TRAF3 negatively regulates calcineurin-NFAT pathway by targeting calcineurin B subunit for degradation

Calcineurin (CN) is the only serine/threonine specific protein phosphatase regulated by Ca(2+) /calmodulin (CaM), which is composed of catalytic A subunit (CNA) and regulatory B subunit (CNB). Tumor necrosis factor (TNF) receptor associated factor 3 (TRAF3) is an essential component in the Toll like receptors and TNF receptors (TNFRs) pathways. The TRAF domain of TRAF3 interacts with a large range of proteins, which share consensus sequences known as TRAF interacting motifs (TIMs). By sequence alignment, we identified two potential TIMs in CNB. However, the relation between TRAF3 and CN has not been reported before. To explore this, we highly expressed the former insoluble TRAF domain of TRAF3 in soluble form by using CaM fusion system for the first time. We demonstrated that the TRAF domain of TRAF3 interacted with CNB. On further investigation, over-expression of TRAF3 inhibited endogenous CN's activity, which decreased NFAT reporter activity and IL-2 production. Knock-down of TRAF3 partially enhanced CN's activity. The possible mechanism was that TRAF3 functioned as ubiquitin E3 ligase for CN and promoted its degradation.