Download MDM2 inhibit p300-mediated p53 acetylation and activation by

MDM2 inhibits p300-mediated p53
acetylation and activation by
forming a ternary complex with the
two protein
Eric Kobet , Xiaoya Zeng , Yong Zhu , David Keller , and Hua Lu
PNAS | November 7, 2000 | vol. 97 | no. 23 | 12547-12552
生科系 04級 賴保諺 891635
p53 : a tumor suppressor gene
which can induce cell growth
arrest and apoptosis
a transcription factor
MDM2 is transcriptionally activated by p53
MDM2 : possess ubiquitin-transferase
activity
MDM2
p53
p53
ubiquitin
ubiquitin
ubiquitination
degradation
DNA
p53
transcription
p53
negative feedback
loop
translation
p53
degradation
p53
ubiquitination
MDM2
p300 : histone acetyl-transferase (HAT)
p300
acetyl - CoA
ace
p53
p53
ace
p53 posttranslation modification
 phosphorylation
by DNA-dependent
protein kinase may contribute to
transactivation and stability
 acetylation
by p300 leading to activation of
sequence-specific DNA binding ability
known
MDM2 could form a complex
with p300 and p53 in vitro.
HeLa Nuclear Extract
and marker
chromatography
western
western
marker
100
90
70
50
40
KD
add antibody
p300 ~ 400kDa
p53 ~ 212kDa
(homotetramer)
MDM2 ~ 90kDa
total
MDM2 can form a ternary complex with p300
and p53 in the nucleus.
~ 700kDa
Whether the purified protein
complex could acetylate p53 ?
western by using
antiacetylated Lys
antibody
The purified p300 was able to acetylate p53-dependent on
acetyl CoA in a dose-dependent fashion.
isotope-labeled acetyl CoA
(autoradiography)
MDM2、p53、
p300 complex
p300, once complexed to MDM2 , may not be able to target
the p53 Lys residues.
MDM2 was preincubated with p300 before adding to the reaction
mixture ( MDM2 interacted with the CH1 domain of p300)
MDM2 can inhibit p53 acetylation by p300 probably
through direct association.
Whether the inhibition requires the association of MDM2 with p53
compare wild-type MDM2 with its N-terminally deleted form
lacking amino acid 58-89 (p53 binding domain)
This suggest that for MDM2 to inhibit p300-mediated p53
acetylation , it must form a ternary complex with p53 and
p300.
Futher investigation
Whether MDM2 could influence the p300 acetylase
activity on different substrates that do not interact
with MDM2.
p73 C-teminal domain (could be acetylated by p300 in vitro)
Association of MDM2 with p53 is required for its inhibitory
effect on p53 acetylation .
What’s the functional consequence of the inhibition of
p300-mediated p53 acetylation by MDM2 ?
We examine the effect of MDM2 on p300-stimulated
p53 sequence-specific DNA-binding activity.
Electrophoretic Mobility-Shift Assay ( EMSA )
add protein antibody
add DNA-binding protein
probe
(DNA)
super shift
shift
p53 antibody
p53
p53 binding
sequence
MDM2 eliminates the ability of p300 to stimulate sequence-specific
DNA-binding activity of p53 in vitro.
Finally, we examine whether MDM2 inhibits p53
acetylation by p300 in vivo .
transfect human lung carcinoma cells with
plasmids coding p53 , MDM2 , p300
Test whether inhibition of p53 acetylatoin by MDM2 affects
sequence–specific DNA-binding activity of p53 in vivo.
p53 antibody
super shift
These results indicate that MDM2 also inhibits sequencespecific DNA-binding activity of p53 in vivo.
MDM2 inhibits p53 acetylation and
activation mediated by p300 in vivo
+
MDM2 can form a ternary complex with p300
and p53 in the nucleus
MDM2 inhibits p300-mediated p53 acetylation
and activation by forming a ternary complex
with the two protein.
UV and γ irradiation differentially regulate
MDM2 expression and p53 acetylation.
MDM2 expression was repressed by UV
(not by γ irradiation).
DNA binding ability of p53 was enhanced by UV
( not by γ irradiation).
DNA was damaged and p53 was activated
and cells may undergo apoptosis.