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

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


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:


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


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:200 μM Ca2+ +10 μM CaM

16HBE14o-(Missiaen et al., 2000)

- CaM

+ CaM


0

20

40

60

- CaM + CaM

Free [Ca2+] (µM) 0.60.30.10.03<0.001

Ca2+

rel

ease

(%

/ 2m

in)


Sf9 (Cardy & Taylor, 1998)


Effects of CaM on IP3 binding:

200 μM Ca2+ +10 μM CaM


- CaM

+ CaM


0

20

40

60

- CaM + CaM

Free [Ca2+] (µM) 0.60.30.10.03<0.001

Ca2+

rel

ease

(%

/ 2m

in)



[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 binding:

CaM (μM)

Lbs-domains (Vanlingen et al., 2000)

Lbs-1 Lbs-2 Lbs-3



- CaM

+ CaM


0

20

40

60

- CaM + CaM

Free [Ca2+] (µM) 0.60.30.10.03<0.001

Ca2+

rel

ease

(%

/ 2m

in)



[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



- 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


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



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


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



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)

regulatory pathways acting on inositol trisphosphate receptor localization and function jan b. parys...

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