2.1 Historical perspective
Malt1 was initially identified as a component of an oncogenic protein fusion commonly expressed in a subset of MALT lymphomas. Several reports identified a chromosomal translocation, t(11;18)(q21;21), which was found to involve the gene encoding the cIAP2 anti-apoptotic protein, and the novel MLT gene [1
]. The resulting protein fusion product combined the C-terminal domain of the MLT protein (hereafter referred to as Malt1 [4
]) with the N-terminus of the cIAP2 protein , a member of the cellular inhibitor of apoptosis protein family (). This protein fusion was found to potently activate NF-κB, a transcription factor of central importance in many biological processes [5
]. Initially, it was unclear how the wild-type Malt1 protein activated NF-κB, as transfection of cells with full-length Malt1 alone did not cause measureable NF-κB activation [5
]. However, early studies revealed that Malt1 binds to the protein Bcl10 [6
], and that over-expressed Bcl10 spontaneously oligomerizes in a manner dependent upon the Bcl10 caspase recruitment domain (CARD) [7
]. Furthermore, induced oligomerization of the Malt1 C-terminal domain was shown to stimulate potent NF-κB activation [6
]. Additional data demonstrated that cIAP2-Malt1 exhibits spontaneous multimerization in a manner dependent on the N-terminal BIR domains, contributed by cIAP2 [8
]. Together, these data suggested a model in which very similar mechanisms of NF-κB activation are employed by cIAP2-Malt1 and wild-type Malt1: namely, spontaneous multimerization of cIAP2-Malt1, and TCR- and Bcl10-dependent multimerization of wild-type Malt1, stimulate NF-κB activation [6
Domain structure of cIAP2, Malt1, and the cIAP2-Malt1 chimera
Further experimental evidence suggested that Bcl10 and Malt1 cooperate with Carma3 [9
] or Carma1 [10
] to form a trimeric protein complex (the CBM), implicated in NF-κB activation. Other data suggested that Malt1 multimerization is Bcl10-dependent, and that formation of Bcl10/Malt1 multimers is required for antigen-receptor mediated activation of NF-κB [11
]. Mechanistically, the model of TCR- and BCR-induced multimerization of the Bcl10/Malt1 complex for transducing NF-κB activating signals is both similar to and consistent with the model of NF-κB activation by steady-state oligomerization of the cIAP2-Malt1 fusion protein [6
]. However, given that the activity of the cIAP2-Malt1 is independent of any upstream activators, while the activity of the wild-type Malt1 protein is strictly dependent on upstream signaling through Bcl10, there is reason to speculate that the mechanisms by which these two molecules effect NF-κB activation may differ significantly. Indeed, accumulating evidence suggests that Malt1 and cIAP2-Malt1 utilize quite distinct mechanisms to regulate NF-κB signal transduction.
2.2 Functional domains of the Malt1 and cIAP2-Malt1 proteins
Malt1 is a member of a family of caspase-like cysteine proteases called paracaspases, which are found in organisms ranging from the slime mold, Dictyostelium
, to man [5
]. As shown in , several distinct functional domains have been identified in the Malt1 protein. The N-terminal region of the protein is involved in interaction with Bcl10, with both the Ig-like domains [6
] and the death domain [14
] contributing to this association. Downstream of the Bcl10-interacting region is the caspase-like “paracaspase” domain, which has homology to the caspase and metacaspase families of cysteine proteases, particularly in regions essential for proteolytic activity [5
]. Although this caspase-like module was recognized shortly after the initial identification of the Malt1 gene, a proteolytic function was only recently documented [16
]. The caspase-like domain of Malt1 (aa 326–567) is sufficient for binding to caspase-8, with Leu359 being essential for biochemical association between Malt1 and caspase-8 [18
]. Malt1 has also been shown to interact with the coiled-coil domain of Carma1, via an undefined sub-domain within the C-terminal two-thirds of the molecule (aa 333–824) [10
]. A binding site for A20, a negative modulator of NF-κB signaling and a target for Malt1 proteolysis, is also within this same region of Malt1 [16
]. C-terminal to the caspase-like domain is another Ig-like domain, which has been reported to bind to the ubiquitin conjugating enzyme, Ubc13 [8
]. The C-terminal region of Malt1 contains two binding sites for TRAF6 and TRAF2, centered around Glu653 and Glu806 [12
], although some data suggest that only the most C-terminal TRAF binding site is functional [15
]. A third TRAF6 binding site was recently identified in the second Ig-like domain in the N-terminus of Malt1, encompassing Glu313 and Glu316 [15
Protein-protein interaction sites within Malt1 and the cIAP2-Malt1 fusion protein
Although the cIAP2-Malt1 fusion lacks the N-terminal Bcl10-binding portion of the wild-type protein, it contains the caspase-like domain, the C-terminal Ig-like domain, and the three TRAF2/6 binding sites described above (). The presence of the Malt1 N-terminal Ig-like domains in the cIAP2-Malt1 fusion is variable, depending on the exact translocation breakpoint (see ). The cIAP2-contributed N-terminus of the fusion protein contains three BIR domains and one ubiquitin-associated (UBA) domain. Among the three BIR domains, only the first, BIR1, is essential for NF-κB activation by cIAP2-Malt1 [8
]. BIR1 has been shown to contain an additional TRAF2/TRAF6 binding site, as well as a region that interacts with the Malt1-contributed C-terminus in a heterotypic oligomerization interaction that contributes to NF-κB activation [13
]. An intact UBA is present in roughly 98% of cIAP2-Malt1 fusions, and a functional UBA is required for efficient cIAP2-Malt1-mediated NF-κB activation, although the precise molecular mechanism has yet to be established [21
]. The N-terminus of cIAP2-Malt1 (aa 1–441) has also been reported to interact with Bcl10, perhaps further enhancing NF-κB activation by the cIAP2-Malt1 chimera [22
The C-terminal domain of cIAP2-Malt1 has been shown to interact with Ubc13 and A20 [8
] via the same domains used by wild-type Malt1. Presumably, cIAP2-Malt1 can also interact with Carma1 and caspase-8, as the defined binding domains [10
] are present in the fusion protein. However, there are currently no published data in which association between the cIAP2-Malt1 chimera and either Carma1 or caspase-8 was directly tested. Data have shown that cIAP2-Malt1 functions independently of upstream activators, suggesting that any association with Carma1 is unlikely to contribute to NF-κB activation. A potential role for caspase-8 in NF-κB activation by cIAP2-Malt1 seems more likely, as Malt1 acts upstream of caspase-8 in TCR activation of NF-κB [18
2.3 NF-κB activation pathways
The activation of NF-κB is a complex biological process, involving numerous independent signaling cascades that activate NF-κB via a number of mechanistically distinct pathways. There are many complexities involved in the activation of NF-κB, many of which remain to be fully understood. In order to understand how Malt1 and cIAP2-Malt1 contribute to NF-κB activation, it is useful to first present an overview of general pathways of NF-κB signal transduction.
NF-κB is not a single entity, but rather a family of transcription factors that are capable of forming homo or heterodimers. The five members of this family are RelA (p65), RelB, cRel, p100/p52, and p105/p50. The term ‘NF-κB’ is typically used to refer to the RelA-p50 heterodimer, which appears to be the most common pairing involved in gene activation. NF-κB proteins are ubiquitously expressed, and NF-κB heterodimers control the expression of genes involved in cellular proliferation, survival, inflammation, stress responses, and a number of other cellular processes. Until recently, there were two known mechanisms by which receptor ligation triggers NF-κB activation, referred to as the canonical and non-canonical pathways. Emerging data now indicate the existence of a third NF-κB activation mechanism, referred to as the linear ubiquitin chain assembly complex (LUBAC) pathway. Although each of these pathways converge on the IKK complex to effect activation of NF-κB, the mechanisms leading to IKK activation are distinct in each case.
The canonical pathway of NF-κB activation is triggered by the engagement of diverse receptors, including T and B cell receptors, the TNF receptor, IL-1R/Toll-like receptors, and a variety of other ligand-activated receptors (reviewed in [23
]). While the receptor-proximal signaling events involved in canonical NF-κB activation depend upon which receptor is engaged, the terminal events in the signaling cascade are well-conserved [23
]. Under resting conditions, the NF-κB heterodimer associates with the IκBα protein. This interaction masks the nuclear localization domains of the NF-κB heterodimer, retaining the complex in the cytoplasm. Upon activation, the IKK complex is activated. The IKK complex consists of 3 subunits: the regulatory IKKγ subunit, and the enzymatically active IKKα and IKKβ subunits. During canonical signaling, the IKKβ subunit phosphorylates the IκBα protein on conserved serine residues, an event which triggers the SCFIκB
E3 ubiquitin ligase-mediated poly-ubiquitination of the IκBα protein on lysine residues. This modification targets the IκBα protein for proteasomal degradation, freeing the NF-κB heterodimer to enter the nucleus and control the expression of target genes [23
The alternative NF-κB pathway appears to be triggered by only a small subset of receptors, including CD40, the BAFF receptor, and the lymphotoxin-β receptor. These receptors activate the kinase NIK, which in turn phosphorylates and activates the IKKα subunit in an IKKγ-independent manner. IKKα then phosphorylates the NF-κB precursor protein, p100, resulting in partial proteolytic processing of p100 to the mature p52 NF-κB subunit. These events promote the nuclear translocation of p52/RelB NF-κB heterodimers, which transactivate target genes [25
The LUBAC-regulated NF-κB pathway was recently identified as an important regulator of TNFα- and IL-1β-mediated NF-κB activation [26
]. Investigators have so far demonstrated that a complex of two enzymes, HOIL-1L and HOIP, catalyze the addition of linear head-to-tail linked polyubiquitin chains to target molecules [27
]. Recently, IKKγ was identified as a target of the LUBAC pathway, and linear polyubiquitination of IKKγ was shown to activate the IKK complex, leading to NF-κB activation. Currently, there is no published information regarding how the TNFα and IL-1β receptors transduce signals to the HOIL-1L/HOIP complex. Similarly, it is not known whether the LUBAC pathway may sometimes function as a standalone NF-κB signaling cascade that is coupled to specific activators, or if it always functions as an enhancer or amplifier of NF-κB activation that operates side-by-side with the canonical NF-κB cascade, as appears to be the case with the TNFα and IL-1β receptors [26
]. Indeed, given the very similar mechanisms of signal transmission employed by the canonical and LUBAC pathways, it is possible that many receptor-triggered pathways of NF-κB activation currently labeled as “canonical” may in fact be entirely or partially dependent on the LUBAC system.
2.4 Antigen receptor activation of the canonical NF-κB cascade
In lymphocytes, T and B cell receptor engagement triggers the rapid tyrosine phosphorylation of a large array of receptor subunits, signaling adaptors, and kinases. These early tyrosine activation events initiate several distinct downstream signaling pathways that activate specific transcription factors, including NFAT, AP-1, and NF-κB, which promote broad changes in gene expression and concomitant lymphocyte activation (reviewed in [28
]). Precisely how these initial tyrosine phosphorylation events are connected to the activation of NF-κB is not fully understood.
Better characterized are several different serine/threonine phosphorylations and lysine polyubiquitinations, for which there is strong evidence of a mechanistic connection to antigen receptor activation of NF-κB. The modification of proteins by polyubiquitin plays an essential role in the transduction of signals that ultimately lead to NF-κB activation (reviewed in [29
]). There are several distinct forms of polyubiquitin chains, with distinct biological properties. K63-linked polyubiquitin consists of individual ubiquitin monomers concatemerized via the lysine at position 63. This linkage results in a relatively linear polyubiquitin conformation, and generally contributes to a change in the function of the modified protein. In contrast, concatemerization of ubiquitin monomers via lysine 48 results in a more globular structure, and marks proteins for proteasomal degradation (reviewed in [31
]). For example, in the canonical NF-κB cascade, the proteasomal degradation of IκBα is triggered by K48 polyubiquitination of IκBα (reviewed in [30
Current model of TCR mediated NF-κB activation
Currently, the earliest known event in a dedicated pathway of antigen receptor-mediated NF-κB activation is the auto-phosphorylation of phosphoinositide-dependent kinase 1 (PDK1) (). Recent studies have shown that the coordinated engagement of the TCR and the T cell co-stimulatory receptor, CD28, leads to the PI-3-kinase-dependent auto-phosphorylation of PDK1 on Thr513. In T cells, phosphorylated PDK1 binds PKCθ, activating this kinase via phosphorylation of Thr538 [34
]. Presumably, an analogous pathway in B cells results in phosphorylation and activation of PKCβ, but this has yet to be demonstrated experimentally. Next, the MAGUK protein, Carma1, is phosphorylated at numerous sites in the “linker” region by either PKCβ in B cells or PKCθ in T cells [36
]. Data are consistent with a model in which this phosphorylation event triggers a conformational change in Carma1, allowing the Carma1 caspase recruitment domain (CARD) to interact with the CARD of Bcl10. Because Bcl10 and Malt1 are constitutively associated in the cytoplasm [38
], the Carma1-Bcl10 interaction also brings Malt1 into the complex, thereby forming the CBM complex () [36
]. Data suggest that the CBM is further stabilized by a direct interaction between the coiled coil domain of Carma1 and the C-terminal portion of Malt1 [10
] (). Analysis of Carma1−/−
], and Malt1−/−
] mice showed very similar defects in lymphocyte proliferation and function, further supporting the hypothesis that these molecules interact and transduce antigen signals to NF-κB.
Coincident with or downstream of formation of the CBM complex, a proportion of cellular Bcl10 and Malt1 becomes oligomerized, as demonstrated by biochemical methodology [12
] and FRET microscopy [11
], and it is these oligomeric species that are active in NF-κB signal transduction [12
]. Microscopic examination of activated T cells has shown that oligomerized Bcl10 and Malt1 are found preferentially in punctate cytosolic structures called POLKADOTS, which may be vesicular in nature [11
]. FRET analyses further demonstrated that POLKADOTS are enriched in Bcl10 in close association with Malt1, as well as other signaling partners, including Carma1 (). Because POLKADOTS are only formed by signaling-competent forms of Bcl10 and Malt1, it is likely that they are intermediates in and/or immediate by-products of signal transmission to NF-κB [11
The active/oligomeric forms of Bcl10 and Malt1 are also associated with specific ubiquitin ligases, particularly TRAF6 [11
] (). TRAF6 is an ubiquitin ligase that adds K63-linked polyubiquitin chains to target molecules [48
]. The ubiquitin conjugating enzyme, Ubc13, and its catalytically inactive cofactor, Uev1A, are also involved in TRAF6-mediated ubiquitination events stimulated by the CBM [12
]. The C-terminal Malt1 Ig-like domain () and/or the TRAF6 RING domain may serve as essential docking sites for Ubc13 [8
TRAF6 is activated by oligomerization [52
], which stimulates auto-ubiquitination on Lys 124. Auto-ubiquitination increases the catalytic activity of TRAF6, enabling TRAF6-mediated K63-polyubiquitination of specific protein targets [53
]. Data suggest that activated TRAF6 polyubiquitinates Malt1 on multiple C-terminal lysines () and Bcl10 on Lys31 and Lys63 [54
]. Mutation of all C-terminal lysines in Malt1 reduced NF-κB activation and IL-2 production, suggesting that Malt1 polyubiquitination is mechanistically involved in TCR activation of NF-κB [54
]. Interestingly, K63-polyubiquitination of Bcl10 is substantially enhanced by the presence of Malt1 [55
], suggesting that Malt1-associated TRAF6 may contribute substantially to K63-polyubiquitin modification of Bcl10 in the CBM complex.
Data from one group suggests that K63-polyubiquitination of Bcl10 is essential for CBM association with the IKK complex and for consequent activation of NF-κB [55
], while another group reports an inability to detect K63-polyubiquitination of CBM-associated Bcl10 [56
]. Overall, data are consistent with a model proposing that IKKγ is able to bind to K63-polyubiquitin chains on both Bcl10 and Malt1, and that blockade of either of these interactions impairs NF-κB activation, by preventing efficient physical association between the CBM and IKK complexes. However, it is also formally possible that ubiquitin modification of Bcl10 and/or Malt1 affects the function of one or both proteins in other as yet unidentified ways that are crucial for maximal activation of the IKK complex and do not contribute directly to physical association with the CBM.
The IKK complex appears to be recruited to the CBM complex through a recently identified ubiquitin binding domain (UBD) in the IKKγ protein. Data suggest that this specialized UBD, called the UBAN (UBD in ABIN proteins and NEMO), facilitates association between the IKK complex and the K63-polyubiquitinated CBM complex [55
]. The association of the CBM and IKK complexes then allows TRAF6 (in cooperation with Ubc13/Uev1A) to add K63-polyubiquitin chains to the C-terminal Zn-finger domain of IKKγ at Lys399 and possibly additional lysines [12
]. Biochemical studies suggest that K63-polyubiquitination of IKKγ enables physical association with the TAK1 kinase/TAB2/TAB3 complex, via the UBD of TAB2. This association allows TAK1 to phosphorylate and activate the IKKβ catalytic subunit [12
] (). However, a more recent T cell study has provided compelling evidence that the phosphorylation of IKKβ is independent of CBM-mediated signaling and IKKγ ubiquitination, suggesting that the mechanism of TAK1 association with the IKK complex remains incompletely defined [61
]. Regardless of the details by which the IKK complex is modified, data from both groups strongly suggest that the combination of IKKγ ubiquitination and IKKβ phosphorylation is required for activation of IKKβ kinase activity. [12
]. IKKβ then phosphorylates IκBα, triggering the terminal activation events of the canonical NF-κB activation cascade, as described above (reviewed in [30
2.5 Challenges to the current model of antigen receptor-stimulated NF-κB activation
Although the above model (or close variants of this model) of antigen receptor-mediated NF-κB activation is generally accepted, there are various experimental observations that appear to be at odds with the currently accepted paradigm. Firstly, in conditional knockouts of Ubc13, which specifically inactivated K63-polyubiquitination pathways in B or T lymphocytes, ligand-mediated activation of NF-κB was diminished, but not abolished [62
]. These data suggest that the LUBAC pathway (or a distinct K63-polyubiquitin-independent pathway) may be able to partially compensate for the absence of K63-polyubiquitination activity [26
]. Additionally, another recent report has shown that murine T cells, particularly CD8+
T cells, deficient in PKCθ, Bcl10, or Malt1 still successfully activate NF-κB and NF-κB dependent functions in response to modest or strong stimulation through the TCR. These observations argue that there is an NF-κB activation pathway that is independent of PKCθ and the CBM complex [65
], which again suggests that the LUBAC pathway may be an important component of antigen receptor signaling to NF-κB. Characterization of antigen receptor signaling to NF-κB in HOIL-1−/−
] may help establish the relative importance of these distinct polyubiquitin-dependent pathways in TCR activation of NF-κB.
2.6 Spontaneous activation of NF-κB by the cIAP2-Malt1 fusion protein
Three recurrent translocations have been identified in Malt lymphoma tumors: The t(11;18)(q21;q21) cIAP-Malt1 translocation, which is the most common; less common are the t(1;14)(p22;q32) and t(14;18)(q32;q21) translocations, which place the Ig heavy chain enhancer upstream of the Bcl10 and Malt1 genes, respectively, causing de-regulated expression of each protein (reviewed in [66
]). Patients with the cIAP2-Malt1 translocation have a poorer clinical prognosis than patients harboring other translocations [67
]. These clinical data may reflect stronger NF-κB activation induced by expression of cIAP2-Malt1 fusions in comparison to over-expression of either Bcl10 or Malt1.
Indeed, initial studies of Malt1 demonstrated that the wild-type protein has no independent ability to activate NF-κB and shows only modest enhancement of Bcl10-mediated NF-κB activation, despite direct physical association with Bcl10 [5
]. In contrast, over-expression of either of the other two members of the CBM complex, CARMA1 and Bcl10, results in potent NF-κB activation [6
]. The weak activation of NF-κB by Malt1 is even more perplexing given data showing that both the cIAP2-Malt1 fusion and an artificially oligomerized Malt1 C-terminus show robust NF-κB activation [5
]. To begin to explain these apparently conflicting observations, it is perhaps most useful to first ask why the expression of the cIAP2-Malt1 fusion protein results in dramatic induction of NF-κB activation independent of external activation signals, while over-expression of the wild-type protein seems only capable of modestly enhancing NF-κB activation by Bcl10.
There are several plausible mechanisms that may account for the behavior of the cIAP2-Malt1 protein fusion. Recent data have shown that cIAP2-Malt1 oligomerizes in a manner dependent on the BIR1 domain [8
], with the BIR1 domain of one protein heterotypically interacting with an undefined site in the Malt1-contributed C-terminal region of another fusion protein [13
] ( and ). Consistent with early data demonstrating that forced oligomerization of the C-terminal portion of Malt1 (including the caspase-like domain and all downstream sequences) can trigger NF-κB activation [6
], spontaneous cIAP2-Malt1 oligomerization makes a measurable contribution to NF-κB activation [13
Molecular mechanisms contributing to receptor-independent cIAP2-Malt1 mediated NF-κB activation
However, recent data indicate that binding to TRAF2 and/or TRAF6 is also essential for NF-κB activation by the cIAP2-Malt1 molecule. A novel TRAF2 binding site was recently identified within BIR1, in a subsequence distinct from the region required for cIAP2-Malt1 multimerization (). Mutation of this binding site or over-expression of a TRAF2 null mutant protein resulted in a striking decrease in cIAP2-Malt1-dependent NF-κB activation [13
]. Interestingly, fusion protein mutants lacking the BIR1 TRAF binding site but retaining the capacity to oligomerize showed a considerable defect in NF-κB activation [13
]. Similarly, mutation of the TRAF6 binding sites in the Malt1-derived C-terminal region also impaired cIAP2-Malt1-mediated NF-κB activation [15
]. These data suggest that both the N-terminal and C-terminal TRAF binding sites are required for cIAP2-Malt1-mediated NF-κB activation. While these data do not conclusively demonstrate TRAF2/TRAF6 activation as a consequence of interaction with the cIAP2-Malt1 fusion protein, they suggest that the ability of the fusion protein to activate NF-κB is primarily dependent on binding to TRAF2/TRAF6 and triggering downstream signaling events. The aggregate data therefore suggest that the oligomerization of the Malt1 C-terminal domain and multiple TRAF2/TRAF6 binding sites are critical determinants of robust NF-κB activation by the cIAP2-Malt1 chimeric protein ().
In addition to binding TRAF2/TRAF6, evidence suggests that the cIAP2-Malt1 protein has further activities that can augment signals to NF-κB. For example, some data suggest that cIAP2-Malt1 itself possesses ubiquitin ligase activity that is capable of K63-polyubiquitinating IKKγ and triggering NF-κB activation [8
]. Also, recent data indicate that the cIAP2-Malt1fusion protein binds to K63-polyubiquitinated IKKγ via the cIAP2-contributed UBA domain [21
] (). It is possible that this interaction protects the IKKγ protein from de-ubiquitination, thereby prolonging activation of the IKK complex. Another possibility is that the UBA-IKKγ interaction stabilizes the interaction between cIAP-Malt1 and the IKK complex, increasing the efficiency of IKKγ K63-polyubiquitination by cIAP-Malt1-associated TRAF2 and TRAF6. Additionally, since the cIAP2-Malt1 UBA is capable of binding both K63-polyubiquitin and linear (head-to-tail) polyubiquitin [21
], an intriguing possibility is that cIAP2-Malt1 mediates IKK activation in part via interaction with the LUBAC pathway, binding to IKKγ that has been modified by linear polyubiquitin chains.
There is also evidence that cIAP2-Malt1 mediates proteolytic cleavage of A20, a negative modulator of NF-κB signaling. A20 binds to an unknown sequence within the Malt1-contributed C-terminus of the chimera, and proteolysis of A20 is mediated by the caspase-like domain ( and ) [16
]. A point mutation that disrupts this proteolytic activity decreases cIAP2-Malt1 NF-κB activation by approximately 3-fold [5
]. Thus, the proteolytic activity of cIAP2-Malt1 appears to augment NF-κB activation by interfering with normal homeostatic mechanisms that serve to limit NF-κB activation. These data are discussed in more detail in section 3, below.
Finally, Bcl10 has been reported to interact with the cIAP2-Malt1 protein fusion and synergistically enhance NF-κB activation in the absence of appropriate stimuli [22
]. In this study, data suggested that Bcl10 interacts with a subfragment of cIAP2 (1–441), which contains the three BIR domains and the UBA domain [21
] (). It is unclear at this time precisely which amino acids of this subregion encode the putative Bcl10 binding site. Bcl10 interaction with the fusion protein may enhance NF-κB activation, as cIAP2-Malt1 expression in the absence of Bcl10 resulted in a 4-fold decrease in NF-κB activation [22
]. Since Bcl10 is thought to act upstream of Malt1 in receptor-mediated signaling to NF-κB, it is unclear how Bcl10 might enhance the ability of the fusion protein to activate NF-κB. It is possible that the Bcl10-fusion protein interaction stabilizes the fusion protein and prevents its degradation, or that Bcl10 prolongs the interactions of the fusion protein with downstream mediators such as TRAF2/TRAF6, thereby extending signaling to NF-κB. However, it is also important to note that at least one report disputes both the physical association of Bcl10 with cIAP2-Malt1 and the enhancement of cIAP2-Malt1-mediated NF-κB activation by Bcl10 [15
], underscoring the need for further studies in this area.
Taken together, the above data strongly suggest that IKK activation by cIAP2-Malt1 is based on cooperative activities resident in both the cIAP2- and Malt1-contributed regions of the chimeric protein. Additionally, these observations suggest a plausible model of cIAP2-Malt1 activation of NF-κB (). It is likely that the constitutive oligomerization of the cIAP2-Malt1 fusion protein contributes to its ability to interact with and oligomerize downstream ubiquitin ligases, such as TRAF2/TRAF6. As in the canonical pathway of NF-κB activation, oligomerization of TRAF2/TRAF6 activates the ubiquitin-ligase function of these proteins. In combination with the ubiquitin conjugating enzyme complex, Ubc13/Uev1A, TRAF2 and TRAF6 trigger the K63-polyubiquitination of the cIAP2-Malt1 fusion and the K63-autoubiquitination of the TRAFs, themselves. The polyubiquitinated cIAP-Malt1 complex is then bound by the IKKγ subunit of the IKK complex, via the UBAN of IKKγ [57
]. This molecular association then allows the TRAFs associated with cIAP2-Malt1 to K63-ubiquitinate IKKγ, recruiting the TAK1/TAB2/TAB3 complex [12
], and triggering the terminal events in the canonical NF-κB activation cascade. Presumably, steady-state oligomerization of the cIAP2-Malt1 fusion protein, perhaps in conjunction with protection of polyubiquitin chains on IKKγ by the cIAP2-Malt1 UBA domain and cleavage of A20 by the caspase-like domain, prevents the termination of IKK activation.
Thus, the chromosomal translocation that generates the cIAP2-Malt1 fusion protein may most accurately be described as a ‘gain of function’ mutation [6
], with the resulting combination of protein domains creating novel functions that neither individual full-length protein possesses. More specifically, the combination of essential functional domains from the N-terminus of cIAP2 and the C-terminus of Malt1 generates a chimeric protein that constitutively signals to IKK and NF-κB, and is apparently refractory to the signal termination mechanisms that normally confine NF-κB activation to a temporally limited period.