E4-ORF1-induced PKB activation depends on Dlg1. (A) E4-ORF1-induced PKB activation is deficient in Dlg1^gt/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 Dlg1^gt/gt MEF. On 6-cm dishes, Dlg1^gt/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.

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 Dlg1^gt/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 Dlg1^gt/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 Dlg1^gt/gt MEF. The latter cells on 6-cm dishes were lipofected with either empty pGW1 (−) or pGW1-HA-PKB (+) (0.4 μg), either empty pCMV_BamNeo (−) or pCMV_BamNeo-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. Dlg1^gt/gt MEF on 6-cm dishes were lipofected with pGW1-HA-PKB (0.4 μg), either empty pCMV_BamNeo (−) or the indicated pCMV_BamNeo-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 Dlg1^gt/gt MEF. The latter cells on 6-cm dishes were lipofected with pGW1-HA-PKB (0.4 μg), either empty pGW1 (−) or pCMV_BamNeo-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.

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).

Model for Dlg1-dependent PI3K activation by the adenovirus E4-ORF1 oncoprotein. See text for details.

Dlg1 is also required for E4-ORF1 to induce anchorage-independent growth in MEF. (A) Dlg1^gt/gt MEF stably expressing E4-ORF1 fail to accumulate activated, endogenous PKB. Dlg1^+/+ or Dlg1^gt/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) Dlg1^gt/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).

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 Dlg1^gt/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 Dlg1^gt/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.

PKB activation by E4-ORF1 requires Ras. (A) Dominant-negative (DN) Ras-N17 inhibits E4-ORF1-induced PI3K activation. NIH 3T3 cells on 6-cm dishes were lipofected with pGW1-HA-PKB (0.5 μg) and the indicated combinations of pGW1-E4-ORF1 (25 ng), pGW1-H-Ras-wt (100 ng), pGW1-H-Ras-N17 (500 ng), pGW1-R-Ras-wt (100 ng), and pCDNA3-myr-p110α coding for constitutively activated PI3K (PI3K^*) (25 ng). Cells were serum starved, and equal amounts of cell extracts were blotted with antibody to (P)Thr308 PKB, HA, or H-Ras. (B) Ras-N17 also inhibits Dlg1-dependent E4-ORF1-induced PKB activation in Dlg1^gt/gt MEF. The latter cells on 6-cm dishes were lipofected with pGW1-HA-PKB (0.4 μg), empty pCMV_BamNeo (−) or pCMV_BamNeo-E4-ORF1 (+) (0.1 μg), empty pGW1 (−) or pGW1-RasN17 (+) (1.0 μg), and empty peGFP (−) or peGFP-Dlg1-I3 (+) (0.4 μg). Cells were serum starved, and an equal amount of cell extract was blotted with antibody to (P)Thr308 PKB, HA, or SAP97. (C) Treatment with the PI3K inhibitor LY unmasks ERK activation by E4-ORF1 in CREF cells. Confluent CREF cells stably transfected with pBABE (vector) or pBABE-E4-ORF1 (E4-ORF1) were serum starved (−) and treated with 50 μM LY (+) for 1 h. An equal amount of cell extract was blotted with antibody to (P)Thr202/Tyr204 p42/44 MAPK, p42/44 MAPK, (P)Thr308 PKB, PKB, or E4-ORF1.