Calcium (Ca2+) is a multifunctional second messenger that regulates lymphocyte differentiation and function. The pathways that initiate Ca2+signaling following antigen receptor engagement in lymphocytes are well delineated. For example, antigen receptor engagement on B cells, activates tyrosine kinases Lyn/Syk and the consequent activation of PLC32 results in the generation of IP3, which activates channels (IP3 receptors) on the endoplasmic reticulum (ER) membrane. Depletion of IP3-sensitive ER Ca2+ stores triggers relocalization of STIM1, the ER Ca2+ sensor, into punctate structures in junctional ER adjacent to the plasma membrane. Orai1, the recently identified CRAC channel pore located in the plasma membrane, also aggregates into puncta following activation, and its subsequent interaction with STIM1 results in """"""""store-operated"""""""" CRAC channel activation. While it has long been thought that IP3-mediated store depletion is both necessary and sufficient for maximal CRAC activation, recent data, including our own data, clearly challenge this notion. Regulation of CRAC activation downstream of IP3-mediated store release was observed in lyn-/-syk-/- B cells following ER Ca2+stores depletion. In support of this idea, our studies demonstrate that CRAC activation is abrogated by either Lyn/Syk inhibitors or neutralizing anti-Lyn and -Syk antibodies introduced into the cytoplasm of single B cells. Our subsequent observation that Orai1 and Syk physically interact led us to hypothesize that Syk is involved in Orai1 redistribution required for CRAC activation. Accordingly, this proposal focuses on the role of Lyn and Syk in the regulation of post-store coupling (Aim1). Another indication that CRAC activity could be independently regulated is provided by our preliminary data that PLC32 directly interacts with Orai1, suggesting. Our data suggest that physical association of PLC32 and Orai1 is critical for CRAC activation and that such interactions can be inhibited by tyrosine phosphorylated TFII-I, a known Btk target. Based on these data and an analogous role for TFII-I in regulating PLC31-dependent activation of TRPC3 Ca2+ channels, we hypothesize that PLC3-2 regulates CRAC channel mediated Ca2+ entry in a lipase-independent manner that is negatively regulated by Btk dependent phosphorylation of TFII-I (tested in Aim 2). Finally, our recent work with Dan Billadeau demonstrates that the adaptor protein WAVE2 regulates Ca2+ entry, but not Ca2+ release from stores following T cell activation. Our preliminary results demonstrate that WAVE2 plays an analogous role in B cells. Furthermore, WAVE2 associates with STIM1 in B cells and dissociates following activation, suggesting a role in orienting STIM1 for interactions with Orai1. We will further explore the mechanistic role of WAVE2 in BCR-induced CRAC activation in Aim 3. Tight regulation of Ca2+signaling is critical for lymphocyte effector function and its dysregulation contributes to immunodeficiency as well as inflammatory diseases. Our proposed studies will identify additional mechanisms by which Ca2+ signaling could go awry in disease states. Results from these studies will also provide insight into approaches to ameliorate immunodeficiency diseases and/or autoimmune and inflammatory conditions.

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Calcium is a multifunctional second messenger that regulates nearly every aspect of lymphocyte differentiation and function and thereby the immune response. The primary objective of these studies is to understand novel mechanisms by which calcium entry into lymphocytes is regulated. Results from these studies will identify targets and strategies both positive and negative manipulation of the immune response to ameliorate immunodeficiency diseases and/or to suppress development of autoimmune and inflammatory disease.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Cellular and Molecular Immunology - B Study Section (CMIB)
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Ferguson, Stacy E
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University of Pennsylvania
Schools of Veterinary Medicine
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Liu, Xiaohong; Berry, Corbett T; Ruthel, Gordon et al. (2016) T Cell Receptor-induced Nuclear Factor ?B (NF-?B) Signaling and Transcriptional Activation Are Regulated by STIM1- and Orai1-mediated Calcium Entry. J Biol Chem 291:8440-52
Han, Ziying; Madara, Jonathan J; Herbert, Andrew et al. (2015) Calcium Regulation of Hemorrhagic Fever Virus Budding: Mechanistic Implications for Host-Oriented Therapeutic Intervention. PLoS Pathog 11:e1005220
Rao, Sheila; Liu, Xiaohong; Freedman, Bruce D et al. (2013) Spleen tyrosine kinase (Syk)-dependent calcium signals mediate efficient CpG-induced exocytosis of tumor necrosis factor ? (TNF?) in innate immune cells. J Biol Chem 288:12448-58
Psathas, James N; Doonan, Patrick J; Raman, Pichai et al. (2013) The Myc-miR-17-92 axis amplifies B-cell receptor signaling via inhibition of ITIM proteins: a novel lymphomagenic feed-forward loop. Blood 122:4220-9
Lu, Jianhong; Qu, Yonggang; Liu, Yuliang et al. (2013) Host IQGAP1 and Ebola virus VP40 interactions facilitate virus-like particle egress. J Virol 87:7777-80
Babich, Alexander; Li, Shuixing; O'Connor, Roddy S et al. (2012) F-actin polymerization and retrograde flow drive sustained PLC?1 signaling during T cell activation. J Cell Biol 197:775-87
Shaffer, Meredith H; Dupree, Renell S; Zhu, Peimin et al. (2009) Ezrin and moesin function together to promote T cell activation. J Immunol 182:1021-32
Zhu, Peimin; Liu, Xiaohong; Treml, Laura S et al. (2009) Mechanism and regulatory function of CpG signaling via scavenger receptor B1 in primary B cells. J Biol Chem 284:22878-87
King, Leslie B; Freedman, Bruce D (2009) B-lymphocyte calcium influx. Immunol Rev 231:265-77
Carrizosa, Esteban; Gomez, Timothy S; Labno, Christine M et al. (2009) Hematopoietic lineage cell-specific protein 1 is recruited to the immunological synapse by IL-2-inducible T cell kinase and regulates phospholipase Cgamma1 Microcluster dynamics during T cell spreading. J Immunol 183:7352-61

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