p53The Master Guardian of the Genome
p53 gene mutations in human tumors
Lung (897) 56% Adrenal (31) 23% Colon (960) 50% Breast (1536) 22% Esophagus (279) 45% Endometrium (224) 22% Ovary (386) 44% Mesothelioma (23) 22% Pancreas (170) 44% Renal (102) 19% Skin (220) 44% Thyroid (299) 13% Gastric (314) 41% He matological (1916) 12% He ad & neck (524) 37% Carcinoid (13) 11% Bladder (308) 34% Melanoma (70) 9% Sarc oma (339) 31% Parathyroid (13) 8% Prostate (87) 30% Cevix (350) 7% He patoce llular (716) 29% Neuroblastoma (212) 1% Brain (456) 25% others (155) 0%
Tumor type (n) p53 mutation Tumor ty pe ( n)
p53 mutation
All tumors: 37%
Greenblatt et al. (1995) Cancer Res. 54:4855
50%
p53(low)
p53(high)
Cell cycle arrest Apoptosis
Genotoxic Stress(e.g. DNA damage)
The Basic Paradigm of p53 Function
INPUTS
OUTPUTS
Proliferative Stress(e.g. oncogenes)
The Discovery of p53
SV40 large T protein binds to p53
T
53 K
61 2 3 4 5
IPNRS anti-T
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 over-expression of p53 and the latter acts as an oncogene
WRONG!!
Cloning of the p53 gene, followed by successive
experiments showed that it is actually a
tumor suppressor geneMoshe
OrenArnold Levine
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 tetramerImagine 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 HighFrequency In the DNA Binding Domain
In 143 families reported:point mutations (85%)deletions (9%)splice mutations (3.5%) insertions (2%)
Ribbon Model Space Filling Model
p53 Binds DNA
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.
p53(low)
p53(high)
Cell cycle arrest Apoptosis
Genotoxic Stress(e.g. DNA damage)
The Basic Paradigm of p53 Function
INPUTS
OUTPUTS
Proliferative Stress(e.g. oncogenes)
Low levels of p53 expression in normal cells
p53 protein levels increase upon exposure to UV (and many other agents)
Campbell et al. Biochemical Society Transactions (2001)
- p53 is a transcription factor, acting as a homotetramer
Summary
- 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
p53-/- mdm2-/-
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
p53
DNA damage
Hyperproliferative stress
Many agents induce p53 activity
Grouped into two classes
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-dependent phosphorylation of the Rad9 adaptor protein is needed for activating Chk1
- Activated Chk1 is released to phosphorylate its effectors
ATR
Rad9
Chk1 (inactive)
Chk1 (active)
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
p53
DNA damage
Hyperproliferative stress
E2Fs
e.g. high activity of 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
LocusChromosome location type of alterations
estimated frequency of alterations
p53
INK4a
17p13
9p21
nucleotide substitution
homozygous deletion nucleotide substitution small deletion/insertion promoter methylation
~ 50%
~ 40%
- p53 is a transcription factor, acting as a homotetramer
Summary
- Transcribed constitutively, but has a very short half life
- DNA damage and a stalled replication fork induce p53 phosphorylation and activation
- Hyperproliferative stress (e.g. oncogenic signaling, hypoxia) activates p53 via ARF
- Ubiquitinated by the E3 ligase MDM2
p53
DNA damage
Cell cycle arrest Apoptosis
What about outputs?
Hyperproliferative stress
(p16)
(p21)
p53 activates transcription of the CKI p21
p53
DNA damage
Cell cycle arrest Apoptosis
What about outputs?
Hyperproliferative stress
Programmed cell death: aka Apoptosis
Specific cells are dying in a programmed manner
http://mathematica.edublogs.org/files/2011/03/C-elegans-2kbu264.JPG
Worms are transparent
C. elegans
One can follow the individual lineage of each of the cells
<1000 cells
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-4No death signal
CED-4 is inactive
Death signal
CED-9
Activates CED-3 caspase for the killing
CED-9CED-4
ced-9 Cell deathced-3ced-4
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
Lineage and programmed
cell deathBob Horvitz John Sulston Sydney Brenner
Physiology and Medicine 2002
Nobel Prize
- Pro-apoptotic signals open up the channels, allowing cyt C to be released to the cytoplasm
A simplified representation of apoptosis
NoxaProapoptotic(BH3-only)
Proapoptotic(multidomain)
Bax
Bcl-2
Cytochrome C
Apaf
Procaspase
Caspase
Mitochondria
All the regulators are from the same protein family (containing BH domains)
Antiapoptotic
http://www.weizmann.ac.il/home/ligivol/apoptosis_project/apoptotic_pathways.html
p53 triggers apoptosis
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