1/Dlg1-I3 complexes translocate to the plasma membrane. 293 T cells cultured on coverslips in 24-well plates were lipofected with the indicated peGFP-Dlg1 isoform (0.5 μg) and either empty pGW1 or pGW1-E4-ORF1 (0.5 μg). Cells were subjected to indirect IF assays with E4-ORF1 antibody and visualized by fluorescence microscopy. (E) E4-ORF1 binding per se triggers Dlg1 translocation to the plasma membrane. 293 T cells cultured on coverslips in 24-well plates were lipofected with peGFP-Dlg1-I3 (0.5 μg) and pGW1-E4-ORF1, -IIIA, or -IIB (0.5 μg). Cells were fixed and visualized by fluorescence microscopy. The efficiency of wt and mutant E4-ORF1 proteins to promote translocation of cytoplasmic GFP-Dlg1-I3 to the plasma membrane was determined by quantifying the fraction of GFP-positive cells showing GFP membrane staining. The efficiency of wt E4-ORF1 was arbitrarily set to 100%, and the efficiency of mutant IIIA or IIB was reported as a percentage of this value. Results represent the average of three independent experiments, where >100 cells were analyzed. The E4-ORF1 PBM or D2 is specifically disrupted in mutant IIIA or IIB, respectively (see text). Download figure Download PowerPoint Binding to E4-ORF1 triggers Dlg1-I3 to translocate to the plasma membrane We also tested whether E4-ORF1 can cause GFP-Dlg1 to accumulate at the plasma membrane of 293T cells. When expressed alone, GFP-Dlg1-I2 and GFP-Dlg1-I3 similarly localized diffusely in the cytoplasm (Figure 5D), as did E4-ORF1, which additionally exhibited characteristic accumulation within cyto