The p53 tumor suppressor
The p53 tumor suppressor
The p53 tumor suppressor
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<strong>The</strong> <strong>p53</strong> <strong>tumor</strong> <strong>suppressor</strong>
1 I II III IV V<br />
Transactivation<br />
domain<br />
<strong>p53</strong><br />
393<br />
NLS I NLS II NLS III<br />
DNA-binding domain Regulatory domain<br />
Tetramerization<br />
domain<br />
• Discovered in 1979 (A. Levine, D. Lane)<br />
(1988, B. Vogelstein)<br />
• bona fide Tumor <strong>suppressor</strong><br />
• Transcriptional activator and repressor<br />
• Mutated on over 50% of human <strong>tumor</strong>s<br />
• Cancer therapeutic target (traditional drugs<br />
side effects)
Discovery of <strong>p53</strong><br />
• Immunocytochemical and immunohistochemical analysis show that the <strong>p53</strong> protein<br />
accumulates in the nucleus of transformed or <strong>tumor</strong> cells. Before 1990, the protein<br />
was believed to be wild type.
<strong>p53</strong> is a <strong>tumor</strong> <strong>suppressor</strong><br />
• <strong>p53</strong> in Friend murine erythroleukemia<br />
• In these <strong>tumor</strong>s induced by the Friend virus, the <strong>p53</strong> gene found in the <strong>tumor</strong> cells is<br />
truncated or mutated. In this <strong>tumor</strong> model, func
<strong>p53</strong> is a <strong>tumor</strong> <strong>suppressor</strong><br />
• Wild type <strong>p53</strong> has an@prolifera@ve proper@es and does not cooperate with Ha-‐ras<br />
• Cotransfec
<strong>p53</strong> is a <strong>tumor</strong> <strong>suppressor</strong><br />
• <strong>p53</strong> gene is mutated in a wide variety of human cancer<br />
• Gene
<strong>p53</strong> mutations in human <strong>tumor</strong>s
<strong>p53</strong> is a <strong>tumor</strong> <strong>suppressor</strong><br />
• Germline muta@on of the <strong>p53</strong> gene are found in Li-‐Fraumeni pa@ents<br />
• This syndrome presents as a familial associa
<strong>p53</strong> is a <strong>tumor</strong> <strong>suppressor</strong><br />
• <strong>p53</strong> in mouse <strong>tumor</strong> development<br />
• Mice homozygous for the null allele appear normal but are prone to the spontaneous<br />
development of a variety of <strong>tumor</strong>s by 6 months of age. <strong>The</strong>se observa
<strong>p53</strong> protein<br />
• Human <strong>p53</strong> protein can be divided into five domains, each corresponding to specific<br />
functions:<br />
• I) <strong>The</strong> amino-terminus part 1-42 contains the acidic transactivation domain and the mdm2<br />
protein binding site. It also contains the Highly Conserved Domain I (HCD I).<br />
II) Region 40-92 contains series repeated proline residues that are conserved in the<br />
majority of <strong>p53</strong>. it also contains a second transactivation domain.<br />
III) <strong>The</strong> central region (101-306) contains the DNA binding domain. It is the target of 90% of<br />
<strong>p53</strong> mutations found in human cancers. It contains HCD II to V.<br />
IV) <strong>The</strong> oligomerization domain (307-355, 4D) consists of a beta-strand, followed by an<br />
alpha-helix necessary for dimerization, as <strong>p53</strong> is composed of a dimer of two dimers. A<br />
nuclear export signal (NES) is localized in this oligomerization domain.<br />
V) <strong>The</strong> C-terminus of <strong>p53</strong> (356-393) contains 3 nuclear localization signals (NLS) and a<br />
non-specific DNA binding domain that binds to damaged DNA. This region is also involved<br />
in downregulation of DNA binding of the central domain.
• <strong>The</strong> amino-‐terminus of <strong>p53</strong><br />
• AD1: ac
• <strong>The</strong> carboxy-‐terminus of <strong>p53</strong><br />
• Tetra (4D): oligomeriza
<strong>p53</strong> is a DNA-‐binding protein<br />
• <strong>The</strong> core domain structure<br />
consists of a beta sandwich<br />
that serves as a scaffold for<br />
two large loops and a loop-‐<br />
sheet-‐helix mo
<strong>p53</strong> structure<br />
<strong>p53</strong> <strong>tumor</strong><br />
<strong>suppressor</strong><br />
binds to DNA<br />
using all four<br />
of its arms.<br />
<strong>p53</strong> is a <strong>tumor</strong> <strong>suppressor</strong>
• <strong>p53</strong> structure<br />
• <strong>p53</strong> <strong>tumor</strong> <strong>suppressor</strong> is a flexible<br />
molecule composed of four iden
• P53/DNA structure<br />
• <strong>p53</strong> <strong>tumor</strong> <strong>suppressor</strong> binds to<br />
DNA using all four of its arms.<br />
• <strong>The</strong> typical binding site for the<br />
whole molecule is composed of<br />
three parts: a specific binding<br />
site for two <strong>p53</strong> domains, a<br />
variable stretch of 0 to 13 base<br />
pairs, and a second specific<br />
binding site for the other two<br />
<strong>p53</strong> domains. .<br />
• <strong>The</strong> tetrameriza
Mdm2 is a repressor of <strong>p53</strong><br />
• MDM2 is a RING-‐finger E3 ubiqui
P53 is a sensor of stress<br />
• <strong>p53</strong> pathway in stress response<br />
• i)<strong>The</strong> stress signals that ac
A Classic model of <strong>p53</strong> stabiliza
<strong>p53</strong> activation by ARF<br />
ARF (known as p14 in<br />
humans and p19 in mice)<br />
was originally identified<br />
as an alternative<br />
transcript of the Ink4a/<br />
ARF <strong>tumor</strong> <strong>suppressor</strong><br />
locus, a gene that<br />
encodes the Ink4a/p16<br />
inhibitor of cyclindependent<br />
kinases. ARF<br />
suppresses aberrant cell<br />
growth in response to<br />
oncogenic stress mainly<br />
by activating the <strong>p53</strong><br />
pathway.<br />
MULE = ARF-BP1<br />
A model of <strong>p53</strong> ac
Mul
P53-mediated growth<br />
arrest:<br />
downstream signaling, G1<br />
arrest via p21 transcription.<br />
<strong>The</strong> CDKI p21 will prevent<br />
Rb phosphorylation via<br />
inhibiation of the CDK4 and<br />
CDK2 kinases.<br />
A model of <strong>p53</strong>-‐mediated cell cycle arrest.
<strong>p53</strong><br />
<strong>p53</strong> and Cell Cycle Arrest<br />
p21<br />
GADD45<br />
14-‐3-‐3σ<br />
cdk2<br />
cdc2<br />
G1<br />
arrest<br />
G2<br />
arrest
<strong>p53</strong><br />
<strong>p53</strong> and Apoptosis<br />
PUMA<br />
Bax<br />
BCL-2<br />
Other <strong>p53</strong> apoptotic targets: Noxa, Fas,<br />
DR5, Apaf-1, <strong>p53</strong>AIP1, PIDD and etc<br />
Apoptosis
Transcrip
Several transcrip
<strong>p53</strong> and Metabolic regulation.<br />
Key targets: SCO2, Hexokinase, GLUT1,3,4, TIGAR and PGM
Regula@on of <strong>p53</strong><br />
Stress<br />
DNA damage<br />
<strong>p53</strong><br />
Ac
Figure 1-Kruse and Gu
Regulation of <strong>p53</strong><br />
Stress<br />
DNA damage<br />
<strong>p53</strong><br />
Ubiquitination<br />
<strong>p53</strong> stabilization<br />
Acetylation<br />
Senescence<br />
Cell growth arrest<br />
Apoptosis<br />
Phosphorylation, Methylation, Sumylation,<br />
Neddylation and etc.
<strong>p53</strong> ubiquitination:<br />
1. Mdm2 Mono vs. Poly<br />
(Ub—Nuclear export)<br />
2. Deubiquitination: HAUSP<br />
3. Mdm2-independent ubiquitination<br />
ARF-BP1, COP1, PIRH2 and etc.
E1 E2 E3<br />
Mono-Ubiquitination<br />
K<br />
Multi Mono-Ubiquitination<br />
K<br />
K-<br />
K<br />
K K<br />
Poly-Ubiquitination<br />
E1 E2 E3<br />
K-<br />
K- K-<br />
K-
Mdm2 can induce both mono- and polyubiquitination of <strong>p53</strong>
Monoubiquitination of <strong>p53</strong> induces nuclear export
Polyubiquitination of <strong>p53</strong> induces nuclear degradation
<strong>p53</strong><br />
Mdm2<br />
Ub<br />
<strong>p53</strong><br />
<strong>p53</strong><br />
<strong>p53</strong><br />
Ub<br />
<strong>p53</strong><br />
Ub<br />
<strong>p53</strong><br />
<strong>p53</strong><br />
<strong>p53</strong> <strong>p53</strong><br />
Nucleus<br />
Mdm2 dependent<br />
Ub Ub<br />
Ub Ub<br />
26S<br />
<strong>p53</strong><br />
Cytoplasm<br />
Ub<br />
PARC/<br />
Cul-7<br />
??
ARF-BP1
Inactivation of ARF-BP1, but not Mdm2,<br />
induces cell growth repression in <strong>p53</strong>-null cells
WT<br />
KO<br />
<strong>p53</strong> activation in vivo in ARF-BP1-null e13.75 embryos<br />
P53 Caspase 3<br />
A B C<br />
D E F<br />
Kon and Gu<br />
unpublished
HAUSP/USP7<br />
HAUSP<br />
207 564 1102<br />
UBP: ubiquitin-specific<br />
process protease
<strong>The</strong> HAUSP Consensus Binding Site on <strong>p53</strong> and Mdm2<br />
(Shi, Y. PLoS 2006)
Ub Ub<br />
Ub Ub<br />
Mdm2<br />
HAUSP<br />
Mdm2<br />
Ub<br />
<strong>p53</strong> <strong>p53</strong><br />
HAUSP<br />
Mdm2<br />
Mdmx<br />
<strong>p53</strong><br />
<strong>p53</strong><br />
Ub Ub<br />
Ub Ub<br />
26S
HAUSP reduction/ablation has a profound effect on <strong>p53</strong>/Mdm2
Analysis of e11.5 embryos from Hausp conditional knockout<br />
Hausp +/cl<br />
Embryo<br />
Hauspko/cl, ERT2<br />
Embryo<br />
H&E Hausp <strong>p53</strong>
Oncogenic stress<br />
HAUSP<br />
<strong>p53</strong><br />
Mdm2<br />
ARF<br />
HAUSP<br />
ARF-BP1/Mule<br />
COP1, PIRH2<br />
and etc.<br />
<strong>p53</strong>
Regulation of <strong>p53</strong><br />
Stress<br />
DNA damage<br />
<strong>p53</strong><br />
Ubiquitination<br />
<strong>p53</strong> stabilization<br />
?<br />
Senescence<br />
Cell growth arrest<br />
Apoptosis
Degradation<br />
MDM2<br />
A CLASSIC MODEL OF P53 ACTIVATION<br />
<strong>p53</strong><br />
<strong>p53</strong> stabilization (I)<br />
ATM/ATR/DNA-PK, Chk1/Chk2<br />
MDM2<br />
P <strong>p53</strong><br />
P <strong>p53</strong><br />
TBP/<br />
TFIID<br />
DNA-binding (II) Transcriptional<br />
Activation (III)
Problems in the model<br />
• Modifica
<strong>p53</strong> and its signaling pathway. Various kinds of stress signals are detected by cells and communicated to the<br />
<strong>p53</strong> protein and its core cons
<strong>p53</strong><br />
Acetyla@on<br />
Phosphoryla@on<br />
Ubiqui@na@on<br />
Neddyla@on<br />
Sumoyla@on<br />
Methyla@on<br />
TAD PRD DBD TD CRD<br />
1 42 63 97 100 300 307 355 393<br />
K120<br />
S6<br />
S9<br />
S15<br />
T18<br />
S20<br />
S33<br />
S36<br />
S37<br />
S46<br />
T55<br />
T81<br />
S149<br />
T150<br />
T155<br />
K164<br />
K305<br />
S315<br />
K320<br />
S376<br />
S378<br />
S392<br />
K319<br />
K320<br />
K321<br />
K351<br />
K357<br />
K370<br />
K372<br />
K373<br />
K381<br />
K382<br />
K386<br />
K320<br />
K321<br />
K370<br />
K372<br />
K373<br />
K381<br />
K382<br />
K386<br />
K370<br />
K372<br />
K373<br />
R333<br />
R335<br />
R337<br />
K370<br />
K372<br />
K373<br />
K386<br />
K382<br />
Modifying<br />
Enzyme<br />
CBP/p300, PCAF, Tip60,<br />
hMOF<br />
ATM, ATR, DNAPK,<br />
CK1, CK2, Chk1/<br />
Chk2, etc<br />
Mdm2, Pirh2,<br />
COP1, ArfBP1,<br />
E4F1, MSL2<br />
Mdm2, FBXO11<br />
PIAS, Topors<br />
Smyd2, Set7/9,<br />
Set8, G9a/Glp,<br />
PRMT5
Regula
<strong>p53</strong> is acetylated at Lysine 120 by Tip60<br />
(Tang et al., 2006; Sykes et al., 2006)
Differen@al effects of Lysine 120 acetyla@on on<br />
growth arrest vs. apoptosis<br />
D
B<br />
<strong>p53</strong> is acetylated at K164 by CBP/p300<br />
1-42 63-97 98-292 300-323 324-355 363-393<br />
Transactivation<br />
domain<br />
N-terminal Central core C-terminal<br />
Proline-rich<br />
domain<br />
K164<br />
DNA-binding domain Tetramerization<br />
domain<br />
C<br />
NLS NES<br />
C-terminal<br />
regulatory<br />
domain
All acetyla@on sites (8K) are involved in p21 ac@va@on
<strong>p53</strong> acetyla@on is essen@al for both growth arrest and apoptosis
Is <strong>p53</strong> acetyla@on essen@al in the DNA damage response?<br />
N-‐Term.
Acetyla@on blocks the forma@on of<br />
<strong>The</strong> <strong>p53</strong>/Mdm2-‐DNA complexes
Acetyla@on of <strong>p53</strong> is cri@cal in the DNA damage response<br />
(even in the absence of phosphoryla@on (Ac@nomycin D))
1. DNA<br />
binding<br />
<strong>p53</strong> Nega@ve feedback<br />
Mdm2, COP1, Pirh2<br />
Mdm2 MdmX<br />
<strong>p53</strong><br />
Acetylation is essential for <strong>p53</strong>-mediated function<br />
2. An@-‐<br />
TFs<br />
repression Ac<br />
RNA pol II<br />
P <strong>p53</strong><br />
P<br />
3. Cofactor<br />
recruitment<br />
TFs<br />
P<br />
cofactor<br />
Ac<br />
<strong>p53</strong><br />
coregulator<br />
RNA pol II<br />
Mechanisms:<br />
DNA binding. Acetyla@on enhances DNA binding<br />
Cell cycle control<br />
p21, GADD45, 14-‐3-‐3σ<br />
Apoptosis<br />
Puma, Noxa, <strong>p53</strong>AIP1, PIG3,<br />
Bax, Fas<br />
Metabolism<br />
TIGAR, Sco2<br />
Autophagy<br />
DRAM, Sestrin-‐1, Sestrin-‐2<br />
An@-‐repression: Acetyla@on affects the Mdm2/<strong>p53</strong> interac@on.<br />
Co-‐factor recruitments: Acetylated <strong>p53</strong> recruits addi@onal cofactors.
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