The regulated activation of NF-κB by antigen receptor signaling is required for normal B and T lymphocyte activation during the adaptive immune response. Dysregulated NF-κB activation is associated with several types of lymphoma, including Diffuse Large B Cell Lymphoma (DLBCL). During normal antigen receptor signaling, the multidomain scaffold protein CARD11 undergoes a transition from a closed, inactive state to an open, active conformation that recruits several signaling proteins into a complex, leading to IKK kinase activation. This transition is regulated by the CARD11 Inhibitory Domain (ID), which participates in intramolecular interactions that prevent cofactor binding to CARD11 prior to signaling, but which is neutralized after receptor engagement by phosphorylation. Several oncogenic CARD11 mutations have been identified in DLBCL that enhance activity and that are mostly found in the Coiled-coil domain. However, the mechanisms by which these mutations cause CARD11 hyperactivity and spontaneous NF-κB activation are poorly understood. In this report, we provide several lines of evidence that oncogenic mutations F123I and L225LI induce CARD11 hyperactivity by disrupting autoinhibition by the CARD11 ID. These mutations disrupt ID-mediated intramolecular interactions, ID-dependent inhibition, and bypass the requirement for ID phosphorylation during T cell receptor signaling. Intriguingly, these mutations selectively enhance the apparent affinity of CARD11 for Bcl10, but not for other signaling proteins that are recruited to CARD11 in an IDdependent manner during normal antigen-receptor signaling. Our results establish a mechanism that explains how DLBCL-associated mutations in CARD11 can initiate spontaneous, receptorindependent activation of NF-κB.The activation of the NF-κB transcription factor by antigen receptor signaling is critical for the proliferation and activation of B and T lymphocytes in the adaptive immune response (1). NF-κB regulates many genes responsible for lymphocyte function, including cytokines, cell surface receptors, and pro-proliferative and anti-apoptotic genes. Mice and humans with mutations that prevent antigen-induced activation of NF-κB fail to mount productive immune responses (2). † This work was supported by RO1AI078980 and PO1AI072677 from the NIH, RSG-06-172-01-LIB from the American Cancer Both T cell receptor (TCR) and B cell receptor (BCR) signaling pathways activate NF-κB through the IKK complex, which is composed of two kinase subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ. The IKK complex phosphorylates IκB proteins that bind and stably retain NF-κB in an inactive state in the cytoplasm. Once phosphorylated, IκB is ubiquitinated and degraded by the 26S proteasome, allowing NF-κB to stably translocate to the nucleus to regulate target genes.The TCR and BCR signaling pathways share many common components that function to activate the IKK complex in a signal-inducible manner. A key scaffold molecule that is required in both pathways is CARD11 (CARMA1, BIMP3...

Immunologic research has benefited tremendously from expression-cloning strategies designed to isolate genes responsible for a wide variety of immunomodulatory activities, including cytokines, receptors, signaling proteins, and transcription factors. Here, we discuss the use of expression-cloning strategies that have been modified to detect cDNAs that influence gene expression as assayed by a transcriptional reporter. We summarize our experience with these screens, review important parameters, and discuss potential modifications.

No abstract