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

Abstract

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-B, 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-B 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-B 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-B.Many signaling pathways that control cell proliferation and differentiation have evolved inhibitory checkpoints that prevent uncontrolled signaling. Checkpoints can involve inhibitory proteins that provide negative feedback once signaling begins or that actively suppress signaling prior to receptor engagement. For several pathways, checkpoint function is achieved by an autoinhibitory domain, which governs the activity of the key pathway component in which it is found. Such autoinhibitory domains have been described for several classes of signaling proteins, including tyrosine (1, 2) and serine/threonine kinases (3-5), guanine nucleotide exchange factors (6), phospholipases (7,8), and E3 ubiquitin ligases (9 -11), among others (12, 13). Although an autoinhibitory domain can provide robust, concentration-independent regulation in cis, the presence of an autoinhibitory domain in a signaling protein can make it especially susceptible to mutations that disrupt autoinhibition and lead to constitutive signaling and potentially pathological cell behavior. A mechanistic understanding of autoinhibitory domain function is thus critical for understanding how the output of signaling pathways is determined and how the dysregulation of autoinhibition by mutation can promote oncogenic transformation.

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

“…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.…”

“…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).…”

“…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).…”

993 CARD14 gain-of-function mutation alone is sufficient to drive IL-23/IL-17-mediated psoriasiform skin inflammation in vivo

“…In response to external stimuli, CARMA proteins are phosphorylated via protein kinase C isoforms (Scudiero et al, 2014). Subsequently, they undergo a conformational change, facilitating recruitment of the interacting partners Bcl10 (via CARD:CARD domain interactions) and Malt1 to form the CARMA:Bcl10:Malt1 (i.e., CBM) signaling complex, or "signalosome" (Bertin, 2001;Gaide et al, 2001;Howes et al, 2016;Jattani et al, 2016;McAllister-Lucas et al, 2001;Scudiero, 2014). CARD11 (CARMA1), expressed in lymphoid cells, is activated upon B-and T-cell receptor engagement, and gain-of-function (GoF) mutations in the genes encoding CARD11, Bcl10, and Malt1 have been associated with a number of lymphoid malignancies (Gaide, et al, 2002;Juilland and Thome, 2016;Pomerantz et al, 2002;Wang et al, 2002).…”