Previous studies have established the contention that IKKβ functions downstream of PKCβ, CARMA1, Bcl10, and MALT1 on the route of BCR-mediated IKK–NF-κB activation (1
). In this study, we present evidence that, in response to BCR activation, IKKβ is not only required for IκB phosphorylation but also modifies assembly of the upstream CARMA1–Bcl10–MALT1 complex. Hence, we propose a model whereby two kinases, PKCβ and IKKβ, sequentially regulate the CARMA1–Bcl10–MALT1 complex. First, PKCβ-mediated phosphorylation of CARMA1 at Ser668 induces the primary CARMA1–Bcl10–MALT1 complex formation and IKKβ activation. Subsequently, IKKβ modifies phosphorylation of CARMA1 on key residues, including Ser578, and enhances the assembly of CARMA1–Bcl10–MALT1, resulting in an increased ability of CARMA1 to activate IKK. Although the data presented here reveal the involvement of PKCβ and IKKβ in the phosphorylation of CARMA1, other kinases also appear to participate in. For instance, phosphorylation of Thr119 still occurred even in the absence of either PKCβ or IKKβ. Moreover, as our study has focused on putative PKCβ-mediated phosphorylation sites on CARMA1, it remains possible that additional phosphorylation sites participate in IKK activation.
Given that the IKK complex is recruited to the CARMA1–Bcl10–MALT1 complex after antigen receptor stimulation, two, although not necessarily mutually exclusive, explanations for the effects of IKKβ on CARMA1 phosphorylation are possible. First, upon recruitment of IKKβ to the CARMA1–Bcl10–MALT1 complex, IKKβ would be activated, thereby directly phosphorylating CARMA1. Alternatively, because other kinases such as PKCβ are also thought to be recruited to the CARMA1–Bcl10–MALT1 complex, the recruited IKKβ to the same complex might regulate these kinases, thereby promoting phosphorylation of CARMA1. In this mechanism, the IKKβ kinase activity might not be necessarily required. In the case of phosphorylation of Ser578, we favor the former idea because of two lines of evidence: (a) the inhibition of phosphorylation on Ser578 in DT40 B cells harboring kinase-inactive IKKβ and (b) the capability of recombinant IKKβ to phosphorylate Ser578. However, the contribution of IKKβ to the initial phosphorylation of Thr119 and Ser668 in a kinase-independent manner suggests that the latter mechanism would also operate. Together, we would like to propose that IKKβ utilizes both mechanisms to promote phosphorylation of the upstream adaptor, CARMA1.
Ser578 does not mach the classical IKK consensus motif (DSΨXXS) found in all IκB proteins, β-catenin, and FOXO3a (14
). But, the conservation of this motif seems to reflect the constraints for recognition of SCF-βTRCP E3 ligase and subsequent proteosomal degradation rather than an IKK phosphoacceptor site. The identification of other IKKβ substrates will allow a better understanding about the molecular parameters for kinase recognition and IKKβ consensus sequences. Although IKKβ is one of the kinases responsible for phosphorylation of Ser578, the residual phosphorylation of this site in IKKβ-deficient DT40 cells was clearly observed, presumably because of PKCβ, because reduction of phosphorylation status at Ser578 was also reproducibly observed in PKCβ-deficient cells.
PDK1-knockdown Jurkat T cells, generated with the use of short hairpin RNA for PDK1, manifested severe defects in CD3/CD28-dependent NF-κB activation, although these knockdown cells apparently had remaining PDK1 (12
). Despite similar levels of remaining PDK1 between knockdown Jurkat cells and DT40 B cells conditionally deficient in PDK1, the mutant DT40 B cells demonstrated normal BCR-mediated NF-κB activation. This difference might reflect a distinct requirement for upstream signaling events in PKCβ and PKCθ activation between B and T cells, respectively; BCR-mediated phospholipase C γ activation might be sufficient for subsequent PKCβ activation, whereas PKCθ activation might require PI3K-mediated PDK1 activity in T cells, probably in addition to phospholipase C γ activation. Supporting this possibility, PDK1 is reported to associate with PKCθ in CD3/CD28-stimulated T cells (12
). Alternatively, as previously proposed (12
), recruitment of CARMA1 and PKCθ into the plasma membrane, presumably raft fractions, requires PDK1 activity in the case of T cell activation. Then, activated PKCθ catalyzes phosphorylation of CARMA1. In contrast, both functions might be exerted by PKCβ in B cells.
Ser575, Ser578, Ser631, Ser660, and Ser668 in the CARMA1 linker region are predicted to be potential PKCβ phosphorylation sites. Among these sites, mutation of Ser578 or Ser668 led to an almost complete defect in BCR-mediated IKK activation, whereas mutation of Ser575 or Ser631 resulted in its partial defect. Although not being formally proven, because of the lack of antibodies toward phospho-Ser575 and phospho-Ser631, the extent of phosphorylation of the S575A or S631A mutant was decreased, as determined by anti–phospho-Ser/-Thr antibody (unpublished data), suggesting that these sites are probably phosphorylated in BCR signaling. In regard to the functional importance of Ser668 (corresponding to Ser657 in mouse and Ser645 in human), two previous papers with Jurkat T cells demonstrated that this site was partially, rather than completely, involved in TCR/CD28-mediated IKK activation (9
). The reason why BCR-mediated IKK activation had a more stringent requirement for phosphorylation of Ser668 in DT40 B cells could be explained by the following three possibilities. First, this difference might simply reflect a species difference between chicken and mouse/human. Second, as discussed in the requirement for PDK1 between B and T cells, this difference might reflect a differential requirement for upstream kinases (PKCβ vs. PKCθ) in B and T cells, respectively. Ser668 could be used more dominantly as an in vivo phosphorylation site by PKCβ in B cells, rather than PKCθ in T cells. Finally, because we used the antigen receptor as a stimulant, in contrast to co-stimulation with antigen receptors and coreceptors (CD3/CD28) in the case of Jurkat T cells, phosphorylation of Ser668 might be used more stringently in the antigen receptor signaling context. As CD28 is known to enhance antigen receptor signaling, presumably through PI3K activation (17
), such augmented PI3K might lower the threshold for the requirement of Ser668 in IKK activation.
The available evidence indicates that Bcl10 is recruited to the CARD of CARMA1 and that MALT1 is recruited to Bcl10 through the binding of the MALT1 immunoglobulin domains to the region of Bcl10 located just C-terminal of the CARD (for review see reference 5
). Indeed, deletion of the CARD of CARMA1 blocked its association with Bcl10 and MALT1. More importantly, deletion or mutation of the CARD of CARMA1 abrogated CARMA1 function to activate NF-κB after TCR stimulation (7
). In the case of DT40 B cells, we also observed that the CARD deletion of CARMA1 almost failed to associate with Bcl10–MALT1 as well as to activate BCR-mediated IKK activation (unpublished data). Thus, the decreased association of the T119A, S578A, or S668A CARMA1 mutant with Bcl10–MALT1 is likely to be one of the major causes for why these mutants could not activate IKK. It has been proposed that the linker domain of CARMA1 in the unstimulated stage might bind to its own CARD through an intramolecular interaction (21
). Once Ser668 is phosphorylated, this interaction could be disrupted, thereby initiating exposure of the CARMA1 CARD to Bcl10. In addition, given the importance of forming homooligomerization of CARMA1 in NF-κB activation, phosphorylation of Ser668 may induce or stabilize CARMA1 oligomerization, which in turn contributes to CARMA1–Bcl10–MALT1 association (6
). Phosphorylation of Ser578 probably utilizes a similar mechanism to that of Ser668 to induce a conformational change of CARMA1. The location of Ser119 (between the N-terminal CARD–coiled-coil domains; ) might provide an insight into the action mechanism of its phosphorylation. Assuming that the coiled-coil domain binds to the CARD in the unstimulated state, this interaction could be disrupted by phosphorylation of Ser119, thereby contributing to exposing the CARD to Bcl10 as well as to forming homooligomerization of CARMA1.
Although we have revealed an as yet unappreciated role of IKKβ in amplifying the NF-κB signal by regulating the upstream complex, our study does not invalidate the role of IKKβ in dampening the NF-κB signal, as proposed in T cells. Recent papers have shown that IKKβ phosphorylates Bcl10, thereby inducing its degradation and disengagement of Bcl10 and Malt1 (13
). Collectively, it is reasonable to anticipate that IKKβ can induce both positive- and negative-feedback loops by phosphorylating upstream signaling molecules. Moreover, these feedback loops are considered to be required for the optimal activation of NF-κB induced by antigen receptor stimulation. Thus, further studies should be aimed at deciphering when and where these loops can operate, thereby changing the strength and duration of NF-κB signals.