Transcript p53 som transkripsjonsfaktor

p53
- guardian or the genome
+ guardian of the tissuse
MBV4230
p53

product of a tumour supressor gene
the most frequently mutated gene in human cancers

393 aa with 4-5 functional domains

biological role as watch dog - “vaktbikkje”


Guardian of the genome - stops the cell cycle upon DNA-damage


Signalling pathway: DNA-damage  enhanced p53  activation of CDKI p21 
G1 arrest  activation of GADD45  stimulated DNA-repair
Guardian of the tissue - facilitates apoptosis if necessary

Signalling pathway: DNA-damage  enhanced p53  apoptosis
MBV4230
p53 mutations
p53 - protein domains
MBV4230
p53 domains
C-terminal allosteric domain
MBV4230
5 distinct domains in p53
1. TAD

TAD N-terminal [aa1-42]





aa 13-23 conserved between species
F19, L22 and W23 necessary for transactivation in vivo
F19, L22 and W23 involved in binding to TAFII70 and TAFII31
TAD negatively regulated through interaction with the MDM2
factor or the E1B-55Kd protein
Structure of the MDM2 N-terminal domain + 13-29 peptid from p53



MDM2 deep hydrophobic pocket
p53 peptide amphipatic helix fitting in the pocket
F19, L22 and W23 involved in binding
MBV4230
Mdm2 - p53
p53-TAD
Mdm2
MBV4230
p53 domains
C-terminal allosteric domain
MBV4230
5 distinct domains in p53
2. Pro-rich domain

Et Pro-rich region between TAD and DBD



PxxP present 5 locations in the region 61-94
deletion of P- rich region  reduced apoptosis-response and reduced
cell cycle arrest, but normal transcriptional response
contains residues that become phosphorylated upon apoptotic response
(HIPK2 phosphorylation of S46)
MBV4230
p53 domains
C-terminal allosteric domain
MBV4230
p53 DBD
2 “-helical loops” that contact
DNA
Two large loops (L2 and L3)
involved in minor groove contact
Scaffold: -sandwich (two
antiparalel -sheets)
Zn++ structuring CHCC-Zn++
MBV4230
5 distinct domains in p53
3. DBD

DBD centrally located [aa102-292]
folded into a “loop-sheet-helix”
motif (LSH)
protease-resistent, independent, Zn++-containing
domain [CHCC-Zn++]
 scaffold: 4- and 5-thread antiparallel -sheet
structure
 2 protruding -helical loops contacting DNA
directly



Two large loops (L2 + L3) involved in minor
groove contact (contact involve R248)


Specific base contact in major groove (K120, C277,
R280)
Several H-bond contacts with sugar-phosphatechain (R273)
Two types of hotspot-mutants in human cancers


disrupts direct DNA-interactions (R248, R273)
disrupts the structure of DBD
MBV4230
DBD mutations

Most of the p53 mutations
that cause cancer are found
in the DNA-binding domain
most common mutation changes
arginine 248 (red), snaking into the
minor groove of the DNA - a strong
stabilizing interaction.
 Other key sites of mutation are shown
in pink, including arginine residues
175, 249, 273 and 282, and glycine
245. Some of these contact the DNA
directly, and others are involved in
positioning other DNA-binding amino
acids.

MBV4230
5 distinct domains in p53
3. DBD

binds DNA as tetramer (dimer of dimer)

DNA recognition sequence reflects this: 4x RRRCW arranged like this:
MBV4230
p53 domains
C-terminal allosteric domain
MBV4230
5 distinct domains in p53
4. Tetramerization

Tetramerization
domain aa 324-355



2 + 2 structure
forms tetramers
linked with DBD via 37aa
flexible linker [aa 287-323]
MBV4230
p53 domains
C-terminal
allosteric domain
MBV4230
5 distinct domains in p53
5. C-terminal allosteric domain

DNA/RNA-binding C-terminal (last 26aa)
open protease-sensitive domain
 Basic region
 binds DNA and RNA non-specifically and can stimulate annealing
 binds DNA ends, internal loops or other loose ends from damaged duplexes
 possible function: (allo)steric regulator of specific DNA-binding
 p53 appears to be present in a latent form inactive in seq.spec. DNAbinding
 Several events in the C-terminal can reactivate p53s central DBD
 deletion of basic C-terminal
 phosphorylation of S378 with PKC
 phosphorylation of S392 with CK2
 binding of C-terminal antibody PAb421
 small singlestranded DNA oligos

Activation of p53
- upstream inputs
MBV4230
Upstream and downstream
Upstream
Signal transduction
pathways
+
..
p53 functions as sensor
of upstream signals
reflecting DNA-damage
/cellular stress
+
p53
Downstream
+
..
activation
MBV4230
Activation of p53 - what happens?

DNA-damage/stress 
 1.



activation of latent p53 [latent form  active form]
enhanced DNA-binding activity
probably also enhanced transactivation activity
post-translational modifications
 2.
stabillization and a rapid increase in protein level
activation of response


activation  level increases 10-100x
Since enhanced levels of p53 may lead to cell cycle-arrest and apoptosis, it is
of critical importance that normal cells keep their p53 levels low
MBV4230
Activated by several signals

types of activating stress


DNA-damage (chain breaks, repairintermediates, recombinationintermediates)
Hypoxia



protective function in tumours (tumour
growth  limited blood supply 
hypoxia  p53 activation  apoptosis
of tumour)
trombospondin appears to be p53
regulated, acts antiangiogenic, will
reduce blood supply further
NTP pool reduced

sufficient NTP-pool for DNAreplication sensed by p53
Activated oncogenes (Myc, Ras, E1A,
ß-catenin)
 Foster defects

MBV4230
The key to stabillization:
the MDM2-p53 coupling

MDM2 associates with p53s TAD (aa 17-27)

MDM2-binding leads to
 1. Repression of transactivation
 2. Destabillization of p53 since MDM2-binding
stimulates degradation of p53

mdm2 knock-out = lethal, rescued by
simultaneous deletion of p53
MBV4230
The key: MDM2-p53 coupling


Mechanisms for stimulated degradation
MDM2 = p53-specific E3 ubiquitin protein
ligase


MDM2 cause transport of p53 from nucleus to cytoplasma,
and export is necessary for degradation
MDM2 = a target gene for p53 being
activated by p53
Negative feedback loop - mechanism for turning off the p53
response
 Induced relatively late - leaves a time window where p53
can function


regulation = f (MDM2-p53 contact)
Via phosphorylation
 Via associated proteins

MBV4230
Several strategies to break the
MDM2-p53 coupling
Before activation
Activated 
phosphorylation 
Broken binding
Activated 
phosphorylation 
inactivated E3-act
Activated 
ARF-binding 
inactivated E3-act
MBV4230
Recent news

More E3 enzymes
suggesting
ubiquitylation
independent of Mdm2
MBV4230
Regulation of MDM2-p53 contact
through phosphorylation of p53 TAD

The ATM kinase
a kinase that is the product of the ATM gene that is lost in
pasients with ataxia-telangiectasia
 phosphorylates S15
 Weakens the p53-MDM2 interaction



CHK2 - recently identified as a S20-kinase
HIPK2 - recently identified as a S46-kinase


activated as response to UV, role in apoptotic response
DNA PK
DNA-dependent protein kinase
 phosphorylates S15
 Weakens p53-MDM2 interaction

MBV4230
upstream signalling pathway
Chk2 is a protein kinase that is
activated in response to DNA
damage and may regulate cell
cycle arrest. Chk2-/- cells were
defective for p53 stabilization
and for induction of p53dependent transcripts such as
p21 in response to gamma
irradiation. Chk2 directly
phosphorylated p53 on serine
20, which is known to interfere
with Mdm2 binding.
MBV4230
Updated: p53 &
DNA damage
p53 functions as a
‘molecular node’ in
the DNA-damage
response.
MBV4230
Recent : HIPK2 binds and phosphorylates
p53 after UV irradiation
UV
HIPK2
…leading to apoptosis
MBV4230
Many covalent modifications of p53
in regulatory N- and C-terminal

Phosphorylation
20

Acetylation

Glycosylation

SUMOylation

Methylation
MBV4230
Several modifications
- complex regulatory mechanisms


The C terminus of p53 is rich in lysines, which are
subjected to acetylation, ubiquitination and
sumoylation.
Acetylation of the C terminus
has been shown to protect p53 from ubiquitination.
 Acetylation of p53 at K373 and K382 increases its DNA-binding activity and
potentiates its interaction with other transcription factors.
 The positive effects of acetylation on p53 activity can be reversed by
deacetylation.


p53 has also been shown to be sumoylated at K386

although the exact role of this modification in the regulation of p53 is not yet
clear.
MBV4230
Acetylation upon p53 activation
p53 stabilization
p300
Mdm2
Phosphorylation
followed by
Acetylation
20
activation
MBV4230
Methylation of p53

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A novel mechanism of p53 regulation through
lysine methylation by Set9 methyltransferase.
Set9 specifically methylates p53 at one residue
within the C-terminal regulatory region.
Methylated p53 is restricted to the nucleus and
the modification positively affects its stability.
Set9 regulates the expression of p53 target genes
in a manner dependent on the p53-methylation
site.
MBV4230
Turning p53 OFF - the hSir2 link

Sir2 - ”silent information regulator”


conserved family identified in silencing in yeast
function as NAD-dep deacetylase
MBV4230
Deacetylation after p53 activation
p300
Mdm2
Phosphorylation
followed by
Acetylation
20
HDAC?
= hSIR2
MBV4230
Model
p53
DNA
damage
Stabilized
Activated
Acetylated
Response ON
hSir2
Growth arrest
Apoptosis
Response OFF
p53
De-acetylated
Alternative activation
- the ARF input
MBV4230
N-terminal control via ARF-binding
ARF (alternative reading frame) from p16INK4a
The INK4A locus (frequently mutated in cancer) → 2 alternatively spliced transcripts →
translated from alternative reading frames → p16 (cdk-inhibitor) + ARF:
• binds MDM2-p53 and inhibits the effect of MDM2s (ligase and shuttling)
• ARF strongly induced by viral oncoproteins and contributes to apoptosis of infected cells
• ARF also induced by Myc
MBV4230
ARF activation  relocalization
In unstressed cells, p53 is degraded following interaction with MDM2 and is exported
to the cytoplasm using nuclear-export signals present in p53 and MDM2.
Inhibition of MDM2-mediated degradation occurs in response to certain stress signals by
activation of ARF expression. When ARF binds to MDM2, the MDM2–ARF complex is
relocalized to the nucleolus using nucleolar-localization signals present in MDM2 and
ARF. This leaves free, transcriptionally active, p53 in the nucleoplasm.
Unstressed cells
p53 degradation
Stressed cells
ARF expression
Stable p53
MBV4230
Localization: A model for PMLmediated recruitment of p53 to NBs.


PML (promyelocytic leukemia) = org.
comp. of nuclear bodies (NBs).
Signal trigger


Modification


Signals from DNA damage such as girradiation (gIR) or oncogene imbalance – e.g.
ras overexpression - trigger SUMOylation (S)
and aggregation of PML into NBs. Factors
including CBP, Rb, Daxx, Sp100 are also
recruited to NBs.
Consequence: phosphorylation (P) and
acetylation (A) of p53. Result in increased and
altered p53 transcriptional activity.
Update
 PML enhances p53 stability by
sequestering Mdm2 to the
nucleolus. After DNA damage,
PML and Mdm2 accumulate in the
nucleolus
MBV4230
At least two main pathways
DNA damage
Kinase activation
Activated
p53
Oncogene activation
ARF activation
MBV4230
Multiple pathways - diverse
responses

Multiple pathways exist to stabilize p53 in response
to different forms of stress


they may involve down-regulation of MDM2 expression or regulation of the
subcellular localization of p53 or MDM2.
Target genes induced by gamma radiation, UV
radiation, and the zinc-induced p53 form distinct sets
and subsets with a few genes in common to all these
treatments.
MBV4230
At least two main pathways
DNA damage
?
Kinase activation
??
Activated
p53
Oncogene activation
ARF activation
?
?
?
The outcomes of
activated p53
- downstream effects
MBV4230
Upstream and downstream
Upstream
Signal transduction
pathways
+
..
p53 functions as sensor
of upstream signals
reflecting DNA-damage
/cellular stress
+
p53
activation
..
Target genes
Activated
Downstream
+
MBV4230
p53 as signal transducer
- downstream response

downstream consequences leading to repair
of damage or apoptosis of damaged cell

Two main types of effects of activated p53

1. Stop/regulation of the cell cycle
2. induction of apoptosis

MBV4230
Downstream response

Transcriptional effects




Target genes inducing cell
cycle arrest
… or DNA damage repair
Target genes promoting
apoptosis
Other types of effects?
?
MBV4230
Cytoplasmic Roles of p53 in
Apoptosis



p53 can initiate apoptosis
in cells in which trx and
translation are inhibited
p53 polyproline domain
(aa 62–91) is necessary to
cause apoptosis
Excluding p53 from the
nucleus causes apoptosis
(> threshold level)

p53 affects mitochondrial
apoptotic regulators

Cytoplasmically localized p53 can either
directly induce Bax oligomerization or liberate
proapoptotic BH3-only proteins bound to
Bcl2/Bcl-XL at the mitochondria. The released
BH3-only proteins can then activate Bax
oligomerization and thereby cause cytochrome
c release leading to apoptosis.
MBV4230
Regulation of the cell cycle
via p53-Rb pathway

Normal cell cycle regulation through four cooperating actors
[p16 - cyclin D1 - cdk4 - Rb] which regulate the G1-S transition
most cancers have one of these four altered
 p16 negative regulator of cyclin D1/cdk4


Signalling pathway: DNA-damage  activated enhanced p53 
activation of CDKI p21 (WAF1, Cip-1)  inhibition of cdk4 
reduced phosphorylation of Rb  G1 arrest
p21 inhibits also several of the other cdks
 p21 binds also PCNA  stop in replication


Signalling pathway: DNA-damage  activated enhanced p53 
activation of GADD45  stimulated DNA-repair
MBV4230
p53-Rb pathway
Apoptosis
Cell cycle arrest
MBV4230
p53 as signal transducer
- several downstream responses



p53 also a role in preventing
gene amplification
p53 also a role in G2/M
checkpoint
Induces a separate
ribonucleotide reductase
(p53R2)

p53R2 encodes a ribonucleotide reductase that
is directly involved in the p53 checkpoint for
repair of damaged DNA.
MBV4230
induction of apoptosis



DNA-damage in p53+/+ cells  apoptosis
DNA-damage in p53-/- cells  no apoptosis
mechanisms far from fully understood
Transcription activation necessary? only partially
 also TF-independent functions involved




Bax induced by p53 - acts pro-apoptotic by counteracting Bcl2
PERP is a novel effector of p53-dependent apoptosis
p53AIP1 (p53-regulated Apoptosis-Inducing Protein 1)

upon severe DNA damage, Ser-46 on p53 is phosphorylated and apoptosis is induced. In
addition, substitution of Ser-46 inhibits the ability of p53 to induce apoptosis and selectively
blocks expression of p53AIP1.
MBV4230
More target genes

Genes induced by gamma radiation, UV
radiation, and the zinc-induced p53 form
distinct sets and subsets with a few genes in
common to all these treatments.

Microarray analysis of 6000 genes showed:

107 induced and 54 repressed genes fell into categories of apoptosis and
growth arrest, cytoskeletal functions, growth factors and their inhibitors,
extracellular matrix, and adhesion genes.
MBV4230
Signalling pathway
Latent p53
activated p53
p53 and cancer
MBV4230
p53 - in cancers

Most frequent gene mutated in cancers


Thousands of mutations from patients sequenced
tumour supressor gene


both alleles must be lost
Common scenario: 1. mutant  2. loss-of-heterozygocity (LOH)


90-95% develop cancer in young age
Two types of DBD-mutations frequent in
tumours


Interferring with prot-DNA contacts
Destabilizing the core structure of DBD