Murine polyomavirus has repeatedly provided insights into tumorigenesis, revealing key control mechanisms such as tyrosine phosphorylation and phosphoinositide 3-kinase (PI3K) signaling. We recently demonstrated that polyomavirus small T antigen (ST) binds YAP, a major effector of Hippo signaling, to regulate differentiation. Here we characterize YAP as a target of middle T antigen (MT) important for transformation. Through a surface including residues R103 and D182, wild-type MT binds to the YAP WW domains. Mutation of either R103 or D182 of MT abrogates YAP binding without affecting binding to other signaling molecules or the strength of PI3K or Ras signaling. Either genetic abrogation of YAP binding to MT or silencing of YAP via short hairpin RNA (shRNA) reduced MT transformation, suggesting that YAP makes a positive contribution to the transformed phenotype. MT targets YAP both by activating signaling pathways that affect it and by binding to it. MT signaling, whether from wild-type MT or the YAP-binding MT mutant, promoted YAP phosphorylation at S127 and S381/397 (YAP2/YAP1). Consistent with the known functions of these phosphorylated serines, MT signaling leads to the loss of YAP from the nucleus and degradation. Binding of YAP to MT brings it together with protein phosphatase 2A (PP2A), leading to the dephosphorylation of YAP in the MT complex. It also leads to the enrichment of YAP in membranes. Taken together, these results indicate that YAP promotes MT transformation via mechanisms that may depart from YAP's canonical oncogenic transcriptional activation functions.

IMPORTANCE The highly conserved Hippo/YAP pathway is important for tissue development and homeostasis. Increasingly, changes in this pathway are being associated with cancer. Middle T antigen (MT) is the primary polyomavirus oncogene responsible for tumor formation. In this study, we show that MT signaling promotes YAP phosphorylation, loss from the nucleus, and increased turnover. Notably, MT genetics demonstrate that YAP binding to MT is important for transformation. Because MT also binds PP2A, YAP bound to MT is dephosphorylated, stabilized, and localized to membranes. Taken together, these results indicate that YAP promotes MT transformation via mechanisms that depart from YAP's canonical oncogenic transcriptional activation functions.