p53 The Master Guardian

R point Cell cycle control involves several checkpoints and checkpoint (molecular breaking) mechanisms

Mutations in p53 can be found in half of all tumors p53 Three lectures in one slide

Discovery of p53 - SV40 DNA tumor virus transforms monkey kidney cells into cancerous cells - The virus is injected into hamsters and the serum is used to precipitate the oncogenic protein: Large T antigen - … and another protein, ~53kD - SV40 DNA tumor virus can also transform murine cells in culture - Both T antigen and p53 are expressed only upon viral infection

Antibodies specific to the large T protein show that T co-immunoprecipitates p53

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

Cloning of the p53 gene, followed by successive experiments showed that it is actually a tumor suppressor gene Moshe Oren Arnold Levine

- T antigen oncogenic activity was carried out by sequestering p53 when the latter was supposed to inhibit tumor progression - p53 is a transcription factor that acts 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 p53 protein levels increase upon exposure to UV (and many other agents) Campbell et al. Biochemical Society Transactions (2001)

# 2) Homozygote mutant mice can complete embryogenesis (but die shortly after) # 3) Low levels of p53 expression in normal cells # 1) Inherited mutations cause distinct cancers

- 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 the ubiquitine system

p53 protein is ubiquitinated by the E3 ligase MDM2

Missense mutations can inhibit p53 from binding to its target genes, including mdm2 (think about expression levels of p53 in these mutants) p53 mutations in human tumors are found with high frequency in the DNA binding domain

Some p53 mutants show over expression of inert p53 protein

p53 DNA damage Hyperproliferative stress Many agents induce p53 activity Grouped into two classes

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-dependent phosphorylation of the Rad9 adaptor protein is needed for activating Chk2 - Activated Chk2 is released to phosphorylate its effectors ATR Rad9 Chk2 (inactive) Chk2 (active)

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

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

Two reactions for the price of one

- 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 arrestApoptosis What about outputs? Hyperproliferative stress

p53 activates the CKI p21 (p16) (p21)

p53 DNA damage Cell cycle arrestApoptosis What about outputs? Hyperproliferative stress