p53
The Master Guardian
Cell cycle control involves several checkpoints and
checkpoint (molecular breaking) mechanisms
R point
Three lectures in one slide
p53
Mutations in p53 can be found in half of all tumors
Discovery of p53
- SV40 DNA tumor virus transforms
monkey kidney cells into cancerous cells
- SV40 DNA tumor virus can also
transform murine cells in culture
- The virus is injected into hamsters and
the serum is used to precipitate the
oncogenic protein: Large T antigen
- … and another protein, ~53kD
- Both T antigen and p53 are
expressed only upon viral infection
Antibodies specific to the large T protein
show that T co-immunoprecipitates p53
NRS
1
2
anti-T
3
4
IP
5
6
T
53 K
Hypothesis
Large T antigen and p53 are oncogenes
- p53, a proto-oncogene, is expressed in low
concentrations in normal cells
- T antigen oncogenic activity leads to overexpression of p53 and the latter acts as an oncogene
Moshe
Oren
Arnold
Levine
Cloning of the p53 gene,
followed by successive
experiments showed that it is
actually a
tumor suppressor gene
- p53 is a transcription factor that acts to inhibit
tumor progression
- T antigen oncogenic activity was carried out by
sequestering p53 when the latter was supposed to
inhibit tumor progression
- T antigen binds also to
pRb, sequestering it from
negatively regulating cell
cycle progression
p53 is a transcription factor, active only as a
homotetramer
p53 acts only as a tetramer
Imagine a scenario:
- One normal copy
- One lof mutated copy, encoding a mutated protein
that can still bind to its partners
Does this mean that +/- heterozygotes do not need a
second mutation for tumor progression?
Not quite, even 1/16 of p53 molecules have some activity
However, missense mutations and not nonsense/frameshift
are the common p53 mutations in cancer patients
p53 mutations in human tumors are found with high
frequency in the DNA binding domain
In 143 families reported:
point mutations (85%)
deletions (9%)
splice mutations (3.5%)
insertions (2%)
How does p53 act?
# 1) Li-Fraumeni syndrome
Inherited germ-line mutations in
p53 cause predisposition for
distinct cancers in variable ages
# 2) Homozygote mutant mice can complete
embryogenesis (but die shortly after)
# 3) Low levels of p53 expression in normal cells
Campbell et al. Biochemical Society Transactions (2001)
p53 protein levels increase upon exposure to
UV (and many other agents)
# 1) Inherited mutations cause distinct cancers
# 2) Homozygote mutant mice can complete
embryogenesis (but die shortly after)
# 3) Low levels of p53 expression in normal cells
Summary
- p53 is a transcription factor, acting
as a homotetramer
- Expressed when cells gone awry
- Two mutated copies in tumors, first is
usually a dominant-negative mutation
- Acts as a tumor suppressor gene
In normal cells we find only low
concentrations of the p53 protein
- p53 protein is actually
synthesized all the time,
but is degraded very fast
via the ubiquitine system
p53 protein is ubiquitinated by the E3 ligase
MDM2
p53 mutations in human tumors are found with high
frequency in the DNA binding domain
Missense mutations can inhibit p53 from binding to its target genes,
including mdm2 (think about expression levels of p53 in these mutants)
Some p53 mutants show over expression of
inert p53 protein
Many agents induce p53 activity
Grouped into two classes
DNA
damage
Hyperproliferative
stress
p53
Subtle DNA damage is sensed by sensor proteins
and repaired by the DNA repair machinery
Extensive DNA damage recruits the
DNA damage response machinery
Two key players:
The protein kinases ATM and ATR
ATM and ATR are recruited to distinct sites and
phosphorylate downstream effectors
ATR is recruited to single - stranded DNA
ATR
Chk2
(inactive)
Rad9
- ATR-dependent phosphorylation of
the Rad9 adaptor protein is needed
for activating Chk2
Chk2
(active)
- Activated Chk2 is released to phosphorylate its effectors
Phosphorylation of p53 (by
ATM/ATR and/or Chk2)
makes it insusceptible to
MDM2 binding
Phosphorylated p53 acts
as a transcription factor
DNA damage response activates p53 by
stabilizing the protein via phosphorylation
(and additional mechanisms)
Additional inhibitory
phosphorylation of MDM2
DNA
damage
Hyperproliferative
stress
p53
e.g. high activity of E2Fs
E2Fs
Hyperproliferative stress response is mediated
through the ARF protein
- E2Fs induce transcription
of the ARF gene
- ARF binds to and sequesters MDM2
- p53 is stabilized
Over activity of oncogenes stimulates apoptosis
through ARF
ARF stands for Alternative Reading Frame
- Found in the same locus of the p16 gene
- Uses an alternative promoter
Two reactions for the price of one
Summary
- p53 is a transcription factor, acting
as a homotetramer
- Transcribed constitutively, but
has a very short half life
- Ubiquitinated by the E3 ligase MDM2
- DNA damage and a stalled replication fork
induce p53 phosphorylation and activation
- Hyperproliferative stress (e.g. oncogenic
signaling, hypoxia) activates p53 via ARF
What about outputs?
DNA
damage
Hyperproliferative
stress
p53
Cell cycle arrest
Apoptosis
p53 activates the CKI p21
(p16)
(p21)
What about outputs?
DNA
damage
Hyperproliferative
stress
p53
Cell cycle arrest
Apoptosis