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

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Genotoxic

Immaflamatory Oncogenic

Metabolism Oxidative

Microbial Temperature

Str

es

s

PARP-1

Mo

dif

ica

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n

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Transcription Replication/Cell Cycle

Me

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DNA Damage

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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

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s øøø

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øøø##

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Starvation

Control SIMA PJ34

MCF7 GFPLC3

iPARP-1 iPARG

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PAR

α tubulin

PARP-1

β actinX 0,341

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0.0

0.5

1.0

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old

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ion

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0

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100

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C3

pu

nc

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d c

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**

##

#

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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

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( %

of

co

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ol )

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**

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øø

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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