Coordinated regulation of scaffold opening and enzymatic activity during CARD11 signaling.

The activation of key signaling pathways downstream of antigen receptor engagement is critically required for normal lymphocyte activation during the adaptive immune response. CARD11 is a multidomain signaling scaffold protein required for antigen receptor signaling to NF-κB, c-Jun N-terminal kinase, and mTOR. Germline mutations in the CARD11 gene result in at least four types of primary immunodeficiency, and somatic CARD11 gain-of-function mutations drive constitutive NF-κB activity in diffuse large B cell lymphoma and other lymphoid cancers. In response to antigen receptor triggering, CARD11 transitions from a closed, inactive state to an open, active scaffold that recruits multiple signaling partners into a complex to relay downstream signaling. However, how this signal-induced CARD11 conversion occurs remains poorly understood. Here we investigate the role of Inducible Element 1 (IE1), a short regulatory element in the CARD11 Inhibitory Domain, in the CARD11 signaling cycle. We find that IE1 controls the signal-dependent Opening Step that makes CARD11 accessible to the binding of cofactors, including Bcl10, MALT1, and the HOIP catalytic subunit of the linear ubiquitin chain assembly complex. Surprisingly, we find that IE1 is also required at an independent step for the maximal activation of HOIP and MALT1 enzymatic activity after cofactor recruitment to CARD11. This role of IE1 reveals that there is an Enzymatic Activation Step in the CARD11 signaling cycle that is distinct from the Cofactor Association Step. Our results indicate that CARD11 has evolved to actively coordinate scaffold opening and the induction of enzymatic activity among recruited cofactors during antigen receptor signaling.

NK Cell Maturation and Cytotoxicity Are Controlled by the Intramembrane Aspartyl Protease SPPL3.

NK cell maturation is critical for normal effector function and the innate immune response to tumors and pathogens. However, the molecular pathways that control NK cell maturation remain largely undefined. In this article, we investigate the role of SPPL3, an intramembrane aspartyl protease, in murine NK cell biology. We find that deletion of SPPL3 in the hematopoietic system reduces numbers of peripheral NK cells, clearance of MHC class I-deficient tumors in vivo, and cytotoxicity against tumor cells in vitro. This phenotype is concomitant with reduced numbers of CD27(+)CD11b(+) and CD27(-)CD11b(+) NK cells, indicating a requirement for SPPL3 in efficient NK cell maturation. NK cell-specific deletion of SPPL3 results in the same deficiencies, revealing a cell-autonomous role for SPPL3 in these processes. CRISPR/Cas9 genomic editing in murine zygotes was used to generate knockin mice with a catalytically compromised SPPL3 D271A allele. Mice engineered to express only SPPL3 D271A in NK cells phenocopy mice deleted for SPPL3, indicating a requirement for SPPL3 protease activity in NK cell biology. Our results identify SPPL3 as a cell-autonomous molecular determinant of NK cell maturation and expand the role of intramembrane aspartyl proteases in innate immunity.

Cooperative Control of Caspase Recruitment Domain-containing Protein 11 (CARD11) Signaling by an Unusual Array of Redundant Repressive Elements.

Several classes of signaling proteins contain autoinhibitory domains that prevent unwarranted signaling and coordinate the induction of activity in response to external cues. CARD11, a scaffold protein critical for antigen receptor signaling to NF-κB, undergoes autoregulation by a poorly understood inhibitory domain (ID), which keeps CARD11 inactive in the absence of receptor triggering through inhibitory intramolecular interactions. This autoinhibitory strategy makes CARD11 highly susceptible to gain-of-function mutations that are frequently observed in diffuse large B cell lymphoma (DLBCL) and that disrupt ID-mediated autoinhibition, leading to constitutive NF-κB activity, which can promote lymphoma proliferation. Although DLBCL-associated CARD11 mutations in the caspase recruitment domain (CARD), LATCH domain, and coiled coil have been shown to disrupt intramolecular ID binding, surprisingly, no gain-of-function mutations in the ID itself have been reported and validated. In this study, we solve this paradox and report that the CARD11 ID contains an unusual array of four repressive elements that function cooperatively with redundancy to prevent spontaneous NF-κB activation. Our quantitative analysis suggests that potent oncogenic CARD11 mutations must perturb autoinhibition by at least three repressive elements. Our results explain the lack of ID mutations in DLBCL and reveal an unusual autoinhibitory domain structure and strategy for preventing unwarranted scaffold signaling to NF-κB.

Intramolecular Interactions and Regulation of Cofactor Binding by the Four Repressive Elements in the Caspase Recruitment Domain-containing Protein 11 (CARD11) Inhibitory Domain.

The CARD11 signaling scaffold transmits signaling between antigen receptors on B and T lymphocytes and the transcription factor NF-κB during the adaptive immune response. CARD11 activity is controlled by an inhibitory domain (ID), which participates in intramolecular interactions and prevents cofactor binding prior to receptor triggering. Oncogenic CARD11 mutations associated with the activated B cell-like subtype of diffuse large B cell lymphoma somehow perturb ID-mediated autoinhibition to confer CARD11 with the dysregulated spontaneous signaling to NF-κB that is required for the proliferation and survival of the lymphoma. Here, we investigate how the four repressive elements (REs) we have discovered in the CARD11 ID function to inhibit CARD11 activity with cooperativity and redundancy. We find that each RE contributes to the maintenance of the closed inactive state of CARD11 that predominates in the absence of receptor engagement. Each RE also contributes to the prevention of Bcl10 binding in the basal unstimulated state. RE1, RE2, and RE3 participate in intramolecular interactions with other CARD11 domains and share domain targets for binding. Remarkably, diffuse large B cell lymphoma-associated gain-of-function mutations in the caspase recruitment domain, LATCH, or coiled coil can perturb intramolecular interactions mediated by multiple REs, suggesting how single amino acid oncogenic CARD11 mutations can perturb or bypass the action of redundant inhibitory REs to achieve the level of hyperactive CARD11 signaling required to support lymphoma growth.

Oncogenic IL7R gain-of-function mutations in childhood T-cell acute lymphoblastic leukemia.

Interleukin 7 (IL-7) and its receptor, formed by IL-7Rα (encoded by IL7R) and γc, are essential for normal T-cell development and homeostasis. Here we show that IL7R is an oncogene mutated in T-cell acute lymphoblastic leukemia (T-ALL). We find that 9% of individuals with T-ALL have somatic gain-of-function IL7R exon 6 mutations. In most cases, these IL7R mutations introduce an unpaired cysteine in the extracellular juxtamembrane-transmembrane region and promote de novo formation of intermolecular disulfide bonds between mutant IL-7Rα subunits, thereby driving constitutive signaling via JAK1 and independently of IL-7, γc or JAK3. IL7R mutations induce a gene expression profile partially resembling that provoked by IL-7 and are enriched in the T-ALL subgroup comprising TLX3 rearranged and HOXA deregulated cases. Notably, IL7R mutations promote cell transformation and tumor formation. Overall, our findings indicate that IL7R mutational activation is involved in human T-cell leukemogenesis, paving the way for therapeutic targeting of IL-7R-mediated signaling in T-ALL.