Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys...
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Transcript of Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys...
Regulatory pathways acting oninositol trisphosphate receptor
localization and function
Jan B. Parys
K.U.Leuven Univ. AlbertaEdmonton20-22 July 2004
(Clapham, 1995)
Intracellular Ca2+ homeostasis
Tetrameric structureGenes: 3Regulation by Ca2+
N
C
IP3
REGULATORY SITESCa2+-binding sitesPKA/PKG phosphoryl.ATP bindingCaspase-3 cleavage
Monomeric type 1 IP3R (IP3R1)
N
C
IP3
REGULATORY SITESCa2+-binding sitesPKA/PKG phosphoryl.ATP bindingCaspase-3 cleavage
Monomeric type 1 IP3R (IP3R1)
N
C
IP3
REGULATORY SITESCa2+-binding sitesPKA/PKG phosphoryl.ATP bindingCaspase-3 cleavage
Monomeric type 1 IP3R (IP3R1)
N
C
IP3
REGULATORY SITESCa2+-binding sitesPKA/PKG phosphoryl.ATP bindingCaspase-3 cleavage
Monomeric type 1 IP3R (IP3R1)
N
C
IP3
PP1
CaM
CaBP
AKAP9
PP1 PKA
RACK1Gβ
HOMERCARP
IRBITTRP
mGluR 1a, 5ShankIP3RRyRCa2+ channels
HAP1A/Htt
ANKYRIN B
4.1N
Spectrin-actincytoskeleton
CASK and/orsyndecan-2
SIG-1R
CHROMOGRANIN A & B
Cyt c
Monomeric type 1 IP3R (IP3R1)
1) Regulation of the IP3R by calmodulin
2) Dynamic regulation of the IP3R byprotein kinase C
3) Activation of an IP3-independentpathway by IP3R1 cleavage throughcaspase-3
1) Regulation of the IP3R by calmodulin
A7r5 (Missiaen et al., 1999)
0
20
40
60
- CaM + CaM
Free [Ca2+] (µM) 0.60.30.10.03<0.001
Ca2+
rel
ease
(%
/ 2m
in)
Effects of CaM on IP3-induced Ca2+ release:
A7r5 (Missiaen et al., 1999)
0
20
40
60
- CaM + CaM
Free [Ca2+] (µM) 0.60.30.10.03<0.001
Ca2+
rel
ease
(%
/ 2m
in)
Cerebellum (Michikawa et al., 1999)
Effects of CaM on IP3-induced Ca2+ release:200 μM Ca2+ +10 μM CaM
A7r5 (Missiaen et al., 1999)
0
20
40
60
- CaM + CaM
Free [Ca2+] (µM) 0.60.30.10.03<0.001
Ca2+
rel
ease
(%
/ 2m
in)
Cerebellum (Michikawa et al., 1999)
Effects of CaM on IP3-induced Ca2+ release:200 μM Ca2+ +10 μM CaM
16HBE14o-(Missiaen et al., 2000)
- CaM
+ CaM
A7r5 (Missiaen et al., 1999)
0
20
40
60
- CaM + CaM
Free [Ca2+] (µM) 0.60.30.10.03<0.001
Ca2+
rel
ease
(%
/ 2m
in)
Cerebellum (Michikawa et al., 1999)
Sf9 (Cardy & Taylor, 1998)
Effects of CaM on IP3-induced Ca2+ release:
Effects of CaM on IP3 binding:
200 μM Ca2+ +10 μM CaM
16HBE14o-(Missiaen et al., 2000)
- CaM
+ CaM
A7r5 (Missiaen et al., 1999)
0
20
40
60
- CaM + CaM
Free [Ca2+] (µM) 0.60.30.10.03<0.001
Ca2+
rel
ease
(%
/ 2m
in)
Cerebellum (Michikawa et al., 1999)
Sf9 (Cardy & Taylor, 1998)
[3 H]I
P3 b
indi
ng (
%)
0
20
40
60
80
100
120
* * **
**
*
0 10
*
0 0.3 1 3 10 0 0.3 1 3 10
Effects of CaM on IP3-induced Ca2+ release:
Effects of CaM on IP3 binding:
CaM (μM)
Lbs-domains (Vanlingen et al., 2000)
Lbs-1 Lbs-2 Lbs-3
200 μM Ca2+ +10 μM CaM
16HBE14o-(Missiaen et al., 2000)
- CaM
+ CaM
A7r5 (Missiaen et al., 1999)
0
20
40
60
- CaM + CaM
Free [Ca2+] (µM) 0.60.30.10.03<0.001
Ca2+
rel
ease
(%
/ 2m
in)
Cerebellum (Michikawa et al., 1999)
Sf9 (Cardy & Taylor, 1998)
[3 H]I
P3 b
indi
ng (
%)
0
20
40
60
80
100
120
* * **
**
*
0 10
*
0 0.3 1 3 10 0 0.3 1 3 10
Effects of CaM on IP3-induced Ca2+ release: Ca2+ dependent
Effects of CaM on IP3 binding: Ca2+ independent
CaM (μM)
Lbs-domains (Vanlingen et al., 2000)
Lbs-1 Lbs-2 Lbs-3
200 μM Ca2+ +10 μM CaM
16HBE14o-(Missiaen et al., 2000)
- CaM
+ CaM
N
C
IP3
CaM
High-affinityCa2+-dependentMutations ineffectiveNot in IP3R3
Low-affinityCa2+-dependentNot in neuronal IP3R1
Low-affinityCa2+-independentResponsible for effects on IP3
binding
Monomeric type 1 IP3R (IP3R1)
Cyt1 Cyt2
Lbs-1
Lbs-1 1-225
1
581226
581
1 159
154
+HIS
+HIS
+GST
A
B
C
E
D
F
1-5-10 1-5-10
1-5-8-14 70% IQ 76% IQ
53% IQ
A B C D E F-0.2
0.0
0.2
0.4
0.6
0.8
1.0
200 µM free Ca 2+
1 mM EGTA
309
• Band-shift experiments on non-denaturing gels
• Interaction with dansyl-CaM
Detailed analysis of the N-terminal CaM-binding site
Cyt1 Cyt2
Lbs-1
Lbs-1 1-225
1
581226
581
1 159
154
+HIS
+HIS
+GST
A
B
C
E
D
F
1-5-10 1-5-10
1-5-8-14 70% IQ 76% IQ
53% IQ
Detailed analysis of the N-terminal CaM-binding site
A B C D E F-0.2
0.0
0.2
0.4
0.6
0.8
1.0
200 µM Ca2+
1mM EGTA
309
Inte
nsi
ty lo
ss (
1-B
/Bo)
Kd 0.1 μM Kd 1 μM
(Sienaert et al., 2002)
A
B
C
E
D
F
1 159
Lbs-1 49-81 106-128
0
20
40
60
80
100
[3 H]I
P 3 bin
din
g t
ov
co
ntr
ole
(%
) controle 10 µM CaMcontrol ∆ B ∆ E
[3H
]IP3 b
indi
ng
CaM -+ ++- -
Both the B and the E sites are necessary for CaM binding
Lbs-1 Lbs-1 N CaM Lbs-1 C Ca Lbs-1 N+C CaM Lbs-1 CaM
0
20
40
60
80
100
[3H]IP3 binding
control controle N-CaM C-CaM N+C-CaM CaM
0
20
40
60
80
45C
a2+
45Ca2+ flux
control
Both the N- and C-terminal parts of CaM are needed
CaM
C-CaMN-CaM
CaM-like Ca2+-binding proteins
Inhibitory
Activatory ???
(Haeseleer et al., 2002)
CaBP1
GSTGST
1-604GST
1-225GST
226-604
Binding of CaBP1 to the same site?
A
B
C
E
D
F
1CaM CaM
159
CaBP1
GSTGST
1-604GST
1-225GST
226-604
Binding of CaBP1 to the same sitebut only to the domain B
A
B
C
E
D
F
1CaM CaM
159
CaBP1
CaB
P1/1 1/2 1/4 1/5 1/6 1/81/3 C
aB
P
Ratio of CaBP: peptide B
0 2 4 6 8 100.0
0.5
1.0
Ban
d in
tens
ity
Peptide B: CaBP
Ca2+
EGTA
1/10
Ca2+
EGTA
CaBP binding is also Ca2+ independent
(Nadif Kasri et al.,2004)
0.5 µM 1 µM 100 µM
Control
CaM
IICR is inhibited in vivo by CaM and CaM1234 …
Time (s)
ATP
600
400
200
Ca2
+i (
nM
)
100075050025000
CaM1234
0.5µM 1µM 100µMATP
control
0 200 400 600 800 10000
Time (s)
1000
200
400
600
800
Ca2+
i (n
M)
… and by both short and long CaBP1
(Nadif Kasri et al., 2004)
sCaBP
lCaBP
0.5µM 1µM 100µMATP
control
0 200 400 600 800 10000
Time (s)
1000
200
400
600
800
Ca2+
i (n
M)
… and by both short and long CaBP1and also by the Ca2+-insensitive CaBP1134
(Nadif Kasri et al., 2004)
sCaBP
lCaBP
CaBP134
Suramin
CaM
In the presence of B In the presence of E
0 100 µM
Suramin interacts with the CaM-binding sites
0 100 µM
0.01 0.1 1 10
0
10
20
30
40
50
60
70
Ca2
+ re
lea
se
vs
A2
31
87
(%
)
IP3 (µM)
(Nadif Kasri et al., in press)
+suramin
aa 1-581
aa 226-581
Calmodulin is not the Ca2+ sensor of the IP3R
(Nadif Kasri et al., in press)
L15 fibroblasts
Model: IP3R structure is dependent on Ca2+
+ Ca2+
- Ca2+
Change in way that CaM,CaBP, … interacts
(Hamada et al., 2003)
1) Dynamics concerning the intracellular localization of the IP3R
2) Dynamic regulation of the IP3R byprotein kinase C
3) Activation of an IP3-independentpathway by IP3R1 cleavage throughcaspase-3
Localization of IP3R1 and IP3R3in A7r5 smooth-muscle cells
IP3R1 IP3R3
(Vermassen et al., 2003)
Redistribution of IP3R1 after prolonged stimulation
Resting cells + AVP
AVP > 1hPLC activationIP3-ester ThapsigarginCPA
[Ca2+]cyt
(Vermassen et al., 2003)
Structure of the endoplasmic reticulum
SERCAPDIER-targeted
EYFP
Control
AVP
Factors participating in IP3R redistribution
PKC activator OAG induction
Staurosporine inhibitionPKC inhibitors Bisindolylmaleimide inhibition
Gö-6876 inhibition
Drugs acting on Nocodazole inhibitionmicrotubuli Taxol inhibition
Action on vesicle Brefeldin A inductiontransport Cooling to 15 ºC inhibition
Do other IP3R isoformsalso redistribute in a similar way?
IP3R3 in
16HBE14o-
cells
Control
Agonist (ATP) OAG PKC
TG Ca2+
Agonist( )
IP3Rredistribution
IP3R redistribution is dependent on the cell status
PKC-mediated phosphorylation of IP3R1
[32P]ATP
Anti-Ser-P
PKC-mediated phosphorylation of IP3R1and IP3R3
[32P]ATP
Anti-Ser-P[32P]ATP
Anti-IP3R
Anti-Ser-P
PKA-mediated phosphorylation of IP3R1stimulates PKC-mediated phosphorylation
Ca2+ inhibits PKC-mediated phosphorylation of IP3R1
Ca2+ and calmodulin differentiallyregulate PKC-mediated phosphorylation of
IP3R1 and IP3R3
Con
trol
0.2
µg/m
l αC
T0.
4 µg
/ml α
CT
Phosphorylatedfragments
273 kDa225 kDa 210 kDa
130 kDa
40 kDa
Determination of the PKC phosphorylationsites on IP3R1 and IP3R3
Blot strippedand
reprobed
273 kDa225 kDa 210 kDa130 kDa
40 kDa
Anti-(1829-1848) Ab
PurifiedIP3R1
N
C
IP3
Monomeric type 1 IP3R (IP3R1)
CaM
High-affinityCa2+-dependentMutations ineffectiveNot in IP3R3
Low-affinityCa2+-dependentNot in neuronal IP3R1
PKA/PKG phosphoryl.
Low-affinityCa2+-independentResponsible for effects on IP3
binding
Potential physiological role ofPKC-mediated phosphorylation of IP3R1
Potential physiological role ofPKC-mediated phosphorylation of IP3R1
CaMor
Preincubation
ACTIVATION
Ca2+
INHIBITION
Ca2+ CaM
PP
- function ?- interaction ?
PKC
1) Dynamics concerning the intracellular localization of the IP3R
2) Dynamic regulation of the IP3R byprotein kinase C
3) Activation of an IP3-independentpathway by IP3R1 cleavage throughcaspase-3
During apoptosis, IP3R1 is cleavedto a 95 kDa C-terminal fragment
IP3R
95K
For expression in IP3R ko cells:
ACT.
IN.
(Δ1-1891)IP3R1 expression does not reduce the amount of releasable Ca2+ from the ER
CN
CN
C
N
in the presence of Ca2+ex
Caspase-3 mediated cleavage of IP3R1leads to an increase in ...
Caspase-3 activity Apoptosis
Apoptosis-related increase in [Ca2+]i requires
caspase-3-mediated cleavage of IP3R1
IP3R-KO IP3R1casp
(1-1891)IP3R1(1-225)IP3R1WT-IP3R1
Staurosporine Anti-chicken IgM
Caspase-3activation
IP3R1cleavage
Apoptosis
IP3-independentactivity
[Ca2+]i
Model: feedback of truncated IP3R on apoptosis in an IP3-independent way
GENERAL CONCLUSIONS CaM and CaBP are prime negative regulators ofIP3R function in the presence of Ca2+, but are not the
Ca2+ sensor.
The localization of IP3Rs can be dynamically
regulated, depending on the physiological context.
The phosphorylation of the various IP3R isoforms
by PKC is differentially regulated by PKA, Ca2+, and CaM, which can contribute to the modulation of the spatio-temporal Ca2+ signals.
Caspase-3-mediated cleavage of IP3R1 leads to an
IP3-independent Ca2+ release, which promotes
apoptotic cell death.
GENERAL CONCLUSIONS
Associated proteins and cellular factors
both contribute (in a direct or an indirect way)
to IP3Rs regulation by modulation
of their structure, function and/or localization.
Geert CALLEWAERT - Humbert DE SMEDT - Ludwig MISSIAEN
Jan B. PARYS
IP3-team (Leuven, Belgium)
Zerihun ASSEFAGeert BULTYNCKIris CARTONSarah KOCKSNael NADIF KASRIJoelle N. CHABWINE
Karolina SZLUFCIKVeerle VANDERHEYDENEsther VENMANSLeen VERBERTElke VERMASSENJan VRIJENS
In collaboration with the groups of:K. MIKOSHIBA (Univ. Tokyo)R.A. FISSORE (Univ. Massachusetts)M. MICHALAK (Univ. Alberta)R. RIZZUTO (Univ. Ferrara)M.J. BERRIDGE – M.D. BOOTMAN – L. RODERICK (Babraham)C.W. TAYLOR (Univ. Cambridge)F. WUYTACK (Physiology - K.U.Leuven)J. GORIS – E. WAELKENS (Biochemistry – K.U.Leuven)
Geert CALLEWAERT - Humbert DE SMEDT - Ludwig MISSIAEN
Jan B. PARYS
IP3-team (Leuven, Belgium)
Zerihun ASSEFAGeert BULTYNCKIris CARTONSarah KOCKSNael NADIF KASRIJoelle N. CHABWINE
Karolina SZLUFCIKVeerle VANDERHEYDENEsther VENMANSLeen VERBERTElke VERMASSENJan VRIJENS
In collaboration with the groups of:K. MIKOSHIBA (Univ. Tokyo)R.A. FISSORE (Univ. Massachusetts)M. MICHALAK (Univ. Alberta)R. RIZZUTO (Univ. Ferrara)M.J. BERRIDGE – M.D. BOOTMAN – L. RODERICK (Babraham)C.W. TAYLOR (Univ. Cambridge)F. WUYTACK (Physiology - K.U.Leuven)J. GORIS – E. WAELKENS (Biochemistry – K.U.Leuven)
Geert CALLEWAERT - Humbert DE SMEDT - Ludwig MISSIAEN
Jan B. PARYS
IP3-team (Leuven, Belgium)
Zerihun ASSEFAGeert BULTYNCKIris CARTONSarah KOCKSNael NADIF KASRIJoelle N. CHABWINE
Karolina SZLUFCIKVeerle VANDERHEYDENEsther VENMANSLeen VERBERTElke VERMASSENJan VRIJENS
In collaboration with the groups of:K. MIKOSHIBA (Univ. Tokyo)R.A. FISSORE (Univ. Massachusetts)M. MICHALAK (Univ. Alberta)R. RIZZUTO (Univ. Ferrara)M.J. BERRIDGE – M.D. BOOTMAN – L. RODERICK (Babraham)C.W. TAYLOR (Univ. Cambridge)F. WUYTACK (Physiology - K.U.Leuven)J. GORIS – E. WAELKENS (Biochemistry – K.U.Leuven)
Geert CALLEWAERT - Humbert DE SMEDT - Ludwig MISSIAEN
Jan B. PARYS
IP3-team (Leuven, Belgium)
Zerihun ASSEFAGeert BULTYNCKIris CARTONSarah KOCKSNael NADIF KASRIJoelle N. CHABWINE
Karolina SZLUFCIKVeerle VANDERHEYDENEsther VENMANSLeen VERBERTElke VERMASSENJan VRIJENS
In collaboration with the groups of:K. MIKOSHIBA (Univ. Tokyo)R.A. FISSORE (Univ. Massachusetts)M. MICHALAK (Univ. Alberta)R. RIZZUTO (Univ. Ferrara)M.J. BERRIDGE – M.D. BOOTMAN – L. RODERICK (Babraham)C.W. TAYLOR (Univ. Cambridge)F. WUYTACK (Physiology - K.U.Leuven)J. GORIS – E. WAELKENS (Biochemistry – K.U.Leuven)