1. Cellular Transforming Processes Sensed by Apoptin Dept. of Biological Chemistry, LIC Leiden University, The Netherlands Dept. of Biochemistry & Molecular Biology Tongji Medical College, HUST, Wuhan, China Dongjun Peng

2. in human tumor cells in human normal cells CAV Apoptin alive dead Phosphorylation

3. Model system: SV40 T antigen induces transforming processes Apoptosis Normal cells + SV40-T N localization apoptin + SV40-T apoptin Phosphorylation apoptin SV40-T antigen can be used to identify the cellular factors involved in apoptin activation P-apoptin

4. SV40 LT/st harbors transforming domains targeting cellular factors nls LT 708aa p53 binding st 174aa Rb binding PP2A binding st unique 136aa J J Hsc70 binding LT136 N-terminal of LT/st is sufficient for apoptin’s activation Apoptin + LT136/st Phosphorylation Nuclear localization Apoptosisinduction yesyesyes

5. LT st 174aa Rb PP2A binding 124aa 124 J J LT136/st/Rb- Control LT136/st P-apoptin apoptin Is nuclear targeting of LT136/st important? LT124/st/nls-/Rb- nls LT st 174aa Rb PP2A binding 136aa 136 J J Rb binding domain mutation Rb is only involved in bringing LT/st into nucleus Nuclear targeting is essential LT124/st/nls-

6. Are both LT136 and st of importance? Control st LT136/st LT136 LT136 + st apoptin st P-apoptin LT136 Both LT136 and st are needed for optimal activation of apoptin st 174aa PP2A binding nls LT 136aa 136 J J nls LT 136aa 136 J

7. Is the J domain important for apoptin’s activation? LT136 D44N + st LT136 + st D44N LT136 D44N + st D44N LT136+st apoptin st P-apoptin LT136 D44N mutation in J domain Both J-domains are involved in apoptin activation nls LT Rb binding 136aa 136 J J PP2A binding st 174aa + nls LT 136aa 136 J J PP2A binding st 174aa + nls LT Rb binding 136aa 136 J J PP2A binding st 174aa + nls LT 136aa 136 J J PP2A binding st 174aa + nls LT Rb binding 136aa 136 J J PP2A binding st 174aa + nls LT 136aa 136 J J PP2A binding st 174aa +

8. Is the PP2A binding domain in st crucial? A  (PP2A) st LT136 Input Strep- LT136/st C103S Strep- LT136/st Actin C103S F9 Control LT136/st C103S LT136/st P-apoptin apoptin st LT136

9. Recently: Both the J domain & PP2A binding domain of st contact and inactivate PP2A Nuclear presence of both J domain & PP2A binding domain are crucial for activation of tumor-related kinase Chen Y, 2007, Nat Struct Mol Biol Cho US, 2007, PLoS Biology

10. Is inactivation of PP2A sufficient to activate apoptin’s tumor-related kinase? control B56-  P-apoptin apoptin B56  RNAi analysis RNAi

11. Inactivation of PP2A’s regulatory subunit B56γ is sufficient to activate apoptin’s tumor-related kinase

12. Acknowledgement Dept. of Biological Chemistry in University Leiden Mathieu Noteborn Maarten de Smit Patrick Voskamp Rhyenne Zimmerman Dept. of Molecular Genetics in University Leiden Claude Backendorf Ying-Hui Zhang Wilbert Vermeij University of Pittsburgh James Pipas KNAW Tongji Medical College Leiden Institute of Chemistry