Cleavage of IP 3 R1 by caspase-3 and intracellular Ca 2+ homeostasis during Apoptosis Laboratory of Physiology, KULeuven, Belgium Assefa Z, Bultynck G, Szlufcik K, Nadif Kasri N, Vermassen E, Goris J, Missiaen L, Callewaert G, Parys JB & De Smedt H

characterized by: - PS exposure - cytoplasmic and nuclear shrinkage - cell membrane blebbing - chromatic condensation - intranucleosomal DNA fragmentation - Fragmentation into membrane-bound apoptotic bodies genetically controlled form of cell death critical for normal embryonic development and adult tissue homeostasis. dysregulation of apoptosis is associated with a number of disease conditions. involves caspase activity. Apoptosis: brief introduction

Ca 2+ cardiolipin calpain calcineurin endonucleases caspase Apaf-1 FADD Pro-caspase-8 caspase-6 caspase-7 caspase-6 caspase-7 Apoptosis: various ways of dying

. are directly implicated in apoptosis: * Expression of antisense cDNA construct of IP 3 R3 blocks dexamethasone- induced apoptosis and increase in [Ca 2+ ] i in lymphocytes. Khan et al., Science 273: 503, 1996 * IP 3 R1 deficient cells were resistant to Fas, dexamethasone and  -irradiation. Jayaraman et al., Mol. Cell Biol. 17: 3005, 1997 * BCR-induced apoptosis in lymphocytes was significantly inhibited only in cells deficient of all three receptors. Sugawara et al., EMBO J. 16: 3078, 1997 * IP 3 R1 is a substrate of caspase-3 during apoptosis. Hirota et al., JBC 274: 34433, 1999 Apoptosis: the role of IP 3 Rs

FKBP12 Caspase-3 cleavageCytochrome C binding CaM  1-225

IP 3 R1 constructs: - the constructs were stably expressed in DT40 cells that lack all three IP 3 Rs. C N ER lumen cytoplasm C N C N

Del(1-225)-IP 3 R1 is no longer functional

(  )IP 3 R1-Mitotracker (  )IP 3 R1 -bodipy TG Localization of (  )IP 3 R1 expressed in IP 3 R triple ko cells

Cleavage of IP 3 R1 in vitro and in cells undergoing apoptosis

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

Secondary necrosis in STS-treated cells

IP 3 R-KO (  )IP 3 R1 IP 3 R1  casp (  1-225)IP 3 R1 WT-IP 3 R1 Fluo-3 Fura red fluorescence intensity (relative units) fluorescence intensity (relative units) Apoptosis-related rise in [Ca 2+ ] i requires caspase cleavage of IP 3 R1.

Caspase-3 activation precedes the increase in [Ca 2+ ] i WT-IP 3 R1

Caspase inhibitors block the rise in [Ca 2+ ] i WT-IP 3 R1 (  1-225)IP 3 R1 Fluo-3 Fura red fluorescence intensity

WT-IP 3 R1 (  1-225)IP 3 R1 (  )IP 3 R1 Ca 2+ -free Normal Apoptotic cell death and the associated rise in [Ca 2+ ] i could be induced in Ca 2+ -free medium

(  )IP 3 R1 expression does not reduce the amount of releasable Ca 2+ from the ER (  )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 Role of IP 3 R1 in apoptotic cell death

CONCLUSIONS: 1. Calcium release through IP 3 R1 is required for the rapid execution of apoptotic cell death; but that doesn’t involve IICR activity but cleavage by caspase The disruption of intracellular Ca 2+ homeostasis during apoptosis seems to be the consequence rather than the primary cause of apoptosis. 3. The specific pattern of changes in [Ca 2+ ] i during apoptosis in different cell types might be related to the relative distribution of IP 3 R1 among the different tissues.

N C IP 3 PP1 AKAP9 PP1PKA RACK1 Gβ Type 1 IP 3 R (IP 3 R1) CaM CaBP -Ca 2+ +Ca 2+ REGULATORY SITES Ca 2+ -binding sites PKA/PKG phosphoryl. ATP binding Caspase-3 cleavage