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The API2-MALT1 fusion exploits TNFR pathway-associated RIP1 ubiquitination to promote oncogenic NF-κB signaling.

Rosebeck S, Rehman AO, Apel IJ, Kohrt D, Appert A, O'Donnell MA, Ting AT, Du MQ, Baens M, Lucas PC, McAllister-Lucas LM - Oncogene (2013)

Bottom Line: We identified receptor interacting protein-1 (RIP1) as a novel API2-MALT1-associated protein, and demonstrate that RIP1 is required for API2-MALT1 to stimulate canonical nuclear factor kappa B (NF-κB).Furthermore, we found that TNF receptor-associated factor 2 (TRAF2) recruitment is required for API2-MALT1 to induce RIP1 ubiquitination, NF-κB activation and cellular transformation.Intriguingly, constitutive RIP1 ubiquitination was recently demonstrated in several solid tumors, and now our study implicates RIP1 ubiquitination as a critical component of API2-MALT1-dependent lymphomagenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, MI, USA.

ABSTRACT
The API2-MALT1 fusion oncoprotein is created by the recurrent t(11;18)(q21;q21) chromosomal translocation in mucosa-associated lymphoid tissue (MALT) lymphoma. We identified receptor interacting protein-1 (RIP1) as a novel API2-MALT1-associated protein, and demonstrate that RIP1 is required for API2-MALT1 to stimulate canonical nuclear factor kappa B (NF-κB). API2-MALT1 promotes ubiquitination of RIP1 at lysine (K) 377, which is necessary for full NF-κB activation. Furthermore, we found that TNF receptor-associated factor 2 (TRAF2) recruitment is required for API2-MALT1 to induce RIP1 ubiquitination, NF-κB activation and cellular transformation. Although both TRAF2 and RIP1 interact with the API2 moiety of API2-MALT1, this moiety alone is insufficient to induce RIP1 ubiquitination or activate NF-κB, indicating that API2-MALT1-dependent RIP1 ubiquitination represents a gain of function requiring the concerted actions of both the API2 and MALT1 moieties of the fusion. Intriguingly, constitutive RIP1 ubiquitination was recently demonstrated in several solid tumors, and now our study implicates RIP1 ubiquitination as a critical component of API2-MALT1-dependent lymphomagenesis.

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The role of the MALT1 moiety in API2-MALT1-induced RIP1 ubiquitination(A) HEK-293T cells were co-transfected with plasmids encoding FLAG-API2-MALT1, the API2 moiety, or the MALT1 moiety and HA-Ub. RIP1 and NEMO ubiquitination were assessed. (B and C) Either WT API2-MALT1 or a C-terminal deletion (1-762) (B) or the proteolytically inactive mutant (C678A) (C) were co-transfected into HEK-293T cells with HA-Ub to compare RIP1 and NEMO ubiquitination and NF-κB activation. (D) Either WT API2-MALT1 (A7M3) or a TRAF6 binding-deficient mutant (M7M-E2A) were co-expressed with HA-Ub to compare RIP1 and NEMO ubiquitination and NF-κB activation. (E) Proposed model of API2-MALT1-induced canonical NF-κB activation.
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Figure 7: The role of the MALT1 moiety in API2-MALT1-induced RIP1 ubiquitination(A) HEK-293T cells were co-transfected with plasmids encoding FLAG-API2-MALT1, the API2 moiety, or the MALT1 moiety and HA-Ub. RIP1 and NEMO ubiquitination were assessed. (B and C) Either WT API2-MALT1 or a C-terminal deletion (1-762) (B) or the proteolytically inactive mutant (C678A) (C) were co-transfected into HEK-293T cells with HA-Ub to compare RIP1 and NEMO ubiquitination and NF-κB activation. (D) Either WT API2-MALT1 (A7M3) or a TRAF6 binding-deficient mutant (M7M-E2A) were co-expressed with HA-Ub to compare RIP1 and NEMO ubiquitination and NF-κB activation. (E) Proposed model of API2-MALT1-induced canonical NF-κB activation.

Mentions: Thus far, our data suggested that the API2 moiety of API2-MALT1 recruits TRAF2 and RIP1 in BIR1 domain-dependent and independent manners, respectively. We next found, however, that expression of the API2 moiety alone is insufficient to induce either RIP1 or NEMO ubiquitination (Figure 7A) or to activate NF-κB (20). The MALT1 moiety is similarly insufficient on its own. This suggests that fusion of the API2 and MALT1 moieties is essential for providing a gain of function to the oncoprotein per se, which culminates in RIP1 ubiquitination and recruitment of the NF-κB-activating kinase complexes. The MALT1 moiety of API2-MALT1 possesses multiple binding sites for the E3 ubiquitin ligase, TRAF6, as well as a proteolytically active caspase-like domain (see Supplementary Figure S1A) (17, 39). We found that expression of a C-terminal deletion mutant of API2-MALT1 (1–762; depicted in Supplementary Figure S1A), which lacks the binding sites for the TRAF6 E3 ubiquitin ligase and the E2 conjugating enzyme, UBC13, and also lacks MALT1 proteolytic activity, failed to induce RIP1 and NEMO ubiquitination and to activate NF-κB (Figure 7B). To specifically determine if the proteolytic activity of the API2-MALT1 caspase-like domain is necessary for RIP1 ubiquitination, we compared wild-type API2-MALT1 to the C678A point mutant, which lacks proteolytic activity but retains TRAF6 binding, and found that loss of MALT1 protease activity does not impair API2-MALT1-induced RIP1 or NEMO ubiquitination, nor does it impair canonical NF-κB activation (Figure 7C). We then sought to determine if MALT1 moiety-dependent recruitment of TRAF6 is required for API2-MALT1-induced RIP1 ubiquitination by comparing wild-type API2-MALT1 with a point mutant of API2-MALT1 that cannot interact with TRAF6 (M7M-E2A), but retains TRAF2 binding (39). Interestingly, expression of the TRAF6 binding-deficient API2-MALT1 mutant still promoted RIP1 ubiquitination, albeit to a lesser degree, but NEMO ubiquitination and subsequent NF-κB activation were completely lost (Figure 7D). We were unable to specifically investigate the impact of TRAF6 deficiency on API2-MALT1-dependent RIP1 ubiquitination because siRNA-mediated knockdown of TRAF6 resulted in dramatically decreased levels of API2-MALT1 (data not shown). Our analysis thus far indicates that two of the known functions attributed to the MALT1 moiety of API2-MALT1, MALT1 proteolytic activity and TRAF6 binding, are not required for API2-MALT1 to induce RIP1 ubiquitination, although TRAF6 binding is required for API2-MALT1 to induce NEMO ubiquitination. Further investigations will be required to not only determine how the MALT1 moiety contributes to API2-MALT1-induced RIP1 ubiquitination but also the nature of the critical interplay between the API2 and MALT1 moieties to promote RIP1 ubiquitination and NF-κB activation.


The API2-MALT1 fusion exploits TNFR pathway-associated RIP1 ubiquitination to promote oncogenic NF-κB signaling.

Rosebeck S, Rehman AO, Apel IJ, Kohrt D, Appert A, O'Donnell MA, Ting AT, Du MQ, Baens M, Lucas PC, McAllister-Lucas LM - Oncogene (2013)

The role of the MALT1 moiety in API2-MALT1-induced RIP1 ubiquitination(A) HEK-293T cells were co-transfected with plasmids encoding FLAG-API2-MALT1, the API2 moiety, or the MALT1 moiety and HA-Ub. RIP1 and NEMO ubiquitination were assessed. (B and C) Either WT API2-MALT1 or a C-terminal deletion (1-762) (B) or the proteolytically inactive mutant (C678A) (C) were co-transfected into HEK-293T cells with HA-Ub to compare RIP1 and NEMO ubiquitination and NF-κB activation. (D) Either WT API2-MALT1 (A7M3) or a TRAF6 binding-deficient mutant (M7M-E2A) were co-expressed with HA-Ub to compare RIP1 and NEMO ubiquitination and NF-κB activation. (E) Proposed model of API2-MALT1-induced canonical NF-κB activation.
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Figure 7: The role of the MALT1 moiety in API2-MALT1-induced RIP1 ubiquitination(A) HEK-293T cells were co-transfected with plasmids encoding FLAG-API2-MALT1, the API2 moiety, or the MALT1 moiety and HA-Ub. RIP1 and NEMO ubiquitination were assessed. (B and C) Either WT API2-MALT1 or a C-terminal deletion (1-762) (B) or the proteolytically inactive mutant (C678A) (C) were co-transfected into HEK-293T cells with HA-Ub to compare RIP1 and NEMO ubiquitination and NF-κB activation. (D) Either WT API2-MALT1 (A7M3) or a TRAF6 binding-deficient mutant (M7M-E2A) were co-expressed with HA-Ub to compare RIP1 and NEMO ubiquitination and NF-κB activation. (E) Proposed model of API2-MALT1-induced canonical NF-κB activation.
Mentions: Thus far, our data suggested that the API2 moiety of API2-MALT1 recruits TRAF2 and RIP1 in BIR1 domain-dependent and independent manners, respectively. We next found, however, that expression of the API2 moiety alone is insufficient to induce either RIP1 or NEMO ubiquitination (Figure 7A) or to activate NF-κB (20). The MALT1 moiety is similarly insufficient on its own. This suggests that fusion of the API2 and MALT1 moieties is essential for providing a gain of function to the oncoprotein per se, which culminates in RIP1 ubiquitination and recruitment of the NF-κB-activating kinase complexes. The MALT1 moiety of API2-MALT1 possesses multiple binding sites for the E3 ubiquitin ligase, TRAF6, as well as a proteolytically active caspase-like domain (see Supplementary Figure S1A) (17, 39). We found that expression of a C-terminal deletion mutant of API2-MALT1 (1–762; depicted in Supplementary Figure S1A), which lacks the binding sites for the TRAF6 E3 ubiquitin ligase and the E2 conjugating enzyme, UBC13, and also lacks MALT1 proteolytic activity, failed to induce RIP1 and NEMO ubiquitination and to activate NF-κB (Figure 7B). To specifically determine if the proteolytic activity of the API2-MALT1 caspase-like domain is necessary for RIP1 ubiquitination, we compared wild-type API2-MALT1 to the C678A point mutant, which lacks proteolytic activity but retains TRAF6 binding, and found that loss of MALT1 protease activity does not impair API2-MALT1-induced RIP1 or NEMO ubiquitination, nor does it impair canonical NF-κB activation (Figure 7C). We then sought to determine if MALT1 moiety-dependent recruitment of TRAF6 is required for API2-MALT1-induced RIP1 ubiquitination by comparing wild-type API2-MALT1 with a point mutant of API2-MALT1 that cannot interact with TRAF6 (M7M-E2A), but retains TRAF2 binding (39). Interestingly, expression of the TRAF6 binding-deficient API2-MALT1 mutant still promoted RIP1 ubiquitination, albeit to a lesser degree, but NEMO ubiquitination and subsequent NF-κB activation were completely lost (Figure 7D). We were unable to specifically investigate the impact of TRAF6 deficiency on API2-MALT1-dependent RIP1 ubiquitination because siRNA-mediated knockdown of TRAF6 resulted in dramatically decreased levels of API2-MALT1 (data not shown). Our analysis thus far indicates that two of the known functions attributed to the MALT1 moiety of API2-MALT1, MALT1 proteolytic activity and TRAF6 binding, are not required for API2-MALT1 to induce RIP1 ubiquitination, although TRAF6 binding is required for API2-MALT1 to induce NEMO ubiquitination. Further investigations will be required to not only determine how the MALT1 moiety contributes to API2-MALT1-induced RIP1 ubiquitination but also the nature of the critical interplay between the API2 and MALT1 moieties to promote RIP1 ubiquitination and NF-κB activation.

Bottom Line: We identified receptor interacting protein-1 (RIP1) as a novel API2-MALT1-associated protein, and demonstrate that RIP1 is required for API2-MALT1 to stimulate canonical nuclear factor kappa B (NF-κB).Furthermore, we found that TNF receptor-associated factor 2 (TRAF2) recruitment is required for API2-MALT1 to induce RIP1 ubiquitination, NF-κB activation and cellular transformation.Intriguingly, constitutive RIP1 ubiquitination was recently demonstrated in several solid tumors, and now our study implicates RIP1 ubiquitination as a critical component of API2-MALT1-dependent lymphomagenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, MI, USA.

ABSTRACT
The API2-MALT1 fusion oncoprotein is created by the recurrent t(11;18)(q21;q21) chromosomal translocation in mucosa-associated lymphoid tissue (MALT) lymphoma. We identified receptor interacting protein-1 (RIP1) as a novel API2-MALT1-associated protein, and demonstrate that RIP1 is required for API2-MALT1 to stimulate canonical nuclear factor kappa B (NF-κB). API2-MALT1 promotes ubiquitination of RIP1 at lysine (K) 377, which is necessary for full NF-κB activation. Furthermore, we found that TNF receptor-associated factor 2 (TRAF2) recruitment is required for API2-MALT1 to induce RIP1 ubiquitination, NF-κB activation and cellular transformation. Although both TRAF2 and RIP1 interact with the API2 moiety of API2-MALT1, this moiety alone is insufficient to induce RIP1 ubiquitination or activate NF-κB, indicating that API2-MALT1-dependent RIP1 ubiquitination represents a gain of function requiring the concerted actions of both the API2 and MALT1 moieties of the fusion. Intriguingly, constitutive RIP1 ubiquitination was recently demonstrated in several solid tumors, and now our study implicates RIP1 ubiquitination as a critical component of API2-MALT1-dependent lymphomagenesis.

Show MeSH
Related in: MedlinePlus