To test our hypothesis, we chose to characterize two of the most active CARD11 oncogenic mutants, F123I and L225LI (17
). The F123I mutation is located in the L1 region of CARD11 that we have previously defined between the CARD and Coiled-coil domains (6
). The L225LI mutation lies within the Coiled-coil domain.
To begin to address whether oncogenic CARD11 mutations interfere with ID function, we quantitatively determined the specific signaling activity of wild-type and mutant CARD11 variants in the absence and presence of the ID. If an oncogenic CARD11 mutation confers hyperactivity by interfering with ID function, then the effect of the mutation should be comparable to that of deleting the ID. Furthermore, the mutation should have a minimal effect in the absence of the ID.
We determined the relative specific activity of wild-type CARD11 and oncogenic mutants F123I and L225LI by titrating the expression of these proteins in an NF-κB activation assay in HEK293T cells that included the NF-κB reporter, Igκ2-IFN-LUC, and the CSK-LacZ normalization control vector. While HEK293T cells do not express CARD11 endogenously, they do express all of the factors that function downstream of CARD11 and that are required for CARD11-mediated NF-κB activation. Each expression vector was titrated under subsaturating conditions, and the signaling activity and levels of protein expression were quantitatively determined as described in Experimental Procedures. As shown in , the F123I mutation increased the specific activity of CARD11 by 12.6 fold, while the L225LI mutation increased activity by 38.1 fold. These effects are comparable to that achieved by the deletion of the ID, which conferred a 27.2 fold increase in specific activity (). The F123I and L225LI mutations had a much smaller effect in the absence of the ID. The ΔID-F123I variant displayed only a 1.9 fold enhanced activity, as compared to the ΔID, while ΔID-L225LI was only 2.0 fold more active than the ΔID (). Since the oncogenic mutations had a much larger effect in the presence of the ID than in the absence of the ID, the results are consistent with the hypothesis that these mutations interfere with the autoinhibitory action of the ID.
Figure 2 Effect of F123I and L225LI oncogenic mutations on CARD11 specific signaling activity. (A–C) HEK293T cells were transfected with 6 ng of pCSK-LacZ and 20 ng of Igκ2-IFN-LUC in the presence of the indicated amounts (in ng) of expression (more ...)
We have previously demonstrated that the ID can inhibit the ΔID when expressed in trans
by associating in a CARD- and Coiled-coil-dependent manner (6
). We tested whether the F123I and L225LI oncogenic mutations, in the context of the ΔID, would confer resistance to the ID inhibition in trans
, as predicted by our hypothesis. We titrated the expression of an ID-GST fusion protein in presence of either ΔID, ΔID-F123I, or ΔID-L225LI in Jurkat T cells, and determined that while the ΔID could readily be inhibited by the ID in trans
in a dose-dependent manner, the ΔID-F123I and ΔID-L225LI variants were not inhibited by the ID in trans
To address the effects of these mutations on the association of the ID with the ΔID, we co-expressed the ID-GST with either ΔID, ΔID-F123I, or ΔID-L225LI in HEK293T cells and determined the effect of the mutations on the ability of the ID-GST to bind the ΔID in a GST-pulldown assay. Both F123I and L225LI mutations reduced the association of the ΔID with the ID (), consistent with the hypothesis that these oncogenic mutations disrupt the intramolecular interaction between the ID and the Coiled-coil domain.
During antigen receptor signaling, the autoinhibitory effect of the ID on CARD11 scaffold activity is neutralized by the inducible phosphorylation of ID residues including S564, S577, and S657 (4
). This phosphorylation is thought to cause the ID to disengage from the CARD and Coiled-coil domains, allowing the recruitment of signaling proteins to CARD11 into a complex that activates the IKK complex. The mutation of either S564, S577, or S657 to alanine prevents their phosphorylation and renders CARD11 activity uninducible by upstream signaling (7
). A prediction of our hypothesis that oncogenic CARD11 mutations disrupt ID-mediated inhibition is that these oncogenic mutations should bypass the effect of serine-to-alanine mutations that render the ID resistant to phosphorylation-mediated neutralization. To test this prediction, we assayed the activity of wild-type and mutant CARD11 variants in an RNAi-rescue assay using Jurkat T cells in which endogenous CARD11 is knocked down by the stable expression of an shRNA (sihCARD11-2) that targets the human CARD11 mRNA. As shown in , the stable knockdown of CARD11 reduced the activation of NF-κB by anti-CD3/anti-CD28 treatment by ~75%, as compared to a control Jurkat T cell line, as previously described (6
). This effect could be rescued by the expression of wild-type murine CARD11, which is resistant to knockdown by this hairpin, but not by the S564A mutant of murine CARD11, which cannot support ID neutralization. As predicted by our hypothesis, the effect of the S564A mutation was completely bypassed by both F123I and L225LI oncogenic mutations. When expressed in the sihCARD11-2 line, the F123I S564A double mutant displayed an enhanced activity that was similar to that observed with the ΔID and the F123I single mutant, and this activity was not further enhanced by anti-CD3/anti-CD28 crosslinking (). The L225LI S564A double mutant also displayed an activity indistinguishable from the L225LI mutant and was also not further enhanced by TCR engagement (). Importantly, the S564A mutation did not affect protein expression levels in the context of wild-type, F123I, or L225LI variants (). These results indicate that both F123I and L225LI mutations obviate the need for the TCR-induced PKCθ-mediated ID phosphorylation and ID neutralization that is required for activation of wild-type CARD11 during signaling. The data support the hypothesis that the oncogenic mutations disrupt the inhibitory intramolecular interaction mediated by the ID.
Figure 4 F123I and L225LI oncogenic mutations bypass the need for ID neutralization in T cells. (A) Wild-type Jurkat T cells, or Jurkat T cells stably expressing the sihCARD11-2 hairpin that targets human CARD11, were transfected with 200ng pCSK-LacZ, 1800 ng (more ...)
Since it has been suggested that oncogenic mutations result in the aggregation of CARD11 (17
) we tested the effect of the F123I and L225LI mutations on the ability of FLAG-CARD11 to associate with myc-CARD11 in an immunoprecipitation assay in HEK293T cells under subsaturating conditions (). The F123I mutation had no apparent effect on the extent of CARD11 oligomerization, while the L225LI mutation displayed a modest, but reproducible three-fold increase in apparent oligomerization. Since one mutation affected oligomerization while the other did not, the data suggest that the enhancement of CARD11:CARD11 association is not an obligate part of the mechanism by which oncogenic mutations in CARD11 increase activity. However, it remains possible that the increased apparent oligomerization of the L225LI variant may contribute to the enhanced activity of that variant.
We have previously demonstrated that the ID controls the recruitment of Bcl10, TAK1, TRAF6, IKKγ, and Caspase-8 to CARD11 (6
). The hyperactive ΔID variant can constitutively associate with these proteins and behaves as a CARD11 in which the ID has been neutralized by upstream signaling. An important prediction of the hypothesis that the oncogenic mutations interfere with ID function is that these mutations should lead to an enhanced ability of CARD11 to recruit signaling proteins whose association is regulated by the ID. Using an immunoprecipitation assay in HEK293T cells, we first tested whether the F123I or L225LI mutations increased the apparent affinity of CARD11 for Bcl10. We expressed FLAG-Bcl10 with myc-tagged wild-type CARD11, ΔID, the F123I mutant, or the L225LI mutant and assayed the extent to which each CARD11 variant coimmunoprecipitated with Bcl10 in an anti-FLAG IP. As shown in , both the F123I and L225LI mutants displayed an enhanced ability to associate with Bcl10, indistinguishable from that of the ΔID ( cf. lanes 1–4).
We then tested whether the F123I or L225LI mutations enhance affinity for Bcl10 in the absence of the ID. Both the ΔID-F123I and ΔID-L225LI variants did display a slightly higher apparent affinity for Bcl10 in the coimmunoprecipitation assay, as compared to the ΔID (, cf. lanes 13–24), indicating that each mutation does modestly enhance Bcl10 association in an ID-independent manner. However, the effect of these mutations was much larger in the presence of the ID (, cf. lanes 1–12), than in the absence of the ID (, cf. lanes 13–24). These data support the hypothesis that the main effect of the mutations is to disrupt ID-mediated intramolecular interactions to expose a surface on CARD11 for Bcl10 binding. The minimal effects in the absence of the ID indicate that the mutations do not dramatically increase the affinity of CARD11 for Bcl10 at the CARD11:Bcl10 protein-protein interface.
We next tested whether the F123I or L225LI mutants displayed enhanced apparent affinities for other protein cofactors that we have previously shown to associate with CARD11 in an ID-regulated manner. Surprisingly, and in contrast to the predictions of our hypothesis, neither F123I nor L225LI mutants displayed enhanced associations with any of the other FLAG-tagged cofactors tested, including TRAF6 ( cf. lanes 5–8), TAK1 ( cf. lanes 5–8), IKKγ ( cf. lanes 5–8), and Caspase-8 ( cf. lanes 5–8). These results indicate that the oncogenic mutations F123I and L225LI selectively disrupt ID function. While the mutations disrupt the ability of the ID to prevent Bcl10 association with CARD11, they do not appear to affect the inhibitory action of the ID on the association of other signaling proteins.
To test whether Bcl10 binding to the F123I or L225LI mutants could influence the recruitment of other cofactors, we also coexpressed FLAG-tagged TRAF6, IKKγ, Caspase-8, or TAK1, with the F123I and L225LI mutants in the presence of overexpressed untagged Bcl10. Even in the presence of high concentrations of Bcl10, none of these cofactors were recruited to the F123I or L225LI mutants at concentrations at which they clearly associated with the ΔID ( cf. lanes 1–4). The results confirm that ID-mediated inhibition of CARD11 binding to TAK1, TRAF6, Caspase-8, and IKKγ is intact in the F123I and L225LI oncogenic mutants, and that these mutants have a selective, enhanced ability to recruit Bcl10.
Since MALT1 is an obligate signaling cofactor that directly associates with Bcl10 (20
), we tested whether Bcl10 could mediate the recruitment of MALT1 to the F123I and L225LI mutants. We coexpressed HA-tagged MALT1 with myc-tagged wild-type CARD11, the ΔID, or the F123I or L225LI mutants in the absence and presence of FLAG-tagged Bcl10 and conducted an anti-HA coimmunoprecipitation assay (). MALT1 did not associate with any of the CARD11 variants in the absence of overexpressed Bcl10 (, cf. lanes 5–8). In the presence of overexpressed Bcl10, MALT1 did not associate with wild-type CARD11, but did associate with the F123I and L225LI mutants to an extent that was comparable to that observed with the ΔID (, cf. lanes 1–4). Thus, the Bcl10 that is selectively recruited to the F123I and L225LI mutants is competent to co-recruit MALT1.
T cell receptor signaling to the IKK complex has been shown to require the ubiquitination of Bcl10 on lysines 31 and 63, which facilitates the transient association of Bcl10 with IKKγ during IKK activation (11
). To test whether the F123I and L225LI CARD11 variants also depend on this modification of Bcl10, we first assessed the NF-κB-inducing activity of these mutants in an HEK293T cell line, KD-Bcl10, which stably expresses an shRNA that targets the Bcl10 mRNA and results in ~90% Bcl10 knockdown (6
). We compared activities in the KD-Bcl10 line to that observed in the KD-GFP HEK293T cell line, which stably expresses an shRNA that targets GFP. As expected, Bcl10 deficiency impaired the ability of both F123I and L225LI mutants to activate the Igκ2
-IFN-LUC reporter, indicating that both variants require Bcl10 for signaling activity (). Assessment of F123I and L225LI activities in KD-GFP and KD-Bcl10 Jurkat T cell lines revealed that Bcl10 was also required for the full activities of these oncogenic mutants in Jurkat T cells (). We next compared wild-type Bcl10 with the K31R, K63R double mutant for the potential to rescue signaling in the KD-Bcl10 HEK293T and Jurkat T cell lines. The Bcl10 K31R, K63R mutant has previously been shown to be functionally deficient in TCR signaling (11
). At comparable levels of expression, the K31R, K63R double mutant was less able than wild-type Bcl10 to rescue signaling downstream of either the F123I or L225LI CARD11 variants in either HEK293T cells () or Jurkat T cells (). These data suggest that both of these oncogenic mutants depend upon the same ubiquitination of Bcl10 that occurs during physiological TCR signaling.
Figure 10 Bcl10 residues K31 and K63 are required for maximal signaling by F123I and L225LI oncogenic mutants. (A) KD-GFP or KD-Bcl10 HEK293T cell lines were transfected as in with 2–8 ng of myc-F123I or 1–8 ng of myc-L225LI, and the indicated (more ...)