Role of PARP-1 and Poly (ADP-Ribosyl)ation in the activation of AMPkα2 and
induction of starvation-induced autophagyRodríguez- Vargas, JM1 ; Rodríguez-Lara MI1; Dantzer, F2; López-Rivas, A3 and Oliver-Pozo FJ1
1 Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC, 18100, Granada.
2. École Supérieure de Biotechnologie de Strasbourg, IREBS-CNRS, 67412 Illkirch Cedex.
3. Andalusian Center for Molecular Biology and Regenerative Medicine, 42092, Sevilla.
The most physiological autophagic stimulus is nutrient deprivation or starvation. In response to nutrient stress, cells start an autophagy program that can lead to adaptation or death. The mechanisms underlying the
signalling from starvation to the initiation of autophagy are not fully understood. Several distinct kinase complexes have been implicated in autophagic pathway. AMP kinase, called the energetic sensor of the cells, is a
Serine/Threonine kinase that promotes energy-producing catabolic pathways, as autophagy, while inhibiting anabolic pathways. As negative regulator of cell growth and proliferation pathways, one of the major targets of AMPk
is the complex mTORC1 kinase. The mTORC1 pathway controls a number of biological process that are important for the normal function of the cell including cell-cycle progression, survival, migration, transcription, translation
and metabolism. Globally, mTORC1 controls the anabolic general pathways. In the context of autophagy regulation AMPk is a positive regulator of autophagy while mTORC1 is a negative regulator o inhibitor of autophagy.
PARP-1 is a nuclear enzyme with 116 kDa that present three structural domains: 1) a DNA binding domain with three Zn fingers which are essentials in the direct interaction with the chromatin and a nuclear localization
sequence (NLS), 2) a central automodification domain which contains several glutamate, aspartate and lysine residues as putative acceptor for auto (ADP-Ribosyl)ation and finally 3) a C-terminal catalytic domain which contains
tryptophan-, glycine- and arginine-rich domain ,or WGR domain, and the “PARP signature” sequence requires for the catalysis of PAR synthesis. PARP-1 catalyzes the covalent attachment of Poly ADP-Ribose (PAR polymer) on
itself and other nuclear protein acceptors in a enzymatic reaction called Poly (ADP-Ribosyl)ation or PARylation, including histones, DNA repair proteins, transcription factors and chromatin modulators, using NAD+ as a donor
of ADP-Ribose and ATP as energetic molecule.
In previous studies we have demonstrated that the absence or inhibition of PARP-1 strongly delays starvation-induced autophagy (Rodríguez-Vargas et al (2012) Cell Research 22:1181-1198). During starvation, ROS-
induced DNA damage activates PARP-1, leading to ATP depletion (an early event after starvation); in this context AMPk is activated and mTORC1 in inhibited, finally autophagy is triggered. The abscense or inhibition of PARP-1
delayed ATP depletion, blunted AMPk activation and prevented the complete loss of mTORC1 activity, leading to a delay in autophagy. PARP-1 depletion favours apoptosis in starved cells, suggesting a pro-survival role of
autophagy and PARP-1 activation during nutrient deprivation.
We have demonstrated that in fed cells PARP-1 form a molecular complex with AMPkα2 isoform in the nucleus. During nutrient deprivation PARP-1/AMPk complex is dissociated in response to PARP-1 activation.
Moreover nuclear AMPkα2 free from PARP-1 complex is modified by Poly (ADP-Ribosyl)ation (PARylated). Short times of starvation induced translocation of PARylated-AMPkα2 protein from nucleus to cytosol where is
necessary for a correct inhibition of mTORC1 and autophagy induction. PAR-AMPkα2 will be an important signal to correct activation, by phosphorylation, of cytosolic AMPk by LKB1 kinase. Phospho-AMPk will be the negative
regulator of mTORC1 and activator of ULK1 kinase. Activated ULK1 is the first autophagy-related protein necessary for autophagosomes formation.
.
Figure 1: (a)Autophagy Pathway, (b) Regulation by AMPk and PARP-1 structure
1a 1b
Figura 3: Levels of PARylation regulates autophagy induction during starvationRESULTS
3a
3b
3a: Effect of PARP-1, PARG silencing and PARP-1 inhibition on starvation-induced autophagy. MCF7 GFPLC3 cells.
3b: Effect of PAR accumulation on starvation-induced autophagy. shVector and shPARG A549 cells.
3c: Effect of ULK1 silencing on starvation-induced autophagy. shVector and shPARG cells.
3d: Endogenous LC3-II conversion in shPARG A549 cells during silencing of ULK1 and PARP inhibitor treatment.
4a: Effect of PARylation inhibition on ATP levels and AMPk activation. Treatment with PJ34 and PARP-1
silencing (60nM) strongly delays starvation-induced loss of ATP levels in MCF7 GFPLC3 cells.
4b: Effect of PAR accumulation on AMPk activation. Accumulation of PAR polymer by specific silencing of
PARG (30nM) hadn’t effect on starvation-induced loss of ATP in starved MCF7 GFPLC3.
4a
5a: Endogenous co-Immunoprecipitation of AMPk and PARP-1 in MCF7 GFPLC3 during starvation.
5b: Confocal microscopy to detect Colocalization between PARP-1 and AMPk.
5c: Interaction AMPkα2 with Catalytic Domain (EF) of PARP-1. Pulldown assay in COS1 cells.
5d: Starvation-dependent interaction of PARP-1 and AMPkα2. Pulldown assay in COS1 cells.
5e: Role of PARylation in PARP-1/AMPkα2 interaction. PARP Inhibitor KUDOS 100 nM.
5f: AMPkα2 is PARylated during starvation-induced autophagy. IP PAR overexpression of FLAG-AMPkα2
in shPARG HeLa cells.
Acknowledgement: This work was supported (in part) by a grant (RD12/0036/0046) from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness
(FIS 00/0948, FIS G03/152, SAF: 2003-01217, SAF2006-01094), Junta de Andalucía (POT-CTS-0239) & Fundación La Caixa (BM06-219-0).
Phagophore Autophagosome
Lysosome
Autolysosome
Nucleation Elongation Fusion
PULK1/2
FIP200ATG101
ATG13
Protein
Synthesis
Autophagy
AMPk
Starvation
TSC1
TSC2
RhebGTP
Raptor
mLST8mTOR
P
P
AMPk
P
P
P
•Nuclear Enzyme 114 KDa
NAD+ Domain 523-1014
523 1014
ACTIVE SITE
859-908
WGR
547-631
N C
1014 Aah PARP-1
FI FII NLS
DNA Binding Domain (DBD) 1-371
DEVD
214
21 53 125 162 207 226
FIII
Automodification Domain 372-522
372 522
BRCT LZ
1c
Genotoxic
Immaflamatory Oncogenic
Metabolism Oxidative
Microbial Temperature
Str
es
s
PARP-1
Mo
dif
ica
tio
n
•Poly (ADP-Ribose) signaling
•PARPs activity
•NAD+↓, ATP↓
Chromatin Modulation Cell Death
DNA Repair Proteasome
Cell Survival Senescence
Transcription Replication/Cell Cycle
Me
ch
an
ism
Ta
rge
t
Pa
thw
ay
UU
U
PS
C PARG
Normal/weak
PARP-1 PARP-1
Overactivation
ExcessiveIrreparable
DNA Damage
Necrosis
Inflamation
Autophagy
Survival
Collapse Energy
NAD+↓, ATP↓
Apoptosis
Cleveaged PARP-1
Caspases 3/7
Apoptosis
PARP-1 Activation
DNA Repair
Survival
2a 2b
Figure 2: Integrated Response of PARP-1 to Cellular Stress
PJ34 10 μM
##
***
MCF7 GFPLC3
0
10
20
30
40
50
60
70
SIMA
iPARP-1 60 nM
iPARG 30 nM
% L
C3
pu
nc
tate
d c
ell
s øøø
*
ø
###
***
øøø##
***
ø
Starvation
Control SIMA PJ34
MCF7 GFPLC3
iPARP-1 iPARG
Starvation 30 min.
PAR
α tubulin
PARP-1
β actinX 0,341
**
0.0
0.5
1.0
X-f
old
mR
NA
exp
ress
ion
*
A549
0
50
100
% L
C3
pu
nc
tate
d c
ell
s
**
##
#
***
Starvation
Vector + PJ34 10μM
shPARG
Vector sh
shPARG + PJ34 10μM
Starvation
A549
0
50
100
% L
C3
pu
nc
tate
d c
ell
s
##*****
#
*****
Vector + iULK1 60 nM
shPARG SIMA
Vector sh SIMA
shPARG + iULK1 60 nM
LC3-I
LC3-II
β actin
shPARG A549
iULK1SIMA
Starved Starved
shPARG A549
PJ34 10 µMSIMA
Starved Starved
3b
3d
Figure 4: Inhibition of PARylation downregulates AMPk pathway
PARP-1
GAPDH
0 10 20 30 40 50 60 70 80 90 1000
102030405060708090
100110120
Untreated
PJ34 10 M
iPARP-1 60 nM
Starvation ( min. )
Intr
ac
ell
ula
r A
TP
Le
ve
ls
( %
of
co
ntr
ol )
**
**
*
MCF7 GFPLC3
Starvation
MCF7 GFPLC3
Starvation
PJ34 10 MUntreatedStarvation
MCF7 GFPLC3
p-AMPk
β actin
AMPk
Starvation
SIMA 60 nM iPARP-1 60 nM
PARP-1
p-ACC
PAR
α tubulin
0 10 20 30 40 50 60 70 80 90 100
0
10
20
30
40
50
60
70
80
90
100
110
120
SIMA 30 nM
iPARG 30 nM
Starvation ( min. )
Intr
ace
llu
lar
AT
P L
eve
ls
( %
of
co
ntr
ol )
MCF7 GFPLC3
**
**
øø
øø
Starvation
iPARG 30 nM SIMA 30 nM
MCF7 GFPLC3
p-ACC
p-AMPk
AMPk
Starvation
β actin
4b
Figure 5: Activation of AMPk requires interaction with PARP-1 and its PARylation
Starvation
MCF7 GFPLC3
IP PARP-1
AMPk
PARP-1
INPUT
p-AMPk
PARP-1
AMPk
IP AMPk
PARP-1
AMPk
MCF7
0
10
20UntreatedStarvation
% C
olo
cali
za
tio
nP
AR
P-1
/AM
Pk
*
Untreated Starved 30
min P1 Domains
FLAG-AMPkα2
AMPkINPUT
GST
AMPk
pBC PARP-1
Starvation
INPUT
GST
pBC EF
FLAG-AMPkα2
AMPk
AMPk
Starvation
5a 5b 5c
5d
INPUT
GST
AMPk
AMPk
pBC PARP-1
FLAG-AMPkα2
UntreatedKUDOS
100 nM
pBC EF
Untreated
KUDOS
100 nMIP PAR INPUT
WB FLAG
WB PAR
5e
5f
3c
Starvation (ROS)
PARP1
AMPkα2
mTORC1
Raptor
mLST8mTOR
ULK1/mATG13/FIP200
ATP
DNA DAMAGE
PARP-1
1. Binding
AMPKα2
PARP1
FED
¿?
AMPKα2 2. PARylation
STARVED
PARP
inhibitor
ATG13
ULK1
FIP200
ATG101
Conclusion
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