p53
The Master Guardian of the Genome
p53 gene mutations in human tumors
Tumor type (n)
Lung (897)
Colon (960)
Esophagus (279)
Ovary (386)
Pancreas (170)
Skin (220)
Gastric (314)
He ad & neck (524)
Bladder (308)
Sarc oma (339)
Prostate (87)
He patoce llular (716)
Brain (456)
p 53
mu tation
56%
50%
45%
44%
44%
44%
41%
37%
34%
31%
30%
29%
25%
Tu mor ty pe ( n)
p 53
mu tation
Adrenal (31)
Breast (1536)
Endometrium (224)
Mesothelioma (23)
Renal (102)
Thyroid (299)
He matological (1916)
Carcinoid (13)
Melanoma (70)
Parathyroid (13)
Cevix (350)
Neuroblastoma (212)
others (155)
23%
22%
22%
22%
19%
13%
12%
11%
9%
8%
7%
1%
0%
All tu mors: 37%
50%
Greenblatt et al. (1995) Cancer Res. 54:4855
The Basic Paradigm of p53 Function
Genotoxic Stress
(e.g. DNA damage)
p53
(low)
Proliferative Stress
(e.g. oncogenes)
INPUTS
p53
(high)
Cell cycle arrest
Apoptosis
OUTPUTS
The Discovery of p53
SV40 large T protein binds to 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
WRONG!!
Moshe
Oren
Arnold
Levine
Cloning of the p53 gene,
followed by successive
experiments showed that it is
actually a
tumor suppressor gene
Autosomal Dominant Li-Fraumeni syndrome
Inherited germ-line mutations in
p53 cause predisposition for
distinct cancers in variable ages
p53 Mutant Mice Develop Cancer
p53 is a transcription factor, active only as a
homotetramer
p53 acts only as a tetramer
Imagine a scenario:
- One normal copy
- One lof 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
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%)
p53 Binds DNA
Ribbon Model
Space Filling Model
The most common mutation changes arginine 248, colored red here. Notice how it snakes
into the minor groove of the DNA (shown in blue and green), forming a strong stabilizing
interaction. When mutated to another amino acid, this interaction is lost. Other key sites of
mutation are shown in pink, including arginine residues 175, 249, 273 and 282, and
glycine 245.
The Basic Paradigm of p53 Function
Genotoxic Stress
(e.g. DNA damage)
p53
(low)
Proliferative Stress
(e.g. oncogenes)
INPUTS
p53
(high)
Cell cycle arrest
Apoptosis
OUTPUTS
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)
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 ubiquitin mediated
proteolysis
p53 protein is ubiquitinated by the E3 ligase
MDM2
Genetic Evidence that Mdm2 Inhibits p53
mdm2-/-
p53-/-
p53-/- mdm2-/-
Mdm2 is a p53 Target Gene
p53 control of Mdm2 transcription is a negative feedback loop
Some p53 mutants show over expression
of inert p53 protein
p53 control of Mdm2 transcription is a negative feedback loop
Many agents induce p53 activity
Grouped into two classes
DNA
damage
Hyperproliferative
stress
p53
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
Chk1
(inactive)
Rad9
- ATR-dependent phosphorylation of
the Rad9 adaptor protein is needed
for activating Chk1
Chk1
(active)
- Activated Chk1 is released to phosphorylate its effectors
Phosphorylation of p53 (by
ATM/ATR and/or Chk2)
makes it unable to bind
MDM2
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
p53 and Ink4a are the two most
frequently mutated genes in human tumors
est ima ted f requency
of alte rat ions
Chromosome
locat ion
ty pe of alte rat ions
p53
17p13
nuc leotide substitution
~ 50%
INK4a
9p2 1
homoz ygous dele tion
nuc leotide substitution
small deletion/insertion
promote r methyla tion
~ 40%
Loc us
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 transcription of the CKI p21
(p16)
(p21)
What about outputs?
DNA
damage
Hyperproliferative
stress
p53
Cell cycle arrest
Apoptosis
Programmed cell death: aka Apoptosis
Specific cells are dying in a
programmed manner
C. elegans
Worms are transparent
One can follow the individual
lineage of each of the cells
<1000 cells
http://mathematica.edublogs.org/files/2011/03/C-elegans-2kbu264.JPG
http://www.imsc.res.in/~sitabhra/research/neural/celegans/
Deciphering the cell lineage of C. elegans
Cell death is also a “cell fate”
Use genetics to identify mutants
Epistasis analysis and biochemical studies
allow the scientists to build a pathway
Genetic control of programmed cell death
ced-9
ced-4
CED-4
No
death
signal
Death
signal
CED-9
CED-9
ced-3
Cell death
CED-4 is
inactive
CED-4
Activates
CED-3
caspase for
the killing
Gain of function mutations in bcl-2 were
associated with human cancers (bcl= b cell
lymphoma resulting from a translocation event)
Our body uses the same apoptotic mechanisms
used in PCD, for killing “bad” or unwanted cells
http://herkules.oulu.fi/isbn9514266676/html/i267425.html
Nobel Prize
Lineage and
programmed
cell death
Physiology and
Medicine 2002
Bob Horvitz John Sulston Sydney Brenner
- Pro-apoptotic signals open
up the channels, allowing cyt
C to be released to the
cytoplasm
A simplified representation of apoptosis
Proapoptotic
(BH3-only)
Noxa
Antiapoptotic
Bcl-2
Procaspase
Apaf
Proapoptotic
(multidomain)
Bax
Caspase
Cytochrome C
Mitochondria
All the regulators are from the same protein family
(containing BH domains)
p53 triggers apoptosis
http://www.weizmann.ac.il/home/ligivol/apoptosis_project/apoptotic_pathways.html