rôles des phosphoinositides dans les pathologies...
TRANSCRIPT
B. Payrastre et al.
INSERM U1048, I2MC,
Toulouse, France
Rôles des phosphoinositides dans les
pathologies cardiovasculaires
PI(4,5)P2
Activated mouse platelet
Phosphoinositides are minor constituents of cell me mbranes with a highly dynamic metabolism
PIP-K IIPI 3-KClass I, II & III
PI K II & IIIPIP-K I
PLC
CH2 CH CH2
O
O C
Arachidonic acid(20:4)
Stearic acid(18:0)
O
C
O
O -
P
O
O
O
100
50%
Relative amounts of phosphoinositides
In general their head group protrudes from the cytosolic leaflet of membranes to get
phosphorylated/dephosphorylated and to bindwith various affinity and specificity to proteins.
cytosol
PA
PI(ER)PI synthase
CTPCDP-DAG
Inositol
CDP-DAG synthase
PPi
PACMP
PI -
TP
PLC(PM) PI PI(4,5)P2PI(4)P DAG
IP3
The PI cycle in the seventies/eighties
USA 1969 UK 1979 Japan 80’s
M. & L. HokinR. Dawson
J. Folch
M. BerridgeR.H. Michell
R. Irvine
Y. NishizukaK. Mikoshiba
Michell R.H. Biochimica Biophysica Acta 1975
PI(4,5)P2PI4P
PI
IP3
DAGPIP5K
Various pathways of phosphoinositide synthesis and interconversions
PI(3,4)P2
PI(3,4,5)P3PI3K identification
Nature (1988) 332, 644-646
Whitman M, Downes CP, Keeler M, Keller T & Cantley L.
PI5P
Rameh L, Tolias KF,Duckworth BC & Cantley L
PI5P identificationNature (1997) 390, 192-196
PI3P
PI(3,5)P2
Dove et al. PI(3,5)P2pathway identification
Nature (1997) 390, 187-192
PIP3-binding PH domainfrom Grp1
PI(3)P-binding FYVE domainfrom EEA1
PI(3)P-binding PX domain from p47phox
Phosphoinositide binding domains
Itoh T & Takenawa T, Cell Signal 2002
PIP2-binding FERM domain from radixin
PIP2-binding ENTH domainfrom epsin
PI5P-binding domainPHD from ING2
Phosphoinositides define organelle identity ?
Botelho, Bioessays, 2009Bebpia et al., Nature 2005Carlton & Cullen, Trends Cell Biol 2005Balla & Varnai, Curr Protoc cell Biol 2009
PI(4,5)P2
PI(3,4)P2PI(3,4,5)P2
PI(3,5)P2
PI3P
PI4P
PI5P
PM
ER
golgi
EE
MVB/LE
Lys/vac
CCV
nucleus
CSK
PHD
PH PX
ββββ-prop
PHPX
PHENTH
GAT EEA1(FYVE)
PI3P
PXFYVE
PLCδ (PH)
PHENTH
FERM
PI(4,5)P2
Phosphoinositides in the dynamics of cell organizat ion and polarity
Di Paolo & Camilli, Nature Rev 2006Sasaki et al., Progress Lipid Res 2009Gassama & Payrastre, Int Rev Cell Mol Biol 2009
AEE
Nucleus
Basolateral
Apical
BEE
Lysosome
RE
ECMPI(3,4,5)P3
PI(4,5)P2
PI3K
PTEN
Epithelial cell polarity
PIP5K
TGN PI4K
Migrating cells :- PI(3,4,5)P3 at the leading edge
Dividing cells : - PI(3,4,5)P3 at the midsection of
the cortex to properly orientatethe spindle
- PI3P at the midbody during cytokinesisto recruit cytokinesis regulatory machinery
- PI(4,5)P2 at the mitotic cortex as a spatial regulator of its stability
The regulation and localization of PI-kinases and - phosphatases is critical to ensure adequate cell response to environmental cues .
60.00 70.00
PI(4)P
Time (min)15.00 25.00
0
500
1000
CTS
PI(3)P
PI(3,5)P2
PI(3,4)P2
PI(4,5)P2
PI(3,4,5)P3
HPLC
PIP2
PIP
origin
MP
PI
PIP3
TLC
deacylationO
CH
CH2 O
C O
C CH2OO
P
O
O
OH P04OH
O
P04
P04
Analysis of phosphoinositides: a specialized lipid biochemistry
Isotopic labelling (3H-inositol or 32Pi) followed by TLC and HPLC techniques :
B. Payrastre, Methods Mol Biol 2004
Analysis of phosphoinositides : towards mass spectr ometry methods
Chemical derivation (phosphate methylation with TMS-diazomethane) coupled to HPLC-MS techniques :
J. Clark et al., Nature Methods 2011Plateau de lipidomique MétaToul
I2MC, Toulouse (http://www.i2mc.inserm.fr)
0,00
2,00
4,00
6,00
8,00
10,00
12,00
PIP
3 (n
g/10
7pl
atel
ets)
WT SHIP1 -/-
**
**
PIP3 level in WT versus SHIP1 -/- platelets:
PIP3 molecular species
Phosphoinositide metabolism and human diseases
PI(4,5)P2PI4P
PI
IP3
DAGPIP5K
PI(3,4,5)P3
PI(3,4)P2
Pendaries et al., FEBS Lett 2003McCrea & De Camilli Physiology 2009
PI(3,5)P2
PI3P PI5P
Lowe syndrome
Myotubular myopathy&
Charcot-Marie-Tooth
oncogenesis
LCContractural S
(PIP5Kγ)γ)γ)γ)
The Shigella flexneri effector IpgD has a motif related to the active site of mammalian phosphoinositide phosphatases
PCWNCKSGKDRTGMQDAEIKREIIRK
AAIHCKAGKG RTGVMICAYLLHRGKF
VLVHCSDGWDRTAQLTSLAML-M-LD
VLVHCSDGWDRTPQIVALAKL-L-LD
IpgD
PTEN
MTM1
MTMR3
The IpgDC438S is an inactive phosphatase
PM
Host cell
Shigella flexneriType III secretionsystem
IpgD
The Shigella flexneri effector IpgD is a PI-phosphatase
IpgD-dependent PI(4,5)P 2 hydrolysis in Hela cellsinfected with Shigella flexneri
12000
PI(4,5)P2
PIP
origin
MP
WT
0
3000
6000
9000PIP
PI(4,5)P2
30’
origin
MP
0’ 15’
∆ ∆ ∆ ∆ IpgD
0
3000
6000
9000
12000
0’ 15’ 30’
PI(4,5)P2
PIP
PI(4,5)P2
PIP
PI5P
PI4Por ?
Time (min)40.00 45.00
0
200
400
600
800
CTS
PI4P PI5P
Time (min)40.00 45.00
0
200
400
600
800
CTS
PI4P
The Shigella effector IpgD is sufficient to increase PI5P levels in host cells
32P-PI(4,5)P2PI5P + PIP-kinase II αααα γ32P-ATPPI4PPI3P
50
100
C IpgD
Expression of GFP-IpgDin Hela cells
IpgD-C438S
pmol
PI5
P /
mg
prot
.
32P-PI(4,5)P2
- BS176 ∆ IpgDWT
pmol
PI5
P /
mg
prot
.
0
100
200
300
400
Hela cells infected with wild typeshigella and mutants
PI5P mass assay
Bacteria
PI5P is produced in host cells at the entry site of S. flexneri
(PHDx2 biot) (Phalloidin)PI5P F-Actin
(LPS)
Biot-GST-2XPHD probe
PHD PHD
Streptavidin-Alexa594
GSTBIOTINE
O. Gozani et al Cell 2003
S. flexneri
IpgD
PI(4,5)P2 PI5P
Actin
Host cell
PI(3,4,5)P3
AKT
Host cellsurvival
Shigella flexneri injects the PI-phosphatase IpgD in the host cell through its Type III secretion system :
a model to study the role of PI5P
Niebuhr et al EMBO J. 2002Pendaries et al. EMBO J. 2006Coronas et al. Biochem Soc Symp 2007Ramel et al. BBRC 2009
?
PI5P integrates membrane and cytoskeleton dynamics
Akt
Prolonged survival signaling
PI5PPP
Lysosomal degradation blockade
RTK activation
Tom1 ?
EE
Bacterial infection
PI(3,5)P2
Chicanne et al Biochem J 2012Dupuis-Coronas et al. J Biol Chem 2011Amoasii et al. PLoS Genetics 2012
PIKfyvePI3P
Directed migration
Tiam1
Rab4
Rac1GTP
Actin dynamics
Recycling
Rac1GTP
Tiam1
Rac1GTP
PI5P
Ramel et al, 2011
Oncogenes
EGF-R
PI3Kββββ in platelet activation and thrombus growth
Plasma membrane
Kinases (Akt,PDK)Phospholipase (PLCγ )
Adaptors (Gab)GEFs (Vav, ARNO)
GrowthProliferation & Survival
MetabolismDifferentiation
Polarisation & Motility
PP
PIP2
PP
P
PIP3
Signal
CancerImmunityDiabetes
Haemostasis
A critical role for PI3K and PIP3 in cell regulatio n
Vanhaesebroeck B. et al., Nature Review 2010 Sasaki et al., Progress Lipid Res 2009Hirsch E. et al., Trends Biochem Sci 2009
PH
Increase protein concentrationto generate a steric environmentfavorable for protein interactions
BLEEDING
THROMBOSIS
Adapted from Brass, Nature 2001
The key role of platelets in haemostasis and thromb osis
resting
activated
Interactions with blood cells and endothelial cells,
secretion of cytokines, growth factors and matrix proteins
- Inflammation- Atherosclerosis- Angiogenesis- Metastasis
NewsFocusScience 2010
BloodFlow
GPIbα
Atherosclerotic plaque
Collagen
FGFGFGFGααααIIbIIbIIbIIbββββ3333
++++TxA2ADP
FGFGFGFG
VWF
GPVI
Platelet activation and ischemic vascular events du ring atherothrombosis
platelet
PI3Ks are activated downstream of most activatory h uman platelet receptors
PI3Ks
Class I PI 3-kinases are drug targets in
cancer and immune diseases
(ex : XL147, XL765, INK1117)
PI3K inhibitors entering in clinic
TXA2,Thrombin
GqG12/13
Gi
ADP
P2Y12
PAR1 & 4, TP, P2Y1
PAR1 & 4, TP
Sol
uble
ago
nist
s
FcRγγγγ
Talin
Fibrinogen
Collagen
vWF
Syk
Src
ααααIIbββββ3 (GpIIbIIIa)
Adh
esiv
e m
olec
ules
Blood platelets contain all the different PI3K
Vanhaesebroeck et al., Nat Rev Mol Cell Biol 2012Laurent P.A. et al., Advance Biol Reg 2013
p110 α, β, δα, β, δα, β, δα, β, δ
p110 γγγγ
Class Ia
Class Ib
PI3KC2 α,β,γα,β,γα,β,γα,β,γ
Vps34p
Class II
Class IIIClass III
PF4Cre-p110ββββlox/lox
PF4Cre-p110ααααlox/lox
PF4Cre-p110β β β β lox/lox
p110ββββ
lung
liver
kidney
platelets
p110ββββ
p110δδδδ
p110γγγγ
p110αααα
actin
platelets
Mouse model of PI3K ββββ invalidation specifically in the megakaryocyte/platelet lineage
not mandatory to induce platelet aggregation in res ponse to thrombin or U46619 stimulation but required for the formation of large platelet aggregates
required for ααααIIbββββ3 -mediated platelet adhesion on fibrinogen under flo w conditions and spreading in static conditions, involved in fib rin clot retraction
PI3Kββββ in platelet activation in vitro
critical role in GPVI-mediated platelet activation (Akt activation, PLC γγγγ2 activation, Ca2+ mobilization, PKC activation, PLA2 activation & TXA2, 12-HETE, 8-HETE synthesis )
Role in vivo ?Jackson et al Nature Med 2005Ragab et al., Blood 2007Gratacap et al. Blood 2009Martin et al., Blood 2010Canobbio et al Blood 2009
A role for PI3K ββββ in arterial thrombus formation
PI3Kββββ-null mice have a normal tail bleeding time but are protected against the formation of occlusive thrombus.
Carotideinjury by FeCl3
Thrombus formation in vivo:(carotide injury and monitoring of blood flow)
Cre- P110ββββ-null
(9) (10)
% o
f a
nim
als
fo
rmin
g t
hro
mb
us
Stable occlusion
Unstable partial occlusion
No occlusion (<25%)
1
Flow probe(transonic)
Ble
edin
g tim
e (s
)
P110β-nullWild-type0
100
200
300
> 1800
Tail bleeding time :
PI3Kββββ regulates thrombus growth and stability under high shear rate
PI3Kβ plays a key role in thrombus stability
Laurent et al., submitted
Laser
Laser injury of mesenteric arteriole
and intravital microscopy :
LP ko
0 120 240 360 480 6000
10000
20000
30000
40000
50000
Time (s)T
hrom
bus
area
(µm
²)0 120 240 360 480 600
0
10000
20000
30000
40000
50000
Time (s)
Thro
mbu
s ar
ea (
µm²)
WT p110βnull
8 s 80 s
McFadyen & Jackson, Thromb Haemost 2013
Thrombus growth and stability is a highly dynamic p rocess
High shear stress
Blood flow
Study of platelet adhesive interactions and thrombus growth in whole blood
under flow conditions micro-capillary coatted with collagen, vWF or
endothelial cells
aspiration
pomp
Ulratfastimagingsystem
Fluorescentplatelets
(whole blood)
Whole blood Flow
Physiological shear (22.5 dyn/cm2)
*
*
22.5 dyn/cm2 (120 s) Thrombus formed
PI3Kββββ regulates thrombus stability under high shear rate
High shear (180 dyn/cm 2)120s 180s
p110ββββnull
WT
180 dyn/cm2 (60 s)Imaging/quantification
180 dyn/cm2 (60s)
wt p110ββββnull0
50000
100000
150000
200000
250000
**
thro
mbi
vol
ume
(µm
3)
wt p110ββββnull0
20
40
60
80
*su
rfac
e co
vera
ge (
%)
Laurent et al., submitted
Flow 22.5 dyn/cm2
(2 min)
p110ββββ-null
control
p-GSK-3 αααα (Ser21)
p-GSK-3 ββββ (Ser9)
p-Akt (Ser473)
WT p110ββββnull
actin
GSK-3ββββ
Akt
PI3Kββββ is required for GSK3 αααα/ββββ inhibitory phosphorylationwithin the thrombus
120sAkt
GSK3αααα/ββββ
PI3Kβ
P
P
P
PDK1/2
Laurent et al., submitted
0
100000
200000
300000
400000
CHIR-99021 (1µM) - + - +
p110ββββnullWT
thro
mbi
vol
ume
(µm
3 )
0
20
40
60
80
CHIR-99021 (1µM) - + - +
p110ββββnullWT
surf
ace
cove
rage
(%
)
180 dyne/cm 2 180 dyne/cm 2
** **
Thrombus formed
at 22.5 dyne/cm2
(120 s)
180 dyn/cm2
(60 s)
Imaging/quantification
Inhibition of GSK3 restores thrombus stability of P I3Kββββ null platelet mice under high shear stress
Laurent et al., submitted
Inhibition of PI3K has little effect on primary hae mostasis and bleeding but
affects thrombus growth and stability in high shear stress conditions.
Targeting PI3K ββββ as a novel antithrombotic strategy ?
PI3Kββββ is expressed in many cell types suggesting potentia l side effects (a
recent phase I clinical trial with AZD6482 by Astra has shown little or no
adverse effect J Thromb Haemost 2012)
PI3Kββββ inhibition destabilizes platelet thrombi leading to the formation of
platelet emboli (distal embolization ?).
Advantage :
Potential disadvantages :
The PI3Kββββ/Akt/GSK3 axis is critical for thrombus stability u nder high shear stress condition
WT
Sepsis Sham
p110ββββnull
PI3Kββββ null platelet mice are protected against thrombus f ormation in lung microcirculation upon peritonitis-induced seps is
liver
Intestine
WT p110ββββnull
Peritonitis-induced severe sepsis in mice
1515
6
Num
ber
of th
rom
bi/n
umbe
r of
ves
sels
P2Y1
TP
Thrombus growthThrombus growthLA
T
Syk
FcRγGPVI
Gq
Gi
PAR1
PAR4
P2Y12
Gi
Gq
G12/13
G12/13
αIIββββ3
SFKPLCγ2
PLCγ2
PI3Kαααα PI3Kββββ
PI3Kββββ
PI3KββββGSK3Thrombus growth and stability under
high shear rate
PI3Kαααα and PI3K ββββ have distinct and important roles in platelet activation and thrombus stability
“Platelet production and function,sign aling and phosphoinositides ”
Inserm U563, Toulouse, France
Collaborations :B. Van Haesebroeck
C. GachetP. Sansonetti
S. SeverinJ. ViaudF. BoalP.A. LaurentA. Antkowiak(D. Ramel & F. Lagarigue)
B. PayrastreF. Gaits-IacovoniH. TronchèreM.P. GratacapA.D. TerrisseG. ChicanneA. RagabJ.M. Xuereb
Animal , lipidomic and Cell Imaging Facilities
Acides biliaires (bile)
Lipoprotéines• Eicosanoides• Phospholipides• Céramides,
Sphingomyéline• Stérol C4• PIP3• Sphingosine-
Phosphate• Isoprostanes• LPA,PA,LPC
• Sphinganine, sphingosine
• Céramides• Sphingomyéline• Ganglioside,
GluCer, GalCer
Instruments analytiques et
molécules analysées
Instruments analytiques et
molécules analysées
• Oestradiol• Stérols• Oxystérols• Acides gras
hydroxylés (non conventionnels)
Lipides neutres- TG- DG- Cholestérol- Chol esters
Acides Gras libres et Totaux
DéveloppementsEssais
� Le personnel du plateau
� Les membres du conseil scientifique
Bernard Payrastre (Président), Xavier Collet, Thierry Levade, Marc Poirot, Hervé Guillou, Nicolas Cénac, Joost Schanstra, Nathalie Auge, François Tercé, Jean Sébastien Saulnier-Blache
Véronique ROQUESTechnicienne UPS
PaulineLE FAOUDER
CDD IR
Aude DUPUYCDD IE
Fabien RIOLSCDD IE
Justine BERTRAND MICHEL
IR, responsable plateau
MetaToul-Lipidomique
Plateau de lipidomique MétaToulI2MC, Toulouse (http://www.i2mc.inserm.fr)
Thrombin 0.5IU/ml
U46619 1µM
Control p110 ββββ-null
0 1 2 3 4 5 6 7
Time (min)
0
50
100
0
50
100
Agg
rega
tion
(%)
Agg
rega
tion
(%)
Control p110ββββ-null
Thrombin0.5 IU/ml0.3 IU/ml0.2 IU/ml
U466191 µM0.5 µM0.25 µM
U46619
0 1 2 3 4 5 6 7
Time (min)
PI3Kβ is necessary for the formation of large aggregates upon thrombin and TXA2 stimulation and is critical for GPVI-induced platelet activation
Role of PI3K ββββ in platelet aggregation in response to various agon ists
Control p110ββββ-null
0
50
100
0
50
100
Agg
rega
tion
(%)
Agg
rega
tion
(%)
Collagen10 µg/ml5 µg/ml2.5 µg/ml
Convulxin
15 nM10 nM
0 1 2 3 4 5 6 7
Time (min)0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
Time (min)
Collagen 10µg/ml
Control p110 ββββ-null
GpVI
YP
YP
Fcγγγγ−−−−Chain
Src
ITA
M
CollagenCVX
Syk
SH
2S
H2
Y 175Y 195Y 235
Y 136
An important role of PI3K ββββ in GPVI-mediated platelet activation
PLCγγγγ2pY
Secreted ADP
P2Y12
Giraft
Secretion / Aggregation
PKC
Ca2+
DAG
IP3
Ragab et al., Blood 2007Gratacap et al. Blood 2009Martin et al., Blood 2010
PI3Kββββ
PIP
2
PIP
3Akt
/α
PIP
3 pr
oduc
tion
(Fol
d in
crea
se)
CVX15 nM
Resting
Control
p110ββββ-null
*
PIP3
Mbr/Csk boundPLCγ2
total PLCγ2
0 30 60 30 60 (s)
p110βnull
A role for PI3K αααα in arterial thrombus formation in vivo
0 30 60 90 120 150 1800
100
200
300
400
500
Time (s)
Thro
mbu
s ar
ea (
µm²)
WT KO0
100
200
300
400
500 *
Thro
mbu
s ar
ea (
µm²)
Area at 30s
Superficial lesion
WT
p110α
0 120 240 360 480 6000
5000
10000
15000
20000
25000
30000
Time (s)
Thr
ombu
s ar
ea (
µm²)
Deep lesion
WT
p110α
WT KO0
2.0××××106
4.0××××106
6.0××××106
8.0××××106
1.0××××107ns
AU
C (
µm2 x
min
)
PI3Kαααα is implicated in thrombus growth in vivo upon superficial arterial injury and is not involved in thrombus sta bility
Laser
Laser injury of mesenteric arteriole
and intravital microscopy :
Thrombin : (IU/ml)
PI3Kα partially contributes to ITAM-mediated Akt activation
P-Akt (Ser473)
actin
- -0.02 0.020.03 0.03
- -0.2 0.20.25 0.250.5 0.5
P-Akt (Ser473)
actin
U46619 :(µM)
p110ααααnullWT
- 1 1.5 2 - 1 1.5 2
P-Akt (Ser473)
actin
CRP :(µg/ml)
Role of PI3K αααα in Akt activation
GpVI
YP
YP
Fcγγγγ−−−−ChainpY
Src/BTK
Src
ITA
M
CollagenCVX
Syk
SH
2S
H2 PLCγγγγ2
PIP
2
Secretion / Aggregation
PIP
2
IP3
DAG PKC
Ca2+
Y PY PY P
Y P
Nieswandt & Watson, Blood 2003
Mechanism of platelet activation via the collagen receptor GPVI
Integrin GpIIbIIIa ( αΙΙαΙΙαΙΙαΙΙbββββ3) signaling is essential for platelet aggregation
ααααIIb ββββ3
(Closed!)
(occupied!)
Inside-out signaling
Primary agonists
Signaling cascade 1
Integrin regulatorsCytoskeletonchanges
Fibrinogen
P
P
Signaling cascade 2
Outside-in signalingFormation of large aggregates
with thightly associated platelets
PI3Kββββ is involved in integrin GpIIbIIIa signaling
Controlp110ββββ-null
0 30 60 120Time (min)
**
***
Delay in the clot retraction : potential role of p110ββββ in organizing an efficient GpIIbIIIa-mediated contractility
Fibrin clot retractionC
lot r
etra
ctio
n (%
ser
um e
xtru
ded
from
clo
t)
***
***
*
Control
p110ββββ-null
Sur
face
cov
ered
(%
)
***
PI3Kββββ is required for GpIIbIIIa-mediated platelet adhesio n on fibrinogen under flow conditions and spreading i n static conditions
Controlp110ββββ-null
Platelet adhesion on fibrinogenunder flow conditions (1500 s-1)
Platelet adhesion on fibrinogen(static conditions)
actintubulin
Control p110β-null
30 60 1200
Time (min)
P-Akt (Ser473)
total Akt
- -0.2 0.20.3 0.30.5 0.5
- -0.25 0.250.5 0.51 1
P-Akt (Ser473)
total Akt
Thrombin : (IU/ml)
U46619 :(µM)
p110ββββnullWT
PI3Kβ plays an essential role in Aktactivation induced by both ITAM and GPCR
-mediated platelet signaling
- 1 1.5 2 - 1 1.5 2
P-Akt (Ser473)
total Akt
CRP :(µg/ml)
Respective roles of PI3-kinases αααα and ββββ in Akt activation
PI3Kβ
ααααIIbββββ3
Fibrinogen
P-MLC
Ca2+-calmodulin
MLCK
PI3KββββSHIP1SHIP1 PtdIns(3,4,5)P3
PtdIns(3,4)P2
PI 3K ββββ and SHIP1 play an important role in the formation o flarge aggregates
Formation of large aggregates with tightly associated platelets
TXA2ADP
TXA2 ADP
CAMs
Eph kinases/Ephrine
GPCR
cytoskeletonorganization
mec
hani
cal
forc
e
Adapted from Brass et al. J. Clin. Invest. 2005
?
PI5P production increases the colocalization of EGFR with early endosome markers
EndosomeEEA1
P P
P
P
P
P
SNX1
Col
ocal
isat
ion
EG
FR
/EE
A1
GFP -IpgD WT
GFP
*
0%
20%
40%
60%
80%
100%
GFP -IpgD WT
GFP
Col
ocal
isat
ion
EG
FR
/SN
X1 *
C EGFP EEA1 EGFR Merge
EGFP
EGFP
-Ipg
D-W
T
A EGFP SNX1 EGFR Merge
EGFP
EGFP
-IpgD
-WT
controle IpgD
IpgD-inducible MEF cells
P-EGFR Y1173
P-EGFR Y1068
EGFR
P-Akt S473
Akt
Ectopic expression of IpgD and PI5P generation acti vate EGFR
IpgD-inducible MEF cells
IpgD
Ctrl
Inp54PI(4,5)P2 PI4P
IpgDPI(4,5)P2 PI5P
GFP -IpgD WT
GFP
Colocalisation P-EGFR/EEA1
0%
20%
40%
60%
80%
100% *
GFP -IpgD WT
GFP
Colocalisation EGFR/P-Akt
0%
20%
40%
60%
80%
100% *
A EGFP EEA1 P-EGFR Y1173 Merge
EGFP
EGFP
-IpgD
-WT
C EGFP EGFRP-Akt S473 Merge
EGFP
EGFP
-IpgD
-WT
PI5P production increases the colocalization of the active EGFR with early endosome markers
Gorvel et al. Cell 1991
- +EE
- +LE
0
5
10
15
20
25
IpgD :
EEA1
BHK cells
Endosome isolationand
biochemical analysis
PI5
P(f
old
incr
ease
)
0
0.5
1
PI3
P(f
old
incr
ease
) 1.5
P-EGFR
P-Akt
PI5P PI(4,5)P2Type II PIP4Ks
PIPLIP ?
(PTPMT1)
PI(3,5)P2
3-P
ME
PI3
K (
I)
Eukaryotic cells have the machinery to produce PI5P
Bacterial phosphatase
PI5
P p
mol
/5x1
09ce
lls
60
30
Emptyplasmid
*
*
0MTM1
WTMTM1C375S
S. Cerevisiae ymr1∆
Bertazzi et al. PLoS Genetics 2012
4-PME(Type I & II 4-PME)
Myotubularins
PI(3,5)P2
MTMR2
IpgD
PI5PPI(4,5)P2
Nucleus
Cell Survival
PI3K
FKHR
P70-S6KGSK3
Akt
?
PI5P-mediated Akt activation requires EGFR
EGFR
Host cell
P-Akt S473
Total Akt
Control IpgD WT
EGFR siRNA (nM): 0 0100 100
EGFR
Control siRNA (nM): 0 0100 100
P-Akt S473
Total Akt
Control IpgD WT
AG 1478 (10 nM): - + - +
P-Akt T308
EGFR
Actin
GFP
IpgD WT
GFP-PI5P4KIIββββ
0’ 60’
EGF Stimulation (200 ng/ml)
- +
- -
+
+
- +
- -
+
+
PI5P production protects EGFR from degradation
EE
LE
Ly
EEA1
SNX1
LAMP-1
PI5P production accumulates the fluid phase markerrhodamine-dextran in early endosomes
0%
20%
40%
60%
80%
100%
GFP -IpgD WT
GFP
Dextran/EEA1
*
0%
20%
40%
60%
80%
100%
GFP -IpgD WT
GFP
Dextran/Lamp1
*
A EGFP Dextran EEA1 Merge
EG
FPE
GFP
-IpgD
-WT
EGFP Dextran Lamp1 Merge
EGFP
EGFP
-IpgD
-WT
C
Pulse-chaseexperiments
PI5P is a new player in trafficking that regulates signal persistence in Shigella flexneri infected cells
IpgD
PI5PPI(4,5)P2
Nucleus
Cell Survival
PI3K
FKHRP70-S6K
GSK3
Akt
?
Host cell
PI5P production via IpgD activatesEGF-R and blocks its degradation to maintain
Akt-mediated survival signals
EGFRProlonged
survivalsignals
LE
RE
Golgi
EE
Ly
PtdIns5P
EGFR
Active EGFR
B
Retrograde pathway
PM
Recycling
Retrograde pathway
Ramel et al, 2011
IpgD
PI5PPI(4,5)P2
Nucleus
Cell Survival
PI3K
FKHR
P70-S6KGSK3
Akt
?
PI5P-mediated Akt activation requires EGFR
EGFR
Host cell
P-Akt S473
Total Akt
Control IpgD WT
EGFR siRNA (nM): 0 0100 100
EGFR
Control siRNA (nM): 0 0100 100
P-Akt S473
Total Akt
Control IpgD WT
AG 1478 (10 nM): - + - +
P-Akt T308
PF4Cre-p110β β β β lox/lox
p110ββββ
lung
liver
kidney
platelets
p110ββββ
p110δδδδ
p110γγγγ
p110αααα
actin
platelets
Mouse model of PI3-kinases αααα and ββββ invalidation specifically in the megakaryocyte/platelet lineage
PI3Kβ
PF4Cre-p110α α α α lox/lox
p110ββββ
p110δδδδ
p110γγγγ
p110αααα
actin
platelets
lung
liver
kidney
platelets
p110αααα
PI3Kα
P-Akt (Ser473)
total Akt
- -0.2 0.20.3 0.30.5 0.5
- -0.25 0.250.5 0.51 1
P-Akt (Ser473)
total Akt
Thrombin : (IU/ml)
U46619 :(µM)
p110ββββnullWT
PI3Kβ plays an essential role in Aktactivation induced by both ITAM and GPCR
-mediated platelet signaling
- 1 1.5 2 - 1 1.5 2
P-Akt (Ser473)
total Akt
CRP :(µg/ml)
Respective roles of PI3-kinases αααα and ββββ in Akt activation
PI3Kβ
Thrombin : (IU/ml)
PI3Kα partially contributes to ITAM-mediated Akt activation
P-Akt (Ser473)
actin
- -0.02 0.020.03 0.03
- -0.2 0.20.25 0.250.5 0.5
P-Akt (Ser473)
actin
U46619 :(µM)
p110ααααnullWT
- 1 1.5 2 - 1 1.5 2
P-Akt (Ser473)
actin
CRP :(µg/ml)
PI3Kα
Stimulation of PI3K ββββ : Tyrosine kinases and G ββββ/γγγγ subunits
RTK
P
PP
P P
P
PIP2
P
PP
PIP3
GTP
α
β/γ
SH3
SH2
SH2
P110ββββ
p85α/β
Ras
PI3-kinase ββββ is activated via GPCR (Guillermet-Guibert et al. PNAS 2008) and/or the
association of the 2 pathways, GPCR & RTK (Hazeki et al. Life Sci 1998; Maier et al. JBC
1999; Tang & Downes JBC 1997; Kubo et al. BJ 2005; Dbouk et al. Science Signaling 2012)
PI3Kββββ
β/γ
The integrine GpIIbIIIa ( ααααIIbβ3)β3)β3)β3) is essential for platelet aggregation and thrombus growth
ITA
M