Polyomavirus Small t Antigen Interacts with Yes-Associated Protein To Regulate Cell Survival and Differentiation

Abstract

Murine polyomavirus small t antigen (PyST) regulates cell cycle, cell survival, apoptosis, and differentiation and cooperates with middle T antigen (MT) to transform primary cells in vitro and in vivo. Like all polyomavirus T antigens, PyST functions largely via its interactions with host cell proteins. Here, we show that PyST binds both Yes-associated protein 1 (YAP1) and YAP2, integral parts of the Hippo signaling pathway, which is a subject of increasing interest in human cancer. The transcription factor TEAD, which is a known target of YAP, is also found in PyST complexes. PyST enhanced YAP association with protein phosphatase 2A (PP2A), leading to decreased YAP phosphorylation. PyST increased YAP levels by decreasing its degradation. This effect was mediated by a reduction in YAP association with ␤-transducin repeat protein (␤TRCP), which is known to regulate YAP turnover in a phosphorylation-dependent manner. Genetic analysis has identified PyST mutants defective in YAP binding. These mutants demonstrated that YAP binding is important for PyST to block myoblast differentiation and to synergize with the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) to promote cell death in 3T3-L1 preadipocytes placed under differentiation conditions. In addition to YAP binding, both of these phenotypes require PyST binding to PP2A. IMPORTANCEThe Hippo/YAP pathway is a highly conserved cascade important for tissue development and homeostasis. Defects in this pathway are increasingly being associated with cancer. Polyomavirus small t antigen is a viral oncogene that cooperates with middle T antigen in transformation. On its own, small t antigen controls cell survival and differentiation. By binding YAP, small t antigen brings it together with protein phosphatase 2A. This work shows how this association of small t antigen with YAP is important for its effects on cell phenotype. It also suggests that PyST can be used to characterize cellular processes that are regulated by YAP.

A Transformation-Defective Polyomavirus Middle T Antigen with a Novel Defect in PI3 Kinase Signaling

“…MT was immunoprecipitated using MT-1, HA-tagged proteins were immunoprecipitated using anti-HA resin, and FLAG-tagged proteins were immunoprecipitated using anti-FLAG resin according to methods published previously (81). SDS-PAGE (10%) (82) was used.…”

Transformation by Polyomavirus Middle T Antigen Involves a Unique Bimodal Interaction with the Hippo Effector YAP

“…The medium was then replaced with fresh propagation medium supplemented with blasticidin (20 g/ml; Santa Cruz) to generate a stable cell line. YAP short hairpin RNA (shRNA) and TAZ/WWTR1 shRNA lentiviral vectors were purchased from Sigma-Aldrich and packaged in 293T cells according to methods reported previously (61). NIH 3T3 cells were infected as described above.…”

“…pInducer20-MT; wild-type FLAG-YAP2 (Addgene plasmid 19045), WW1 FLAG-YAP mutant (Addgene 17795), and WW1/WW2 FLAG YAP mutants, gifts from Marius Sudol (69); and HA-TAZ, a gift from Kunliang Guan (Addgene plasmid 32839) (70), were transiently expressed in 293T cells according to a method reported previously (61).…”

“…MT was immunoprecipitated by using MT-1, HA-tagged proteins were immunoprecipitated by using anti-HA resin, and FLAG-tagged proteins were immunoprecipi-tated by using anti-FLAG resin according to methods reported previously (61). SDS-PAGE (10%) analysis was described previously (73).…”

“…We previously demonstrated that polyomavirus small T antigen (ST) binds YAP to affect differentiation (61). The sequence identity between ST and the N terminus of MT, together with the knowledge that MT binds the YAP relative TAZ (62,63), led us to hypothesize that MT may interact with YAP.…”