Structure-function analysis of the inositol trisphosphate receptor and its role in cell life and cell death

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1 Structure-function analysis of the inositol trisphosphate receptor and its role in cell life and cell death Jan B. Parys School of Medicine K.U.Leuven INTRACELLULAR Ca 2+ SIGNALING (Clapham, 1995) 1

2 IP 3 R activation (Berridge, 2008) IP 3 R regulation and function Isoform diversity (expression, localization, properties) Regulation by Ca 2+, ATP, Regulation by regulatory proteins (CaM, IRBIT, ) Regulation by phosphorylation (PKC, MAPK, Plk1, ) Function oocyte maturation/fertilisation, cancer, cell death, Our research group, last 20 years: > 300 papers 2

3 ONGOING RESEARCH PROJECTS 1) Regulation of IP 3 R by protein kinases and phosphatases 2) Role of IP 3 R activity during apoptosis and autophagy 3) Role of the interaction between IP 3 R, polycystin-1 and -2 in autosomal dominant polycystic kidney disease 4) Regulation of connexin and pannexin hemichannels RECENT PUBLICATIONS (selection) Szado et al. (2008). Phosphorylation of inositol 1,4,5-trisphosphate receptors by protein kinase B/Akt inhibits Ca 2+ release and apoptosis. Proc. Nat. Ac. Sci. USA 105, Rong et al. (2008). Targeting Bcl-2-IP 3 receptor interaction to reverse Bcl-2's inhibition of apoptotic calcium signals. Mol. Cell 31, Ito et al. (2008). Inositol 1,4,5-trisphosphate receptor 1, a widespread Ca 2+ channel, is a novel substrate of polo-like kinase 1 in eggs. Dev. Biol. 320, Vanderheyden et al., (2009). Regulation of inositol 1,4,5-trisphosphate-induced Ca 2+ release by reversible phosphorylation and dephosphorylation. Biochim. Biophys. Acta 1793, Vanderheyden et al. (2009) Regulation of inositol 1,4,5-trisphosphate receptor type 1 function during oocyte maturation by MPM-2 phosphorylation. Cell Calcium 46, de Mattia et al. (2009). Human golgi antiapoptotic protein modulates intracellular calcium fluxes. Mol. Biol. Cell 20, Rong et al. (2009). The BH4 domain of Bcl-2 inhibits ER calcium release and apoptosis by binding the regulatory and coupling domain of the IP 3 receptor. Proc. Nat. Ac. Sci. USA 106, Lee et al. (2009) Inositol 1,4,5-trisphophate receptor 1 degradation in mouse eggs and impact on [Ca 2+ ] i oscillations. J. Cell. Physiol. (in press). Sammels et al. (2009). Polycystin-2 activation by IP 3 -induced Ca 2+ release requires its direct association with the inositol 1,4,5-trisphosphate receptor in a signalling microdomain. FASEB J. (submitted). 3

4 UNIVERSIDAD DE CHILE Calcium Signaling : C. Hidalgo and E. Jaimovich Cell death: S. Lavandero and A. Stutzin Unfolded protein response: C. Hetz Pontificia Universidad Católica de Chile Connexins: JC. Saez Ca 2+ signals regulate both cell death and survival (adapted from Rong and Distelhorst, 2008) 4

5 The anti-apoptotic protein Bcl Bcl-2 / Bcl-X L Bax / Bak (adapted from Reed, 1998) Bcl-2 inhibits Ca 2+ signals in Jurkat cells Expression of Bcl-2 Actin control + 20 μg/ml anti-cd3 5

6 Bcl-2 from Jurkat cell extracts interacts with IP 3 R1 GST + domain IP contr. anti- IgG IP 3 R1 Bcl-2 Input Bcl-2 interacts with a unique site in the regulatory region of IP 3 R1 Pep1: MDENSPLMYHIHLVELLAVC Pep2: NVYTEIKCNSLLPLDDIVRV Ctrl: DLNEVTCSLIVDRINPVKLY IP anti- anti- contr. anti- IP 3 R1 IP 3 R1 IgG IP 3 R1 + + Ctrl Pep2 Bcl-2 Input 6

7 The BH4 domain is responsible for IP 3 R1 interaction no addition + 40 μm BH4 + BH4 + Pep μm contr. Fractional loss (%/2 min) IP 3 IP 3 -induced Ca 2+ release (%) contr. BH4 Time (min) [Peptide] (μm) CONCLUSIONS - The anti-apoptotic proteins Bcl-2 and Bcl-Xl interact with the IP 3 R. -These interactions participate in the pro-survival response. -The BH4 domain of Bcl-2 interacts with part of the regulatory domain of the IP 3 R and this interaction decreases IP 3 -induced Ca 2+ release. - Specific disruption of the interaction leads to increased IP 3 -induced Ca 2+ release. - It is our working hypothesis that targeted disruption of the interaction can lead to controlled apoptosis, e.g. during abnormal cell proliferation (cancer). - Bax-inhibitor-1 and Beclin-1 also interact with both IP 3 Rs and Bcl-2 (not shown). 7

8 Laboratory of Molecular and Cellular Signaling K.U.Leuven, Leuven, Belgium 8

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