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

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.

A dual role for the API2 moiety in API2-MALT1-dependent NF-kappaB activation: heterotypic oligomerization and TRAF2 recruitment.

Mucosa-associated lymphoid tissue (MALT) lymphoma is the most common extranodal lymphoid neoplasm. Chromosomal translocation t(11;18)(q21,q21) is found in 30% of gastric MALT lymphomas and is associated with a failure to respond to standard treatment and a tendency to disseminate. This translocation generates a chimeric protein composed of N-terminal sequences of Inhibitor of Apoptosis 2 (API2, also known as BIRC3 and cIAP2) fused to C-terminal sequences of MALT1. API2-MALT1 promotes cell survival and proliferation via activation of nuclear factor-kappaB (NF-kappaB). Here, we investigate the mechanism by which the API2 moiety contributes to NF-kappaB stimulation. We find that the API2 moiety mediates oligomerization of API2-MALT1 as well as interaction with tumor necrosis factor receptor-associated factor 2 (TRAF2). Surprisingly, oligomerization does not occur via homotypic interaction; rather, the API2 moiety of one monomer interacts with the MALT1 moiety of another monomer. Further, the specific region of the API2 moiety responsible for mediating oligomerization is distinct from that mediating TRAF2 binding. Although deletion or mutation of the TRAF2 binding site does not inhibit oligomerization, it does lead to dramatically decreased NF-kappaB activation. Deletion of both TRAF2 binding and oligomerization regions results in near-complete loss of NF-kappaB activation. Thus, API2 moiety-mediated heterotypic oligomerization and TRAF2 binding both contribute to maximal API2-MALT1-dependent NF-kappaB stimulation.

NF-kappa B activation mediates doxorubicin-induced cell death in N-type neuroblastoma cells.

Neuroblastoma is the most common extracranial solid tumor of childhood. N-type neuroblastoma cells (represented by SH-SY5Y and IMR32 cell lines) are characterized by a neuronal phenotype. N-type cell lines are generally N-myc amplified, express the anti-apoptotic protein Bcl-2, and do not express caspase-8. The present study was designed to determine the mechanism by which N-type cells die in response to specific cytotoxic agents (such as cisplatin and doxorubicin) commonly used to treat this disease. We found that N-type cells were equally sensitive to cisplatin and doxorubicin. Yet death induced by cisplatin was inhibited by the nonselective caspase inhibitor z-Val-Ala-Asp-fluoromethylketone or the specific caspase-9 inhibitor N-acetyl-Leu-Glu-His-Asp-aldehyde, whereas in contrast, caspase inhibition did not prevent doxorubicin-induced death. Neither the reactive oxygen species nor the mitochondrial permeability transition appears to play an important role in this process. Doxorubicin induced NF-kappa B transcriptional activation in association with I-kappa B alpha degradation prior to loss of cell viability. Surprisingly, the antioxidant and NF-kappa B inhibitor pyrrolidine dithiocarbamate blocked doxorubicin-induced NF-kappa B transcriptional activation and provided profound protection against doxorubicin killing. Moreover, SH-SY5Y cells expressing a super-repressor form of I-kappa B were completely resistant to doxorubicin killing. Together these findings show that NF-kappa B activation mediates doxorubicin-induced cell death without evidence of caspase function and suggest that cisplatin and doxorubicin engage different death pathways to kill neuroblastoma cells.

Bimp1, a MAGUK family member linking protein kinase C activation to Bcl10-mediated NF-kappaB induction.

Bcl10 and MALT1, products of distinct chromosomal translocations in mucosa-associated lymphoid tissue lymphoma, cooperate in activating NF-kappaB. Mice lacking Bcl10 demonstrate severe immunodeficiency associated with failure of lymphocytes to activate nuclear factor kappaB (NF-kappaB) in response to antigen receptor stimulation and protein kinase C activation. We characterize Bimp1, a new signaling protein that binds Bcl10 and activates NF-kappaB. Bimp1-mediated NF-kappaB activation requires Bcl10 and IkappaB kinases, indicating that Bimp1 acts upstream of these mediators. Bimp1, Bcl10, and MALT1 form a ternary complex, with Bcl10 bridging the Bimp1/MALT1 interaction. A dominant negative Bimp1 mutant inhibits NF-kappaB activation by anti-CD3 ligation, phorbol ester, and protein kinase C expression. These results suggest that Bimp1 links surface receptor stimulation and protein kinase C activation to Bcl10/MALT1, thus leading to NF-kappaB induction.

Bcl10 and MALT1, independent targets of chromosomal translocation in malt lymphoma, cooperate in a novel NF-kappa B signaling pathway.

At least two distinct recurrent chromosomal translocations have been implicated in the pathogenesis of MALT lymphoma. The first, t(1;14), results in the transfer of the entire Bcl10 gene to chromosome 14 wherein Bcl10 expression is inappropriately stimulated by the neighboring Ig enhancer. The second, t(11;18), results in the synthesis of a novel fusion protein, API2-MALT1. Until now, no common mechanism of action has been proposed to explain how the products of these seemingly unrelated translocations may contribute to the same malignant process. We show here that Bcl10 and MALT1 form a strong and specific complex within the cell, and that these proteins synergize in the activation of NF-kappaB. The data support a mechanism of action whereby Bcl10 mediates the oligomerization and activation of the MALT1 caspase-like domain. This subsequently activates the IKK complex through an unknown mechanism, setting in motion a cascade of events leading to NF-kappaB induction. Furthermore, the API2-MALT1 fusion protein also strongly activates NF-kappaB and shows dependence upon the same downstream signaling factors. We propose a model whereby both the Bcl10.MALT1 complex and the API2-MALT1 fusion protein activate a common downstream signaling pathway that originates with the oligomerization-dependent activation of the MALT1 caspase-like domain.

Isolation and characterization of cDNAs encoding PDE5A, a human cGMP-binding, cGMP-specific 3',5'-cyclic nucleotide phosphodiesterase.

Human cGMP-binding, cGMP-specific 3',5'-cyclic nucleotide phosphodiesterase (PDE5A) cDNAs were isolated. A 3.1-kb composite DNA sequence assembled from overlapping cDNAs encodes an 875-amino-acid protein with a predicted molecular mass of 100012 Da (PDE5A1). Extracts prepared from yeast expressing human PDE5A1 hydrolyzed cGMP. This activity was inhibited by the selective PDE5 inhibitors zaprinast and DMPPO. PDE5A mRNA is expressed in aortic smooth muscle cells, heart, placenta, skeletal muscle and pancreas and, to a much lesser extent, in brain, liver and lung. A 5'-splice variant, PDE5A2, encodes an 833-amino-acid protein with eight unique amino acids at the amino terminus. PDE5A maps to chromosome 4q 25-27.

Identification of key amino acids in a conserved cGMP-binding site of cGMP-binding phosphodiesterases. A putative NKXnD motif for cGMP binding.

cGMP-binding phosphodiesterases contain two kinetically distinct cGMP-binding sites (a and b), and each site contains a conserved N(K/R)XnFX3DE sequence. N276A, K277A, K277R, D289A, and E290A mutants in the N276KX7FX3DE290 sequence of site a (higher affinity site) of bovine cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE or PDE5A) were expressed in High Five cells and purified. The cGMP-binding affinities of three mutants [K277A (Kd approximately 12 microM), D289A (Kd approximately 24 microM), and N276A (Kd approximately 60 microM)] were decreased in comparison with wild-type enzyme (Kd = 1.3 microM), which suggested an important role for Asn276, Lys277, and Asp289 in cGMP binding. These residues could be presented as a putative NKXnD motif, and their functions were predicted based on analogy with the canonical NKXD motif in GTP-binding proteins. No marked differences in catalytic functions such as specific activity, Km for cGMP, and IC50 for zaprinast or 3-isobutyl-1-methylxanthine were found among wild-type and mutant cGB-PDEs. This suggested that cGMP binding to site a does not influence the catalytic properties of cGB-PDE.

An essential aspartic acid at each of two allosteric cGMP-binding sites of a cGMP-specific phosphodiesterase.

The amino acid sequences of all known cGMP-binding phosphodiesterases (PDEs) contain internally homologous repeats (a and b) that are 80-90 residues in length and are arranged in tandem within the putative cGMP-binding domains. In the bovine lung cGMP-binding, cGMP-specific PDE (cGB-PDE or PDE5A), these repeats span residues 228-311 (a) and 410-500 (b). An aspartic acid (residue 289 or 478) that is invariant in repeats a and b of all known cGMP-binding PDEs was changed to alanine by site-directed mutagenesis of cGB-PDE, and wild type (WT) and mutant cGB-PDEs were expressed in COS-7 cells. Purified bovine lung cGB-PDE (native) and WT cGB-PDE displayed identical cGMP-binding kinetics, with approximately 1.8 microM cGMP required for half-maximal saturation. The D289A mutant showed decreased affinity for cGMP (Kd > 10 microM) and the D478A mutant showed increased affinity for cGMP (Kd approximately 0.5 microM) as compared to WT and native cGB-PDE. WT and native cGB-PDE displayed an identical curvilinear profile of cGMP dissociation which was consistent with the presence of distinct slowly dissociating (koff = 0.26 h-1) and rapidly dissociating (koff = 1.00 h-1) sites of cGMP binding. In contrast, the D289A mutant displayed a single koff = 1.24 h-1, which was similar to the calculated koff for the fast site of WT and native cGB-PDE, and the D478A mutant displayed a single koff = 0.29 h-1, which was similar to that calculated for the slow site of WT and native cGB-PDE. These results were consistent with the loss of a slow cGMP-binding site in repeat a of the D289A mutant cGB-PDE, and the loss of a fast site in repeat b of the D478A mutant, suggesting that cGB-PDE possesses two distinct cGMP-binding sites located at repeats a and b, with the invariant aspartic acid being crucial for interaction with cGMP at each site.

Zinc interactions and conserved motifs of the cGMP-binding cGMP-specific phosphodiesterase suggest that it is a zinc hydrolase.

cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) binds tightly to a Zn(2+)-chelate column (Francis, S. H., and Corbin, J. D. (1988) Methods Enzymol. 159, 722-729). Using three different approaches, Zn2+ is now shown to bind to cG-BPDE, and the Kd is determined to be approximately 0.5 microM, with a binding stoichiometry of approximately 3 mol of Zn2+/mol of monomer. A similar concentration range of Zn2+ (0.05-1 microM Zn2+) also supports phosphodiesterase (PDE) catalytic activity. The Zn2+ binding to cG-BPDE is not diminished by, nor is catalysis supported by, relatively high concentrations of Cu2+, Cd2+, Ca2+, or Fe2+. Neither cGMP nor 3-isobutyl-1-methylxanthine affects Zn2+ binding under the conditions used. Mn2+, Co2+, or Mg2+ supports catalysis, but only at significantly higher concentrations (4-, 15-, and 250-fold, respectively) than that required for Zn2+. Two tandem amino acid sequences, which are conserved in the catalytic domains of all characterized mammalian PDEs, resemble the single sequence motif that has been shown to coordinate Zn2+ in the catalytic sites of Zn2+ hydrolases such as thermolysin.

The structure of a bovine lung cGMP-binding, cGMP-specific phosphodiesterase deduced from a cDNA clone.

Polymerase chain reaction (PCR) methodology and cDNA library screening were used to isolate a cDNA clone encoding a cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE) from bovine lung. Degenerate oligonucleotides based on cGB-PDE peptide sequences were used as primers for a PCR reaction with bovine lung cDNA as the template. An 824-base pair PCR product was recovered and used as a probe to screen a bovine lung cDNA library. A 4.5-kilobase pair cDNA clone encoding a full-length cGB-PDE was isolated. The open reading frame of this cDNA predicted an 875 amino acid (AA), 99,525-Da polypeptide. By Northern analysis, the cGB-PDE cDNA hybridized to a single lung 6.9-kilobase mRNA. The identity of the cGB-PDE cDNA was verified by comparison of the deduced AA sequence with several peptide sequences obtained from cGB-PDE. COS-7 cells transfected with cGB-PDE cDNA overexpressed cGMP-binding and cGMP-PDE activities characteristic of lung cGB-PDE. The sequence of cGB-PDE contained a segment (AA 578-812) that was homologous to the putative catalytic region conserved among all mammalian PDEs and a segment (AA 142-526) that was homologous to the putative cGMP binding region of the cGMP-stimulated PDE and the photoreceptor PDEs. As noted also for these PDEs, two internally homologous repeats were contained within the putative cGMP binding region of cGB-PDE. The amino-terminal 142 residues of cGB-PDE showed no significant homology to other PDEs and contained the serine (AA 92) which is phosphorylated by cGMP-dependent protein kinase.