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Molecular and Cellular Biology, January 2002, p. 41-56, Vol. 22, No. 1
0270-7306/01/$04.00+0     DOI: 10.1128/MCB.22.1.41-56.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Cytoplasmic Death Signal Triggered by Src-Mediated Phosphorylation of the Adenovirus E4orf4 Protein

Marie-Claude Gingras, Claudia Champagne, Mélanie Roy, and Josée N. Lavoie*

Centre de Recherche en Cancérologie de l’Université Laval, L’Hôtel-Dieu de Québec, CHUQ, Québec, G1R 2J6, Canada

Received 10 May 2001/ Returned for modification 25 June 2001/ Accepted 2 October 2001


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ABSTRACT
 
In transformed cells, the adenovirus E4orf4 death factor works in part by inducing a Src-mediated cytoplasmic apoptotic signal leading to caspase-independent membrane blebbing and cell death. Here we show that Src-family kinases modulate E4orf4 phosphorylation on tyrosine residues. Mutation of tyrosines 26, 42, and 59 to phenylalanines inhibited Src-induced phosphorylation of E4orf4 in vivo and in vitro but had no effect on the molecular association of E4orf4 with Src. However, in contrast to wild-type E4orf4, the nonphosphorylatable E4orf4 mutant was unable to modulate Src-dependent phosphorylation and was deficient in recruiting a subset of tyrosine-phosphorylated proteins. Indeed, the Src substrates cortactin and p62dok were found to associate with wild-type E4orf4 but not with the nonphosphorylatable E4orf4. Importantly, the nonphosphorylatable mutant E4orf4 was preferentially distributed in the cell nucleus, was unable to induce membrane blebbing, and had a highly impaired killing activity. Conversely, an activated form of E4orf4 was obtained by mutation of tyrosine 42 to glutamic acid. This pseudophosphorylated mutant E4orf4 was enriched in the cytoplasm and plasma membrane, showed increased binding to phosphotyrosine-containing proteins, and induced a dramatic blebbing phenotype associated with increased cell death. Altogether, our findings strongly suggest that Src-mediated phosphorylation of adenovirus type 2 E4orf4 is critical to promoting its cytoplasmic and membrane localization and is required for the transduction of E4orf4-Src-dependent induction of membrane blebbing. We propose that E4orf4 acts in part by uncoupling Src-dependent signals to drive the formation of a signaling complex that triggers a cytoplasmic death signal.


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INTRODUCTION
 
Apoptosis is a cell suicide program that plays a crucial role in the maintenance of cellular integrity (77). Two classical pathways for induction of apoptosis exist in mammalian cells, the intrinsic, or mitochondrial, and extrinsic, or death receptor, pathways, and both involve the activation of caspases, a family of cysteine proteases with aspartate specificity (3, 4, 28, 39). This self-amplifying caspase cascade culminates in the proteolytic inactivation of critical components of survival pathways and activation of proapoptotic functions, which altogether lead to the disassembly of the cell. Despite the general role of caspases in apoptotic processes, several modes of caspase-independent induction of cell death also exist, but the mechanisms involved are poorly understood. In general, the cytosolic hallmarks of apoptosis predominate (e.g., rounder and shrunken morphology, deformations of the plasma membrane, and membrane blebs) and are associated with DNA condensation but not with classical DNA degradation. This programmed cell death response has been termed type II apoptosis. Notably, such suicide programs can be driven by the growth suppressor PML (63), the c-myc-interacting protein Bin1 (17), the Fas-binding protein Daxx (7), and the adenoviral death factor E4orf4 (42). Caspase-independent death programs appear to be evolutionarily conserved, as classic apoptotic inducers such as Bax, Bak, or Apaf-1 elicit death in yeast cells with similar features, despite the fact that yeast lacks caspases (reviewed in reference 25). Regardless of the biochemical pathways involved, study of the cytoplasmic apoptotic events (the extranuclear phase of apoptosis) has lagged, and it is still unclear how cell shape and apoptosis signaling are integrated. Blebbing is almost invariably observed during apoptosis and may contribute to the recognition of apoptotic cells or to mix cell compartments as part of cellular packaging or as a prerequisite for apoptotic body formation (53). Whatever the case, evidence indicates that actin dynamics, which are widely regulated through the Rho GTPases (reviewed in reference 5), regulate the process of blebbing (10, 34, 40, 54, 68), and Rho GTPases can signal the cell death machinery (8, 20–22, 44, 46, 66, 74, 75).

Expression of adenoviral E4orf4 death factor in several mammalian cell lines induces a p53-independent death program (42, 49, 69). We have shown that E4orf4-induced cell death is associated with classic apoptotic hallmarks (DNA condensation, cell shrinkage, and externalization of phosphatidylserines) but does not require activation of the z-VAD-inhibitable caspases either in CHO cells (42) or in a variety of transformed human lines (our unpublished data). E4orf4 appears to be a multifunctional protein that may have several roles during adenoviral infection. The first molecular target of E4orf4 identified was protein phosphatase 2A (PP2A). The direct interaction between E4orf4 and the B55 subunit of PP2A is believed to decrease AP-1 transcriptional activity in adenovirus-infected cells (37, 56) and to trigger autoregulation of E4 transcription, as E4orf4 down-modulates E4 expression (6). Other evidence indicates that E4orf4 binding to PP2A contributes to redirection of the specificity of the host splicing machinery late during the infection cycle, by triggering the dephosphorylation and inactivation of Ser-Arg proteins (23, 36). It is believed that one of the functions of E4orf4 is to cooperate in the killing of adenovirus-infected cells at the end of the infectious cycle, thus promoting dissemination of viral progeny and escaping host immunity (49, 50, 65), but the underlying mechanisms are unknown. Mutagenesis studies indicated that E4orf4 binding to PP2A is involved in induction of p53-independent cell death in E4orf4-expressing cells (48, 70). Nevertheless, these studies indicated that additional functions are also required for cell killing. Notably, some of the E4orf4 mutants were deficient in killing activity, despite their ability to associate with PP2A (48). The downstream targets of E4orf4-PP2A complexes in the death pathway remain to be identified. Recent evidence suggested that the E4orf4-PP2A complex could act, at least in yeast cells, by targeting the anaphase-promoting complex-cyclosome to promote an irreversible growth arrest (38). It remains to be determined if this could play a role in E4orf4-induced cell death in mammalian cells. The nonclassical mechanisms underlying E4orf4-induced cell death in mammalian cells are of great interest for cancer therapy, as E4orf4 proapoptotic activity is higher in transformed cells than in normal cells (70).

Our recent work indicated that E4orf4 expression is first manifested in transformed cells by the early appearance of morphological and actin changes, which rapidly lead to dramatic membrane blebbing. These cytosolic events were found to precede the induction of DNA condensation, indicating that changes in actin dynamics lie upstream in the death pathway (40). Importantly, E4orf4 was found to associate with Src kinases and to modulate Src-dependent phosphorylation of specific cellular proteins. That Src is involved in the transduction of E4orf4 death signal is supported by the finding that inhibition of Src activity decreases E4orf4-dependent blebbing and DNA condensation, whereas activated Src promotes E4orf4-dependent apoptosis (40). In the present study, we report that adenovirus type 2 (Ad2) E4orf4 is phosphorylated on tyrosine residues and that Src kinases can modulate E4orf4 phosphorylation. Furthermore, we provide strong evidence that the tyrosine phosphorylation of E4orf4 is critical for the regulation of the subcellular distribution of E4orf4 and for the formation of a cytoplasmic signaling complex that triggers membrane blebbing and cell death in transformed cells.


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MATERIALS AND METHODS
 
Expression vectors and mutagenesis. E4orf4 mutants were generated by the PCR method using the QuickChange site-directed mutagenesis kit (Stratagene) following the manufacturer’s recommendations. The Flag-E4orf4 construct (40) was used as the template for each single mutant. The primers were designed to replace tyrosine residue (Y) with either phenylalanine (F) or glutamic acid (E). The single mutants were made using sense and antisense primers as follows: Y26F, 5'-GGT GTG GCT TTT TCT GCG GTG GTG GAT GTT-3' and 5'-CAC CGC AGA AAA AGC CAC ACC CAG CCA ACC-3'; Y42F, 5'-GAA GGA GTT TTC ATA GAA CCC GAA GCC AGG-3' and 5'-GGG TTC TAT GAA AAC TCC TTC ATG CGC CGC-3'; Y59F, 5'-GAG TGG ATA TTC TAC AAC TAC TAC ACA GAG-3' and 5'-GTA GTT GTA GAA TAT CCA CTC TCT CAA AGC-3'; Y63F, 5'-TAC AAC TAC TTC ACA GAG CGA GCT AAG CGA-3' and 5'-TCG CTC TGT GAA GTA GTT GTA GTA TAT CCA-3'; Y89F, 5'-TTC AGG AAA TTT GAC TAC GTC CGG CGT TCC-3' and 5'-GAC GTA GTC AAA TTT CCT GAA GCA AAA CCA-3'; Y42E, 5'-CAT GAA GGA GTT GAG ATA GAA CCC GAA GCC-3' and 5'-TTC GGG TTC TAT CTC AAC TCC TTC ATG CGC-3'. (Boldface indicates nucleotide substitution.) Double and triple mutations were generated using single- and double-mutant Flag-E4orf4 constructs as templates, respectively. All mutations were confirmed by DNA sequence analysis.

The expression vector encoding the Flag-E4orf4-green fluorescent protein (GFP) fusion was described previously (40). Expression vectors for mutants E4orf4(3YF) and E4orf4(Y42E) fused to GFP were similarly constructed. The mouse c-src sequence subcloned into a pCI-neo expression vector (Promega) was used as a template for all glutathione S-transferase (GST)-Src constructs (40). The GST-c-src construct was previously described (40). The Src homology 2 (SH2) domain of c-src was excised by PCR using the sense and antisense oligonucleotides 5'-CAGGATCCAGGAATTCG GAC TCC ATC CAG GCT GAG GAG-3' and 5'-TTGCGGCCGCCC CTA GAG GCG GTG ACA CAG GC-3'. The resulting DNA fragment was digested with EcoRI and NotI and inserted into the EcoRI-NotI sites of pGEX4T-3 (Pharmacia) to generate pGEX4T-3/SH2c-src. The GST-c-src{Delta}SH2 construct was produced by the PCR extension overlap technique (31). The c-src SH2 domain was deleted from the 5' sequence by PCR using the primers 5'-CCACAGGTGTCCACT-3' and 5'-TAC GGT AGT AGC CTG GAT GGA GTC GGA GGG-3' and from the 3' sequence using the primers 5'-ATC CAG GCT ACT ACC GTA TGT CCC ACA TCC-3' and 5'-CTAGTTGTGGTTTGTCC-3'. The resulting 5' and 3' c-src sequences were annealed (to join amino acids A147 in the 5' and T244 in the 3' sequence), and a second PCR was performed with the primers 5'-CCACAGGTGTCCACT-3' and 5'-CTAGTTGTGGTTTGTCC-3'. The resulting c-src{Delta}SH2 fragment was digested with EcoRI and NotI and inserted into EcoRI-NotI sites of pGEX4T-3. The Myc-FAK expression vector was provided by J. Thomas Parsons (University of Virginia School of Medicine, Charlottesville), middle-T expression vector was a gift from Stephen M. Dilworth (Royal Postgraduate Medical School, Hammersmith Hospital, London, United Kingdom), and chicken c-src(Y527F) and c-src(K295R) were a gift from Joan S. Brugge (Harvard Medical School, Boston, Mass.).

Cell culture, transfections, immunolocalization, in vivo morphological assays, and survival assays. 293T cells were derived from human embryonic kidney cells and express Ad5 E1A and E1B proteins and large-T antigen (27), and H1299 are non-small-cell lung carcinoma p53-/- cells (55). 293T cells were maintained in Dulbecco’s modified Eagle medium and H1299 cells were cultured in {alpha}-modified Eagle medium, both supplemented with 10% fetal bovine serum and streptomycin sulfate-penicillin (100 U/ml). Cells were grown in a humidified 5% CO2 atmosphere at 37°C. Transfections were performed by the calcium phosphate method as described previously (40), using 4 µg of total plasmid DNA in six-well plates or 15 µg of total plasmid DNA in 10-cm culture dishes. For microinjection, H1299 cells were plated on culture slides coated with 0.2% gelatin-phosphate-buffered saline (PBS) (Sigma).

Cell microinjection was performed with a microinjection system (5171; Eppendorf Scientific, Westbury, N.Y.), mounted on a Nikon TE-300 inverted microscope equipped with a CO2 thermoregulated environmental chamber. Plasmid DNA encoding E4orf4-GFP proteins (0.1 mg/ml) were injected into the nuclei of ~50 cells (pressure, ~100 hPa; time, 0.1 s). The blebbing-inducing activity of E4orf4 proteins was measured in 293T cells transfected with the relevant E4orf4 plasmid DNA together with Flag-GFP at a ratio of 20:1 or in H1299 cells microinjected with E4orf4-GFP plasmid DNA 2 to 3 h after injection, as described previously (40). For in vivo time-lapse analysis, the culture medium was buffered by adding HEPES (pH 7.35) to a final concentration of 50 mM 30 min before analysis. Cells were observed with a Nikon TE-300 inverted microscope equipped with a 60x 0.85 MA objective, and images were captured as 16-bit TIFF files with a Micromax 1300YHS (B/W) cooled charge-coupled device camera (-30°C) (Princeton Instruments, Trenton, N.J.) driven by Metamorph software version 4.5 (Universal Imaging Corp., Downingtown, Pa.). Confocal microscopy was performed using a Bio-Rad MRC-1024 imaging system mounted on a Nikon Diaphot-TMD equipped with a 20x objective lens.

For apoptosis assays, cells were transferred on fibronectin-coated culture slides (Falcon Biocoat; Becton Dickinson Co.) 16 h after transfection. At various times, cells were gently washed in PBS containing 1 mM MgCl2, fixed in 3.7% formaldehyde-PBS for 20 min, and then permeabilized in 0.2% Triton X-100-PBS for 5 min. Immunodetection of E4orf4 proteins was performed using purified rabbit polyclonal 2418 anti-E4orf4 (40), followed by ALEXA-594-labeled goat anti-rabbit immunoglobulin G (Molecular Probes), and DNA was stained with 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI; Molecular Probes). The proapoptotic effect of E4orf4-GFP fusion proteins was analyzed in microinjected H1299 cells by DNA staining with DAPI after cell fixation, 7 h after microinjection. Data are representative of at least four independent transfection experiments in which an average for at least 300 cells was counted for each condition (>1,000 cells) or are the means (and standard errors [SE]) of two or three independent microinjection experiments with H1299 cells in which at least 50 positive cells were evaluated. Survival was assessed by colony-forming assays in 293T cells cotransfected with a plasmid carrying a puromycin resistance gene (pGKpuro) and either empty Flag-pCDNA3 vector, wild-type Flag-E4orf4, Flag-E4orf4(3Y-F), or Flag-E4orf4(Y42E) at a plasmid DNA ratio of 1:20. Twenty-four hours after transfection, cells were lifted, aliquots of cell cultures were kept for Western analysis of E4orf4 expression levels, and the remaining cells were allowed to grow in the presence of puromycin (3 µg/ml) for 10 days to select for transfected cells. The number of surviving cells was evaluated by counting the total number of colonies.

GST fusion proteins and in vitro binding assays. GST-E4orf4 fusions were produced as described previously (40). The fusion proteins were eluted from beads with 100 mM Tris-HCl (pH 8.0)-120 mM NaCl-20 mM reduced glutathione and used for in vitro kinase assays. The pGEX4T-3/c-src and pGEX4T-3/c-src{Delta}SH2 recombinant plasmids were introduced into Epicurian Coli BL21-CodonPlus-(DE3)-RIL. The fusion proteins were produced by growing 50-ml bacterial cultures to an optical density at 600 nm of 0.8 and then treating the cultures with 0.1 mM IPTG (isopropyl-ß-D-galactopyranoside) for 8 h at 30°C. The recombinant plasmid pGEX4T-3/SH2c-src was introduced into Epicurian Coli BL21, and the fusion protein was produced by growing 50-ml bacterial cultures to an optical density at 600 nm between 0.6 and 0.8, followed by addition of 0.5 mM IPTG for 2 h at 37°C. Cells were recovered by centrifugation and kept at -20°C for the night. The bacterial pellets were thawed at room temperature for 15 min and then resuspended in 2 ml of ice-cold lysis buffer (50 mM HEPES [pH 7.4], 450 mM NaCl, 5 mM dithiothreitol, 1 mg of lysozyme per ml, and a protease inhibitor cocktail [Complete; Boehringer Manheim]). The bacterial suspension was incubated with 1% Triton X-100 and 10 µg of DNase I at room temperature for 20 min and then cleared by centrifugation at 35,000 rpm in a Beckman 70.1 Ti rotor for 15 min at 4°C. The supernatants were incubated with glutathione-Sepharose 4B beads overnight at 4°C. The beads were washed five times in washing buffer (PBS, 1% Triton X-100, 1 mM EDTA) and used for in vitro binding assays. The amount of purified fusions was evaluated on a Coomassie-stained gel.

For in vitro binding assays, transfected cells from 10-cm plates were harvested 24 h after transfection, washed in PBS, and resuspended in 2 ml of lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.5 mM Na3VO4, 1.5 mM MgCl2, 1% Nonidet P-40, 10% glycerol, 15 µg of leupeptin per ml, 5 µg of aprotinin per ml, 1 µg of pepstatin per ml). After centrifugation at 18,000 x g, supernatants were precleared with glutathione-Sepharose 4B for 60 min at 4°C. The Sepharose was then removed, and the supernatants were incubated with 5 to 10 µg of freshly prepared immobilized GST fusions overnight at 4°C. The beads were recovered by centrifugation, washed once in lysis buffer and twice in lysis buffer containing only 0.1% Nonidet P-40, and boiled in sodium dodecyl sulfate (SDS) sample buffer. The amounts of wild-type Flag-E4orf4, Flag-E4orf4(3Y-F), Flag-GFP, middle-T antigen, and Myc-FAK absorbed on the GST fusions were analyzed by Western blotting with mouse anti-Flag M2 (Sigma-Aldrich), mouse Pab 762 anti-middle T (gift from Stephen M. Dilworth) (14), and mouse 9E10 anti-c-myc antibody (Sigma-Aldrich), respectively.

Immunoprecipitations, cell fractionation, and blotting. For immunoprecipitations of Flag-E4orf4 proteins and analysis of tyrosine phosphorylation, transfected cells were harvested 24 h after transfection, washed in PBS, and resuspended in 250 µl of boiling SDS buffer (10 mM Tris-HCl [pH 7.4], 1% SDS, 1 mM Na3VO4) per 10-cm culture dish. Lysates were boiled for 5 min and passed through a 26-gauge needle five times. A 750-µl volume of water and 1 ml of 2x immunoprecipitation buffer (IP buffer) (100 mM Tris-HCl [pH 7.4], 300 mM NaCl, 2 mM MgCl2, 4 mM EDTA, 1% sodium deoxycholate, 2% NP-40, 20% glycerol, 2 mM Na3VO4, 100 mM NaF, 20 mM ß-glycerophosphate, 30 µg of leupeptin per ml, 10 µg of aprotinin per ml, 2 µg of pepstatin A per ml) were added to the denatured lysates, and equal amounts of proteins were precleared with protein G Sepharose for 1 h at 4°C. The Sepharose was removed, the supernatants were incubated with mouse anti-Flag M2 for 2.5 h at 4°C, and protein G Sepharose was added to the lysates. After a 1-h incubation at 4°C, the beads were recovered, washed three times with IP buffer, and boiled in SDS sample buffer. Metabolic labeling with [32P]orthophosphate (Amersham Pharmacia) was achieved in Earle’s balanced salt solution lacking phosphate and containing sodium pyruvate (110 mg/liter), glutamine (290 mg/liter), and bovine serum albumin (0.8 g/liter). Cultures were usually transferred to this medium 30 min before addition of 100 µCi of the labeled precursor per ml for 3 h. When indicated, Na3VO4 (0.1 mM) or a selective inhibitor of Src kinases, PP2 (50 µM; Calbiochem) was added during the labeling.

For coprecipitations, cells on 10-cm plates were lysed with 0.5 ml of modified radioimmunoprecipitation (RIPA) buffer (50 mM HEPES [pH 7.4], 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton, 1% sodium deoxycholate, 0.1% SDS, 10% glycerol, 50 mM NaF, 10 mM ß-glycerophosphate, 1 mM Na3VO4, 15 µg of leupeptin per ml, 5 µg of aprotinin per ml, 1 µg of pepstatin A per ml), and lysates were immediately diluted with 0.5 ml HNTG buffer (50 mM HEPES [pH 7.4], 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol), as described previously (71). The lysates were incubated with protein G Sepharose for 1 h at 4°C and cleared by centrifugation. Immunoprecipitates were carried out with mouse anti-Flag M2 (Sigma-Aldrich) for 3 h at 4°C, collected on protein G Sepharose, and washed three times in modified RIPA buffer containing only 1% Triton X-100 before analysis by SDS-polyacrylamide gel electrophoresis (PAGE). Equal amounts of immune complexes were resolved by SDS-8 or 11% PAGE, transferred to nitrocellulose or polyvinylidene difluoride, and processed for immunoblotting with either mouse anti-Flag M2 to detect E4orf4, mouse PY20 antiphosphotyrosine (ICN Biomedicals Inc.) or mouse RC20-HRP (Transduction Laboratories) antiphosphotyrosine antibodies, rabbit SRC2 anti-Src antibody (Santa Cruz Biotechnology, Inc.), rabbit anti-phospho-Src(Y416) (Cell Signaling Technology Inc.), rabbit M-276 anti-Dok-1 (Santa Cruz Biotechnology, Inc.), or mouse 4F11 anticortactin (Upstate Biotechnology).

Equal amounts of total cell lysates were analyzed by Western blotting with the same antibodies. To disrupt the antigen-antibody complex before reprobing, immunoblots were incubated in stripping buffer (62.5 mM Tris-HCl [pH 6.7], 2% SDS, 100 mM ß-mercaptoethanol) for 30 min at 60°C, washed in PBS-Tween (0.1%) at room temperature, and reprocessed for immunoblotting. In vitro kinase assays were performed using Src immunoprecipitates with mouse Ab-1 anti-v-src (Calbiochem-Novabiochem). The Src immune complexes were recovered with protein G Sepharose, washed three times in modified RIPA buffer and once in kinase buffer (50 mM HEPES [pH 7.4], 10 mM MgCl2, 0.1% ß-mercaptoethanol, 0.1 mM Na3VO4), and resuspended in 10 µl of kinase buffer (10 µl of packed beads) containing 2.5 µg of freshly prepared GST fusions, 0.1 mM ATP, and 10 µCi of [{gamma}-32P]ATP. The reactions were allowed to proceed for 20 min at room temperature and then stopped by adding 10 µl of 3x SDS sample buffer. Labeled samples were resolved by SDS-PAGE and visualized by autoradiography. Biochemical fractionations were performed 24 h after transfection. Cells from 10-cm plates were resuspended in 1.2 ml of hypotonic buffer (5 mM Tris-HCl [pH 7.4], 5 mM KCl, 1.5 mM MgCl2, 2.5 mM EDTA, 0.5 mM Na3VO4, 15 µg of leupeptin per ml, 5 µg of aprotinin per ml, 1 µg of pepstatin A per ml), and allowed to swell on ice for 15 min. Cells were disrupted with a Wheaton glass homogenizer fitted with a B pestle (50 strokes), and fractions were obtained by serial centrifugation as described previously (40). Equal volumes of the cell fractions containing nuclei and unbroken cells (P1) and cellular membranes and organelles (P2) and of the soluble fraction (S) were analyzed by SDS-PAGE to determine the relative amount of Flag-E4orf4 proteins, SP1, and ERK in each fraction by immunoblotting with mouse anti-Flag M2 (Sigma Aldrich), mouse IC6 anti-SP1 (Santa Cruz Biotechnology, Inc.), and rabbit anti-ERK (35), respectively. Proteins were detected immunologically after electrotransfer on nitrocellulose membranes as described previously (41), and horseradish peroxidase-linked goat anti-rabbit or anti-mouse immunoglobulin G (Jackson ImmunoResearch Laboratories), revealed by the enhanced chemiluminescence detection system (Renaissance; NEN Life Science Products), was used for detection. Quantification was performed by densitometric analyses with the software program NIH Image.


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RESULTS
 
Src-family kinases modulate Ad2 E4orf4 phosphorylation on tyrosines 26, 42, and 59. While studying the effect of Ad2 E4orf4 on Src-dependent tyrosine phosphorylation of cellular proteins, we observed that E4orf4 was itself phosphorylated on tyrosine. 293T cells were transfected with Flag-E4orf4 alone or together with kinase-deficient c-src(K295M), wild-type c-src, or v-src, and E4orf4 was immunoprecipitated with anti-Flag antibody. Analysis of E4orf4 immune complexes using antiphosphotyrosine antibody revealed the presence of a low fraction of tyrosine-phosphorylated E4orf4 when expressed alone, which was dramatically increased by coexpression with c-src and even more by v-src but not by c-src(K295M) (Fig. 1A). Similar results were obtained with myc-E4orf4 and nontagged E4orf4 (data not shown). To obtain a more quantitative measure of E4orf4 phosphorylation and its modulation by Src kinases, analyses were performed with cells metabolically labeled with [32P]orthophosphate. Coexpression of wild-type c-src produced a threefold increase in E4orf4 phosphorylation, and a similar increase was observed upon inhibition of tyrosine phosphatases by incubating cells with vanadate during the labeling period (Fig. 1B). Coexpression of activated Src kinases, either c-src(Y527F) or v-src, induced a dramatic increase in E4orf4 phosphorylation (more than 17- to 30-fold). Conversely, when 293T cells expressing Flag-E4orf4 only were incubated in the presence of a selective inhibitor of Src kinases (PP2), the basal level of E4orf4 phosphorylation was inhibited by more than 85%. Src-mediated phosphorylation of E4orf4 was also observed in vitro. Immune complexes of activated Src efficiently phosphorylated a GST-E4orf4 fusion protein, whereas immune complexes of kinase-deficient c-src were unable to phosphorylate the purified protein (Fig. 1C). Immunoblot analysis of the in vitro-phosphorylated GST-E4orf4 using antiphosphotyrosine antibody confirmed the presence of phosphorylated tyrosine residues. Thus, the results indicate that E4orf4 phosphorylation can be modulated by Src kinase activity and suggest that E4orf4 is a substrate for Src kinases.



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  		FIG. 1. Modulation of Ad2 E4orf4 tyrosine phosphorylation by Src kinase activity. (A) 293T cells were transfected with empty vector (EV) or Flag-E4orf4 with or without c-src, kinase-deficient c-src(K295M), or v-src. Twenty-four hours after transfection, immunoprecipitations were performed with anti-Flag antibody. E4orf4 immune complexes were analyzed by Western blotting using anti-Flag and antiphosphotyrosine (P-Tyr; PY20) antibodies. (B) Twenty-four hours after transfection of 293T cells with the indicated constructs, cells were metabolically labeled with 32P, and when indicated, an inhibitor of tyrosine phosphatases (Na3VO4, 0.1 mM) or a selective Src kinase inhibitor (PP2, 50 µM) was added during the labeling period. Cells were then processed for immunoprecipitation, and phosphorylation levels were detected by autoradiography. The levels of immunoprecipitated E4orf4 were determined by Western blotting using anti-Flag antibody, and phosphorylation levels were quantified with NIH Image and corrected for E4orf4 levels in immune complexes. R.U., relative units; DMSO, dimethyl sulfoxide. (C) In vitro kinase assays were performed with either precipitated v-src or kinase-deficient c-src(K295R) from transfected 293T cells, incubated with either purified GST or GST-E4orf4. Labeled reaction mixtures were resolved by SDS-PAGE and visualized by autoradiography ([{gamma}-32P]ATP) or analyzed by Western blotting after electrotransfer onto nitrocellulose using antiphosphotyrosine antibody (P-Tyr).

Ad2 E4orf4 is a small protein (114 amino acids) that contains eight tyrosines, and two of them, tyrosines 42 and 59, lie within sequence motifs which resemble the consensus sequence for phosphorylation by Src kinases (EEEIY[G/E]EFD) (72). To identify the tyrosine(s) whose phosphorylation is modulated by Src activity, we performed site-directed mutagenesis to replace various tyrosines with nonphosphorylatable phenylalanine residues either individually or in combination. The mutant Flag-E4orf4 proteins were all expressed as efficiently as the wild-type Flag-E4orf4 when transfected into 293T cells, using the same amount of plasmid DNA in transfection (Fig. 2A). Some of them were expressed even more efficiently than wild-type E4orf4, indicating that the mutations did not interfere with the stability of E4orf4. The effects of the various Y-F mutations on Src-induced phosphorylation of E4orf4 were determined by cotransfection of Flag-E4orf4 mutants with activated Src, either v-src or c-src(Y527F), followed by Western analysis of the level of tyrosine-phosphorylated E4orf4 in Flag-E4orf4 immune complexes with antiphosphotyrosine antibody. Among the five single Y-F mutations performed, three of them, at positions 42, 26, and 59, decreased Src-induced phosphorylation of E4orf4. Mutation of tyrosine 42 had the major inhibitory effect, decreasing by more than 50% the level of E4orf4 tyrosine phosphorylation. Tyrosines 26 and 59 each appeared to account for approximately 25% of the overall Src-induced phosphorylation, whereas mutation of either tyrosine 63 or 89 had no inhibitory effect (Fig. 2B). Various double mutations gave consistent inhibitory effects relative to the effect of mutating the individual tyrosine, and importantly, the triple mutation of tyrosines 26, 42, and 59 to phenylalanines removed most of the Src-induced phosphorylation of E4orf4. To confirm that the identified tyrosines were accessible for phosphorylation by Src, a mutant GST-E4orf4 fusion was produced in which tyrosines 26, 42, and 59 were replaced by phenylalanines [GST-E4orf4(3Y-F)]. In vitro kinase assays were performed with either wild-type GST-E4orf4 or GST-E4orf4(3Y-F), together with activated c-src(Y527F) precipitated from transfected 293T cells. Consistent with the in vivo phosphorylation data, the triple mutation dramatically inhibited Src-induced phosphorylation of GST-E4orf4 (Fig. 2C). Thus, the results strongly suggest that tyrosines 26, 42, and 59 account for most of the Src-dependent phosphorylation of E4orf4.



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  		FIG. 2. Mutation of tyrosines 26, 42, and 59 to phenylalanines inhibits Src-mediated phosphorylation of Ad2 E4orf4. (A) Various E4orf4 proteins were obtained by mutation of the indicated tyrosine residue(s) to phenylalanine by site-directed mutagenesis. The Flag-E4orf4 constructs were transfected into 293T cells, and 24 h after transfection, E4orf4 expression levels were analyzed by Western blotting of equal amounts of total cell lysates using anti-Flag antibody. Ponceau staining of the gel is shown as a loading control. EV, empty vector. (B) 293T cells were transfected with empty vector (EV), wild-type Flag-E4orf4 (WT), or mutant Flag-E4orf4 bearing Y-F substitution(s) of the indicated tyrosine residue(s), together with v-src or activated c-src(Y527F). Twenty-four hours after transfection, equal amounts of cell extracts were processed for immunoprecipitation with anti-Flag antibody. E4orf4 immune complexes were resolved by SDS-PAGE, and tyrosine-phosphorylated E4orf4 (P-Tyr) was detected by Western blotting with antiphosphotyrosine antibody (PY20). The levels of Flag-E4orf4 in immune complexes (IP), and levels of transfected v-src in total lysates (TL) were analyzed by Western blotting with anti-Flag M2 and anti-Src (Ab-1), respectively. The phosphotyrosine levels of E4orf4 mutants were quantified from scanned enhanced chemiluminescence-derived images by densitometric analysis with NIH Image and were corrected for the amount of precipitated Flag-E4orf4 proteins. The results are expressed as percent tyrosine-phosphorylated E4orf4 relative to wild-type (WT) E4orf4 and are the means ± SE of five independent experiments. (C) In vitro kinase assays were performed with precipitated c-src(Y527F) from transfected 293T cells incubated with either purified GST, wild-type GST-E4orf4, or mutant GST-E4orf4(3Y-F). Labeled reaction mixtures were resolved by SDS-PAGE and visualized by autoradiography ([{gamma}-32P]ATP).

Tyrosine phosphorylation of E4orf4 is not required to mediate E4orf4-Src association. We have shown previously that E4orf4 can associate with Src kinases, both by in vitro binding assays and by coprecipitations in various cell lines (with both transfected and endogenous Src kinases) (40). Several binding partners of Src, e.g., p130Cas, AFAP-110, and Sin, contain binding sites for both the SH3 and SH2 domains of Src, which mediate binding to proline-rich motifs and phosphorylated tyrosine residues, respectively (2, 29, 58, 62). The SH3 domain-mediated binding is thought to recruit Src, which then phosphorylates and binds to the SH2-binding site. The E4orf4 sequence contains an N-terminal proline-rich motif which can mediate E4orf4 binding to the v-src SH3 domain in vitro (our unpublished data). We asked whether the tyrosine phosphorylation of E4orf4 contributes to E4orf4-Src association by creating SH2-binding sites. In vitro binding assays were performed with a GST-c-src fusion protein and cell lysates from 293T transfected with either wild-type Flag-E4orf4 or the mutant Flag-E4orf4 bearing the triple Y-F substitution. Lysates from 293T transfected with either polyomavirus middle-T antigen, a known Src partner (12, 15), or GFP were used as positive and negative controls for Src binding, respectively. E4orf4(3Y-F) bound to GST-c-src as efficiently as wild-type E4orf4 (Fig. 3A), despite the fact that it was barely phosphorylated by Src in vivo and in vitro. No major increase in binding was detected when lysates from 293T cells cotransfected with activated c-src to increase wild-type E4orf4 phosphorylation were used (data not shown). Furthermore, a GST-c-src in which the SH2 domain had been deleted (GST-{Delta}SH2) bound the transfected wild-type and mutant E4orf4 proteins to similar degrees (Fig. 3A, right panels). The use of a GST fusion containing only the c-src SH2 domain (GST-SH2) further indicated that neither wild-type E4orf4 nor E4orf4(3Y-F) could bind to the Src SH2 domain. As expected, a large amount of transfected FAK, which interacts with Src SH2 domain (9, 16, 67, 79), bound to the SH2 domain of c-src but not to a Src protein lacking the SH2 domain. Coimmunoprecipitations of overexpressed wild-type E4orf4 and kinase-deficient c-src, as well as coprecipitations of E4orf4(3Y-F) and kinase-deficient c-src in 293T cells, further indicated that neither the tyrosine phosphorylation of E4orf4 nor Src kinase activity was required for the molecular association of E4orf4 with Src (Fig. 3B). Although we cannot exclude the possibility that phosphorylation of E4orf4 may contribute to stabilization of E4orf4 association with Src kinases in vivo by changing E4orf4 conformation, the results indicate that E4orf4 phosphorylation is not required to mediate E4orf4-Src interaction.



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  		FIG. 3. Src-mediated phosphorylation of Ad2 E4orf4 is not required for molecular association of E4orf4 with Src. (A) Pull-down assays were performed with lysates of 293T cells transfected with either wild-type Flag-E4orf4 (WT), Flag-E4orf4(3Y-F), polyomavirus middle-T antigen, or Flag-GFP (left panels) or cotransfected with activated c-src(Y527F) and either Flag-E4orf4, Flag-E4orf4(3Y-F), or Myc-FAK (right panels). Cell lysates were incubated with 10 µg of purified GST or the indicated GST-c-src fusion proteins bound to glutathione-Sepharose. The bound material was analyzed by Western blotting with anti-Flag M2, anti-middle-T antigen, or anti-c-myc to detect the amounts of absorbed E4orf4 or GFP, middle-T antigen, and FAK, respectively. The input lanes (TL) represent 2% of the total extracts. (B) 293T cells were transfected with kinase-deficient c-src(K295R) together with either vector alone (EV), wild-type Flag-E4orf4, or Flag-E4orf4(3Y-F). Twenty-four hours after transfection, immunoprecipitations were performed with anti-Flag M2, and equal amounts of immune complexes were analyzed by Western blotting using anti-Flag M2 and anti-Src (SRC2) to reveal E4orf4 proteins and coprecipitated kinase-deficient c-src, respectively. The amounts of transfected c-src(K295R) in total cell lysates (TL) are shown.

Tyrosine phosphorylation of E4orf4 regulates its association with specific substrates of Src. We previously observed that E4orf4 expression was associated with a rapid and specific modulation of Src-dependent tyrosine phosphorylation of cellular proteins both in cells expressing E4orf4 only and in cells coexpressing activated forms of Src (40). In agreement with our previous findings, when wild-type E4orf4 and activated c-src were overexpressed in 293T cells, a decrease in the tyrosine phosphorylation of some proteins (200- and 125-kDa ranges) and increased phosphorylation of others (80- to 85- and 62- to 68-kDa ranges) was detected in total cell lysates, relative to lysates from Src-only-transfected cells (Fig. 4A). In contrast, when E4orf4(3Y-F) was coexpressed with activated c-src, no noticeable difference in the pattern of Src-induced phosphorylation was detected compared to that obtained with Src-only-transfected cells, despite the presence of a similar level of E4orf4(3Y-F). This suggests that although hypophosphorylated E4orf4 can still associate with Src, the complex cannot functionally transduce E4orf4-dependent signals.



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  		FIG. 4. Modulation of Src-dependent phosphorylation by Ad2 E4orf4 requires tyrosine phosphorylation of E4orf4. (A) 293T cells were transfected with activated c-src alone (EV) or together with either wild-type E4orf4 (WT) or nonphosphorylatable E4orf4(3Y-F). Equal amounts of total cell extracts 24 h after transfection were analyzed by Western blotting using antiphosphotyrosine (P-Tyr, RC20-HRP), anti-Src (SRC2), or anti-Flag M2. Arrows indicate proteins whose Src-dependent phosphorylation is upregulated in cells expressing wild-type E4orf4 but not the nonphosphorylatable E4orf4(3Y-F). (B) 293T cells were transfected as for panel A, and 24 h after transfection, cells were lysed in modified RIPA buffer. Equal amounts of cell lysates were processed for immunoprecipitation of E4orf4 proteins using anti-Flag M2, and immune complexes were analyzed by Western blotting using anti-Flag M2, anti-Src (SRC2), and antiphosphotyrosine (RC20-HRP) to reveal E4orf4 proteins, activated c-src, and phosphotyrosine proteins, respectively. Antiphosphotyrosine blots were stripped and reprobed with either anti-phospho-Src (Y416), anti-Dok-1 (M-276), or anticortactin (4F11). Levels of transfected c-src(Y527F) and endogenous p62dok and cortactin (p80) in total lysates (TL) are shown. (C) 293T cells were transfected with vector alone (EV), wild-type Flag-E4orf4 (WT), or mutant Flag-E4orf4(3YF) and immunoprecipitations were performed using anti-Flag M2. Immune complexes were analyzed by Western blotting with anti-Flag M2, SRC2, M-276, and 4F11. Protein levels in total lysates prior to immunoprecipitation are shown (TL).

To further address the role of tyrosine phosphorylation of E4orf4 in the modulation of Src-dependent signaling, activated c-src was overexpressed together with either the wild-type E4orf4 or E4orf4(3Y-F), and immunoprecipitations of E4orf4 were performed. Analysis of E4orf4 immune complexes using anti-Src antibody revealed that similar amounts of activated c-src were associated with the wild-type and the mutant E4orf4 (Fig. 4B). When E4orf4 immune complexes were analyzed with antiphosphotyrosine antibody, other phosphotyrosine-containing proteins were detected. Remarkably, major bands appeared in the 62- to 68-kDa range, a region where increased tyrosine phosphorylation was observed in lysates of cells expressing wild-type E4orf4 but not E4orf4(3Y-F) (Fig. 4A and B). This suggests that E4orf4 can potentially recruit and associate with specific substrates of Src, thus promoting their phosphorylation. Importantly, the amounts of most associated tyrosine phosphorylated proteins were markedly lower or even barely detectable in immune complexes of E4orf4(3Y-F) (Fig. 4B). In attempts to clarify the identity of some of these phosphotyrosine-containing proteins, antiphosphotyrosine immunoblots were stripped and reprobed for the presence of activated c-src autophosphorylated on tyrosine 416 or for that of p62dok, a RasGAP-binding protein that is a common target of tyrosine kinases (18). Since it was previously found that E4orf4 leads to a dramatic increase in Src-dependent phosphorylation of cortactin, a p80-p85 actin-binding protein (40), we also looked for the presence of cortactin in E4orf4 immune complexes. Reprobing the membranes with anti-phospho-Src(Y416) further confirmed that equivalent amounts of active c-src were associated with wild-type and mutant E4orf4 (Fig. 4B, right panels). Importantly, endogenous p62dok and cortactin (p80) were detected in immune complexes of wild-type E4orf4, but both substrates of Src were barely detected in immune complexes of the nonphosphorylatable E4orf4(3Y-F). This indicated that Src-induced tyrosine phosphorylation of E4orf4 was critical for the recruitment of these Src substrates. Furthermore, E4orf4 association with endogenous Src kinases, p62dok, and cortactin was also detected in cells expressing E4orf4 only (Fig. 4C), suggesting that the formation of such complexes is of potential relevance to E4orf4-induced cell death. In contrast, no significant association between the nonphosphorylatable mutant E4orf4(3Y-F) and p62dok or cortactin was detected, despite the mutant’s ability to associate with endogenous Src kinases. Altogether, the results strongly suggest that E4orf4 association with specific substrates of Src such as p62dok and cortactin (18, 57) relies on the tyrosine phosphorylation of E4orf4 and, thus, that E4orf4 phosphorylation is required for the proper formation of an active E4orf4-Src signaling complex.

Tyrosine phosphorylation of E4orf4 regulates its subcellular distribution. Ad2 E4orf4 is normally distributed in both the cytoplasm and the nucleus (40, 42). Src kinase activity can modulate the subcellular distribution of E4orf4, increasing the cytoplasmic-cytoskeletal over the nuclear fraction (40). To address the role of Src-mediated phosphorylation in the regulation of E4orf4 localization, the subcellular distribution of E4orf4(3Y-F) was compared to that of wild-type E4orf4 in transfected 293T cells by immunofluorescence and confocal microscopy. Consistent with previous work, wild-type E4orf4 was distributed both in the nucleus and cytoplasm (Fig. 5A, panel a) and in cells showing membrane blebbing, E4orf4 was enriched in the cytoplasm (Fig. 5A, panel a'). In contrast, the nonphosphorylatable mutant E4orf4 protein was enriched in the nucleus, as most of the transfected cells presented a very strong nuclear staining with weak cytoplasmic staining relative to wild-type E4orf4 (Fig. 5A, panels b and b'). Indeed, when the numbers of cells presenting mainly nuclear staining (n), mainly cytoplasmic staining (c), and/or plasma membrane staining (m) in cells expressing wild-type E4orf4 were compared to those in cells expressing E4orf4(3Y-F), a reproducible increase (approximately 2.7-fold) in nuclear E4orf4 was observed in cells expressing the nonphosphorylatable E4orf4. Furthermore, a similar change in the cellular distribution of E4orf4(3Y-F) was detected by cell fractionation. Equivalent amounts of wild-type E4orf4 were distributed between the P1 and P2 fractions, and a smaller amount was recovered in the S fraction (Fig. 5B). Remarkably, the majority of the transfected E4orf4(3Y-F) was recovered in the P1 fraction, and protein amounts detected in the P2 and S fractions were decreased more than twofold relative to those of the wild-type protein. As expected, endogenous SP1 protein was recovered in the nuclear fraction whereas endogenous ERK protein was enriched in the soluble fraction, and no difference in their subcellular distribution was observed.



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  		FIG. 5. The tyrosine phosphorylation of Ad2 E4orf4 promotes its accumulation in the cytoplasm and its translocation to the plasma membrane. (A) 293T cells were transfected either with wild-type Flag-E4orf4 (a and a'), nonphosphorylatable Flag-E4orf4(3Y-F) (b and b'), or pseudophosphorylated Flag-E4orf4(Y42E) (c and c'), and immunostaining of E4orf4 proteins was performed 24 h after transfection by using rabbit anti-E4orf4. Specimens were analyzed by fluorescence confocal microscopy. Arrows are designating typical staining: c, cytoplasm mainly; n, nucleus mainly; m, clear plasma membrane accumulation. With these criteria, the numbers of cells that presented clear accumulation of E4orf4 in the different cell compartments were determined and are presented as means ± SE of four independent counts performed on different populations of transfected cells (>1,000 cells). (B) 293T cells were transfected with wild-type Flag-E4orf4 or Flag-E4orf4(3Y-F), and cell fractionation was performed 24 h after transfection. Equal proportions of P1, P2, and S fractions were analyzed by Western blotting using anti-Flag M2, anti-SP1, and anti-ERK. The relative amounts of E4orf4 proteins were quantified from scanned enhanced chemiluminescence-derived images by densitometric analysis with NIH Image and are expressed as the percentage of E4orf4 in each fraction relative to the total amount of transfected E4orf4. (C) 293T cells were cotransfected with wild-type c-src and vector alone (EV) or together with Flag-E4orf4 (WT) or the pseudophosphorylated mutant Flag-E4orf4(Y42E). Twenty-four hours after transfection, immunoprecipitations (IP) were performed with anti-Flag M2, and equal amounts of immune complexes were analyzed by Western blotting with anti-Flag-M2 and antiphosphotyrosine (RC20-HRP). The antiphosphotyrosine blot was stripped and reprobed with anti-phospho-Src(Y416) to reveal the amount of active autophosphorylated c-src in immune complexes of E4orf4. The amounts of transfected wild-type c-src in total lysates (TL) were detected by immunoblotting with anti-Src (SRC2).

The results obtained by both immunostaining and biochemical fractionation strongly suggested that hypophosphorylated E4orf4 was preferentially distributed in the nucleus and that tyrosine phosphorylation of E4orf4 could promote its accumulation in cytoplasm-membrane regions. To further address this possibility, tyrosine 42, which accounts for 50% of Src-induced phosphorylation, was mutated to a glutamic acid (Y42E) to mimic the effect of a constitutive phosphorylation. This mutant E4orf4 was expressed to levels comparable to those obtained for the wild-type E4orf4 by using the same amount of plasmid DNA in transfection (Fig. 6A and 7B). Quantitative analysis of the localization of E4orf4(Y42E) indicated that less than 10% of the transfected cells accumulated the pseudophosphorylated E4orf4 in the nucleus (Fig. 5A), in marked contrast to what was observed for the nonphosphorylated E4orf4. Remarkably, the number of cells that presented a clear accumulation of E4orf4 protein at the plasma membrane was increased more than threefold relative to cells expressing the wild-type E4orf4 (Fig. 5A, panel c', shows a representative example of E4orf4 accumulation at the plasma membrane). Importantly, E4orf4 redistribution to cytoplasm-membrane regions was associated with a dramatic increase in the ability of E4orf4(Y42E) to recruit tyrosine-phosphorylated proteins relative to the wild-type E4orf4. Indeed, when E4orf4 proteins were coexpressed with wild-type c-src, higher levels of tyrosine-phosphorylated proteins were detected in immune complexes of E4orf4(Y42E), and furthermore, a higher level of active c-src (autophosphorylated on Y416) was found to be associated with the pseudophosphorylated protein (Fig. 5C). Taken together, the results strongly suggest that Src-mediated tyrosine phosphorylation of E4orf4 can promote its stable accumulation in the cytoplasm and its translocation to the plasma membrane and that E4orf4 translocation is rate limiting for the formation of active E4orf4-Src signaling complexes.



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  		FIG. 6. Tyrosine phosphorylation of Ad2 E4orf4 is required for E4orf4-dependent induction of membrane blebbing. (A) The morphology of 293T cells cotransfected with Flag-GFP and the vector alone or together with the indicated Flag-E4orf4 constructs, at a plasmid DNA ratio of 1:20, was analyzed by fluorescence confocal microscopy 12 h after transfection. Representative GFP-positive cells coexpressing various E4orf4 proteins are shown, and arrows indicate membrane blebs typically observed in wild-type-E4orf4-expressing cells, compared to cells expressing equivalent amounts of the pseudophosphorylated E4orf4(Y42E). Percentages of blebbing cells were determined by counting GFP-positive cells presenting more than one bleb per cell 24 h after transfection and are expressed relative to the total number of GFP-positive cells. The data are representative of at least four independent experiments in which a minimum of 300 cells per condition were evaluated (means ± SE). After morphological analysis, cells were lysed in SDS sample buffer, and expression levels of the transfected proteins (Flag-E4orf4 and GFP proteins) were determined by Western blotting analysis of equal amounts of cell lysates, using anti-Flag M2 and anti-Src (SRC2) to detect endogenous Src proteins as loading controls. (B) In vivo time lapse analyses of 293T blebbing cells transfected with GFP together with either wild-type E4orf4 or pseudophosphorylated E4orf4(Y42E) were performed by taking pictures of the same cells every 5 s over a period of 2.5 min, 12 to 24 h after transfection. Representative series of pictures are presented for cells expressing either wild-type (WT) E4orf4 or E4orf4(Y42E). Arrows indicate blebbing events (either protrusion or retraction of a bleb), and the numbers of events per cell per min are shown as means ± SE of the blebbing movement estimated in at least 40 cells expressing each of the E4orf4 proteins. (C) 293T transfected with GFP together with either wild-type or pseudophosphorylated E4orf4, with and without c-src, were analyzed by fluorescence microscopy. The occurrence of uropod-like structures was determined by counting the number of giant protrusions (arrows pointing to cell 1) in each GFP-positive cell population 24 h after transfection, relative to the total number of GFP-positive cells. The data are means ± SE of three independent experiments in which at least 300 GFP-positive cells were analyzed. Western analysis of Flag-E4orf4 expression levels and Src (endogenous and transfected) is shown, and Ponceau staining of the gel is presented as a loading control.



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  		FIG. 7. The killing activity of Ad2 E4orf4 is regulated by its tyrosine phosphorylation level. (A) 293T cells were plated on fibronectin-coated slides and transfected with either vector only (EV), wild-type E4orf4, nonphosphorylatable E4orf4(3Y-F), or pseudophosphorylated E4orf4(Y42E), using equivalent amounts of plasmid DNA. At the indicated times after transfection, cells were fixed and immunostaining of E4orf4 proteins was performed, coupled to DNA staining with DAPI. Specimens were analyzed by fluorescence microscopy, and representative phenotypes are presented. The percent apoptotic nuclei was obtained by counting at least 300 E4orf4-positive cells, and data are expressed as the number of cells presenting nuclear condensation (arrows) relative to the total number of E4orf4-positive cells. The data are representative of at least four independent experiments (means ± SE). (B) 293T cells were transfected with pGKpuro together with either the vector only (EV), wild-type E4orf4, E4orf4(3Y-F), or E4orf4 (Y42E), using a plasmid DNA ratio of 1:20, and cell survival assays were performed as described in Material and Methods. Aliquots of transfected cells were kept for Western blot analyses of expression levels 24 h after transfection, before selection of transfected cells. Percentages of cells surviving were obtained by counting the number of resulting colonies and are expressed relative to the total number of colonies obtained in cells transfected with the vector only (control). Data are representative of three independent experiments (means plus SE).

Tyrosine phosphorylation of Ad2 E4orf4 is required to induce membrane blebbing and regulates E4orf4 killing activity. E4orf4 expression is first manifested in transformed cells by the appearance of changes in cell morphology and actin organization that rapidly lead to dramatic membrane blebbing, and Src kinase activity was found to regulate these E4orf4-dependent processes (40). To determine whether Src-mediated tyrosine phosphorylation of E4orf4 was involved, the blebbing-inducing activity of various mutant E4orf4 proteins was compared to that of the wild-type E4orf4 by using GFP as a reporter in cotransfection assays to visualize the cellular morphology in live cells. Overexpression of similar levels of the various E4orf4 proteins in 293T cells led to drastically different cell phenotypes which appeared as early as 8 h after transfection. Quantification of the number of GFP-positive blebbing cells 24 h after transfection revealed that appearance of blebbing in cells expressing the various E4orf4 proteins correlated with the ability of E4orf4 proteins to show Src-dependent tyrosine phosphorylation (Fig. 6A). Indeed, induction of blebbing was dramatically inhibited in cells expressing the nonphosphorylatable mutant E4orf4(3Y-F), whereas it was decreased by 50% in cells expressing a mutant E4orf4 bearing the double Y26-42F substitution. Kinetics analysis of the number of blebbing cells revealed that 48 h after transfection, the number of wild-type-expressing cells undergoing blebbing declined, presumably as a result of increased cell death, whereas in cells expressing the nonphosphorylatable E4orf4, membrane blebbing was still inhibited (data not shown). In marked contrast, overexpression of the pseudophosphorylated mutant E4orf4(Y42E) increased the number of GFP-positive cells showing membrane blebbing relative to cells expressing the wild-type E4orf4. Even more evident was the dramatic increase in the extent of blebbing within individual cells, in terms of both number and size of blebs (Fig. 6A). In vivo time-lapse analysis of blebbing cells was performed, using a charge-coupled device camera under the control of a software program allowing the rapid collection of multiple-image data. More than 40 cells coexpressing GFP and either wild-type E4orf4 or E4orf4(Y42E) were analyzed by capturing images every 5 s over a period of 2.5 min. This allowed us to estimate the occurrence of blebbing (the number of events as defined by either protrusion or retraction of a bleb) as an average of 1.7/cell/min in cells expressing the wild-type E4orf4, compared to 3.0/cell/min in cells expressing the pseudophosphorylated E4orf4(Y42E), at similar E4orf4 expression levels (Fig. 6B).

The increase in both the occurrence of blebbing cells and the extent of blebbing in cells expressing the E4orf4(Y42E) was reminiscent of that previously observed upon coexpression of activated Src and wild-type E4orf4 (40). Furthermore, we observed similar increases in the occurrence of uropod-like structures, which are occasionally observed in cells undergoing dramatic E4orf4-dependent blebbing (Fig. 6C). Uropods are defined as long (at least one-third of the cell body), large bulbs transiently protruding from the cell surface (61). In 293T cells expressing wild-type E4orf4, the transient protrusion of similar giant structures was noticed in a relatively low proportion of cells at a given time. When c-src was overexpressed with wild-type E4orf4, the occurrence of uropod-like structures was increased two- to threefold relative to that in cells expressing wild-type E4orf4 only (Fig. 6C), further indicating that an increase in Src kinase activity can promote the effects of E4orf4 on actin dynamics. The number of cells expressing the E4orf4(Y42E) that presented these dramatic structures was already increased threefold, and importantly, it could not be further increased by coexpression of c-src (Fig. 6C. Altogether, the results strongly suggest that Src-mediated tyrosine phosphorylation of E4orf4 is required and is a rate-limiting step in the process of E4orf4-mediated changes in actin dynamics that leads to membrane blebbing.

Finally, the effect of E4orf4 phosphorylation on its killing activity was measured by looking at DNA condensation and clonogenic survival in cells expressing equivalent levels of wild-type E4orf4, E4orf4(3Y-F), or E4orf4(Y42E). In a finding consistent with previous work, a close correlation was observed between the extent of E4orf4-induced blebbing and E4orf4-dependent DNA condensation (40). Again, a marked increase in the number of cells with apoptotic nuclei was observed in 293T cells expressing the E4orf4(Y42E) relative to cells expressing the wild-type E4orf4 (Fig. 7A). As a result, clonogenic survival was reduced more than twofold (Fig. 7B). Conversely, in cells overexpressing a similar level of the nonphosphorylatable mutant E4orf4(3Y-F), the appearance of apoptotic nuclei was strongly inhibited (more than 50% inhibition), and this correlated with a twofold increase in cell survival (Fig. 7A and B). The results strongly suggest that the tyrosine phosphorylation of E4orf4, which is required for E4orf4-induced blebbing, regulates E4orf4-dependent cell killing.

We then investigated whether the phosphorylation-dependent extranuclear activity of E4orf4 could be generalized to cells that are not transformed by viral oncogenes. The subcellular distribution and proapoptotic activity of the various mutant E4orf4 proteins were analyzed in p53-deficient human non-small-cell lung carcinoma H1299 cells, which are highly sensitive to E4orf4 killing (40, 48). Plasmid DNA encoding either wild-type E4orf4, nonphosphorylatable E4orf4(3Y-F), or pseudophosphorylated E4orf4(Y42E) fused to GFP was microinjected into the cell nucleus, and early effects on cell morphology were monitored in vivo. This allowed us to monitor the in vivo dynamics of E4orf4 distribution by monitoring the subcellular location of the various E4orf4-GFP fusion proteins soon after they were first expressed. Quantification analysis revealed that wild-type E4orf4-GFP preferentially accumulated in the nuclei of a high proportion of cells 2 h postinjection (50%) and translocated into the cytoplasm as cells progressively accumulated higher amounts of E4orf4, in agreement with previous analysis with transfected 293T cells (40). Indeed, a twofold increase in the number of cells that accumulated high levels of E4orf4 in the cytoplasm was observed 3 h postinjection (Fig. 8A). Consistent with what was observed in 293T cells, the nonphosphorylatable E4orf4(3Y-F)-GFP was highly enriched in nuclear regions and was apparently more tightly retained in the nucleus, as no significant redistribution of the mutant protein was observed over a 1-h period. In marked contrast, the pseudophosphorylated E4orf4(Y42E)-GFP was retained in the cytoplasm and less than 10% of the cells accumulated the protein in the cell nucleus 3 h postinjection. Not surprisingly, the blebbing-inducing activity of the various E4orf4-GFP proteins correlated with their ability to accumulate in the cytoplasm 2 h postinjection (Fig. 8B). Finally, analysis of the nuclear morphology 7 h after microinjection revealed that the proapoptotic activity of the nonphosphorylatable E4orf4(3Y-F)-GFP was decreased, whereas that of the pseudophosphorylated E4orf4(Y42E)-GFP was increased relative to wild-type E4orf4-GFP, consistent with what was observed in 293T cells (Fig. 8B). Thus, the results indicated that tyrosine phosphorylation of E4orf4 can regulate its killing function in human tumor cells, presumably by orchestrating the formation of a cytoplasmic death signaling complex. Indeed, E4orf4 was found to associate with and modulate Src kinases in many human transformed lines, including H1299, and cortactin is also associated with E4orf4 in transfected H1299 cells (reference 40 and unpublished results).



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  		FIG. 8. The proapoptotic activity of Ad2 E4orf4 is regulated by tyrosine phosphorylation in human tumor H1299 cells. (A) Plasmid DNA encoding either Flag-GFP (50 ng/µl), wild-type E4orf4 fusion protein E4orf4-GFP (100 ng/µl), nonphosphorylatable E4orf4(3Y-F)-GFP (100 ng/µl), or pseudophosphorylated E4orf4(Y42E)-GFP (100 ng/µl) was microinjected into the nucleus of H1299 cells. Two hours after injection, the subcellular distribution of E4orf4-GFP proteins and their effects on cell morphology were analyzed by fluorescence microscopy in vivo, and representative effects are shown. The subcellular localization of E4orf4-GFP proteins was evaluated 2 and 3 h after injection by counting the number of cells expressing E4orf4 either mainly in the nucleus (n), in the nucleus and the cytoplasm (n/c), or mainly in the cytoplasm (c). The data are means ± SE and are representative of two independent experiments in which at least 50 positive cells were evaluated in each condition. (B) The blebbing-inducing activity of the various E4orf4-GFP proteins was evaluated by counting the number of GFP-positive blebbing cells in vivo 2 h after injection. The killing activity of the various E4orf4-GFP proteins was determined 7 h after microinjection following cell fixation and DNA staining with DAPI. Data are means ± SE of three independent experiments in which at least 50 positive cells were evaluated for each condition.


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DISCUSSION
 
We have shown recently that E4orf4-induced killing in transformed cells is closely linked to the early induction of changes in actin dynamics leading to dramatic membrane blebbing (40). Importantly, we provided strong evidence that this process relies on E4orf4 ability to associate with and to modulate the activity of Src-family kinases. The present study provides further evidence supporting the role of Src kinase activity in the regulation of E4orf4-induced killing. Indeed, our findings strongly suggest that Src-dependent phosphorylation of E4orf4 itself is critical for induction of blebbing and cell death in human transformed cells.

This study provides the first evidence that Ad2 E4orf4 is phosphorylated on tyrosine residues and that a strong modulation of E4orf4 phosphorylation can be driven by Src-family kinases in vivo. Furthermore, tyrosine phosphorylation of purified E4orf4 by active Src isolated from cells was observed in vitro, indicating that E4orf4 is a Src substrate or a substrate for a tyrosine kinase activated by and coprecipitated with Src. We believe that the tyrosine phosphorylation of E4orf4 is likely mediated directly by Src. First, our mutagenesis studies indicated that tyrosine 42 and, to a lesser extent, tyrosines 26 and 59 accounted for Src-induced E4orf4 phosphorylation both in vivo and in vitro. Importantly, two of these tyrosines (42 and 59) are within sequence motifs resembling the amino acid sequence preferentially phosphorylated by Src kinases (72). Second, E4orf4 is found in complex with Src kinases and can associate with Src in vitro (40; this study). Whatever the case, we obtained strong evidence that the tyrosine phosphorylation of E4orf4 is required and is rate limiting for the transduction of the cytoplasmic apoptotic signal. This is supported by data showing that inhibition of Src-induced phosphorylation of E4orf4, upon mutation of tyrosines 26, 42, and 59 to phenylalanines, led to a dramatic inhibition of E4orf4 blebbing-inducing activity that greatly reduced its killing activity. Furthermore, a gain of function was observed upon mutation of tyrosine 42 to glutamic acid to mimic a constitutive phosphorylation on the major site. This led to an increase in the occurrence and dynamics of blebbing, associated with increased cell killing. Indeed, the number of blebbing cells and the extent of blebbing were similarly increased in cells expressing the pseudophosphorylated E4orf4(Y42E) relative to cells coexpressing activated Src and wild-type E4orf4 (40; this study). A Src-dependent increase in the dynamic of blebbing was evidenced by the formation of dramatic protrusions resembling uropods, an effect also mimicked by expression of E4orf4(Y42E) alone, which could not be further potentiated by coexpression of Src. Similar results were obtained in human cells transformed with viral oncogenes (293T) and human p53-/- tumor cells (H1299), indicating that the tyrosine phosphorylation level of E4orf4 may generally modulate its proapoptotic activity in transformed cells. Nevertheless, a limited cell death response was still observed upon overexpression of the nonphosphorylatable mutant E4orf4(3Y-F) (around 50%). This may indicate that a low level of tyrosine phosphorylation on another residue(s) targeted in the context of the triple mutant could account for the residual killing effect. Alternatively, this may reveal the existence of another death-inducing activity of E4orf4, which could involve other cellular targets of the multifunctional Ad2 E4orf4 protein (23, 37, 48, 70). This is likely, considering that the residual killing activity was observed in the absence of significant membrane blebbing induction.

How does tyrosine phosphorylation of E4orf4 act to promote E4orf4-induced blebbing and cell death? Our immunolocalization and fractionation analyses indicate that tyrosine phosphorylation of E4orf4 is critical for its stable accumulation and retention in the cytoplasm and its translocation to the cell membrane, a rate-limiting step for the formation of stable and active E4orf4-Src signaling complexes. In fact, coprecipitations of E4orf4 and Src from a membrane cell fraction were observed when wild-type and pseudophosphorylated E4orf4 were expressed but not with nonphosphorylatable E4orf4 (40) (data not shown). Analysis of the in vivo subcellular distribution of the E4orf4-GFP fusion in microinjected cells clearly revealed that initially, E4orf4 preferentially accumulates in the cell nucleus, but it can shuttle between the nucleus and the cytoplasm. Importantly, the nonphosphorylatable mutant E4orf4(3Y-F) was found mostly retained in the cell nucleus in vivo, whereas pseudophosphorylation on tyrosine 42 promoted E4orf4 retention in the cytoplasm and/or plasma membrane, in both 293T and H1299 cells. Based on our findings, it appears that phosphorylation on tyrosine 42 (or addition of a negative charge) can induce a change in the conformation of E4orf4 that promotes its retention in the cytoplasm and/or inhibits its nuclear import. The subcellular distribution of E4orf4 is likely regulated by specific protein interaction, as the 14-kDa protein always shows a specific distribution instead of being localized diffusely like other small proteins, e.g., GFP (40, 42; this study). Indeed, the second half of the E4orf4 sequence contains the molecular determinants of its nuclear localization (unpublished results). It is possible that structural changes induced by tyrosine phosphorylation of E4orf4 (requiring phosphotyrosine 42) might unmask and/or cover up protein interaction domains, which favor the retention of E4orf4 in the cytoplasm and trigger its translocation at the plasma membrane. Accordingly, the ability of Src kinase activity to promote E4orf4 association with the membrane and cytoskeleton likely relies on E4orf4 phosphorylation (40).

As summarized in a working model (Fig. 9), we believe that Src-mediated phosphorylation of E4orf4 drives a conformational change in E4orf4 protein, allowing its stable accumulation in the cytoplasmic and cytoskeletal compartments. The tyrosine phosphorylation of E4orf4 is also thought to play a role in the recruitment of signaling proteins, presumably involved in the regulation of actin dynamics. Src-mediated phosphorylation of E4orf4 may provide SH2-binding sites for specific substrates of Src, or alternatively, specific substrates might bind to E4orf4 or Src following a conformational change triggered by E4orf4 phosphorylation on tyrosine(s). Indeed, wild-type E4orf4 was found to associate with a subset of phosphotyrosine-containing proteins whose phosphorylation is increased in cells coexpressing activated Src, whereas the nonphosphorylatable E4orf4(3Y-F) was highly deficient in recruiting this subset of proteins. Conversely, higher levels of these tyrosine-phosphorylated proteins were associated with the pseudophosphorylated E4orf4(Y42E) relative to the wild-type protein, further indicating that E4orf4 translocation to the cytoplasm and/or membrane is a rate-limiting step for the transduction of the Src-dependent signal. Importantly, we found that wild-type E4orf4 can associate with p62dok and cortactin, two substrates of Src kinases, in a phosphorylation-dependent manner. Indeed, the nonphosphorylatable mutant E4orf4(3Y-F) was barely associated with these phosphotyrosine-containing proteins. This finding is consistent with the idea that tyrosine phosphorylation of E4orf4 is critical for the recruitment of potential substrates of Src, such as p62dok and cortactin, which, once phosphorylated, would signal actin remodeling and cell death. Interestingly, tyrosine phosphorylation of p62dok was shown to promote the recruitment of RasGAP, a negative regulator of Ras and a p190RhoGAP-binding protein, another potential substrate for Src kinases (18, 19, 32, 33, 59, 80). The RasGAP/p190RhoGAP complex appears to regulate actin cytoskeletal dynamics (43, 51, 64). Accordingly, E4orf4-mediated recruitment and phosphorylation of p62dok may play a central role in the local recruitment of regulators of actin dynamics. Recently, p62dok was also shown to inhibit Src-induced cellular transformation, suggesting that it can play a central role in negative signaling (73). Cortactin, which is also recruited by E4orf4 and whose Src-mediated tyrosine phosphorylation is increased in E4orf4-expressing cells, likely contributes to local actin remodeling (polymerization and contraction) for membrane blebbing induction (26, 40, 45, 78). In future work, we hope to elucidate the potential role of these substrates of Src in the transduction of E4orf4-induced membrane blebbing and cell death, as this may shed light on the physiological role of these signaling proteins and their potential involvement in other extranuclear apoptotic responses.



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  		FIG. 9. Working model. Tyrosine phosphorylation of Ad2 E4orf4 orchestrates the formation of a blebbing-inducing signaling complex and is required to initiate a Src-dependent cytoplasmic death signal. The data indicated that tyrosine phosphorylation of Ad2 E4orf4 is critical for the stable accumulation of E4orf4 in the cytoplasm, presumably by inducing a change in E4orf4 conformation. Based on the data, Src-mediated phosphorylation of E4orf4 is also believed to contribute to the recruitment of substrates of Src (proteins X, Y, and possibly cortactin and p62dok) involved in the modulation of actin dynamics by providing SH2-binding sites or by triggering a change in Src conformation that would promote its binding to specific substrates. Accordingly, Ad2 E4orf4-induced dysregulation of Src kinases may lead to the formation of a signaling complex promoting Src-dependent morphogenic events. Such an imbalance in Src signals is proposed to trigger a cytoplasmic death signal, leading to membrane blebbing and cell death. Symbols: arrows, stimulation; broken arrows, inhibition; squares, hypophosphorylated E4orf4; ovals, hyperphosphorylated E4orf4; YP, phosphotyrosines; XP and YP, tyrosine-phosphorylated substrates of Src.

There is precedent for viral proteins that deregulate Src kinases and use the kinase activity to assemble cellular proteins into an active signaling complex (reviewed in references 13 and 52). Such is the case for polyomavirus middle-T antigens, whose phosphorylation by Src-related kinases at specific tyrosine residues is a prerequisite for the subsequent recruitment and phosphorylation of cellular proteins, which signal cell transformation. How then can E4orf4 similarly use tyrosine kinases, which are known to transduce growth-promoting signals, to drive a cell death response? The ubiquitously expressed Src-family members play redundant roles in a wide array of cellular functions, including cell proliferation, adhesion and/or migration, survival, and actin dynamics (1, 76). The full oncogenic potential of Src kinases likely involves multiple pathways, and the balance of those may be essential to the outcome (survival or demise) (47, 60), as observed for other oncogenes (11, 24, 30). We have shown previously that E4orf4 expression is associated with reduced phosphorylation and activation of FAK and focal adhesion-associated proteins and increased phosphorylation of other substrates of Src (e.g., cortactin) (40). As a consequence of dysregulation of Src kinases by E4orf4, hypophosphorylated FAK may fail to transduce a cell survival signal and contribute to the disorganization of focal complexes observed in E4orf4-expressing cells (34). We propose that Src-dependent morphogenic events (driven by phosphorylation of p62dok, cortactin, and/or proteins X and Y) (Fig. 9) become proapoptotic when uncoupled from Src-dependent survival signals (inhibition of FAK and perhaps others) and that E4orf4 may induce apoptosis, in part, by causing such uncoupling in Src signals. Deciphering the role of specific downstream targets of E4orf4-Src complexes in the cell death process may unravel important cellular functions linking cell proliferation to the cell death machinery, which can transduce caspase-independent cell death in transformed cells.


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ACKNOWLEDGMENTS
 
We thank Aroussen Laflamme for contributing to the construction of E4orf4(3Y-F)-GFP and E4orf4(Y42E)-GFP expression vectors. We are most grateful to André Lévesque for his major contribution in establishing the microinjection technique as well as for his assistance in all microscopic and imaging analyses. We thank Stephen M. Dilworth, Thomas J. Parsons, and Joan S. Brugge for providing us with expression vectors for middle-T antigen, FAK, and chicken c-src, respectively, and Jacques Landry for helpful discussions and critical reading of the manuscript.

This work was supported by a Terry Fox Research Grant (no. 009058) from the National Cancer Institute of Canada (NCIC) and partly by a research grant from the Canadian Institutes of Health Research (CIHR) (MOP-49450). J. N. Lavoie is a Scholar of the Canadian Institutes of Health Research (CIHR).


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FOOTNOTES
 
* Corresponding author. Mailing address: CRC, L’Hôtel-Dieu de Québec, CHUQ, St-Patrick, 9 Rue McMahon, Québec, Qc., Canada, G1R 2J6. Phone: (418) 525-4444, ext. 5120. Fax: (418) 691-5439. E-mail: josee.lavoie{at}crhdq.ulaval.ca. Back


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Molecular and Cellular Biology, January 2002, p. 41-56, Vol. 22, No. 1
0270-7306/01/$04.00+0     DOI: 10.1128/MCB.22.1.41-56.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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