The Role of Phosphorylation in Mediating Cellular Responses
to DNA Damage: The ATM-Mediated DNA Damage Response
Professor Yosef Shiloh
The Role of Phosphorylation in Mediating
Cellular Responses to DNA Damage:
The ATM-Mediated DNA Damage Response
Professor Yosef Shiloh
Sackler School of Medicine
Tel Aviv University, Israel
1
The DNA molecule
2
DNA damage
3
The Role of Phosphorylation in Mediating Cellular Responses
to DNA Damage: The ATM-Mediated DNA Damage Response
Professor Yosef Shiloh
Estimated endogenous DNA damage in mammalian cells
Damage Events per cell/day
Single-strand break 55,200
Depurination 12,000
Depyrimidination 600
06-methylguanine 3,120
Cytosine deamination 192
Glucose-6-phosphate adduct 3
Thymine glycol 270
Thymidine glycol 70
Hydroxymethyluracil 620
8-oxo-G 178
Interstrand cross-link 8
Double-strand break (DSB) 8
Compiled by Dr. Vilhelm Bohr 4
DSB repair pathways
5
Nonhomologous Homologous recombination
end-joining (NHEJ) (HR)
The DNA damage response
DNA repair
Cell cycle checkpoints
Affects numerous
cellular processes
Modulates gene expression
Concerted action of numerous,
interlocked signaling pathways
creating an intricate web
Activated instantaneously
and vigorously,
particularly by DSBs
Pericentriolar material
Mother centriole
6
Daughter centriole
Replicated DNA
Chromosome
The Role of Phosphorylation in Mediating Cellular Responses
to DNA Damage: The ATM-Mediated DNA Damage Response
Professor Yosef Shiloh
Cellular responses to DNA damage
DNA Endogenous Exogenous
turnover agents agents
DNA
damage
Sensors /Activators
Transducers
activation,
signalling
Effectors
DNA Cell cycle Stress
repair arrest responses
Apoptosis
7
үH2AX foci үH2AX foci
Foci show where
the damage occurred
This is a good marker
for the induction
of double-strand breaks
and their disappearance
Sophisticated techniques
can be used to induce the DNA
damage in specific portions
of the nucleus
Many damage response proteins
are rapidly recruited to the damage sites
8
P P P P
P P
P ATM ATM P
RAP80BRCC36 RAP80BRCC36
P
NBS1 NBS1 P
UBC13
P
MDC1
P P P P RNF8
P
P BRCA1 P P
P P MDC1 P BRCA1 P P
P P
P
P ATM P P P
RNF20
P P ATM P P
RNF20
P 53BP1 53BP1 RNF40 P
RNF40 P
Ub Ub Ub Ub Ub Ub Ub
Ub Ub Ub Ub
Ub Ub Ub Ub
Ub Ub Ub Ub
Ub Ub Ub Ub
Ub Ub Ub Ub
9
The Role of Phosphorylation in Mediating Cellular Responses
to DNA Damage: The ATM-Mediated DNA Damage Response
Professor Yosef Shiloh
Defects in maintenance of genome stability
Monogenic disorders combining sensitivity
to genotoxic stresses, tissue degeneration,
cancer predisposition
Chromosomal and genomic aberrations
The DNA damage response as a barrier
to cancer development
Bloom syndrome
Milder mutations or heterozygosity for severe
Fanconi’s anemia
mutations may contribute to chronic conditions:
Sensitivity to acute doses of genotoxic stress
Premature aging
Immunodeficiency
Neurodegeneration
Nijmegen breakage Cancer predisposition Xeroderma pigmentosum
syndrome
Reduced ability to cope with environmental stresses
Load on public health caused by mutations
affecting genomic stability may be heavier
than previously estimated
10
Disorders flag important players Ataxia-telangiectasia
Werner syndrome
in the DNA damage response
Ataxia-telangiectasia (A-T)
1926: Syllaba and Henner described 3 siblings
with progressive ataxia and ocular telangiectasias
1941: Louis-Bar described a Belgian girl
with a similar syndrome
1957: Sedgwick and Boder reported a series
of American patients and called the disease
ataxia-telangiectasia (A-T)
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Ataxia-telangiectasia (A-T) (2)
Autosomal recessive
Cerebellar ataxia (loss of Purkinje cells),
severe neuromotor dysfunction
Telangiectasia
Immunodeficiency (B&T cells);
recurrent infections
Thymic and gonadal dysgenesis
Retarded growth
Chromosomal instability
Cancer predisposition
Acute sensitivity to ionizing radiation
A profound defect in cellular responses
to double strand breaks (DSBs) in the DNA
Responsible gene: ATM
12
The Role of Phosphorylation in Mediating Cellular Responses
to DNA Damage: The ATM-Mediated DNA Damage Response
Professor Yosef Shiloh
Cellular responses to DNA damage
DNA Endogenous Exogenous
turnover agents agents
DNA
damage
ATM Sensors /Activators
Transducers
activation,
signalling
Effectors
DNA Cell cycle Stress
13
Apoptosis
repair arrest responses
The ATM protein
Completely missing or inactivated in A-T
patients (reduced levels in “mild A-T”)
A large (370 kDa), nuclear phosphoprotein
A carboxy-terminal PI3-kinase-like motif
Protein kinase activity (serine-threonine)
ATM targets: preference for SQ or TQ
The primary activator of the cellular response
to double strand breaks (DSBs)
Activated by DSBs
Phosphorylates key
DDR players
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ATM activation
DSBs
Inactive dimers turn
into active monomers;
ATM ATM autophosphorylation
Recruitment (Bakkenist & Kastan, 2003)
to DSB sites Several autophosphorylations,
(Andegeko et al., 2001) Tip60-mediated acetylation
Activated (Kozlov et al., 2006, Sun et al., 2007)
ATM
Role for autophosphorylations
Phosphorylation in ATM retention at DSB sites
(So et al., 2009)
Additional post-translational Effectors
modifications?
DSB Cell cycle Stress
15
Apoptosis
repair checkpoints responses
The Role of Phosphorylation in Mediating Cellular Responses
to DNA Damage: The ATM-Mediated DNA Damage Response
Professor Yosef Shiloh
ATM-mediated DNA damage responses
There are two levels
of phosphorylations
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PI3-kinase-related protein kinases (PIKKs)
1 3056
ATM
1 2644
ATR
1 3031
hSMG-1
1 4128
DNA-PKcs
1 2549
mTOR/FRAP
1 3830
TRRAP
DOMAINS: FAT PI3K FATC 17
p53 activation and stabilization
ATM
S395 26S
Additional T68
modifications
Mdm2
Chk2
S342
S403S367 ATM has a very strong grip
S15 S20 Mdmx HAUSP of this pathway
S387 p53 T42 S337
COP1 ATM can fine tune this pathway
USP10
S342
*p53 *p53 S403S367
*p53 *p53 *p53 Mdmx 14-3-3
26S
HAUSP
poly-Ub
26S
Apoptosis Cell cycle
checkpoints
18
R.I.P
The Role of Phosphorylation in Mediating Cellular Responses
to DNA Damage: The ATM-Mediated DNA Damage Response
Professor Yosef Shiloh
Research directions
ATM
ATM activation
Identification of novel ATM
ATM-mediated pathways
Function of target proteins:
Cellular level
Organismal level Other Protein Protein Gene Cell DNA
stress degradationsynthesis regulation cycle repair
Overview of the DNA response checkpoints
damage response using
systems biology approach
Search for new treatment
modalities for A-T
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New branches of the ATM-mediated
DNA damage response
Substrate identification
Proteomic methods:
ATM
• ATM-associated proteins
• Proteomic screens for ATM substrates
• Damage-induced dynamics
of the cellular phosphoproteome
Candidate approach
Functional analysis of specific substrates and the corresponding pathways
Function of a single protein, single phosphorylation,
in a complex signaling network
Identification of the “proteomic environment”
surrounding the substrate in undamaged
and damaged cells
“Protein replacement” followed
by analysis of cellular phenotype
Gene targeting in mice
20
ATM functional links: a SPIKE map, September 2010
http://www.cs.tau.ac.il/~spike/
ATM
21
The Role of Phosphorylation in Mediating Cellular Responses
to DNA Damage: The ATM-Mediated DNA Damage Response
Professor Yosef Shiloh
High-throughput identification of ATM/ATR/DNA-PK substrates
Most of these
phosphorylations
are real and play
a role in the DNA
damage response 22
Insights into the ATM-mediated DDR
Control of specific processes via many
ATM-mediated pathways
Recruitment to damage sites of proteins that function
in damage processing/signaling, in their “regular”
or different capacities
Broad interface between the DDR and processes driven
by the ubiquitin family of proteins
Intimate relationships with telomere maintenance, meiotic
and V(D)J recombination and RNA metabolizing systems
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Understanding A-T
Defective repair of DNA double strand breaks
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The Role of Phosphorylation in Mediating Cellular Responses
to DNA Damage: The ATM-Mediated DNA Damage Response
Professor Yosef Shiloh
ATM-mediated signaling facilitating DSB repair
Direct targeting of repair proteins
Creating appropriate microenvironment for repair
within chromatin
25
PNKP – polynucleotide kinase 3’-phosphatase
Dual activity enables
restoration of “legitimate” ends
26
ATM-mediated phosphorylation of PNKP
FHA Phosphatase Kinase
5 114126 521
Segal-Raz et al., 2011
Targets: S114 and S126
Induced by DSB-inducing agents
ATM-dependent
Cells in which endogenous
Phosphatase assay
PNKP is replaced by ectopic, 0.6
0.5
non-phosphorylatable PNKP
% Activity
0.4 wt
show intermediate 0.3 x2
radiosensitivity between 0.2
those of PNKP-/- and wild 0.1
0.0
27
type cells 0 15 30 60
Time (min)
The Role of Phosphorylation in Mediating Cellular Responses
to DNA Damage: The ATM-Mediated DNA Damage Response
Professor Yosef Shiloh
Allowing repair to happen within chromatin
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ATM-dependent phosphorylation of KAP-1 mediates
global chromatin relaxation following DNA damage
B B Coiled- HP1BD
RING box box PHD Bromo
coil
Ziv et al., 2006
S824
Known as transcription corepressor
Rapidly phosphorylated by ATM at DSB sites
Quickly migrates throughout chromatin
and mediates chromatin decondensation
Facilitation of DSB repair
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Chromatin reorganization
Prerequisite for all DNA transactions
Major mediator: changes in histone
post-translational modifications
Examples for histone PTMs associated
with the DDR:
H2AX phosphorylation/dephosphorylation
H2A monoubiquitination mediated by RNF8 and RNF168
Monoubiquitination of histone H2B
– an ongoing, dynamic PTM
30
The Role of Phosphorylation in Mediating Cellular Responses
to DNA Damage: The ATM-Mediated DNA Damage Response
Professor Yosef Shiloh
RNF20/40- and ATM-mediated H2B monoubiquitination
Upon DSB induction RNF20/40 is recruited Moyal et al., 2011
P P
to damaged sites and phosphorylated by ATM RNF
20
H2B monoubiquitination at damaged ATM RNF
sites is important for timely recruitment 40
P
of both NHEJ and HR repair proteins
and subsequent DSB repair NHEJ
Formation of appropriate chromatin
microenvironment for DNA repair
Example of a protein machinery that is normally
active in a non-DDR context and is recruited
HR
to the damaged sites to carry out the same
function at the damaged sites
31
Exploration of the interface between the ubiquitin
family and the DNA damage response
32
Players in ubiquitin-related processes
33
~1600 proteins
The Role of Phosphorylation in Mediating Cellular Responses
to DNA Damage: The ATM-Mediated DNA Damage Response
Professor Yosef Shiloh
Overlap between the ubiquitin arena
and ATM substrates
Ub arena ATM substrates
1600 145 900
34
Phosphoproteome dynamics after DNA damage
Untreated DNA damage
A. Bensimon et al., 2010
Prepare nuclei
Lyse nuclei, digest p
p p
p
p
p
p
p
Phosphopeptide p p
p p
enrichment p
p
p
p
LC-MS/MS
Label-free
quantification Computational
35
analysis
Damage-induced protein phosphorylation
Hundreds of proteins phosphorylated
or dephosphorylated in response to DSB induction
Many new phosphorylations, not on SQ/TQ
Only 60% of the phosphorylations are ATM-dependent
ATM “switch off” after damage induction switches off
many ATM-dependent phosphorylations -> ATM
sustained activity is required to maintain them
No ATM inhibitor
With ATM inhibitor
36
The Role of Phosphorylation in Mediating Cellular Responses
to DNA Damage: The ATM-Mediated DNA Damage Response
Professor Yosef Shiloh
Functional networks of phosphorylated
and dephosphorylated proteins
Phosphorylated
Dephosphorylated
37
Both
Ub ATM
1600 145 900
88 63
299
“General”
phosphorylation
38
Functional screens for novel DDR players
Screening of siRNA
libraries using assays
based on DDR readouts
Nuclear foci of 53BP1
KAP-1 phosphorylation
39
The Role of Phosphorylation in Mediating Cellular Responses
to DNA Damage: The ATM-Mediated DNA Damage Response
Professor Yosef Shiloh
Ub ATM
1600
186 15 900
8
299
“General”
phosphorylation
40
The role of phosphorylation in mediating
cellular responses to DNA damage:
the ATM-mediated DNA damage response
ATM
41
42