CARMA1 in the Development and Function in T and B Lymphocytes
Genetic studies using knockout mice of CARMA1, BCL10, and MALT1 as well as a CARMA1-deficient Jurkat cell line have demonstrated an essential role of CBM complex in antigen receptor signaling (Fig. 2a). The T-cell phenotype of CARMA1-deficient (CARD11−/−) mice closely resembles that of BCL10-deficient (BCL10−/−) and of MALT1-deficient (MALT1−/−) mice (Hara et al. 2003; Ruefli-Brasse et al. 2003; Ruland et al. 2001, 2003). Peripheral mature T cells from these knockout mice show almost complete abrogation of proliferation and cytokine production upon stimulation through TCR or with a direct protein kinase C (PKC) activator, PMA plus Ca2+-ionophore (P/I). Accordingly, these mice exhibit severely impaired T-cell immunity. The loss of CBM molecules abrogates TCR-or P/I-induced NF-κB activation owing to defective activation of I-κB kinase (IKK), whereas calcium mobilization and proximal tyrosine phosphorylation are unaffected. In addition, CARMA1 and MALT1 deficiency affect the activation of the MAPK JNK, particularly JNK2, but not the other MAPKs, Erk, and p38.
The CBM deficiency does not affect overall development of thymocytes, with normal numbers of the CD4+CD8+, CD4+CD8−, and CD4−CD8+ cells, but increased CD4−CD8− cells for unclear reasons (Hara et al. 2003; Ruland et al. 2001). No overt developmental defects in conventional CD4 and CD8 T cells are observed in the peripheral lymphoid organs of these knockout mice; however, the number of natural-occurring regulatory T cells (nTregs) is markedly reduced both in the thymus and periphery (Molinero et al. 2009; Schmidt-Supprian et al. 2004). This phenotype is likely attributed to the reduced expression of the transcription factor Foxp3, the master regulator of Treg development, in thymic nTreg precursor cells in CARD11−/− mice because the TCR-induced NF-κB activation directly promotes the transcription of Foxp3 (Long et al. 2009).
Deficiency of CBM molecules in B cells, similar to that in T cells, results in abrogated BCR-induced NF-κB activation and thereby defects in B-cell proliferation and survival, although one line of MALT1−/− mice has exhibited only mild defects in B-cell activation (Fig. 2a) (Hara et al. 2003; Ruefli-Brasse et al. 2003; Ruland et al. 2001, 2003). Reduced follicular (FO) and marginal zone (MZ) B cells in spleen and an almost complete absence of peritoneal B-1 B cells have been consistently observed in CARD11−/−, BCL10−/−, and MALT1−/− mice. CARMA1 and probably BCL10 control JNK activation through BCRs, whereas MALT1 might be dispensable for it. CD40-induced proliferation is also defective in splenic B cells with CBM deficiency, possibly owing to the defective development of MZ B cells, which are the major cells responding to CD40 stimulation.
The involvement of CBM complex in toll-like receptor (TLR) signaling in B cells has been suggested, although it remains a controversial issue. CARD11−/− whole splenic B cells show impaired proliferation in response to LPS (Hara et al. 2003). A study that compared FO and MZ splenic B cells revealed that BCL10 deficiency affected only MZ B cells responding to LPS due to impaired NF-κB activation (Fig. 2a) (Xue et al. 2003).
In addition to the essential role of CBM complex in canonical NF-κB signaling downstream of TCRs and BCRs, it also acts in the noncanonical NF-κB pathway through B-cell activation of the TNF family (BAFF) receptor, which regulates the survival of MZ B cells (Fig. 2a). Lack of MALT1 impairs BAFFR-induced phosphorylation and degradation of NF-κB2 precursor p100 (Tusche et al. 2009). The MALT1−/− MZ but not FO B cells exhibit reduced survival and anti-apoptotic gene induction in response to BAFF in vitro, likely owing to the elevated expression and defective BAFFR-induced downregulation of TRAF3, a negative modulator of the BAFFR-induced survival signal particularly in MZ B cells. The phenotypes of BAFF-Tg mice, including increased basal serum Ig, MZ B cells and B1 B cells, spontaneous germinal center formation, and Ig deposition in the kidney, all disappear in the absence of MALT1 or BCL10.
CARMA1 in NK-Cell Development and Function
Upon triggering of activating NKRs, NK cells attack targets through two defined effector functions: the cytotoxicity and the production of pro-inflammatory cytokines and chemokines. Studies have revealed that CARMA1, BCL10, and MALT1 are essential for production of cytokines and chemokines induced by multiple activating NKRs, including FcγRIII, NK1.1, Ly49H, Ly49D, and NKG2D; in contrast, the cytotoxicity of NK cells induced by these activating NKRs is not affected by CBM deficiency (Fig. 2b) (Gross et al. 2008; Hara et al. 2008). CBM deficiency does not influence either maturation or the repertoire formation of peripheral NK cells. The loss of CBM results in impaired NF-κB activation following activation of NKRs, whereas Vav1 phosphorylation and Ca2+ mobilization, both of which regulate exocytosis of lytic granules, are unaffected. Contribution of CBM to MAPK activation remains controversial. Similar to T cells, PKCθ activity is required for NF-κB activation through activating-NKR. TNF- or IL-18R-mediated NF-κB activation does not require CBM in NK cells.
Signaling Regulation of CBM
Multiple regulation mechanisms, involving phosphorylation, ubiquitylation, oligomerization, caspase activation, and recruitment to plasma membrane, have been proposed to control CBM-signaling (Fig. 1) (Hara et al. 2010).
Upon activation of antigen receptors, CARMA1 and BCL10 are phosphorylated by several kinases. Phosphorylation of CARMA1 by PKCθ (mainly in T cells) and PKCβ (mainly in B cells) in the PKC-regulated domain (PRD) likely transform CARMA1 to an active one that is accessible to BCL10 and other downstream molecules. Phosphorylation of CARMA1 by CaMKII facilitates the interaction between CARMA1 and BCL10. CaMKII also phosphorylates BCL10 but this phosphorylation is involved in the attenuation of the signaling. HPK1 phosphorylates CARMA1 within the PRD and is involved in both JNK and NF-κB activation although the precise mechanism is unclear. Phosphorylation of CARMA1 by IKKβ promotes signaling activation by enhancing the assembly of CBM complex. IKKβ also phosphorylates BCL10 within the MALT1-interacting S/T-rich domain and within CARD upon TCR stimulation. The former interferes with IKK ubiquitination by causing disengagement of BCL10 from MALT1, and the latter induces BCL10 degradation in the proteasome, thus negatively regulating the signaling. CK1α associates with the PRD of CARMA1after TCR stimulation and contributes to initial NF-κB activation; however, subsequent phosphorylation of the PRD by CK1α contributes to the negative feedback of the signaling.
Upon receptor stimulation, CARMA1 recruits downstream molecules and triggers oligomerization and ubiquitination cascades. BCL10-dependent MALT1 oligomerization induces activation of the E3 ubiquitin ligase TRAF6, which in turn activates the IKK complex through lysine (K) 63-linked ubiquitylation of the regulatory subunit of IKK NEMO. BCL10 and MALT1 also undergo K63-linked ubiquitination in the CARD domain and the C-terminal region, respectively, upon T-cell activation, which provide docking surfaces for the recruitment of NEMO. MALT1 itself has an E3-ligase activity and targets MALT and NEMO for ubiquitination. In contrast, ubiquitination also acts for signaling inhibition. CARMA1 is K48-linked-polyubiqutinated after receptor stimulation, leading to degradation of CARMA1 in the proteasome, which is dependent on the phosphorylation by PKCs on PRD. cIAP likely targets this ubiquitination of CARMA1. The E3 Cbl-b promotes mono-ubiquitination of CARMA1, which is involved in the anergy induction in NK T cells. BCL10 undergoes degradation following ubiquitination of CARD after receptor stimulation, which contributes to the termination of signaling. NEDD, cIAP, β-TrCP, and Itch have been suggested as E3 ubiquitin ligases of BCL10. The CNS5 and CNS2 of the COP9 signalosome fine-tune IKK activation by interfering with the polyubiquitination and degradation of BCL10. The deubiquitinating enzyme A20 catalyzes the removal of the K63-linked ubiquitin chains on MALT1 and therefore regulates the duration and strength of signals.
MALT1 and CARMA1 interact with Caspase-8 and thereby regulate the Caspase-8-c-FLIPL-mediated NF-κB activation pathway. Paracaspase activity of MALT1 fine-tunes CBM-signaling by cleaving BCL10 and A20. The BCL10 cleavage is required for TCR-induced cell adhesion to the extracellular matrix protein fibronectin. The cleavage of A20 by MALT1 disrupts the inhibitory effect of A20.
Membrane recruitment of signaling components is a crucial event in CBM-mediated NF-κB activation. BCL10, PKCθ, PKCβ, MALT1, pro-caspase-8, c-FLIPL, and the IKK complex are recruited into lipid rafts after antigen receptor stimulation. CARMA1 resides in both the cytoplasm and lipid rafts in resting cells, but the amount in lipid rafts increases after activation. CARMA1 controls the recruitment of PKCθ, BCL10-MALT1, and IKK complexes to lipid rafts. The adapter protein ADAP acts as a linker between the TCR-ZAP-70-SLP-76 signaling complex and CBM by binding to CARMA1. PDK1 recruits PKCθ and CARMA1 to lipid rafts upon TCR stimulation.
CBM in Lymphomas
The chromosomal translocations, t(11;18)(q21;q21), t(1;14)(p22;q32) and t(14;18)(q32;q21), have been well characterized in MALTL. MALT1 gene was originally identified in the break point of t(11;18)(q21;q21) (Du 2007). This translocation generates API2-MALT1 fusion products comprising the N-terminus of API2 and the C-terminus of MALT1. The fusion product, but neither API2 nor MALT1 alone, is capable of activating NF-κB. The translocations, t(1;14)(p22;q32) and t(14;18)(q32;q21), bring the BCL10 and MALT1 genes under the regulatory control of the Ig heavy chain (IgH) enhancer, respectively, leading to dysregulated expression of these genes and aberrant NF-κB activation. In addition, BCL10 gene amplification has been reported in pancreatic cancer and nodal diffuse large B-cell lymphoma (DLBCL). Similarly, MALT1 gene amplification was found in cell lines of MZ B-cell lymphoma and DLBCL. While normal B cells express BCL10 in the cytoplasm, MALTL cells bearing t(11;18)(q21;q21) and t(1;14)(p22;q32) express the protein predominantly in the nucleus, indicating a possible relationship between aberrant BCL10 nuclear localization and tumorigenesis.
Among the subtypes of DLBCL, the least curable activated-B-cell-like (ABC) subtype DLBCLs, but not the germinal center B-cell-like (GCB) subtype, rely on constitutive NF-κB signaling for survival (Hara et al. 2010). A loss-of-function RNA interference screen for genes required for survival of ABC DLBCLs revealed that CARMA1 is a key upstream signaling component responsible for the constitutive IKK activation in ABC DLBCLs but not GCB DLBCLs (Ngo et al. 2006). In line with this, oncogenic missense mutations of CARMA1 gene, all within exons encoding the C-C, have been found in ABC DLBCLs (Lenz et al. 2008). These mutations constitutively activate the NF-κB pathway and enhance antigen receptor signaling to NF-κB, possibly owing to aggregate the formation of the mutant proteins. The oncogenic forms of CARMA1 promote proteolytic activity of MALT1. Inhibition of MALT1 activity with the inhibitor z-VRPR-fmk specifically affects the growth and survival of ABC DLBCLs.
The CBM-regulated BAFFR signaling also contributes to the development of B lymphomas. BCL10-transgenic mice elevate BAFF expression and specifically promote survival of MZ B cells, and some mice develop splenic MZ lymphomas (MZL) (Li et al. 2009). BAFF overexpression, with concomitant nuclear expression of BCL10 and NF-κB activation, is associated with Helicobacter pylori-independent growth of gastric DLBCL with histological evidence of MALTL (Kuo et al. 2008).
NF-κB plays a central role in the activation and survival of lymphocytes. CARMA1 is a CARD-MAGUK family adaptor protein originally found as a binding partner of BCL10 via CARD-CARD interaction. CARMA1 and the MALTL-related proteins BCL10 and MALT1 form so-called CBM complex following receptor stimulation. CBM complex is essential for the canonical NF-κB activation signaling through TCRs, BCRs, and activating NKRs, as well as for the noncanonical NF-κB signaling through BAFF, thereby regulating proliferation, survival, and effector functions of T, B, and NK cells. Multiple regulation mechanisms involving phosphorylation, ubiquitylation, oligomerization, caspase activation, and recruitment to plasma membrane have been proposed to control NF-κB activation signaling through CARMA1. The chromosomal translocations and the amplification of MALT1 and BCL10 genes are associated with the development of MALTL and DLBCL. ABC DLBCLs, but not GCB DLBCLs, rely on constitutive NF-κB activation via CBM-signaling for its survival. Oncogenic missense mutations of CARMA1 gene within the coiled-coil domain, leading to constitutive activation of the NF-κB pathway and enhancing antigen receptor signaling to NF-κB, have been found in ABC DLBCL. The CBM-regulated BAFF signaling also contributes to the development of MALTL, MZL, and DLBCL. Thus, CBM-signaling may be a promising therapeutic target of specific B lymphomas.
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