Cellular Transforming Processes

Sensed by Apoptin

Dept. of Biological Chemistry, LICLeiden University, The Netherlands

Dept. of Biochemistry & Molecular BiologyTongji Medical College, HUST, Wuhan, China

Dongjun Peng

in human tumor cellsin human normal cells

CAV

Apoptin

alive dead

PhosphorylationPhosphorylation

Model system: SV40 T antigen induces transforming processesModel system: SV40 T antigen induces transforming processes

ApoptosisNormal cells

+ SV40-T

N localization

apoptin + SV40-TapoptinPhosphorylation

apoptin apoptin apoptin

SV40-T antigen can be used to identify the cellular factors involved in apoptin activation

P-apoptin

SV40 LT/st harbors transforming domains targeting cellular factors

nls

LT 708aa

p53 binding

st 174aa

Rb binding

PP2Abinding

st unique

136aa

J

J

Hsc70binding

LT136

N-terminal of LT/st is sufficient for apoptin’s activationN-terminal of LT/st is sufficient for apoptin’s activation

Apoptin + LT136/stApoptin + LT136/st

PhosphorylationPhosphorylation NuclearNuclear localizationlocalizationApoptosisApoptosis

inductioninduction

yesyes yesyes yesyes

LT

st 174aa

Rb

PP2Abinding

124aa

124 J

J

LT13

6/st

/Rb-

Contro

lLT

136/

st

P-apoptin

apoptin

Is nuclear targeting of LT136/st important?Is nuclear targeting of LT136/st important?

LT12

4/st

/nls

-/Rb-

nls

LT

st 174aa

Rb

PP2Abinding

136aa

136 J

J

Rb binding domain mutation

• Rb is only involved in bringing

LT/st into nucleus

• Nuclear targeting is essential

LT12

4/st

/nls

-

Are both LT136 and st of importance?Are both LT136 and st of importance?

J PP2Abindingst 174aa

Con

trol

st LT13

6/st

LT13

6

LT13

6 +

st

apoptin

st

P-apoptin

LT136

Both LT136 and st are needed for optimal activation of apoptinBoth LT136 and st are needed for optimal activation of apoptin

st 174aaPP2Abinding

nls

LT

136aa

136 J

J

nls

LT

136aa

136 J

Is the J domain important for apoptin’s activation?Is the J domain important for apoptin’s activation?

LT13

6 D

44N

+ s

t

LT13

6 +

st D

44N

LT13

6 D

44N

+ s

t D44

N

LT13

6+st

apoptin

st

P-apoptin

LT136

D44N mutation in J domain

Both J-domains are involved

in apoptin activation

nls

LT Rbbinding

136aa

136 J

J PP2Abindingst 174aa

+nls

LT

136aa

136 J

J PP2Abindingst 174aa

+nls

LT Rbbinding

136aa

136 J

J PP2Abindingst 174aa

+nls

LT

136aa

136 J

J PP2Abindingst 174aa

+nls

LT Rbbinding

136aa

136 J

J PP2Abindingst 174aa

+nls

LT

136aa

136 J

J PP2Abindingst 174aa

+

Is the PP2A binding domain in st crucial?Is the PP2A binding domain in st crucial?

A (PP2A)

st

LT136

Inpu

t

Stre

p-LT

136/

st

C10

3S

Stre

p-LT

136/

st

Actin

nls

LT

st 174aa

Rbbinding

PP2Abinding

136aa

136 J

J

C103S

F9 Con

trol

LT13

6/st

C10

3S

LT13

6/st

P-apoptin

apoptin

stLT136

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 BiolCho US, 2007, PLoS Biology

Is inactivation of PP2A sufficient to activate apoptin’s Is inactivation of PP2A sufficient to activate apoptin’s tumor-related kinase?tumor-related kinase?

cont

rol

B56

-

P-apoptin

apoptin

B56

RNAi analysis

RNAi

Inactivation of PP2A’s regulatory subunit Inactivation of PP2A’s regulatory subunit

B56B56γγ is sufficient to activate apoptin’s is sufficient to activate apoptin’s

tumor-related kinasetumor-related kinase

Acknowledgement Acknowledgement

Dept. of Biological Chemistry in University LeidenDept. of Biological Chemistry in University Leiden

Mathieu NotebornMathieu Noteborn

Maarten de SmitMaarten de Smit

Patrick VoskampPatrick Voskamp

Rhyenne ZimmermanRhyenne Zimmerman

Dept. of Molecular Genetics in University LeidenDept. of Molecular Genetics in University Leiden

Claude BackendorfClaude Backendorf

Ying-Hui ZhangYing-Hui Zhang

Wilbert VermeijWilbert Vermeij

University of PittsburghUniversity of Pittsburgh

James Pipas James Pipas

KNAW

Tongji Medical College

Leiden Institute of Chemistry