T cell receptor-induced nuclear factor κb (NF-κB) signaling and transcriptional activation are regulated by STIM1- and Orai1-mediated calcium entry

T cell activation following antigen binding to the T cell receptor (TCR) involves the mobilization of intracellular Ca2⁺ to activate the key transcription factors nuclear factor of activated T lymphocytes (NFAT) and NF-κB. The mechanism ofNFATactivation by Ca2⁺ has been determined. However, the role of Ca2⁺ in controlling NF-κB signaling is poorly understood, and the source of Ca2⁺ required for NF-κB activation is unknown. We demonstrate that TCR- but not TNF-induced NF-κB signaling upstream of IκB kinase activation absolutely requires the influx of extracellular Ca2⁺ via STIM1-dependent Ca2⁺ release-activated Ca2⁺/Orai channels. We further show that Ca2⁺ influx controls phosphorylation of the NF-κB protein p65 on Ser-536 and that this posttranslational modification controls its nuclear localization and transcriptional activation. Notably, our data reveal that this role for Ca2⁺ is entirely separate from its upstream control of IκBκ degradation, thereby identifying a novel Ca2⁺-dependent distal step in TCR-induced NF-κB activation. Finally, we demonstrate that this control of distal signaling occurs via Ca2⁺-dependent PKC α-mediated phosphorylation of p65. Thus, we establish the source of Ca2⁺ required for TCR-induced NF-κB activation and define a new distal Ca2⁺-dependent checkpoint in TCR-induced NF-κB signaling that has broad implications for the control of immune cell development and T cell functional specificity.