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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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ShRNA-mediated Dlg1 depletion diminishes E4-ORF1-induced recruitment of PI3K to the plasma membrane.Indirect immunofluorescence assays were performed and analyzed as described in the legend to Figure 9 using the indicated MCF10A lines.
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ppat-1004102-g010: ShRNA-mediated Dlg1 depletion diminishes E4-ORF1-induced recruitment of PI3K to the plasma membrane.Indirect immunofluorescence assays were performed and analyzed as described in the legend to Figure 9 using the indicated MCF10A lines.

Mentions: We next tested the cell lines for co-localization of p85 and Dlg1 (Figure 9). In vector and V125A cells, p85 exhibited a cytoplasmic distribution but, due to an absence at the plasma membrane, it failed to co-localize with Dlg1. In wtORF1 cells, p85 was similarly distributed in the cytoplasm and, more importantly, was additionally detected at the plasma membrane where it co-localized with Dlg1. Furthermore, the latter effect was decreased by shRNA-mediated Dlg1 depletion in wtORF1 cells (Figure 10). These data, together with those presented in Figure 8, indicated that PBM-mediated binding of the wt E4-ORF1 protein to Dlg1 functions not only to assemble the Dlg1:E4-ORF1:PI3K ternary complex but also to localize PI3K to the plasma membrane.


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)

ShRNA-mediated Dlg1 depletion diminishes E4-ORF1-induced recruitment of PI3K to the plasma membrane.Indirect immunofluorescence assays were performed and analyzed as described in the legend to Figure 9 using the indicated MCF10A lines.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004102-g010: ShRNA-mediated Dlg1 depletion diminishes E4-ORF1-induced recruitment of PI3K to the plasma membrane.Indirect immunofluorescence assays were performed and analyzed as described in the legend to Figure 9 using the indicated MCF10A lines.
Mentions: We next tested the cell lines for co-localization of p85 and Dlg1 (Figure 9). In vector and V125A cells, p85 exhibited a cytoplasmic distribution but, due to an absence at the plasma membrane, it failed to co-localize with Dlg1. In wtORF1 cells, p85 was similarly distributed in the cytoplasm and, more importantly, was additionally detected at the plasma membrane where it co-localized with Dlg1. Furthermore, the latter effect was decreased by shRNA-mediated Dlg1 depletion in wtORF1 cells (Figure 10). These data, together with those presented in Figure 8, indicated that PBM-mediated binding of the wt E4-ORF1 protein to Dlg1 functions not only to assemble the Dlg1:E4-ORF1:PI3K ternary complex but also to localize PI3K to the plasma membrane.

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