Post-transcriptional modifications of p53

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(No Transcript)
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Post-transcriptional modifications of p53
Ubiquitin mediated Protealysis
CSN Phosphorylation
ATM etc Phosphorylation
Ubiquitin mediated Protealysis
Mdm2 interaction
O-GlcNac
P53
Ser149
Ser15
Thr18
Ser20
Ser155
Mdm2 binding region
Mdm2 a member of E3 ubiquitin-ligase CSN(COP9
signalosome) a conserved protein complex that
evolved in parallel with the ubiquitin-proteasome
system
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Two chemicals used

• Dox(doxorubicin) a DNA-damaging agent that is
  known to stabilize p53
• STZ(streptozotocin) an O-GlacNacase inhibitor.

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Effiect of Dox/STZ treatment on accumulation and
O-GlcNAcylation of p53
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Effiect of Dox/STZ treatment on accumulation and
O-GlcNAcylation of p53
Transcriptional level?
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O-GlcNAcylation of p53 by STZ treatment reduces
p53 ubiquitination and p53Mdm2 interactions in
MCF-7 cells
Total ubiquitin level
ALLN a proteasome inhibitor
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O-GlcNAcylation of p53 by STZ treatment
inhibitsphosphorylation of p53 at Thr 155 in
MCF-7 cells
CNS-p53 interaction
CNS levels
Thr 155
CNS Kinase activity
Thr 155
In-vitro assay IP CNS His-tag purify p53 as
substrate
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p53 is modified by O-GlcNAc at Ser 149
bn residue masses1 ynresidue massesH2O1
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Mutation of the p53 O-GlcNAcylation site (Ser
149) abrogates STZ induced p53 accumulation
?
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Mutation of the Ser 149 abrogates STZ induced p53
accumulation in p53-knockout H1299 cells
Exogenously expressed p53 retains biological
activity
?
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Conclusions

• Ser 149 of p53 is O-GlcNAcylated
• This modification associated decreased
  phosphorylation at Thr 155, which is a site of
  COP9 signalsome.
• Thus stabilize p53 by blocking ubiquitin-dependent
  proteolysis