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PNAS 108 (47): 19072-19077

Copyright © 2011 by the National Academy of Sciences.


CD95 triggers Orai1-mediated localized Ca2+ entry, regulates recruitment of protein kinase C (PKC) β2, and prevents death-inducing signaling complex formation

Nadine Khadraa,1, Laurence Bresson-Bepoldinb,1, Aubin Pennac, Benjamin Chaigne-Delalanded, Bruno Séguie, Thierry Levadee, Anne-Marie Vacherb, Josy Reiffersb, Thomas Ducretf, Jean-François Moreaud, Michael D. Cahalanc,2, Pierre Vacherb,3, and Patrick Legembrea,2,3

aUniversité Rennes-1, Institut de Recherche sur la Santé, l'Environnement et le Travail/Equipe Accueil Signalisation et Réponse aux agents infectieux et Chimiques 35043 Rennes, France; bUniversité Bordeaux Ségalen, Institut National de la Santé et de la Recherche Médicale U916, Institut Bergonié, 33076 Bordeaux, France; cDepartment of Physiology and Biophysics and Center for Immunology, University of California, Irvine, CA 92697; d Center National de la Recherche Scientifique UMR5164, 33076 Bordeaux, France; eUniversité Paul Sabatier, Institut National de la Santé et de la Recherche Médicale UMR1037, Centre de Recherches en Cancérologie de Toulouse, 31432 Toulouse, France; and fInstitut National de la Santé et de la Recherche Médicale U1045, Centre de Recherche Cardio-Thoracique de Bordeaux, 33076 Bordeaux, France

Contributed by Michael D. Cahalan, October 13, 2011 (sent for review September 22, 2011)

Abstract: The death receptor CD95 plays a pivotal role in immune surveillance and immune tolerance. Binding of CD95L to CD95 leads to recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates caspase-8 and caspase-10. Efficient formation of the CD95/FADD/caspase complex, known as the death-inducing signaling complex (DISC), culminates in the induction of apoptosis. We show that cells exposed to CD95L undergo a reorganization of the plasma membrane in which the Ca2+ release-activated Ca2+ channel Orai1 and the endoplasmic reticulum-resident activator stromal interaction molecule 1 colocalize with CD95 into a micrometer-sized cluster in which the channel elicits a polarized entry of calcium. Orai1 knockdown and expression of a dominant negative construct (Orai1E106A) reveal that on CD95 engagement, the Orai1-driven localized Ca2+ influx is fundamental to recruiting the Ca2+-dependent protein kinase C (PKC) β2 to the DISC. PKCβ2 in turn transiently holds the complex in an inactive status, preventing caspase activation and transmission of the apoptotic signal. This study identifies a biological role of Ca2+ and the Orai1 channel that drives a transient negative feedback loop, introducing a lag phase in the early steps of the CD95 signal. We suggest that these localized events provide a time of decision to prevent accidental cell death.

Key Words: Fas • lymphocytes

Author contributions: N.K., L.B.-B., P.V., and P.L. designed research; N.K., L.B.-B., A.P., B.C.-D., A.-M.V., and T.D. performed research; B.S., T.L., J.R., and M.D.C. contributed new reagents/analytic tools; N.K., L.B.-B., P.V., and P.L. analyzed data; and L.B.-B., J.-F.M., M.D.C., P.V., and P.L. wrote the paper.

1N.K. and L.B.B. contributed equally to this work.

3P.V. and P.L. contributed equally to this work.

The authors declare no conflict of interest.

This article contains supporting information online at

2To whom correspondence may be addressed. E-mail: mcahalan{at} or patrick.legembre{at}

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