Transcript p53 activates transcription of genes - California State University, Los

Structural Basis of the DNA binding
Domain of the p53 tumor suppressor
protein
May 25, 2006
MOLECULAR DIAGNOSTICS
Dr. Sandra Sharp
Jake Leon
Department of Biology and Microbiology
California State University Los Angeles
5151 State University Drive
Los Angeles, CA 90032
Normal Cells are able to prevent cancer by activating a natural
defense mechanism
Cancer:
• DNA damage
• DNA damage activates gene expression
• Genes code proteins
• Proteins participate in response to DNA damage
• p53 acts as a transcription factor that transactivates
genes to stop tumors by causing apoptosis, repairing DNA, or
by preventing cell proliferation.
Role of p53 in tumor suppression
p53 knockout mice develop tumors spontaneously
(Donehower et al., 1992)
p53 binds to specific sequences on the DNA
(Bargonetti et al., 1991; El-Deiry et al., 1992)
p53 activates transcription of genes
(Levine, 1997; Giaccia and Kastan, 1998)
In human cancers, p53 is frequently observed to show
mutations that inhibit its ability to bind to DNA
Outline of Next Series of Slides
• Normal p53 in the cell when there is no
detectable DNA damage
• Normal p53 in response to DNA damage
– 2 slides – repairable damage
• Phopshorylation of p53 blocking its degradation and
• Causing p53 to function as a transcription factor to stop
the cell cycle
– 2 slides – irreparable damage
• Phosphorylation of p53 blocking its degradation and
• Causing p53 to function as a transcription factor to
induce apoptosis
Latent p53
Half-life: ~20 min
p53
Nucleus
This normally rapid turnover prevents normal cells from
entering into cell cycle arrest
MDM2
or
undergoing apoptosis
Murine Double Minute 2
26 S Proteosome
Momand et al., 2005
ATP
ATM
Ataxia Telangiectasia Mutagenesis
S15
ADP
p53
CHK2
Checkpoint Kinase 2
S20
ADP
ATP
MDM2
p21
GADD45
p53
TAF70
TAF31
RNA
polymerase
ATP
DNA-PK
DNA-dependent protein kinase
S15
ADP
p53
These genes participate in the activation of
APOPTOSIS
p53
TAF70
Bax
NOXA
TAF31
PUMA RNA
polymerase
KILLER/DR5
Fas/Apo1
Apoptosis Induction by p53
• P53 also responds to unrepaired DNA
damage by inducing expression of genes
that trigger apoptosis (programmed cell
death) of the injured cell.
– This ultimately leads to cell death.
• DNA-PKc (catalytic subunit) is part of the
enzymatic machinery for
– VDJ rearrangement
– Non-homologous end joining
Apoptosis –
in response to irreparable DNA damage
Bax
Note the role of
tumor suppressor
p53.
What is p53?
• A protein of ~53 kilodaltons
• A nuclear phosphoprotein




DNA viruses and their oncogenes.
• E6 is produced by Human Papilloma Virus (HPV) and can
contribute to cervical cancer.
• E1b is produced by Adenovirus.
– Human cells are permissive for adenovirus.
• Causes the common cold.
– Adenovirus transforms rodent (non-permissive) cells
• Human virus JC is similar to SV40 and may be associated
with certain cancers, but a causative role has yet to be
confirmed.
– JC virus T antigen causes tumors in nude mice.
• All these proteins are products of “early” genes in their viral
replication cycles.
• A productive infection of these viruses leads to lysis of the
host cell. A non-productive infection allows the cells to live.
What is p53?
• Transcriptional regulator
– Binds to 12 bp recognition sequence in the
promoters (regulatory regions) of the genes it
regulates
– Activates transcription by interacting with RNA
polymerase complex


What is p53?
• Acts as a tetramer
– Individual molecules associate at
tetramerization region
– Oligomerization of mutated p53 with wt p53 
inactive p53 complex

What is p53?
• Detection of damaged DNA by p53 causes p53 to be
stabilized and accumulate in the cell.
• DNA damage activates the kinase ATM, which
phosphorylates p53.
• When damaged DNA is not present, p53 is turned over
rapidly and does not accumulate because
– the protein MDM2 binds to the transcription-activation region of p53
and targets p53 for degradation by a proteosome.

(TAD)
 Note: MDM2 binds when the TAD is LESS phosphorylated.
p53 as a transcriptional regulator
• If DNA damage is detected by binding of DNA fragments to the
non-specific DNA binding region of p53, p53 stops DNA
synthesis until the damage is repaired.
• If DNA damage is detected, then
– p53 is phosphorylated by a protein known as ATM
– MDM2 is released from being bound to the transcriptional activation
domain of p53 and
– p53 is able to act as a transcriptional activator and turn on genes for
• cyclin dependent kinase inhibitor p21, which
– stops or prevents DNA synthesis
• DNA repair
– Example: GADD45
• If DNA damage is extensive and can not be repaired, p53
induces genes for apoptosis (programmed cell death).
p53 as a transcriptional
regulator
• p53 activates the gene for MDM2
– MDM2
• targets p53 for degradation and prevents inappropriate
build up
• prevents transcriptional activation by p53
– So, it’s a negative feedback loop!
• p53 also turns expression of some genes off.
How does p53 inhibit DNA
synthesis? Let’s work backwards.
• E2F transcription factor turns on
transcription of genes for DNA synthesis.
• E2F can’t turn on genes if it is bound to Rb1,
a tumor suppressor.
• Rb1 can’t bind E2F if it is heavily
phosphorylated.
• Rb1 is phosphorylated by cyclin-dependent
kinases (CDKs).
How does p53 inhibit DNA
synthesis?
• Cylin dependent kinases can be inhibited by
cyclin dependent kinase inhibitors (CDKIs).
If CDKs are inhibited
– Rb1 won’t be phosphorylated
– E2F will be bound by Rb1
– DNA synthesis genes will not be transcribed
• And remember . . . .
P53 induces expression of CDKI p21, a
cyclin dependent kinase inhibitor!
• Check out the next slide for a visual of these
pathways.
Phosphorylation of Rb
Figure legend on next slide.
p53 Mutations - where are they?
This magnification of mutations in the DNA binding region of
p53 gives more information regarding how the mutation
affects p53. Note particularly that some mutations cause p53
to be misfolded (denatured) and others do not.
Have you figured it out?
For our assay, the samples are cell extracts from two mouse cell lines,
BC3H1 and C2C12.
One line is wild type for p53; one is mutant.
One accumulates detectable levels of p53; one doesn’t.
Based on this lecture and your assay results, have you figured out
which cell line does what?
Have you thought about why?
There is one explanation confirmed in the literature and at least one
additional plausible contribution to what you observe.
(Hint: P53 is not accumulating in either of these cell lines in response
to DNA damage. DNA damage is a temporary condition which is
repaired immediately. If it is not repaired, the cell soon dies as a
result of apoptosis. Mutations may be the result of incorrect repair of
DNA damage, but they are no longer considered damage because
they are perfectly base-paired.)
p53 DBD Folding
Z
n
Cho, Y., Gorina, S., Jeffrey, P.D. and Pavletich, N.P. (1994) Science 265: 346-355
Crystal Structure of p53 DBD-DNA Complex
PDB file 1tsr
Cho, Y., Gorina, S., Jeffrey, P.D. and Pavletich, N.P. (1994) Science 265: 346-355
Crystal Structure of p53 DBD-DNA Complex
PDB file 1tsr
Cho, Y., Gorina, S., Jeffrey, P.D. and Pavletich, N.P. (1994) Science 265: 346-355
Crystal Structure of p53 DBD-DNA Complex
PDB file 1tsr
Cho, Y., Gorina, S., Jeffrey, P.D. and Pavletich, N.P. (1994) Science 265: 346-355
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