NF-B activation downstream of antigen receptor engagement is a highly regulated event required for lymphocyte activation during the adaptive immune response. The pathway is often dysregulated in lymphoma, leading to constitutive NF-B activity that supports the aberrant proliferation of transformed lymphocytes. To identify novel regulators of antigen receptor signaling to NF-B, we developed bioluminescence resonance energy transfer-based interaction cloning (BRIC), a screening strategy that can detect protein-protein interactions in live mammalian cells in a high-throughput manner. Using this strategy, we identified the RING finger protein RNF181 as an interactor of CARD11, a key signaling scaffold in the antigen receptor pathway. We present evidence that RNF181 functions as an E3 ubiquitin ligase to inhibit antigen receptor signaling to NF-B downstream of CARD11. The levels of the obligate signaling protein Bcl10 are reduced by RNF181 even prior to signaling, and Bcl10 can serve as a substrate for RNF181 E3 ligase activity in vitro. Furthermore, RNF181 limits the proliferation of human diffuse large B cell lymphoma cells that depend upon aberrant CARD11 signaling to NF-B for growth and survival in culture. Our results define a new regulatory checkpoint that can modulate the output of CARD11 signaling to NF-B in both normal and transformed lymphocytes.T he activation of the NF-B transcription factor downstream of antigen receptor engagement is a critical event required for lymphocyte activation during the adaptive immune response (1, 2). In humans and mice, genetic defects in components of the antigen receptor signaling pathway result in immunodeficiency syndromes in which the failure to mount an effective immune response confers enhanced susceptibilities to infection and impaired clearance of pathogens (3, 4).CARD11 is a critical signaling scaffold protein that functions to transmit signals from triggered antigen receptors to the IB kinase (IKK) complex, which inducibly phosphorylates inhibitory IB proteins, leading to their ubiquitinylation and degradation, which allows NF-B heterodimers to stably translocate to the nucleus (5-14). During antigen receptor signaling, CARD11 undergoes a transition from a closed, inactive state to an open, active scaffold that recruits several signaling cofactors into a complex that induces IKK kinase activity. CARD11 is kept inactive prior to receptor engagement by an inhibitory domain (ID) that participates in intramolecular interactions that involve the CARD, LATCH, and coiled-coil (CC) domains (15, 16). Triggering of the T cell receptor (TCR) or B cell receptor (BCR) results in the phosphorylation of the ID on specific serines (17-21), which neutralizes its inhibitory activity and allows the signal-induced recruitment of obligate signaling cofactors to CARD11, including Bcl10, MALT1, TRAF6, IKK␥, and caspase-8 (15). The formation and stability of the CARD11-nucleated multiprotein complex dictate the extent and duration of IKK kinase activity and NF-B activation following antig...
