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

The central role of calcium (Ca) signaling in lymphocyte development and acquisition of functional immunity and tolerance is well established. Ca signals are initiated upon antigen binding to cognate receptors on lymphocytes that trigger store operated Ca 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 Ca to enter the cell across the plasma membrane. Importantly, STIM/Orai-dependent Ca 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 Ca regulates NFAT activation and signaling is well known. By contrast, a mechanistic understanding of how Ca 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 Ca 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 Ca dependent initiation of NF-kB signaling, (2) Ca dependent induction of NF-κB heterodimer activation and nuclear localization, and (3) and how Ca regulates NF-κB dependent expression of target genes and proteins.