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The human adenovirus E4-ORF1 protein subverts discs large 1 to mediate membrane recruitment and dysregulation of phosphatidylinositol 3-kinase.

Kong K, Kumar M, Taruishi M, Javier RT - PLoS Pathog. (2014)

Bottom Line: At this site, Dlg1 also co-localizes with the activated PI3K effector protein Akt, indicating that the ternary complex mediates PI3K signaling.Signifying the functional importance of the ternary complex, the capacity of E4-ORF1 to induce soft agar growth and focus formation in cells is ablated either by a mutation that prevents E4-ORF1 binding to Dlg1 or by a PI3K inhibitor drug.These results demonstrate that E4-ORF1 interacts with Dlg1 and PI3K to assemble a ternary complex where E4-ORF1 hijacks the Dlg1 oncogenic function to relocate cytoplasmic PI3K to the membrane for constitutive activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
Adenoviruses infect epithelial cells lining mucous membranes to cause acute diseases in people. They are also utilized as vectors for vaccination and for gene and cancer therapy, as well as tools to discover mechanisms of cancer due to their tumorigenic potential in experimental animals. The adenovirus E4-ORF1 gene encodes an oncoprotein that promotes viral replication, cell survival, and transformation by activating phosphatidylinositol 3-kinase (PI3K). While the mechanism of activation is not understood, this function depends on a complex formed between E4-ORF1 and the membrane-associated cellular PDZ protein Discs Large 1 (Dlg1), a common viral target having both tumor suppressor and oncogenic functions. Here, we report that in human epithelial cells, E4-ORF1 interacts with the regulatory and catalytic subunits of PI3K and elevates their levels. Like PI3K activation, PI3K protein elevation by E4-ORF1 requires Dlg1. We further show that Dlg1, E4-ORF1, and PI3K form a ternary complex at the plasma membrane. At this site, Dlg1 also co-localizes with the activated PI3K effector protein Akt, indicating that the ternary complex mediates PI3K signaling. Signifying the functional importance of the ternary complex, the capacity of E4-ORF1 to induce soft agar growth and focus formation in cells is ablated either by a mutation that prevents E4-ORF1 binding to Dlg1 or by a PI3K inhibitor drug. These results demonstrate that E4-ORF1 interacts with Dlg1 and PI3K to assemble a ternary complex where E4-ORF1 hijacks the Dlg1 oncogenic function to relocate cytoplasmic PI3K to the membrane for constitutive activation. This novel mechanism of Dlg1 subversion by adenovirus to dysregulate PI3K could be used by other pathogenic viruses, such as human papillomavirus, human T-cell leukemia virus type 1, and influenza A virus, which also target Dlg1 and activate PI3K in cells.

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E4-ORF1 activates PI3K and upregulates PI3K protein levels in a PBM-dependent manner but does not activate the MAP kinases ERK1 and ERK2.MCF10A lines transduced with empty retroviral vector (vector cells) or the vector encoding wt Ad9 E4-ORF1 (wtORF1 cells), PBM mutant Ad9 E4-ORF1 V125A (V125A cells), or rasV12 (rasV12 cells) were treated with either DMSO vehicle (−) or 100 µM LY294002 (LY) (+) for 30 min. Cell extracts were analyzed in an immunoblot assay with antibodies to the indicated proteins.
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ppat-1004102-g002: E4-ORF1 activates PI3K and upregulates PI3K protein levels in a PBM-dependent manner but does not activate the MAP kinases ERK1 and ERK2.MCF10A lines transduced with empty retroviral vector (vector cells) or the vector encoding wt Ad9 E4-ORF1 (wtORF1 cells), PBM mutant Ad9 E4-ORF1 V125A (V125A cells), or rasV12 (rasV12 cells) were treated with either DMSO vehicle (−) or 100 µM LY294002 (LY) (+) for 30 min. Cell extracts were analyzed in an immunoblot assay with antibodies to the indicated proteins.

Mentions: Consistent with previous results in rodent fibroblast lines [7], immunoblots of extracts from the cell lines revealed higher levels of activated PI3K effector Akt dually phosphorylated at T308 and S473 (P-Akt) in wtORF1 and rasV12 cells than in vector and V125A cells (Figure 2, compare lanes 1–2 to lanes 3 and 5; Figures 3A and 3B, compare lane 1 and lane 3), as well as higher levels of activated, phosphorylated MAP kinases ERK-1 and -2 (P-ERK1/2) in rasV12 cells than in other cells (Figure 2, compare lane 5 to lanes 1–3) [34]. Compared to vector and V125A cells, wtORF1 and rasV12 cells also displayed higher levels of p85α, p85β, and p110α (Figure 2, compare lanes 1–2 to lanes 3 and 5). Moreover, treatment of wtORF1 and rasV12 cells with the PI3K inhibitor drug LY294002 (LY) returned the high P-Akt levels to those of vector cells, yet had little or no effect on the high p85 and p110 levels (Figure 2, compare lane 1 to lanes 3–4 and 5–6). In contrast, stable expression of a short hairpin RNA (shRNA) that depleted Dlg1 decreased the high levels of P-Akt, p85, and p110 in wtORF1 cells but not in rasV12 cells (compare Figure 3A, lanes 3–4 to Figure 3B, lanes 3–4). From cumulative immunoblots, we quantified pertinent protein level differences for wtORF1 or rasV12 cells versus vector cells, as well as for wtORF1 cells transduced with the Dlg1 shRNA vector versus the scrambled shRNA vector (Tables S2, S3, and S4). Taken together, the data showed that while E4-ORF1 and rasV12 similarly elevate p85α, p85β, and p110α levels and activate Akt in a PI3K-dependent manner in human epithelial cells, E4-ORF1 differs from rasV12 in its dependence on both a PBM and Dlg1 for these activities and in not activating the Raf/MAP kinase pathway.


The human adenovirus E4-ORF1 protein subverts discs large 1 to mediate membrane recruitment and dysregulation of phosphatidylinositol 3-kinase.

Kong K, Kumar M, Taruishi M, Javier RT - PLoS Pathog. (2014)

E4-ORF1 activates PI3K and upregulates PI3K protein levels in a PBM-dependent manner but does not activate the MAP kinases ERK1 and ERK2.MCF10A lines transduced with empty retroviral vector (vector cells) or the vector encoding wt Ad9 E4-ORF1 (wtORF1 cells), PBM mutant Ad9 E4-ORF1 V125A (V125A cells), or rasV12 (rasV12 cells) were treated with either DMSO vehicle (−) or 100 µM LY294002 (LY) (+) for 30 min. Cell extracts were analyzed in an immunoblot assay with antibodies to the indicated proteins.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4006922&req=5

ppat-1004102-g002: E4-ORF1 activates PI3K and upregulates PI3K protein levels in a PBM-dependent manner but does not activate the MAP kinases ERK1 and ERK2.MCF10A lines transduced with empty retroviral vector (vector cells) or the vector encoding wt Ad9 E4-ORF1 (wtORF1 cells), PBM mutant Ad9 E4-ORF1 V125A (V125A cells), or rasV12 (rasV12 cells) were treated with either DMSO vehicle (−) or 100 µM LY294002 (LY) (+) for 30 min. Cell extracts were analyzed in an immunoblot assay with antibodies to the indicated proteins.
Mentions: Consistent with previous results in rodent fibroblast lines [7], immunoblots of extracts from the cell lines revealed higher levels of activated PI3K effector Akt dually phosphorylated at T308 and S473 (P-Akt) in wtORF1 and rasV12 cells than in vector and V125A cells (Figure 2, compare lanes 1–2 to lanes 3 and 5; Figures 3A and 3B, compare lane 1 and lane 3), as well as higher levels of activated, phosphorylated MAP kinases ERK-1 and -2 (P-ERK1/2) in rasV12 cells than in other cells (Figure 2, compare lane 5 to lanes 1–3) [34]. Compared to vector and V125A cells, wtORF1 and rasV12 cells also displayed higher levels of p85α, p85β, and p110α (Figure 2, compare lanes 1–2 to lanes 3 and 5). Moreover, treatment of wtORF1 and rasV12 cells with the PI3K inhibitor drug LY294002 (LY) returned the high P-Akt levels to those of vector cells, yet had little or no effect on the high p85 and p110 levels (Figure 2, compare lane 1 to lanes 3–4 and 5–6). In contrast, stable expression of a short hairpin RNA (shRNA) that depleted Dlg1 decreased the high levels of P-Akt, p85, and p110 in wtORF1 cells but not in rasV12 cells (compare Figure 3A, lanes 3–4 to Figure 3B, lanes 3–4). From cumulative immunoblots, we quantified pertinent protein level differences for wtORF1 or rasV12 cells versus vector cells, as well as for wtORF1 cells transduced with the Dlg1 shRNA vector versus the scrambled shRNA vector (Tables S2, S3, and S4). Taken together, the data showed that while E4-ORF1 and rasV12 similarly elevate p85α, p85β, and p110α levels and activate Akt in a PI3K-dependent manner in human epithelial cells, E4-ORF1 differs from rasV12 in its dependence on both a PBM and Dlg1 for these activities and in not activating the Raf/MAP kinase pathway.

Bottom Line: At this site, Dlg1 also co-localizes with the activated PI3K effector protein Akt, indicating that the ternary complex mediates PI3K signaling.Signifying the functional importance of the ternary complex, the capacity of E4-ORF1 to induce soft agar growth and focus formation in cells is ablated either by a mutation that prevents E4-ORF1 binding to Dlg1 or by a PI3K inhibitor drug.These results demonstrate that E4-ORF1 interacts with Dlg1 and PI3K to assemble a ternary complex where E4-ORF1 hijacks the Dlg1 oncogenic function to relocate cytoplasmic PI3K to the membrane for constitutive activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
Adenoviruses infect epithelial cells lining mucous membranes to cause acute diseases in people. They are also utilized as vectors for vaccination and for gene and cancer therapy, as well as tools to discover mechanisms of cancer due to their tumorigenic potential in experimental animals. The adenovirus E4-ORF1 gene encodes an oncoprotein that promotes viral replication, cell survival, and transformation by activating phosphatidylinositol 3-kinase (PI3K). While the mechanism of activation is not understood, this function depends on a complex formed between E4-ORF1 and the membrane-associated cellular PDZ protein Discs Large 1 (Dlg1), a common viral target having both tumor suppressor and oncogenic functions. Here, we report that in human epithelial cells, E4-ORF1 interacts with the regulatory and catalytic subunits of PI3K and elevates their levels. Like PI3K activation, PI3K protein elevation by E4-ORF1 requires Dlg1. We further show that Dlg1, E4-ORF1, and PI3K form a ternary complex at the plasma membrane. At this site, Dlg1 also co-localizes with the activated PI3K effector protein Akt, indicating that the ternary complex mediates PI3K signaling. Signifying the functional importance of the ternary complex, the capacity of E4-ORF1 to induce soft agar growth and focus formation in cells is ablated either by a mutation that prevents E4-ORF1 binding to Dlg1 or by a PI3K inhibitor drug. These results demonstrate that E4-ORF1 interacts with Dlg1 and PI3K to assemble a ternary complex where E4-ORF1 hijacks the Dlg1 oncogenic function to relocate cytoplasmic PI3K to the membrane for constitutive activation. This novel mechanism of Dlg1 subversion by adenovirus to dysregulate PI3K could be used by other pathogenic viruses, such as human papillomavirus, human T-cell leukemia virus type 1, and influenza A virus, which also target Dlg1 and activate PI3K in cells.

Show MeSH
Related in: MedlinePlus