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Cellular Transforming Processes Sensed by Apoptin

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

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Cellular Transforming Processes Sensed by Apoptin

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

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

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

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

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

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

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

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

  9. Nuclear presence of both J domain & PP2A binding domain are crucial for activation of tumor-related kinase Recently: Both the J domain & PP2A binding domain of st contact and inactivate PP2A 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? RNAi analysis control B56- RNAi B56 P-apoptin apoptin

  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 Leiden Institute of Chemistry Tongji Medical College

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