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

Abstract

The CARD11 signaling scaffold transmits signaling between antigen receptors on B and T lymphocytes and the transcription factor NF-B 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-B 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.

Molecular Determinants of Scaffold-induced Linear Ubiquitinylation of B Cell Lymphoma/Leukemia 10 (Bcl10) during T Cell Receptor and Oncogenic Caspase Recruitment Domain-containing Protein 11 (CARD11) Signaling

“…8A). Prior to TCR engagement, the REs in the CARD11 ID prevent the association of Bcl10 (17) and HOIP to CARD11. Upon receptor triggering, the autoinhibitory roles of the REs are neutralized, allowing HOIP recruitment to CARD11 in an interaction that involves the CARD11 coiled-coil and two separate regions of HOIP, one containing Ring Finger 1 in the RBR domain, and another containing the two Ndl4-like zinc fingers.…”

“…Expression Constructs-CARD11 constructs, including CARD11⌬ ID and double deletion constructs (15), and constructs encoding oncogenic CARD11 mutants (31) and RE mutants (16,17) have been previously described. pGEX4T-1-NEMO-UBAN (amino acids 242-350) was kindly provided by Jonathan D. Ashwell (NCI, National Institutes of Health, Bethesda), and this vector was modified by standard cloning techniques to generate pGEX4T-1-NEMO-UBAN 2 .…”

“…This neutralization of the ID causes it to disengage from the CARD, LATCH, and coiled-coil, which then allows these domains to recruit other signaling cofactors to CARD11, including Bcl10, TRAF6, NEMO, and caspase-8 (15). Recently, we reported that the inhibitory activity of the ID is accomplished by four REs, which function cooperatively with redundancy (16,17). The fact that HOIP and CARD11 associated in a signal-dependent manner suggested that the CARD11 ID and the REs within it might control CARD11 binding to HOIP.…”

“…In addition CARD11 is mutated with gain-of-function mutations in ϳ10% of human ABC DLBCL cases, which can account for their observed receptor-independent signaling to NF-B (24). These oncogenic gain-of-function CARD11 mutations are located in the CARD, LATCH, and coiled-coil domains of CARD11 (24 -29), and previous studies have demonstrated that they hyperactivate CARD11 activity by disrupting autoinhibition by the CARD11 ID (16,17,30,31). Interestingly, the most active oncogenic CARD11 mutations selectively enhance the ability of the protein to associate with Bcl10 but not to other signaling cofactors so far examined (30,31).…”

“…Prior to receptor signaling, the ID maintains CARD11 in a closed inactive state through intramolecular interactions that require the CARD and coiled-coil domains of CARD11 and that prevent the recruitment of signaling cofactors (15). The ID achieves its autoinhibition through four repressive elements (REs) that function cooperatively with redundancy (16,17). Upon antigen receptor engagement, the inhibitory activity of the REs is neutralized, in part through the phosphorylation of specific serines within the ID, allowing CARD11 to assemble a multiprotein signaling complex (15,18,19).…”