Analysis of Calcium Control of Canonical NF-κB Signaling in B Lymphocytes

The central role of calcium (Ca²⁺) signaling in lymphocyte development and acquisition of functional immunity and tolerance is well established. Ca²⁺ signals are initiated upon antigen binding to cognate receptors on lymphocytes that trigger store operated Ca²⁺ entry (SOCE). The underlying mechanism of SOCE in lymphocytes involves TCR and BCR mediated activation of Stromal Interaction Molecule 1 and 2 (STIM1/2) embedded in the ER membrane. Once activated, STIM proteins oligomerize and re-localize to ER domains juxtaposed to the plasma membrane where they activate Orai channels to allow Ca²⁺ to enter the cell across the plasma membrane. Importantly, STIM/Orai-dependent Ca²⁺ signals guide antigen induced lymphocyte development and function principally by regulating the activity of transcription factors.The most widely studied of these transcription factors is the Nuclear Factor of Activated T cells (NFAT). NFAT is expressed ubiquitously and the mechanism by which Ca²⁺ regulates NFAT activation and signaling is well known. By contrast, a mechanistic understanding of how Ca²⁺ signals also shape the activation and specificity of NF-κB to control the expression of pro-inflammatory genes has lagged. Here we discuss the methodology used to investigate Ca²⁺ dependent mechanisms of NF-κB activation in lymphocytes. Our approach focuses on three main areas of signal transduction and signaling: (1) antigen receptor engagement and Ca²⁺ dependent initiation of NF-kB signaling, (2) Ca²⁺ dependent induction of NF-κB heterodimer activation and nuclear localization, and (3) and how Ca²⁺ regulates NF-κB dependent expression of target genes and proteins.