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, 89 (21), 10774-85

Adenovirus E4-ORF1 Dysregulates Epidermal Growth Factor and Insulin/Insulin-Like Growth Factor Receptors To Mediate Constitutive Myc Expression

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Adenovirus E4-ORF1 Dysregulates Epidermal Growth Factor and Insulin/Insulin-Like Growth Factor Receptors To Mediate Constitutive Myc Expression

Kathleen Kong et al. J Virol.

Abstract

The E4-ORF1 protein encoded by human adenovirus stimulates viral replication in human epithelial cells by binding and activating cellular phosphatidylinositol 3-kinase (PI3K) at the plasma membrane and cellular Myc in the nucleus. In this study, we showed that E4-ORF1 hijacks the tyrosine kinase activities of cellular epidermal growth factor receptor (EGFR) and insulin receptor (InsR)/insulin-like growth factor receptor 1 (IGF1R), as well as the lipid kinase activity of PI3K, to mediate constitutive Myc protein expression. We additionally demonstrated that EGFR contributes to constitutive Myc expression through the capacity of E4-ORF1 to induce ligand-independent EGFR activation and stimulation of the Ras/Mek/Erk pathway, the latter activity of which was conserved by human adenoviruses. Results further suggested that EGFR normally forms a complex with the cellular PDZ protein Discs Large 1 (Dlg1), a component of the Dlg1:E4-ORF1:PI3K ternary complex that mediates E4-ORF1-induced PI3K activation, and that E4-ORF1 binds the Dlg1:EGFR complex and promotes the association of EGFR with InsR and IGF1R. In addition to its role in constitutive Myc expression, InsR/IGF1R also negatively regulates EGFR autophosphorylation and EGFR-mediated Ras/Mek/Erk signaling, and data suggested that E4-ORF1 binding to Dlg1 antagonizes these activities. Collectively, our findings suggest that in human epithelial cells, E4-ORF1 targets EGFR, InsR/IGF1R, and PI3K at the plasma membrane to activate cytosolic signaling pathways that sustain Myc protein levels in the nucleus. We postulate that E4-ORF1-induced constitutive Myc expression functions to ensure the formation of nuclear E4-ORF1:Myc complexes, which have been shown to activate Myc and to enhance adenovirus replication.

Importance: While human adenoviruses primarily produce self-limited acute infections in humans, these agents are associated with life-threatening diseases in immunocompromised patients and in otherwise healthy individuals infected with certain virulent serotypes. The adenovirus E4-ORF1 protein enhances viral replication by activating the cellular lipid kinase PI3K and the cellular transcription factor Myc. Here we report that E4-ORF1 usurps the functions of the cellular tyrosine kinase receptors EGFR and InsR /: IGF1R, as well as PI3K, to sustain Myc protein expression in cells. Furthermore, sustained Myc expression depended on E4-ORF1-induced ligand-independent EGFR activation that stimulated Ras/Mek/Erk signaling, a function found to be conserved by human adenoviruses. Given the established roles of PI3K, the Ras/Mek/Erk pathway, and Myc in the adenovirus life cycle, our findings may aid in the development of safer, more effective therapeutic strategies to treat severe adenovirus infections as well as improved adenovirus vectors for use in vaccination and gene and cancer therapy.

Figures

FIG 1
FIG 1
E4-ORF1 activates the Ras/Mek/Erk pathway. (A) E4-ORF1 increases Erk1/2 activation in MCF10A cells cultured in complete medium (CM) by a primarily D2-dependent mechanism. Cell extracts prepared from the indicated MCF10A lines at 24 h or 72 h postplating in complete medium were immunoblotted for the indicated proteins. (B) E4-ORF1-induced Erk1/2 activation in MCF10A cells cultured in complete medium requires Mek1/2. The indicated MCF10A lines cultured in complete medium were treated with DMSO or U0126 (20 μM) for 5.5 h, when cell extracts were prepared and immunoblotted for the indicated proteins. (C) Erk1/2 activation is a conserved function of human adenoviruses. Extracts prepared from MCF10A cells that were mock infected or infected with the indicated Ad at a multiplicity of infection of 1 for 48 h were immunoblotted for the indicated proteins. (D) Mek1/2 is required for Erk1/2 activation induced by Ad9 infection and RasV12. MCF10A cells infected with Ad9, as described above for panel C, or RasV12 cells were treated with DMSO or U0126 (20 μM) for 5.5 h, when cell extracts were prepared and immunoblotted for the indicated proteins. (E) E4-ORF1 also induces D2-dependent Erk1/2 activation in MCF10A cells incubated in serum and growth factor-depleted medium. The indicated MCF10A lines cultured in complete medium were incubated in minimal medium (MM) for the indicated times, when cell extracts were prepared and immunoblotted for the indicated proteins.
FIG 2
FIG 2
EGFR mediates E4-ORF1-induced Erk1/2 activation. (A) The EGFR inhibitor AG1478 blocks EGF-induced EGFR autophosphorylation and Erk1/2 activation. Vector cells preincubated in minimal medium for 1 h were stimulated with EGF (5 ng/ml) mixed with either DMSO or AG1478 (1478) (0.5 μM) for 15 min, when cell extracts were prepared and immunoblotted for the indicated proteins. PY-EGFR detected with antiphosphotyrosine antibody PY20 comigrated with EGFR. (B) EGFR activity is required for E4-ORF1-induced Erk1/2 activation in MCF10A lines cultured in complete medium (CM). The indicated MCF10A lines cultured in complete medium were treated with DMSO or AG1478 (1478) (0.5 μM) for 1 h, when extracts were prepared and immunoblotted for the indicated proteins. (C) EGFR is also required for E4-ORF1-induced Erk1/2 activation in MCF10A cells incubated in serum and growth factor-depleted medium. wtORF1 cells cultured in complete medium were incubated in minimal medium (MM) containing either DMSO, LY294002 (LY) (100 μM), or AG1478 (0.5 μM) for 1 h, when cell extracts were prepared and immunoblotted for the indicated proteins. PY-EGFR was detected as described above for panel A.
FIG 3
FIG 3
E4-ORF1 does not enhance EGF-dependent EGFR and Erk1/2 activation. The indicated MCF10A lines cultured in complete medium were incubated in minimal medium containing EGF (5 ng/ml) for the indicated times, when cell extracts were prepared and immunoblotted for the indicated proteins. PY-EGFR was detected as described in the Fig. 2A legend.
FIG 4
FIG 4
E4-ORF1 and Dlg1 associate with tyrosine-phosphorylated proteins p170 and p120. (A) E4-ORF1 and Dlg1 bind to tyrosine-phosphorylated 170-kDa (PY-p170) and 120-kDa (PY-p120) cellular proteins. Cell extracts (400 μg of protein) from the indicated MCF10A lines were subjected to IP with either Dlg1 or E4-ORF1 antibody. Recovered proteins and cell extracts were immunoblotted for the indicated proteins. The expanded antiphosphotyrosine blot showing PY-p170 and PY-p120 covers the molecular mass range from 200 kDa to 100 kDa. PY-p170 and PY-p120 were detected with antiphosphotyrosine antibody 4G10. (B) The level of PY-p170, but not that of PY-p120 or PY-p95, is diminished by the EGFR inhibitor AG1478. Cell extracts (1 mg of protein) from the indicated MCF10A lines were subjected to IP with E4-ORF1 antibody. Recovered proteins and cell extracts were immunoblotted for the indicated proteins. PY-p170, PY-p120, and PY-p95 were detected with antiphosphotyrosine antibody PY20.
FIG 5
FIG 5
E4-ORF1 promotes association of EGFR with InsR and IGF1R. Cell extracts (350 μg of protein) from the indicated MCF10A lines were subjected to IP with EGFR antibody. Recovered proteins and cell extracts were immunoblotted for the indicated proteins.
FIG 6
FIG 6
InsR/IGF1R dampens EGFR autophosphorylation and Erk1/2 activation. (A) AG1024 inhibits insulin-induced InsR/IGF1R autophosphorylation and activation of Akt and Erk1/2. Vector cells preincubated in minimal medium (MM) for 1 h were stimulated with insulin (10 μg/ml) mixed with DMSO, AG1478 (1478) (0.5 μM), or AG1024 (1024) (50 μM) for 15 min, when cell extracts were prepared and immunoblotted for the indicated proteins. PY-InsR and PY-IGF1R detected with antiphosphotyrosine antibody 4G10 comigrated with InsR and IGF1R, respectively. (B) InsR/IGF1R dampens EGFR autophosphorylation and Erk1/2 activation in MCF10A cells cultured in complete medium (CM). The indicated MCF10A lines cultured in complete medium were treated with DMSO or AG1024 (50 μM) for 1 h, when cell extracts were prepared and immunoblotted for the indicated proteins. PY-EGFR was detected as described in the Fig. 2A legend. (C) InsR/IGF1R inhibits EGFR autophosphorylation in MCF10A cells incubated in serum and growth factor-depleted medium. The indicated MCF10A lines cultured in complete medium were incubated in minimal medium containing DMSO or AG1024 (50 μM) for 1 h, when cell extracts were prepared and immunoblotted for the indicated proteins. PY-EGFR was detected as described in the Fig. 2A legend. (D) InsR/IGF1R dampens D2-dependent E4-ORF1-induced Erk1/2 activation in MCF10A cells incubated in serum and growth factor-depleted medium. The indicated MCF10A lines cultured in complete medium were incubated in minimal medium containing DMSO or AG1024 (50 μM) for 1 h, when cell extracts were prepared and immunoblotted for the indicated proteins. (E) InsR/IGF1R dampens EGFR-mediated Erk1/2 activation induced by E4-ORF1. wtORF1 cells cultured in complete medium were incubated in minimal medium containing DMSO or either AG1478 (0.5 μM) or AG1024 (50 μM) alone or in combination for 1 h, when cell extracts were prepared and immunoblotted for the indicated proteins. PY-EGFR was detected as described in the Fig. 2A legend.
FIG 7
FIG 7
E4-ORF1 promotes constitutive Myc protein expression in MCF10A cells by a mechanism that requires the activities of EGFR, InsR/IGF1R, and PI3K. (A) E4-ORF1 prevents Myc downregulation in MCF10A cells incubated in serum and growth factor-depleted medium. The indicated MCF10A lines cultured for 24 h in complete medium (CM) were incubated in minimal medium (MM) for the indicated times, when cell extracts were prepared and immunoblotted for the indicated proteins. (B) E4-ORF1 also prevents Myc downregulation in MCF10A cells cultured for 72 h in complete medium. Extracts of the indicated MCF10A lines cultured for 72 h in complete medium were immunoblotted for the indicated proteins. (C) InsR/IGF1R, EGFR, PI3K, and Mek1/2 activities are required for E4-ORF1-induced constitutive Myc expression in MCF10A cells incubated in serum and growth factor-depleted medium. wtORF1 cells cultured in complete medium were incubated in minimal medium containing DMSO, AG1024 (1024) (50 μM), AG1478 (1478) (0.5 μM), LY294002 (LY) (100 μM), or U0126 (10 μM) for 3 h, when cell extracts were prepared and immunoblotted for the indicated proteins. (D) The activities of InsR/IGF1R and PI3K, but not EGFR, are required for Myc expression in MCF10A cells cultured in complete medium. The indicated MCF10A lines cultured in complete medium were treated with DMSO, AG1024 (50 μM), AG1478 (0.5 μM), LY294002 (100 μM), or U0126 (10 μM) for 1 h, when cell extracts were prepared and immunoblotted for the indicated proteins. (E) AG1478 or U1026 treatment for 1 h reduces E4-ORF1-induced constitutive Myc expression in wtORF1 cells incubated in serum and growth factor-depleted medium. Extracts of wtORF1 cells cultured in complete medium were incubated for 3 h in minimal medium containing DMSO, AG1478 (0.5 μM), or U0126 (10 μM) during the last 1 h of incubation, at which time the cell extracts were prepared and immunoblotted for the indicated proteins. (F) E4-ORF1-induced constitutive Myc expression depends on Dlg1. At 72 h post-cell plating in complete medium, extracts of wtORF1 cells expressing the Dlg1 shRNA (+) or control scrambled shRNA (−) were prepared and immunoblotted for the indicated proteins.
FIG 8
FIG 8
Hypothesized model for E4-ORF1-induced constitutive Myc protein expression mediated by EGFR, InsR/IGF1R, and PI3K. (A) In normal MCF10A cells, EGFR and Dlg1 form a Dlg1:EGFR complex at the plasma membrane. When activated by ligand, EGFR autophosphorylates tyrosine (Y) residues that directly mediate Ras/Mek/Erk signaling (black) and also those that augment Ras/Mek/Erk signaling (white). InsR/IGF1R activated by ligand specifically downregulates EGFR autophosphorylation on white Y residues that augment Ras/Mek/Erk signaling. (B) E4-ORF1 assembles two different protein complexes at the plasma membrane to activate three separate signaling pathways. E4-ORF1 forms the E4-ORF1:Dlg1:EGFR:InsR:IGF1R quinary complex by binding to the Dlg1:EGFR complex and promoting EGFR dimerization and EGFR association with InsR and IGF1R. In the quinary complex, the E4-ORF1 D2 element promotes EGFR autophosphorylation on black Y residues to activate Ras/Mek/Erk signaling, whereas the E4-ORF1 PBM via binding to Dlg1 enhances the latter signaling by antagonizing InsR/IGF1R-mediated suppression of EGFR autophosphorylation on white Y residues. An additional consequence of E4-ORF1 forming the quinary complex is activation of InsR/IGF1R (denoted by asterisks) and undetermined downstream signaling pathways. E4-ORF1 also binds both Dlg1 and PI3K to form the Dlg1:E4-ORF1:PI3K ternary complex, which activates the PI3K/Akt/mTOR pathway. (C) The three signaling pathways (Ras/Mek/Erk, InsR/IGF1R, and PI3K/Akt/mTOR) activated by E4-ORF1 converge in the nucleus to promote constitutive Myc expression, which enhances Ad replication by ensuring the formation of sufficient quantities of E4-ORF1:Myc complexes to stimulate anabolic glucose metabolism and nucleotide biosynthesis. See the text for details about steps 1 to 8 in the model.

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