p53 - guardian or the genome

+ guardian of the tissuse

MBV4230

Odd S. Gabrielsen

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 watchdog - “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

Odd S. Gabrielsen

p53 mutations

p53 - protein domains

MBV4230

Odd S. Gabrielsen

p53 domains

C-terminal allosteric domain

MBV4230

Odd S. Gabrielsen

5 distinct domains in p531. 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

p53 TAD

MBV4230

Odd S. Gabrielsen

Mdm2 - p53

Mdm2

p53-TAD

MBV4230

Odd S. Gabrielsen

p53 domains

C-terminal allosteric domain

MBV4230

Odd S. Gabrielsen

5 distinct domains in p532. 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

Odd S. Gabrielsen

p53 domains

C-terminal allosteric domain

MBV4230

Odd S. Gabrielsen

p53 DBD

2 “α-helical loops” that contact DNA

Zn++ structuring CHCC-Zn++

Two large loops (L2 and L3) involved in minor groove contact

Scaffold: β-sandwich (tw

o antiparalel β-sheets)

MBV4230

Odd S. Gabrielsen

5 distinct domains in p533. 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 β-sheet

structure 2 protruding α-helical loops contacting DNA

directly Specific base contact in major groove (K120, C277,

R280) Two large loops (L2 + L3) involved in minor

groove contact (contact involve R248) Several H-bond contacts with sugar-phosphate-chain

(R273) Two types of hotspot-mutants in human cancers

disrupts direct DNA-interactions (R248, R273) disrupts the structure of DBD

MBV4230

Odd S. Gabrielsen

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

Odd S. Gabrielsen

p53 p53

5 distinct domains in p533. DBD

binds DNA as tetramer (dimer of dimer) DNA recognition sequence reflects this: 4x RRRCW arranged like this:

p53

RNA pol II

p53

MBV4230

Odd S. Gabrielsen

p53 domains

C-terminal allosteric domain

MBV4230

Odd S. Gabrielsen

5 distinct domains in p534. Tetramerization Tetramerization

domain aa 324-355 2β + 2α structure forms tetramers linked with DBD via 37aa

flexible linker [aa 287-323]

MBV4230

Odd S. Gabrielsen

p53 domains

C-terminal allosteric domain

MBV4230

Odd S. Gabrielsen

5 distinct domains in p535. 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. DNA-binding

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

Odd S. Gabrielsen

Upstream and downstream

p53

Signal transductionpathways

+ +..

+ ..

Upstream

Downstream

p53 functions as sensor of upstream signals reflecting DNA-damage /cellular stress

activation

MBV4230

Odd S. Gabrielsen

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

p53

inactive

active

DNAdamage

p53p53

p53

p53

MBV4230

Odd S. Gabrielsen

Activated by several signals

types of activating stress DNA-damage (chain breaks, repair-

intermediates, recombination-intermediates)

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 DNA-replication

sensed by p53 Activated oncogenes (Myc, Ras, E1A,

ß-catenin) Foster defects

MBV4230

Odd S. Gabrielsen

The key to stabillization: the MDM2-p53 coupling

MDM2 associates with p53s TAD (aa 17-27) MDM2-binding leads to

1. Repression of transactivation2. Destabillization of p53 since MDM2-binding

stimulates degradation of p53 mdm2 knock-out = lethal, rescued by

simultaneous deletion of p53

p53

Mdm2

MBV4230

Odd S. Gabrielsen

The key: MDM2-p53 coupling Mechanisms for stimulated

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

MDM2 = p53-specific E3 ubiquitin protein ligase

MBV4230

Odd S. Gabrielsen

The key: MDM2-p53 coupling MDM2 = p53-specific E3 ubiquitin

protein ligase regulation = f (MDM2-p53 contact)

Via phosphorylation Via associated proteins

MBV4230

Odd S. Gabrielsen

Several strategies to break theMDM2-p53 coupling

Before activation

Activated phosphorylation

Broken binding

Activated phosphorylation inactivated E3-act

Activated ARF-binding

inactivated E3-act

MBV4230

Odd S. Gabrielsen

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

Odd S. Gabrielsen

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 p53-dependent 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

Odd S. Gabrielsen

Updated: p53 & DNA damage

p53 functions as a ‘molecular node’ in the DNA-damage response.

MBV4230

Odd S. Gabrielsen

p53 - switch from inactive to active

Remember - two important changes upon activation - increased levels + altered state

30

p53

inactive

active

DNAdamage

p53p53

p53

p53Ac

P

Ac

Ac

Ac

Ac

P

P

P

MBV4230

Odd S. Gabrielsen

Many covalent modifications of p53 in regulatory N- and C-terminal

Phosphorylation

Acetylation

Glycosylation

SUMOylation

Methylation

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MBV4230

Odd S. Gabrielsen

Several modifications - complex regulatory mechanisms The C terminus of p53 is rich in lysines, which are

subjected to acetylation, ubiquitylation, sumoylation and neddylation.

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

Odd S. Gabrielsen

Acetylation upon p53 activation

Phosphorylationfollowed by

Acetylation

20

Mdm2

p300

p53 stabilization

activation

MBV4230

Odd S. Gabrielsen

Methylation of p53

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

Odd S. Gabrielsen

Turning p53 OFF - the hSir2 link

Sir2 - ”silent information regulator” conserved family identified in silencing in yeast function as NAD-dep deacetylase

MBV4230

Odd S. Gabrielsen

Deacetylation after p53 activation

Phosphorylationfollowed by

Acetylation

20

p300

Mdm2

HDAC? = hSIR2

MBV4230

Odd S. Gabrielsen

Model

DNAdamage

p53StabilizedActivatedAcetylated

Growth arrestApoptosis

Response ON

Response OFFhSir2

p53De-acetylated

MBV4230

Odd S. Gabrielsen

Triggering p53 .... and turning it off again

Summary

38

Alternative activation- the ARF input

MBV4230

Odd S. Gabrielsen

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

Odd S. Gabrielsen

ARF activation → relocalization

Unstressed cells Stressed cellsARF expression

p53 degradation Stable p53

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.

MBV4230

Odd S. Gabrielsen

At least two main pathways

DNA damage

Kinase activation

Activatedp53

Oncogene activation

ARF activation

MBV4230

Odd S. Gabrielsen

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

Odd S. Gabrielsen

At least two main pathways

DNA damage

Kinase activation

Activatedp53

Oncogene activation

ARF activation?? ? ?

??

The outcomes of activated p53 - downstream effects

MBV4230

Odd S. Gabrielsen

Upstream and downstream

p53

Signal transductionpathways

+ +..

+ ..

Upstream

Downstream

p53 functions as sensor of upstream signals reflecting DNA-damage /cellular stress

activation

Target genesActivated

MBV4230

Odd S. Gabrielsen

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

Odd S. Gabrielsen

Downstream target genes:1. Cell cycle arrest p53 activate Cdk-inhibitors, leading to

cell cycle arrest

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Odd S. Gabrielsen

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

Odd S. Gabrielsen

p53-Rb pathway

DNA damage

Cell cycle arrestApoptosis

MBV4230

Odd S. Gabrielsen

Downstream target genes:2. Promoting apoptosis p53 activate target genes that

promote programmed cell death - apoptosis PUMA (p53-upregulated mediator of apoptosis) - a

protein that belongs to the BCL2 family and that promotes mitochondrial outer membrane permeabilization and apoptosis. PUMA is a crucial mediator of apoptosis in response to p53.

Noxa - same family

Mitochondrial action Induction of apoptosis: p53 seems predominantly to

influence the “intrinsic pathway”, leading to a perturbation of mitochondrial membrane potential, and so the release of apoptogenic factors from the mitochondrial intermembrane space into the cytoplasm. This triggers a cascade of events leading to caspase activation and cell death. 51

MBV4230

Odd S. Gabrielsen

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

Odd S. Gabrielsen

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

Odd S. Gabrielsen

Summary: upstream + downstream

54

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Odd S. Gabrielsen

Stress signal integration

Integration of stress responses p53 has a key role in integrating the cellular responses (pink boxes) to

different types of stress (blue boxes). Activation of p53 can result in a number of cellular responses, and it is

possible that different responses are induced by different stress signals.

55

p53 and cancer

MBV4230

Odd S. Gabrielsen

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