Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton 20-22 July 2004.

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Presentation transcript:

Regulatory pathways acting on inositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. Alberta Edmonton July 2004

(Clapham, 1995) Intracellular Ca 2+ homeostasis Tetrameric structure Genes: 3 Regulation by Ca 2+

N C IP 3 REGULATORY SITES Ca 2+ -binding sites PKA/PKG phosphoryl. ATP binding Caspase-3 cleavage Monomeric type 1 IP 3 R (IP 3 R1)

N C IP 3 REGULATORY SITES Ca 2+ -binding sites PKA/PKG phosphoryl. ATP binding Caspase-3 cleavage Monomeric type 1 IP 3 R (IP 3 R1)

N C IP 3 REGULATORY SITES Ca 2+ -binding sites PKA/PKG phosphoryl. ATP binding Caspase-3 cleavage Monomeric type 1 IP 3 R (IP 3 R1)

N C IP 3 REGULATORY SITES Ca 2+ -binding sites PKA/PKG phosphoryl. ATP binding Caspase-3 cleavage Monomeric type 1 IP 3 R (IP 3 R1)

N C IP 3 PP1 CaM CaBP AKAP9 PP1PKA RACK1 Gβ HOMER CARPIRBIT TRP mGluR 1a, 5 Shank IP 3 R RyR Ca 2+ channels HAP1A/Htt ANKYRIN B 4.1N Spectrin-actin cytoskeleton CASK and/or syndecan-2 SIG-1R CHROMOGRANIN A & B Cyt c Monomeric type 1 IP 3 R (IP 3 R1)

1) Regulation of the IP 3 R by calmodulin 2) Dynamic regulation of the IP 3 R by protein kinase C 3) Activation of an IP 3 -independent pathway by IP 3 R1 cleavage through caspase-3

1) Regulation of the IP 3 R by calmodulin

A7r5 (Missiaen et al., 1999) Effects of CaM on IP 3 -induced Ca 2+ release:

A7r5 (Missiaen et al., 1999)Cerebellum (Michikawa et al., 1999) Effects of CaM on IP 3 -induced Ca 2+ release: 200 μM Ca μM CaM

A7r5 (Missiaen et al., 1999)Cerebellum (Michikawa et al., 1999) Effects of CaM on IP 3 -induced Ca 2+ release: 200 μM Ca μM CaM 16HBE14o- (Missiaen et al., 2000) - CaM + CaM

A7r5 (Missiaen et al., 1999)Cerebellum (Michikawa et al., 1999) Sf9 (Cardy & Taylor, 1998) Effects of CaM on IP 3 -induced Ca 2+ release: Effects of CaM on IP 3 binding: 200 μM Ca μM CaM 16HBE14o- (Missiaen et al., 2000) - CaM + CaM

A7r5 (Missiaen et al., 1999)Cerebellum (Michikawa et al., 1999) Sf9 (Cardy & Taylor, 1998) Effects of CaM on IP 3 -induced Ca 2+ release: Effects of CaM on IP 3 binding: CaM (μM) Lbs-domains (Vanlingen et al., 2000) Lbs-1 Lbs-2 Lbs μM Ca μM CaM 16HBE14o- (Missiaen et al., 2000) - CaM + CaM

A7r5 (Missiaen et al., 1999)Cerebellum (Michikawa et al., 1999) Sf9 (Cardy & Taylor, 1998) Effects of CaM on IP 3 -induced Ca 2+ release: Ca 2+ dependent Effects of CaM on IP 3 binding: Ca 2+ independent CaM (μM) Lbs-domains (Vanlingen et al., 2000) Lbs-1 Lbs-2 Lbs μM Ca μM CaM 16HBE14o- (Missiaen et al., 2000) - CaM + CaM

N C IP 3 CaM High-affinity Ca 2+ -dependent Mutations ineffective Not in IP 3 R3 Low-affinity Ca 2+ -dependent Not in neuronal IP 3 R1 Low-affinity Ca 2+ -independent Responsible for effects on IP 3 binding Monomeric type 1 IP 3 R (IP 3 R1)

Cyt1 Cyt2 Lbs-1 Lbs-1  HIS +GST A B C E D F % IQ 76% IQ 53% IQ ABCDEF 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  HIS +GST A B C E D F % IQ 76% IQ 53% IQ Detailed analysis of the N-terminal CaM-binding site ABCDEF µM Ca 2+ 1mM EGTA 309 Intensity loss (1-B/B o ) K d 0.1 μM K d 1 μM (Sienaert et al., 2002)

A B C E D F 1159 control∆ B∆ E [ 3 H]IP 3 binding CaM Both the B and the E sites are necessary for CaM binding

Both the N- and C-terminal parts of CaM are needed CaM C-CaM N-CaM

CaM-like Ca 2+ -binding proteins Inhibitory Activatory ??? (Haeseleer et al., 2002)

CaBP1 GST GST GST Binding of CaBP1 to the same site? A B C E D F 1 CaM 159

CaBP1 GST GST GST Binding of CaBP1 to the same site but only to the domain B A B C E D F 1 CaM 159 CaBP 1

CaBP 1/11/21/41/51/61/81/3 CaBP Ratio of CaBP: peptide B Band intensity Peptide B: CaBP Ca 2+ EGTA 1/10 Ca 2+ EGTA CaBP binding is also Ca 2+ independent (Nadif Kasri et al., 2004)

0.5 µM1 µM100 µM Control CaM IICR is inhibited in vivo by CaM and CaM 1234 … Time (s) ATP Ca 2+ i (nM) CaM 1234

0.5µM 1µM 100µM ATP control Time (s) Ca 2+ i (nM) … and by both short and long CaBP1 (Nadif Kasri et al., 2004) sCaBP lCaBP

0.5µM 1µM 100µM ATP control Time (s) Ca 2+ i (nM) … and by both short and long CaBP1 and also by the Ca 2+ -insensitive CaBP1 134 (Nadif Kasri et al., 2004) sCaBP lCaBP CaBP 134

Suramin CaM In the presence of B In the presence of E µM Suramin interacts with the CaM-binding sites µM (Nadif Kasri et al., in press) +suramin aa aa

Calmodulin is not the Ca 2+ sensor of the IP 3 R (Nadif Kasri et al., in press) L15 fibroblasts

Model: IP 3 R structure is dependent on Ca 2+ + Ca 2+ - Ca 2+ Change in way that CaM, CaBP, … interacts (Hamada et al., 2003)

1) Dynamics concerning the intracellular localization of the IP 3 R 2) Dynamic regulation of the IP 3 R by protein kinase C 3) Activation of an IP 3 -independent pathway by IP 3 R1 cleavage through caspase-3

Localization of IP 3 R1 and IP 3 R3 in A7r5 smooth-muscle cells IP 3 R1IP 3 R3 (Vermassen et al., 2003)

Redistribution of IP 3 R1 after prolonged stimulation Resting cells+ AVP AVP > 1h PLC activation IP 3 -ester Thapsigargin CPA [Ca 2+ ] cyt (Vermassen et al., 2003)

Structure of the endoplasmic reticulum SERCAPDI ER-targeted EYFP Control AVP

Factors participating in IP 3 R redistribution PKC activatorOAGinduction Staurosporineinhibition PKC inhibitorsBisindolylmaleimideinhibition Gö-6876inhibition Drugs acting onNocodazoleinhibition microtubuliTaxolinhibition Action on vesicleBrefeldin Ainduction transportCooling to 15 ºCinhibition

Do other IP 3 R isoforms also redistribute in a similar way? IP 3 R3 in 16HBE14o- cells Control Agonist (ATP) OAG  PKC TG  Ca 2+

Agonist ( ) IP 3 R redistribution IP 3 R redistribution is dependent on the cell status

PKC-mediated phosphorylation of IP 3 R1 [ 32 P]ATP Anti-Ser-P

PKC-mediated phosphorylation of IP 3 R1 and IP 3 R3 [ 32 P]ATP Anti-Ser-P [ 32 P]ATP Anti-IP 3 R

Anti-Ser-P PKA-mediated phosphorylation of IP 3 R1 stimulates PKC-mediated phosphorylation

Ca 2+ inhibits PKC-mediated phosphorylation of IP 3 R1

Ca 2+ and calmodulin differentially regulate PKC-mediated phosphorylation of IP 3 R1 and IP 3 R3

Control 0.2 µg/ml αCT0.4 µg/ml αCT Phosphorylated fragments 273 kDa 225 kDa210 kDa 130 kDa 40 kDa Determination of the PKC phosphorylation sites on IP 3 R1 and IP 3 R3 Blot stripped and reprobed 273 kDa 225 kDa 210 kDa 130 kDa 40 kDa Anti-( ) Ab Purified IP 3 R1

N C IP 3 Monomeric type 1 IP 3 R (IP 3 R1) CaM High-affinity Ca 2+ -dependent Mutations ineffective Not in IP 3 R3 Low-affinity Ca 2+ -dependent Not in neuronal IP 3 R1 PKA/PKG phosphoryl. Low-affinity Ca 2+ -independent Responsible for effects on IP 3 binding

Potential physiological role of PKC-mediated phosphorylation of IP 3 R1

CaM or Preincubation ACTIVATION Ca 2+ INHIBITION Ca 2+ CaM PP - function ? - interaction ? PKC

1) Dynamics concerning the intracellular localization of the IP 3 R 2) Dynamic regulation of the IP 3 R by protein kinase C 3) Activation of an IP 3 -independent pathway by IP 3 R1 cleavage through caspase-3

During apoptosis, IP 3 R1 is cleaved to a 95 kDa C-terminal fragment IP 3 R 95K For expression in IP 3 R ko cells: A C T. IN.

(Δ1-1891)IP 3 R1 expression does not reduce the amount of releasable Ca 2+ from the ER C N C N C N in the presence of Ca 2+ ex

Caspase-3 mediated cleavage of IP 3 R1 leads to an increase in... Caspase-3 activityApoptosis

Apoptosis-related increase in [Ca 2+ ] i requires caspase-3-mediated cleavage of IP 3 R1 IP 3 R-KO IP 3 R1  casp (  )IP 3 R1(  1-225)IP 3 R1 WT-IP 3 R1

StaurosporineAnti-chicken IgM Caspase-3 activation IP 3 R1 cleavage Apoptosis IP 3 -independent activity [Ca 2+ ] i Model: feedback of truncated IP 3 R on apoptosis in an IP 3 -independent way

GENERAL CONCLUSIONS CaM and CaBP are prime negative regulators of IP 3 R function in the presence of Ca 2+, but are not the Ca 2+ sensor. The localization of IP 3 Rs can be dynamically regulated, depending on the physiological context. The phosphorylation of the various IP 3 R isoforms by PKC is differentially regulated by PKA, Ca 2+, and CaM, which can contribute to the modulation of the spatio-temporal Ca 2+ signals. Caspase-3-mediated cleavage of IP 3 R1 leads to an IP 3 -independent Ca 2+ release, which promotes apoptotic cell death.

GENERAL CONCLUSIONS Associated proteins and cellular factors both contribute (in a direct or an indirect way) to IP 3 Rs regulation by modulation of their structure, function and/or localization.

Geert CALLEWAERT - Humbert DE SMEDT - Ludwig MISSIAEN Jan B. PARYS IP 3 -team (Leuven, Belgium) Zerihun ASSEFA Geert BULTYNCK Iris CARTON Sarah KOCKS Nael NADIF KASRI Joelle N. CHABWINE Karolina SZLUFCIK Veerle VANDERHEYDEN Esther VENMANS Leen VERBERT Elke VERMASSEN Jan 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 IP 3 -team (Leuven, Belgium) Zerihun ASSEFA Geert BULTYNCK Iris CARTON Sarah KOCKS Nael NADIF KASRI Joelle N. CHABWINE Karolina SZLUFCIK Veerle VANDERHEYDEN Esther VENMANS Leen VERBERT Elke VERMASSEN Jan 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 IP 3 -team (Leuven, Belgium) Zerihun ASSEFA Geert BULTYNCK Iris CARTON Sarah KOCKS Nael NADIF KASRI Joelle N. CHABWINE Karolina SZLUFCIK Veerle VANDERHEYDEN Esther VENMANS Leen VERBERT Elke VERMASSEN Jan 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 IP 3 -team (Leuven, Belgium) Zerihun ASSEFA Geert BULTYNCK Iris CARTON Sarah KOCKS Nael NADIF KASRI Joelle N. CHABWINE Karolina SZLUFCIK Veerle VANDERHEYDEN Esther VENMANS Leen VERBERT Elke VERMASSEN Jan 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)