title: PP2A inactivation is a crucial step in triggering apoptin-induced tumor-selective cell killing.
abstract: Apoptin (apoptosis-inducing protein) harbors tumor-selective characteristics making it a potential safe and effective anticancer agent. Apoptin becomes phosphorylated and induces apoptosis in a large panel of human tumor but not normal cells. Here, we used an in vitro oncogenic transformation assay to explore minimal cellular factors required for the activation of apoptin. Flag-apoptin was introduced into normal fibroblasts together with the transforming SV40 large T antigen (SV40 LT) and SV40 small t antigen (SV40 ST) antigens. We found that nuclear expression of SV40 ST in normal cells was sufficient to induce phosphorylation of apoptin. Mutational analysis showed that mutations disrupting the binding of ST to protein phosphatase 2A (PP2A) counteracted this effect. Knockdown of the ST-interacting PP2A-B56γ subunit in normal fibroblasts mimicked the effect of nuclear ST expression, resulting in induction of apoptin phosphorylation. The same effect was observed upon downregulation of the PP2A-B56δ subunit, which is targeted by protein kinase A (PKA). Apoptin interacts with the PKA-associating protein BCA3/AKIP1, and inhibition of PKA in tumor cells by treatment with H89 increased the phosphorylation of apoptin, whereas the PKA activator cAMP partially reduced it. We infer that inactivation of PP2A, in particular, of the B56γ and B56δ subunits is a crucial step in triggering apoptin-induced tumor-selective cell death.
authors.affiliation: Department of Molecular Genetics, Leiden Institute for Chemistry, Leiden University, Leiden, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Zimmerman
authors.forename: D J
authors.initials: DJ
authors.lastname: Peng
authors.forename: H
authors.initials: H
authors.lastname: Lanz
authors.forename: Y H
authors.initials: YH
authors.lastname: Zhang
authors.forename: A
authors.initials: A
authors.lastname: Danen-Van Oorschot
authors.forename: S
authors.initials: S
authors.lastname: Qu
authors.forename: C
authors.initials: C
authors.lastname: Backendorf
authors.forename: M
authors.initials: M
authors.lastname: Noteborn
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D009369:Neoplasms / Q000378:metabolism / Q000473:pathology; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017354:Point Mutation; D011485:Protein Binding; D054648:Protein Phosphatase 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: C471621:AKIP1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D000952:Antigens, Polyomavirus Transforming; D051017:Apoptosis Regulatory Proteins; D007546:Isoquinolines; D009687:Nuclear Proteins; D021122:Protein Subunits; D034741:RNA, Small Interfering; D013449:Sulfonamides; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D054648:Protein Phosphatase 2; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide