Session 3: Calcium toolkit

Lectures L3.2 L3.1 Role of zinc-calcium interplay in structure and function of human S100A12 Structural basis for intracellular Olga V. Moroz1, Elena V. Blagova1, calcium sensing and regulation Tony J. Wilkinson1, Keith S. Wilson1, Igor B. Bronstein2 Le Zheng1, Peter B. Stathopulos1, Rainer Schindl2, 1Structural Biology Laboratory, Chemistry Department, University of Guang-Yao Li1, Christoph Romanin2, Mitsu Ikura1 York, United Kingdom; 2School of Biomedical and Health Sciences, King’s College London, United Kingdom 1Division of Signaling Biology, Ontario Cancer Institute and Department e-mail: Olga Moroz of Medical Biophysics, University of Toronto, Toronto, ON, Canada M5G 1L7; 2 Institute of Biophysics, University of Linz, Linz, Austria Human S100A12 is a member of the S100 family of cal- e-mail: Mitsu Ikura cium-modulated EF-hand proteins. It has a role in inflam- mation and host parasite responses, and is linked to major Calcium ion (Ca2+) is a dynamically regulated cell signal diseases such as diabetes, cystic fibrosis, rheumatoid arthri- which controls a wide variety of cellular processes such as tis and atherosclerosis. Like several other S100 proteins, neuronal signaling, contraction, lymphocyte activation, cell S100A12 binds zinc in addition to calcium. Zinc binding differentiation, and fertilization. My laboratory is interested to S100A12 significantly enhances the calcium affinity [1]. in the elucidation of the structural basis on Ca2+ sensing Previous studies on the S100A12 interactions with one and regulatory mechanisms and in the most recent years of its receptors RAGE (the Receptor for the Advanced we have been focusing on the mechanism of the store op- Glycation End products) show that calcium and zinc de- erated Ca2+ entry (SOCE). Early studies showed that the pendent oligomerization is essential for target recognition stromal interaction molecule-1 (STIM1) is a key activator by S100A12 [2]. Our X-ray structures of S100A12 in sev- of SOCE in response to diminished luminal Ca2+ levels. eral states - apo, calcium, calcium+copper, and zinc in the Subsequently, we have determined the solution structure absence of calcium- suggested an explanation for zinc-in- of the Ca2+-sensing region of STIM1 consisting of the EF- duced increase in the affinity to calcium [3]. Basing on the hand and sterile a motif (SAM) domains (EF-SAM). More structures, a model could be proposed for metal-dependent recently, we have investigated STIM2, a homologoue of changes in the oligomerization state of the protein. The human STIM1, and identified key structural features which role of both zinc and calcium in target binding by S100A12 contribute to the properties of the Ca2+ sensory function during host parasite responses is confirmed by experiments of STIM proteins. We found that EF-hand and SAM do- with paramyosin from the tropical parasites Onchocerca volvu- mains cooperate in transmitting the luminal Ca2+ signal into lus and Brugia malayi. Possible common features of calcium the activation of SOCE and that structural stability and and zinc-dependent modulation of oligomerisation and Ca2+ affinity of those isoforms are well balanced in order to target interactions for the other S100 family members will execute their respective functions. We provide atomic reso- also be discussed. lution insight into the molecular basis for STIM-mediated References: SOCE initiation and show that the folded/unfolded state 1. Dell’Angelica EC et al. (1994) J Biol Chem 269: 28929–28936. of the Ca2+ sensing region of STIM is crucial to SOCE 2. Moroz OV et al. (2009) BMC Biochem 10: 1. regulation. 3. Moroz OV et al. (2009) J Mol Biol 391: 536–551. Acknowledgements: Supported by CIHR and CFI. Vol. 57 11th Meeting of the European Calcium Society, 2010 19 L3.3 L3.4 Calmodulin and calmodulin- Structural basis of S100-RAGE interaction binding proteins in cancer Michael Koch1, Christine Betz2, Seth Chatiyat3, Colin D. White, Zhigang Li, David B. Sacks Walter J. Chazin3, Guenter Fritz1,2 Brigham and Women’s Hospital and Harvard Medical School, 1University of Konstanz, Biology, Germany; 2University of Freiburg, Department of Pathology, USA Neuropathology, Germany; 3Vanderbilt University, Center for Structural e-mail: David B. Sacks Biology, USA e-mail: Günter Fritz A substantial body of evidence supports a fundamental role for calmodulin in cell proliferation and cell cycle progres- S100 proteins constitute the largest subgroup within the sion [1]. Calmodulin is implicated in neoplastic transfor- family of EF-hand calcium-binding proteins. Most S100 mation, with altered expression levels of calmodulin and proteins form homo- and heterodimers under physiological selected calmodulin-binding proteins documented in hu- conditions but also larger assemblies like tetra-, hexa-, and man malignancy. The steroid hormone estrogen promotes octamers are reported. Several S100 proteins are secreted proliferation of human breast epithelial cells by interacting to the extracellular space where they exert neurotrophic, with the estrogen receptor (ER). ER is routinely measured proinflammatory or antimicrobial functions. Prominent in tissue from patients with breast cancer as it has impor- examples are S100B and S100A12, which act via the recep- tant implications for both therapy and prognosis. We have tor for advanced glycation endprodcuts (RAGE). We inves- shown that calmodulin interacts directly with ER, thereby tigate the interactions between RAGE and S100 proteins enhancing ER stability [2]. Further analysis revealed that by in vitro binding studies and X-ray crystallography. The calmodulin reduces proteasome-dependent degradation X-ray structure of human Ca(II)-loaded S100B revealed of ER. In addition, binding to calmodulin is necessary for that S100B can assemble into large multimers, which were normal transcriptional function of ER. Therefore, the mo- confirmed in human brain and by recombinant expression lecular interaction between calmodulin and ER could be a in E. coli [1]. Such multimers exhibit decreased dissociation target for therapeutic intervention in patients with breast from RAGE resulting most likely in a sustained activation cancer. of the receptor signal cascade. The ErbB family of receptor tyrosine kinases comprises To further characterize S100-RAGE interaction we deter- four members, namely the epidermal growth factor recep- mined the X-ray structure of RAGE ligand-binding do- tor (EGFR), HER2/Neu, ErbB3 and ErbB4/ErbB1 [3]. main at 1.85 Å resolution [2]. The arrangement of RAGE ErbB members are potent mediators of normal cell growth ectodiomain molecules in the crystal and complementary and development, and aberrant receptor expression or biochemical studies suggest a role for self-association in functioning plays a crucial part in the development and ev- RAGE function.The S100 interactiong surface of RAGE olution of cancer [3]. For example, HER2 is overexpressed was mapped onto the structure from titrations with S100B in ~25% of invasive breast cancer and is associated with re- monitored by heteronuclear NMR spectroscopy. These duced survival. Published evidence reveals that calmodulin NMR chemical shift perturbations were used as input for binds EGFR and HER2 [4]. IQGAP1, a scaffold protein restrained docking calculations to generate a model for the that integrates signalling pathways, binds to and regulates VC1-S100B complex [2]. numerous proteins [5]. Accumulating evidence suggest IQ- References: GAP1 is an oncogene that is important for tumourigenesis 1. Ostendorp O. et al. (2007) Embo J 26: 3868–3878. [6]. Calmodulin binds to and regulates the function of IQ- 2. Koch M. et al. (2010) Structure (in press). GAP1. Importantly, we have observed that IQGAP1 binds directly to both EGFR and HER2, thereby altering func- tion of the receptors. Analogous to other IQGAP1 bind- ing partners, calmodulin modulates the interaction between IQGAP1 and HER2. Collectively, these data suggest that calmodulin antagonists may be of value in the treatment of breast cancer. References: 1. Lu KP et al. (1993) Endocr Rev 14: 40–58. 2. Li Z et al. (2001) J Biol Chem 276: 17354–17360. 3. Baselga J et al. (2009) Nat Rev Cancer 9: 463–475. 4. Sanchez-Gonzalez P et al. (2010) Febs J 277: 327–342. 5. Brown MD et al. (2006) Trends Cell Biol 16: 242–249. 6. White CD et al. (2009) FEBS Lett 583: 1817–1824. 20 Abstracts 2010 L3.5 Posters Membrane targetting of the EF-hand P3.1 containing calcium sensing proteins Calneurons I and II (CaBP8 and CaBP7) Fine-tuning of cerebellar Purkinje Hannah V. McCue, Robert D. Burgoyne, Lee P. Haynes cells by calbindin D-28k University of Liverpool, Physiology Department, United Kingdom Jarosław J. Barski1, 2 e-mail: Hannah McCue 1Medical University of Silesia, Center for Experimental Medicine, Katowice, Poland; 2Medical University of Silesia, Department of Physiology, Katowice, Poland Calneuron I and Calneuron II (also known as CaBP8 and e-mail: Jaroslaw Barski CaBP7 respectively) make up a subfamily of the calcium binding protein (CaBP) family of calcium sensors. The Long-term depression (LTD) of Purkinje cell-parallel fiber CaBPs are a family of Calmodulin-like, EF-hand contain- synaptic transmission is a critical determinant of normal ing proteins which have recently emerged as regulators of cerebellar function. Impairment of LTD through, for ex- various important neuronal processes. The family com- ample, disruption of the metabotropic glutamate receptor/ prises of six genes and their splice isoforms, each harbour- IP3/calcium signaling cascade in mutant mice, results in se- ing distinct biochemical characteristics that may mediate vere deficits of both synaptic transmission and cerebellar specific interactions at different subcellular localisations. motor control. Here we demonstrate that selective genetic The calneurons, which are abundantly expressed in the deletion of the calcium-binding protein calbindin D-28k brain and at lower levels in other tissues, show the greatest (calbindin) from cerebellar Purkinje cells results in distinctly sequence divergence compared to the rest of the CaBPs. different cellular and behavioral alterations. These mutants In particular they display a distinctive pattern of EF-hand display marked permanent deficits of motor coordination inactivation and possess a 38 amino acid extension at their and sensory processing. This occurs in the absence of al- C-termini which is unique amongst the CaBPs. This C-ter- terations in a form of LTD implicated in the control of be- minal extension is highly hydrophobic and was predicted havior. Analysis of synaptically-evoked calcium transients to form a transmembrane domain and appears to mediate in spines and dendrites of Purkinje cells demonstrated an membrane association of the calneurons. Analysis of the alteration of time-course and amplitude of fast calcium subcellular localisation of these proteins using overexpres- transients after parallel or climbing fiber stimulation. By sion of fluorescently tagged proteins has shown colocalisa- contrast, the delayed mGluR-mediated calcium transients tion with both trans-Golgi and lysosomal markers. Muta- were normal. Our results reveal a unique role of Purkinje tion or removal of the predicted transmembrane domain cell calbindin in a specific form of motor control, and sug- disrupts normal targeting to these compartments resulting gest that rapid calcium buffering may directly control be- in loss of membrane association and increased cytosolic haviourally relevant neuronal signal integration. fluorescence. Trypsin protection assays, used to investigate References: the topology of this hydrophobic region, confirmed the ex- Barski J et al. (2003) J Neurosci 23: 3469–3477. istence of a C-terminal transmembrane spanning domain. Barski J et al. (2002) Genesis 32: 165–168. These results indicate that the calneurons associate with Barski J et al. (2002) Genesis 28: 93–98. membranes via a C-terminal transmembrane domain which Airaksinen M et al. (1997) Proc Natl Acad Sci USA 94: 1488–1493. is essential for their localisation to the trans-Golgi network and lysosomes in cells. Vol. 57 11th Meeting of the European Calcium Society, 2010 21 P3.2 P3.3 Characterization of S100A8/ S100P is a novel interaction partner A9-RAGE interaction and regulator of IQGAP1 Christine Betz1, David Liberg2, Günter Fritz1 Annika Heil1, Ali Reza Nazmi1, Nicole Assard2, Jacques Baudier2, Volker Gerke1 1University of Freiburg, Department of Neuropathology, Freiburg, Germany; 2Active Biotech Research Lund, Sweden 1Institute of Medical Biochemistry, Centre for Molecular Biology of e-mail: Christine Betz Inflammation, University of Muenster, Germany;2 Institut National de la Santé et de la Recherche Médicale, Laboratoire Transduction du Signal, S100A8 and S100A9 are small EF-hand proteins which act Grenoble, France as central pro-inflammatory mediators in acute and chronic e-mail: Annika Heil inflammation. The proteins forms preferentially -a het erodimer which is secreted by neutrophils and phagocytes. Ca2+-binding proteins of the S100 family participate in Extracellularly, the heterodimer acts as an endogenous intracellular Ca2+-signalling by binding to and regulating agonist for Toll-like receptor 4 (TLR-4) [1] and the recep- specific cellular targets in their Ca2+-loaded conformation. tor for advanced glycation end products (RAGE). The Since information on specific cellular targets of different binding to TLR-4 and RAGE is dependent on Ca2+ and S100 proteins is still limited, we developed an affinity ap- largely enhanced in the presence of Zn2+ [1, 2]. Interest- proach that selects for protein targets only binding to the ingly, S100A9-RAGE interaction and strongly inhibited by physiologically active dimer of a S100 protein. Using this quinoline-3-carboxamides [2]. The goal of this project is to approach we here identify IQGAP1 as a novel and dimer- determine conformational changes required for S100A8/ specific target of S100P, a member of the S100 family en- A9 receptor interaction by X-ray crystallography. Both riched in the cortical cytoskeleton. The interaction between S100A8/A9 and RAGE ectodomain are expressed as re- S100P and IQGAP1 is strictly Ca2+-dependent and charac- combinant proteins in E. coli at large scale and purified to terized by a dissociation constant of 0.08 µM. Binding oc- homogeneity. Currently we monitor complex formation by curs primarily through the IQ domain of IQGAP1 and the fluorescence spectroscopy and size exclusion chromatog- first EF hand loop of S100P, thus identifying a novel struc- raphy and screen S100A8/A9-Zn2+, S100A8/A9-RAGE tural principle of S100-target protein interactions. Upon ectodomain and S100A9-quinoline-3-carboxamides com- cell stimulation S100P and IQGAP1 colocalize at or in plexes for crystallization. Successful crystallization and close proximity to the plasma membrane and complex for- structure determination will shed new light into S100- mation can be linked to altered signal transduction prop- RAGE interactions. erties of IQGAP1. Specifically, the EGF-induced tyrosine References: phosphorylation of IQGAP1 that is thought to function 1. Vogl T. I (2007) Nat Med 13: 1042–1049. in assembling signalling intermediates at IQGAP1 scaf- 2. Björk P. et al. (2009) PLoS Biol 7: e97. folds in the subplasmalemmal region is markedly reduced in cells overexpressing S100P but not in cells expressing a S100P mutant deficient in IQGAP1 binding. Furthermore, B-Raf binding to IQGAP1 and MEK1/2 activation occur- ing downstream of IQGAP1 in EGF-triggered signalling cascades are compromised at elevated S100P levels. Thus, S100P is a novel Ca2+-dependent regulator of IQGAP1 that can downregulate the function of IQGAP1 as signal- ling intermediate by direct interaction. 22 Abstracts 2010 P3.4 P3.5

Non-opioid influence of BD on Interactions of Munc13-1 with Ca2+- cell membrane conductivity calmodulin: novel 1-5-8-26 binding

1 motif and additional interaction with Oleksandra Ivashchenko , Volodymyr Khmyz, Oleg 2+ Krishtal, Oleksandr Maximyuk2, Georgy Bakalkin3 C1 domain: implications for Ca regulation of short-term plasticity 1Taras Shevchenko National University of Kyiv, Radiophysical Faculty, Medical Radiophysics Department, Ukraine; 2Bogomoletz Institute Mitcheell Maestre-Martinez1, Fernando Rodriguez- of Physiology, Department of Cellular Membranology, Ukraine; 1 1 1 3 Castaneda , Donghan Lee , Nicolas Coudevylle , Kalina International Center of Molecular Physiology, Ukraine; Karolinska 2 2 2 1 Institute, Department of Clinical Neuroscience, Sweden Dimova , Noa Lipstein , Olaf Jahn , Stefan Becker , Nils e-mail: Oleksandra Ivashchenko <[email protected]> Brose2, Teresa Carlomagno1,3, Christian Griesinger1 1MPI for Biophysical Chemistry, NMR-based Structural Biology, Am Faßberg 11, 37077 Göttingen, Germany; 2MPI for Experimental Dynophins are endogenous ligands of K-opioid receptors. Medicine, Molecular Neurobioly, Hermann-Rein-Str. 3, 37075 Göttingen, They are similar to cell permiable peptides in their ability to Germany; 3present address: European Molecular Biology Laboratory, cross cellular membrane. We have demonstrated influence of Meyerhofstr.1, 69117 Heidelberg, Germany big dinorphin (BD) on neuronal (cells of dorzal ganglia and e-mail: Mitcheell Maestre Martinez hippocampus) and non-neuronal (НЕК-293) cells. 1 µM concentration of BD caused the changes in conductivity of Ca2+ signaling in neurons through calmodulin (CaM) has the investigated cells with an amplitude of transmembrane a prominent function in the regulation of neurotransmit- current in the range of –20 to –350 pA. Influence of BD ter release [1]. Ca2+-CaM binds to a conserved region in was reversible. 5 µM concentration of naloxone (antago- the priming proteins Munc13-1 and ubMunc13-2 and thus nist of dynorphin) had no influence on BD-induced mem- regulates synaptic neurotransmitter release in neurons in brane conductivity changes. Increase of extracellular Ca2+ response to residual Ca2+ signals [2]. We solved the struc- 2+ concentration caused decrease of BD-induced membrane ture of Ca 4-CaM in complex with the CaM-binding do- conductivity changes (2 µМ Са2+-maximal transmembrane main (CaMBD) of Munc13-1, which reveals a novel 1-5- current amplitude –2000 pA, 10 µM Са2+-maximal current 8-26 CaM-binding motif with two separated structural amplitude –200 pA). Increase of extracellular concentra- modules, each involving a CaM domain, connected by tion of Mg2+ did not have any significant effect (2 µM flexible linkers [3]. The C-module features an amphiphilic Ca2+ 2 µM Mg2+-maximal current amplitude –700 pA, helix of Munc13-1 bound to the C-terminal CaM domain, 2 µM Ca2+ 10 µM Mg2+ –400 pA, 10 µM Ca2+ 2 µM Mg2+ with hydrophobic residues at positions 1-5-8 acting as an- –80 pA). Amplitude and frequency of BD-induced changes chors. The N-module consists of a tryptophan at position of conductivity depended a lot on holding membrane po- 26 attached to the N-terminal CaM domain. Photoaffinity tential value. With the change of membrane potential value labeling data confirms the NMR structure and also reveals from –100 on –60 mV transmembrane current was de- the same modular architecture in the complex with the ub- creased from –2500 to –150 pA. Our results demonstrated Munc13-2 isoform [3, 4]. The N-module can be dissociat- 2+ non-opioid influence of big dynorphin on cell membrane ed with EGTA to form a half-loaded Munc13/Ca 2-CaM conductivity which became apparent on neuronal and non- complex, which interconverts readily into the fully Ca2+- neuronal cells and depended a lot on sustained membrane loaded species. The Ca2+ regulation of these Munc13 iso- potential value and extracellular concentration of Ca2+. forms can therefore be explained by the modular nature of the Munc13/Ca2+-CaM interactions, where the C-module provides a high affinity interaction activated at nanomolar 2+ [Ca ]i and the N-module acts as a sensor at micromolar 2+ [Ca ]i. Furthermore, NMR titrations revealed that the C1 domain of Munc13-1 poses an additional interaction site for the N-terminal CaM domain. Our structural model for this interaction shows that the first EF-hand loop of CaM contacts one of the diacylglycerol binding loops of the C1 domain. This suggests that CaM perturbs the binding of diacylglycerol to the C1 domain. References: 1. Neher E, Sakaba T (2008) Neuron 59: 861–872. 2. Junge HJ et al. (2004) Cell 118: 389–401. 3. Rodriguez-Castaneda F et al. (2010) EMBO J 29: 680–691. 4. Dimova K et al. (2009) Biochemistry 48: 5908–5921. Vol. 57 11th Meeting of the European Calcium Society, 2010 23 P3.6 P3.7 Defining protein-protein networks Phospholipase C-η2 is activated by at the structural level by saturation elevated intracelllar Ca2+ levels and requires transfer difference spectroscopy both the PH and C2 domains for activity Małgorzata Palczewska1, Britt Mellström2, Petra Popovics, William E. Beswick, Alan J. Stewart 2 1 Jose R. Naranjo , Patrick Groves School of Medicine, University of St Andrews, St Andrews, KY16 9TS, 1Instituto de Tecnologia Química e Biológica (ITQB-UNL), Oeiras, United Kingdom Portugal; 2Centro Nacional de Biotecnología (CNB), CSIC, Madrid, Spain e-mail: Petra Popovics e-mail: Małgorzata Palczewska Phospholipase C-η (PLC-η) enzymes are a recently discov- Calcium-sensor proteins function in calcium-signaling ered family of mammalian PLCs of which there are two pathways through calcium-dependent protein-protein in- members: PLCη1 and PLCη2. More attention has been teraction networks. X-ray crystallography, a range of NMR focused on PLCη2 due to its expression in regions of the methods and theoretical modeling are commonly used to brain associated with memory and cognition. Here we show structurally define protein complexes. While these methods that PLCη2 is activated by elevated intracellular Ca2+ levels. have helped to consolidate many calcium-signaling path- Inositol phosphate accummulation was measured in COS7 ways, they are not fail-safe. For example, the apo forms cells transiently expressing PLCη2. Activity increased 5- to of many EF-hand proteins contain dynamic hydrophobic 6-fold in response to monensin (a Na+ ionophore). Mon- cores (molten-globule states) that are resistant to crystal- ensin had no effect upon the activity of PLCη1 or PLCδ1. lography and result in poorly dispersed NMR spectra. How The increase in PLCη2 activity was due largely to Na+-in- can we obtain structural information on such systems? duced mitochondrial Ca2+ release as activity was reduced Saturation Transfer Difference spectroscopy (STD) is an 47% by CGP37157, a specific inhibitor of mitochondrial NMR method that reports which part of a small ligand Na+/Ca2+ exchange. PLCη2 activity was also measured in (millimolar concentration) interacts with a larger biomol- transfected cells at various free Ca2+ concentrations and ex- ecule (often at a micromolar concentration that is too low hibited a large increase between 0.1 µM and 1 µM Ca2+. The to be actually observed by NMR). STD can be used to roles of the conserved pleckstrin homology (PH) and C2 structurally define how a peptide derived from one protein domains in regulating activity were also examined. A con- interacts with a full-length protein of undefined structure. struct encoding a PH domain-lacking enzyme (ΔPH) was The EF-hand protein DREAM is a multi-function protein generated. Also three C2 domain mutants were made en- and oligomerization plays a key role in regulating its func- compassing substitutions at key residues (D787A, D787N tion in the apo state. The NMR spectra of apo DREAM and D846A). The ΔPH and D846A proteins did not re- shows a poor dispersion of signals characteristic of a poor- spond to monensin (or Ca2+). Moreover, D787N exhibited ly folded or molten-globule like state. Yeast two hybrid re- reduced enzyme activity (0.65-fold) relative to wild-type. vealed two peptide regions that were important for head-to- A 0.5-fold increase in activity was observed with D787A, tail interactions (the STD screening of a synthetic peptide but the protein expression was also elevated (4-fold). The library might be used in the future). STD is normally ap- Ca2+-sensitivity of D787A was similar to wild-type PLCη2 plied to larger proteins (> 30 kDa) and small ligands (<0.5 however, sensitivity of D787N to Ca2+ was reduced ~10 kDa). Considerable optimization was required to obtain fold suggesting a potential role for this domain in regu- high quality STD spectra for an N-terminal peptide ligand lating Ca2+-activation. Studies with GFP-PLCη2 revealed (~2 kDa) in the presence of unlabeled, N terminal trun- a punctate cellular distribution and partial co-localization cated DREAM (residues 71-256, ~20 kDa). However, we with mitochondria. Cellular localization of C2 domain were successful and the obtained data allowed us to calcu- mutants mirrored that of wild-type but the ΔPH enzyme late an interaction score for each proton of each residue in exhibited diffuse cytosolic staining indicating it to be defec- the peptide. These scores, in turn, allowed us to design and tive in membrane binding. Collectively, this work highlights test loss-of-function mutants by assays testing functionality a new component of the Ca2+ signaling toolkit and enhanc- of DREAM protein (oligomerization, transcription repres- es our understanding of neuronal Ca2+ signaling pathways, sion in vivo). Although STD does not provide detailed 3D particularly those involving efflux of mitochondrial Ca2+. structures, the method experimentally determines and/or PLCη2 is exceptionally Ca2+-sensitive and therefore likely verifies the interaction surfaces between proteins. to facilitate the amplification or crosstalk of transient cel- lular Ca2+ signals in vivo. 24 Abstracts 2010 P3.8 Sphingosine kinases regulate NOX2 activity via p38 MAPK-dependent translocation of S100A8/A9 Véronique Schenten1, Chantal Melchior1, Natacha Steinckwich1,2, Eric J. Tschirhart1, Sabrina Bréchard1 1Life Sciences Research Unit, University of Luxembourg, 162a, Avenue de la Faïencerie, L-1511 Luxemburg; 2Present address: Laboratory of Signal Transduction and National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA e-mail: Véronique Schenten

Neutrophils play a fundamental role in host defense by neutralizing pathogens through the generation of reac- tive oxygen species by the NADPH oxidase (NOX2). It is well established that extracellular Ca2+ entry participates to NOX2 activation. In non-excitable cells, Ca2+ influx is predominantly mediated via store-operated Ca2+ entry (SOCE), a complex mechanism in which intracellular Ca2+ store-depletion from the endoplasmic reticulum results in Ca2+ entry through store-operated Ca2+ channels at the plasma membrane. Sphingosine 1-phosphate (S1P) produced by sphingosine kinases (SphKs) has been involved in Ca2+ homeostasis and thus, could intervene in NOX2 regulation. The aim of our study was to characterize the importance of SphKs in NOX2 activation and the signaling cascade involved in this mechanism in neutrophil-like HL-60 cells. Inhibition of fMLF-induced H2O2 production by DHS, a sphingosine kinase inhibitor and SphKs siRNA underlined the role of SphKs in the oxidative response. Using thapsigargin and PMA, we demonstrated that SphK-regulated NOX2 ac- tivity relies on intracellular Ca2+ store depletion. Since it has been previously shown that the translocation of the Ca2+-binding protein complex S100A8/A9 participates to NOX2 activation and depends on Ca2+ store depletion, we examined whether SphKs have a role in this S100A8/A9- dependent pathway. We provide evidence that SphKs regu- late S100A8/A9 membrane recruitment and p38 MAPK activity, known to induce S100A9/A8 translocation. In conclusion, we have defined a pathway leading to NOX2 activation in which p38 MAPK-mediated S100A8/A9 translocation is regulated by Ca2+ store depletion-depend- ent SphK activation.