The human adenovirus serotype 5 E4ORF1 (Ad5E4ORF1) protein promotes primary endothelial cell survival and angiocrine functions by hijacking the cellular phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway. However, the mechanism by which E4ORF1 activates PI3K in vascular cells remains largely unknown. Here we show that Ad5E4ORF1 recruits multiple host scaffold proteins, including DLG1, which facilitates AKT activation in response to both Ad5E4ORF1 and endogenous receptor agonists. Furthermore, Ad5E4ORF1 specifically engages the human PI3K isoform p110α-p85β through multi-domain interactions exclusively with p110α. Notably, E4ORF1 proteins from different adenoviral serotypes differentially interact with p110α, resulting in varying levels of AKT activation in endothelial cells. We propose that E4ORF1 specifically recognizes and allosterically activates p110α-p85β via direct multi-site contacts with p110α.
