CARD11 is a critical adapter molecule that functions to couple the recognition of antigen and costimulatory signals at the surface of the T cell to the induction of NF-κB activity that is required for T-cell activation and proliferation in the adaptive immune response. As a signaling adapter, CARD11 must contain determinants that allow it to receive pathway-specific upstream signals and transmit the information to non-pathway-specific molecules that are used widely for the activation of the IKK complex and NF-κB.
Our results illuminate how CARD11 is converted from an inactive state to an active signaling scaffold so that upstream TCR signaling is translated into the assembly of an active signaling complex (Fig. ). Prior to signaling, the ID interacts with the CARD and the coiled-coil domain to prevent the recruitment of signaling cofactors to CARD11. The interaction of the ID with the CARD and the coiled-coil domain may occur within the same CARD11 molecule or between two or more oligomerized molecules of CARD11. Signaling downstream of the TCR results in the neutralization of the activity of the ID, which requires the phosphorylation of the ID on serine residues 564 and 657 by PKCθ and on serine 577 by an undetermined kinase (32
). Once the ID is neutralized, the CARD, L1, and coiled-coil domains present surfaces for the recruitment of Bcl10, TAK1, TRAF6, caspase-8, and IKKγ. MALT1 is presumably recruited through its interaction with Bcl10, and caspase-8 stably associates during signaling in a Bcl10- and MALT1-dependent manner. These factors then function to promote the activation of the IKK complex and NF-κB, which other investigators have shown to involve the TRAF6-mediated ubiquitination of MALT1 (43
) and IKKγ (59
), the MALT1-mediated ubiquitination of IKKγ (73
), the ubiquitination of Bcl10 by an undetermined ligase (71
), the TAK1-mediated phosphorylation of IKKβ (52
), and the proteolytic activity of caspase-8 on an undefined substrate (58
FIG. 15. Model of the signal-induced conversion of CARD11 to an active signaling scaffold. Prior to TCR engagement, the ID of CARD11 interacts with the CARD and the coiled-coil (CC) domain to prevent the association of signaling cofactors. Although it is depicted (more ...)
It is important to note that our association studies do not distinguish between direct and indirect association between CARD11 and these cofactors since the experiments were performed using cell lysates. Other proteins that are present in HEK293T and Jurkat T cells may bridge the interactions we studied.
The determinants in CARD11 that control its association with Bcl10, TAK1, TRAF6, IKKγ, and caspase-8 lie within the CARD-L1-coiled-coil region (Fig. ). The ΔID hyperactive variant, which corresponds to activated CARD11 in which the ID has been neutralized, required both the CARD and the coiled-coil domain to associate with Bcl10. The TAK1 association required only the CARD, while the TRAF6 association required the CARD, L1, and coiled-coil domains. The association of IKKγ and caspase-8 required the coiled-coil domain and, to a lesser extent, the CARD. Bcl10, TAK1, and IKKγ have previously been shown to associate with CARD11 in an inducible manner during antigen receptor signaling (17
). Our observation that the ΔID had an enhanced ability to associate with TRAF6 and caspase-8 compared to wild-type CARD11 predicted that both of these proteins would also associate with CARD11 in T cells in a signal-dependent manner. This was indeed the case (Fig. ). Caspase-8 and TRAF6 have been shown to interact, and this interaction may be important for signaling events that occur subsequent to their recruitment to CARD11 (4
Although the ID regulates the association of multiple cofactors with CARD11, it does not appear to regulate CARD11 oligomerization. Both wild-type and ΔID variants of CARD11 appeared to have equivalent capacities to self-associate (Fig. ). We have shown that the coiled-coil domain is targeted by the ID, and Tanner et al. have demonstrated that the coiled-coil domain is required for CARD11 oligomerization (62
). This indicates that CARD11 oligomerization is likely mediated by surfaces of the coiled-coil domain that are not engaged by the ID and is not influenced by signals that neutralize activity of the ID.
Recent models have proposed that during TCR signaling, Bcl10 is recruited to CARD11 and functions to recruit other signaling cofactors into a CARD11-containing complex so that IKK complex activation may proceed (43
). Our data in HEK293T cells indicate that once the ID is neutralized, CARD11 has an intrinsic enhanced affinity for TAK1, TRAF6, caspase-8, and IKKγ and can recruit these molecules in a Bcl10-independent manner (Fig. to ).
In Jurkat T cells, the signal-induced recruitment of TRAF6 and the IKK complex to CARD11 were largely Bcl10 and MALT1 independent (Fig. and ), consistent with a Bcl10- and MALT1-independent ability of CARD11 to associate with these cofactors once the ID is neutralized by upstream signaling. In contrast, the inducible association of caspase-8 with CARD11 in T cells exhibited a much greater dependence on Bcl10 and MALT1 than the other factors examined (Fig. ). This observation was surprising since the ΔID could associate with caspase-8 C360S in HEK293T cells in a manner that was not affected by the ~90% reduction in Bcl10 or MALT1 levels. The difference might be explained by the fact that we assayed association in HEK293T cells using the catalytically inactive C360S variant of caspase-8. Caspase-8 is known to undergo conformational changes after the activation of its proteolytic activity (6
). Since caspase-8 is activated during TCR signaling, it is likely that the associations we detected in Jurkat T cells involved catalytically active caspase-8. It is possible that the inactive conformation of caspase-8 can be recruited to CARD11 during signaling in a Bcl10-independent manner and then convert to the active conformation which stably associates with CARD11 through Bcl10 and MALT1. We were not able to reproducibly assay the signal-induced recruitment of TAK1 to CARD11 in Jurkat T cells with the available anti-TAK1 antibodies. However, the CARD11-TAK1 association was not as inhibited by the ID in cis
as were the other cofactor associations, and TAK1 required only the CARD for association with the ΔID, while the other factors required other domains. These data strongly suggest that TAK1 is recruited to CARD11 in a manner that is independent of the other factors we studied.
The association of Bcl10, TAK1, TRAF6, IKKγ, and caspase-8 with the CARD and with the L1 and coiled-coil domains explains why these domains of CARD11 are required after the inhibitory activity of the ID is neutralized by upstream signals. We also demonstrated that each of the L3, SH3, L4, and GUK domains of CARD11 is also necessary for signaling after ID neutralization. Each of these domains is required for PMA/ionomycin-mediated signaling to NF-κB in T cells, and each is required for constitutive signaling by the CARD11 ΔID (Fig. ).
The L3, SH3, L4, and GUK domains may mediate the recruitment of other factors required for IKK complex activation, may target CARD11 and associated factors to a cellular locale that is required for signaling, or may promote conformational states of the CARD and the coiled-coil domain that are required to bind cofactors that we did not study. All of these domains are required for the activity of the ΔID in both lymphoid and nonlymphoid cells, indicating that they function in concert with factors that are not specific to the antigen-receptor signaling pathway and do not depend on the specific spatial architecture of the immunological synapse. The SH3 domain has been shown to be involved in membrane recruitment of CARD11 (67
), and it is likely that membrane recruitment of CARD11 is required for PKCθ to act on the ID. However, it is not clear whether SH3-mediated membrane association or a distinct function of the SH3 domain is required for events downstream of PKCθ action. The PDZ-L3-SH3 region of CARD10 (also called CARMA3) has been demonstrated in a yeast two-hybrid assay to present a binding surface for IKKγ (56
); however, our results suggest that these domains in CARD11 are not required for IKKγ association, which requires the coiled-coil domain and CARD instead. The CARD11 GUK domain has been shown to bind PDK1 (27
), a kinase that functions in this pathway in PKCθ activation and in recruitment of CARD11 to lipid rafts in T cells. Since we demonstrate a role for the GUK domain subsequent to ID neutralization, other GUK-binding proteins are likely to be involved in this pathway. Finally, the adhesion and degranulation-promoting adapter protein (ADAP) has been shown to interact with the C-terminal half of CARD11 containing the PDZ, L3, SH3, L4, and GUK domains (33
). Since ADAP is required for TCR signaling to NF-κB, this protein may explain the requirement for one or more of these CARD11 domains in this pathway in T cells. However, since ADAP is expressed in a tissue-restricted fashion and since L3, SH3, L4, and GUK domains function in both lymphoid and nonlymphoid cells, other proteins are likely to interact with these domains during CARD11-mediated signaling.
The integrative signaling function of CARD11 that is required for normal antigen receptor signaling is also likely to play a critical role in the progression of some types of cancer. Lymphomas of mucosa-associated lymphoid tissue (MALT lymphoma) are associated with chromosomal translocations that lead to the overexpression of Bcl10 or MALT1 or to the expression of a hyperactive API2-MALT1 fusion protein (1
). It is possible that in MALT lymphoma the association of these molecules with CARD11 is required for their dysregulated signaling to NF-κB, which is thought to contribute to lymphoma progression. The activated-B-cell-like subtype of diffuse large B-cell lymphoma displays aberrant activation of the IKK complex and NF-κB in a manner that is required for proliferation and survival (10
). Recently, CARD11 has been shown to be a required upstream signaling molecule in this lymphoma (42
), and missense mutations in CARD11 have been found in ~10% of activated-B-cell-like diffuse large B-cell lymphoma tumor biopsies (28
). Intriguingly, several of these missense mutations increase the signaling ability of CARD11 and map to the coiled-coil domain (28
). Based upon our studies, we suggest that oncogenic coiled-coil mutations could lead to enhanced signaling activity, most likely by disrupting the inhibitory interaction that we have described between the ID and the coiled-coil domain. Alternatively, one or more of these mutations could enhance the affinity of CARD11 for Bcl10, IKKγ, TRAF6, or caspase-8. Each of the surfaces that CARD11 uses to coordinate signaling to NF-κB may provide a target for therapeutic amelioration of lymphomas that depend on CARD11 signaling. Importantly, since CARD11 appears to be critical in a limited number of NF-κB-inducing pathways, pharmacological agents that target the scaffolding function of CARD11 might not perturb the important biological effects of the diverse array of physiological stimuli that regulate NF-κB in other contexts.