Novel Mechanisms of Calcium Signaling in B lymphocytes

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DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.