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-B. 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-B induction. Furthermore, the API2-MALT1 fusion protein also strongly activates NF-B and shows dependence upon the same downstream signaling factors. We propose a model whereby both the Bcl10⅐MALT1 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.

Stimulation of the T cell receptor (TCR) complex initiates multiple signaling cascades that lead to the activation of several transcription factors, including the NF-kappa B family members. Although various proximal signaling components of the TCR have been intensively studied, the distal components that mediate TCR-induced NF-kappa B activation remain largely unknown. Using a somatic mutagenesis approach, we cloned a CARMA1-deficient T cell line. Deficiency in CARMA1 (originally known as CARDII) resulted in selectively impaired activation of NF-kappa B induced by the TCR and a consequent defect in interleukin-2 (IL-2) production. Reconstitution of the CARMA1-deficient cells with CARMA1 fully rescued this signaling defect. Together, our results show that CARMA1 is an essential signaling component that mediates TCR-induced NF-kappa B activation.

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Angiotensin II (Ang II) is a peptide hormone that, like many cytokines, acts as a proinflammatory agent and growth factor. After injury to the liver, the hormone assists in tissue repair by stimulating hepatocytes and hepatic stellate cells to synthesize extracellular matrix proteins and secrete secondary cytokines and by stimulating myofibroblasts to proliferate. However, under conditions of chronic liver injury, all of these effects conspire to promote pathologic liver fibrosis. Much of this effect of Ang II results from activation of the proinflammatory NF-B transcription factor in response to stimulation of the type 1 Ang II receptor, a G protein-coupled receptor. Here, we characterize a previously undescribed signaling pathway mediating Ang II-dependent activation of NF-B, which is composed of three principal proteins, CARMA3, Bcl10, and MALT1. Blocking the function of any of these proteins, through the use of either dominant-negative mutants, RNAi, or gene targeting, effectively abolishes Ang II-dependent NF-B activation in hepatocytes. In addition, Bcl10 ؊/؊ mice show defective hepatic cytokine production after Ang II treatment. Evidence also is presented that this pathway activates NF-B through ubiquitination of IKK␥, the regulatory subunit of the I B kinase complex. These results elucidate a concrete series of molecular events that link ligand activation of the type 1 Ang II receptor to stimulation of the NF-B transcription factor. These findings also uncover a function of the CARMA, Bcl10, and MALT1 proteins in cells outside the immune system. G protein-coupled receptor ͉ hepatocyte ͉ IkB kinase ͉ inflammation ͉ ubiquitination

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Proper regulation of nuclear factor-κB (NF-κB) transcriptional activity is required for normal lymphocyte function, and deregulated NF-κB signaling can facilitate lymphomagenesis. We demonstrate that the API2-MALT1 fusion oncoprotein created by the recurrent t(11;18)(q21;q21) in mucosa-associated lymphoid tissue (MALT) lymphoma induces proteolytic cleavage of NF-κB inducing kinase (NIK) at Arg325. NIK cleavage requires the concerted actions of both fusion partners and generates a C-terminal NIK fragment that retains kinase activity and is resistant to proteasomal degradation. The resulting deregulated NIK activity is associated with constitutive noncanonical NF-κB signaling, enhanced B-cell adhesion, and apoptosis resistance. Our study reveals the gain-of-function proteolytic activity of a fusion oncoprotein and highlights the importance of the noncanonical NF-κB pathway in B-lymphoproliferative disease.

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 cGMPbinding 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 ϳ1.8 M cGMP required for half-maximal saturation. The D289A mutant showed decreased affinity for cGMP (K d > 10 M) and the D478A mutant showed increased affinity for cGMP (K d Ϸ 0.5 M) 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 (k off ‫؍‬ 0.26 h ؊1 ) and rapidly dissociating (k off ‫؍‬ 1.00 h ؊1 ) sites of cGMP binding. In contrast, the D289A mutant displayed a single k off ‫؍‬ 1.24 h ؊1 , which was similar to the calculated k off for the fast site of WT and native cGB-PDE, and the D478A mutant displayed a single k off ‫؍‬ 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. Cyclic nucleotide phosphodiesterases (PDEs)1 constitute a complex family of enzymes which catalyze the hydrolysis of 3Ј:5Ј-cyclic nucleotides to the corresponding nucleoside 5Ј-monophosphates. The multiple PDEs differ in their substrate specificities, sensitivities to inhibitors, modes of regulation, and tissue distributions. Most PDEs are chimeric multidomain proteins, possessing distinct catalytic and regulatory domains (1). A 250-amino acid segment of sequence, which is conserved among all mammalian PDEs and is located in the more carboxyl-terminal portions of the PDE molecules, contains the catalytic site of these enzymes (1-4). Domains of the PDEs which interact with allosteric/regulatory factors are thought to be located within the more amino-terminal regions (1, 5, 6).The cGMP-binding PDEs comprise a heterogeneous subgroup of PDEs, all of which exhibit allosteric cGMP-binding sites that are distinct from the sites of cyclic nucleotide hydrolysis. This group consists of at least three classes of PDEs: the cGMP-stimulated PDEs (cGS-PDEs, or PDE2s 2 ) (7), the photoreceptor PDEs (rod outer segment PDE (ROS-PDE; PDE6A/B) (8) and cone PDE (PDE6C) (9)), and the cGMP-binding, cGMPspecific PDE (cGB-PDE; PDE5A) (10). The s...

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-LeuGlu-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-B transcriptional activation in association with I-B␣ degradation prior to loss of cell viability. Surprisingly, the antioxidant and NF-B inhibitor pyrrolidine dithiocarbamate blocked doxorubicin-induced NF-B transcriptional activation and provided profound protection against doxorubicin killing. Moreover, SH-SY5Y cells expressing a super-repressor form of I-B were completely resistant to doxorubicin killing. Together these findings show that NF-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.