The Carma1-Bcl10-Malt1 signaling module bridges TCR signaling to the canonical IκB kinase (IKK)/NF-κB pathway. Covalent attachment of regulatory ubiquitin chains to Malt1 paracaspase directs TCR signaling to IKK activation. Further, the ubiquitin-editing enzyme A20 was recently suggested to suppress T cell activation, but molecular targets for A20 remain elusive. In this paper, we show that A20 regulates the strength and duration of the IKK/NF-κB response upon TCR/CD28 costimulation. By catalyzing the removal of K63-linked ubiquitin chains from Malt1, A20 prevents sustained interaction between ubiquitinated Malt1 and the IKK complex and thus serves as a negative regulator of inducible IKK activity. Upon T cell stimulation, A20 is rapidly removed and paracaspase activity of Malt1 has been suggested to cleave A20. Using antagonistic peptides or reconstitution of Malt1−/− T cells, we show that Malt1 paracaspase activity is required for A20 cleavage and optimal IL-2 production, but dispensable for initial IKK/NF-κB signaling in CD4+ T cells. However, proteasomal inhibition impairs A20 degradation and impedes TCR/CD28-induced IKK activation. Taken together, A20 functions as a Malt1 deubiquitinating enzyme and proteasomal degradation and de novo synthesis of A20 contributes to balance TCR/CD28-induced IKK/NF-κB signaling.

Diffuse large B cell lymphoma (DLBCL) is the most common type of lymphoma in humans. The aggressive activated B cell–like (ABC) subtype of DLBCL is characterized by constitutive NF-κB activity and requires signals from CARD11, BCL10, and the paracaspase MALT1 for survival. CARD11, BCL10, and MALT1 are scaffold proteins that normally associate upon antigen receptor ligation. Signal-induced CARD11–BCL10–MALT1 (CBM) complexes couple upstream events to IκB kinase (IKK)/NF-κB activation. MALT1 also possesses a recently recognized proteolytic activity that cleaves and inactivates the negative NF-κB regulator A20 and BCL10 upon antigen receptor ligation. Yet, the relevance of MALT1 proteolytic activity for malignant cell growth is unknown. Here, we demonstrate preassembled CBM complexes and constitutive proteolysis of the two known MALT1 substrates in ABC-DLBCL, but not in germinal center B cell–like (GCB) DLBCL. ABC-DLBCL cell treatment with a MALT1 protease inhibitor blocks A20 and BCL10 cleavage, reduces NF-κB activity, and decreases the expression of NF-κB targets genes. Finally, MALT1 paracaspase inhibition results in death and growth retardation selectively in ABC-DLBCL cells. Thus, our results indicate a growth-promoting role for MALT1 paracaspase activity in ABC-DLBCL and suggest that a pharmacological MALT1 protease inhibition could be a promising approach for lymphoma treatment.

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) represents a very aggressive human lymphoma entity. Constitutive NF-κB activation caused by chronic active B-cell receptor (BCR) signaling is common feature of many ABC DLBCL cells; however, the pathways linking BCR signaling to the NF-κB prosurvival network are largely unknown. Here we report that constitutive activity of PI3K and the downstream kinase PDK1 are essential for the viability of two ABC DLBCL cell lines that carry mutations in the BCR proximal signaling adaptor CD79B. In these cells, PI3K inhibition reduces NF-κB activity and decreases the expression of NF-κB target genes. Furthermore, PI3K and PDK1 are required for maintaining MALT1 protease activity, which promotes survival of the affected ABC DLBCL cells. These results demonstrate a critical function of PI3K-PDK1 signaling upstream of MALT1 protease and NF-κB in distinct ABC DLBCL cells and provide a rationale for the pharmacologic use of PI3K inhibitors in DLBCL therapy.

No abstract

Uncoating of clathrin-coated vesicles requires the Jdomain protein auxilin for targeting hsc70 to the clathrin coats and for stimulating the hsc70 ATPase activity. This results in the release of hsc70-complexed clathrin triskelia and concomitant dissociation of the coat. To understand the complex role of auxilin in uncoating and clathrin assembly in more detail, we analyzed the molecular organization of its clathrin-binding domain (amino acids 547-813). CD spectroscopy of auxilin fragments revealed that the clathrin-binding domain is almost completely disordered in solution. By systematic mapping using synthetic peptides and by site-directed mutagenesis, we identified short peptide sequences involved in clathrin heavy chain and AP-2 binding and evaluated their significance for the function of auxilin. Some of the binding determinants, including those containing sequences 674 DPF and 636 WDW, showed dual specificity for both clathrin and AP-2. In contrast, the two DLL motifs within the clathrin-binding domain were exclusively involved in clathrin binding. Surprisingly, they interacted not only with the N-terminal domain of the heavy chain, but also with the distal domain. Moreover, both DLL peptides proved to be essential for clathrin assembly and uncoating. In addition, we found that the motif 726 NWQ is required for efficient clathrin assembly activity. Auxilin shares a number of proteinprotein interaction motifs with other endocytic proteins, including AP180. We demonstrate that AP180 and auxilin compete for binding to the ␣-ear domain of AP-2. Like AP180, auxilin also directly interacts with the ear domain of ␤-adaptin. On the basis of our data, we propose a refined model for the uncoating mechanism of clathrin-coated vesicles.Clathrin-coated vesicles are involved in numerous membrane transport processes. After their formation, they rapidly shed their protein coats to allow fusion of the vesicle membranes with endosomes and to recycle the coat components. The disassembly of the clathrin coat is mediated by the molecular chaperone hsc70 and requires a cofactor of the DnaJ protein family known as auxilin (1). Auxilin is a neuron-specific protein involved in the removal of the clathrin coat from endocytosed synaptic vesicle membranes and thus in the recycling of synaptic vesicles (1-3), whereas the homolog auxilin-2, also referred to as cyclin G-associated kinase, is more broadly distributed (4, 5). Both proteins contain a C-terminal J-domain preceded by a centrally located clathrin-binding domain and an N-terminal phosphatase-and tensin-like domain with unknown function. In addition to these domains, auxilin-2 also possesses an N-terminal Ark-type kinase domain that has been shown to autophosphorylate auxilin-2 and to phosphorylate the -subunit of the adaptor protein complexes AP-1 and AP-2 (4).According to the current model for the uncoating of clathrincoated vesicles, it is believed that auxilin first binds to clathrin coats and then recruits hsc70 in an ATP-dependent reaction via its J-domain. The interacti...

The Carma1–Bcl10–Malt1 (CBM) complex bridges T‐cell receptor (TCR) signalling to the canonical IκB kinase (IKK)/NF‐κB pathway. NF‐κB activation is triggered by PKCθ‐dependent phosphorylation of Carma1 after TCR/CD28 co‐stimulation. PKCθ‐phosphorylated Carma1 was suggested to function as a molecular scaffold that recruits preassembled Bcl10–Malt1 complexes to the membrane. We have identified the serine–threonine protein phosphatase PP2A regulatory subunit Aα (PPP2R1A) as a novel interaction partner of Carma1. PPP2R1A is associated with Carma1 in resting as well as activated T cells in the context of the active CBM complex. By siRNA‐mediated knockdown and in vitro dephosphorylation, we demonstrate that PP2A removes PKCθ‐dependent phosphorylation of Ser645 in Carma1, and show that maintenance of this phosphorylation is correlated with increased T‐cell activation. As a result of PP2A inactivation, we find that enhanced Carma1 S645 phosphorylation augments CBM complex formation, NF‐κB activation and IL‐2 or IFN‐γ production after stimulation of Jurkat T cells or murine Th1 cells. Thus, our data define PP2A‐mediated dephosphorylation of Carma1 as a critical step to limit T‐cell activation and effector cytokine production.

CVAK104 is a novel

The Carma1-Bcl10-Malt1 (CBM) complex connects T-cell receptor (TCR) signalling to the canonical IjB kinase (IKK)/NF (nuclear factor)-jB pathway. Earlier studies have indicated that the COP9 signalosome (CSN), a pleiotropic regulator of the ubiquitin/26S proteasome system, controls antigen responses in T cells. The CSN is required for the degradation of the NF-jB inhibitor IjBa, but other molecular targets involved in T-cell signalling remained elusive. Here, we identify the CSN subunit 5 (CSN5) as a new interactor of Malt1 and Carma1. T-cell activation triggers the recruitment of the CSN to the CBM complex, and CSN downregulation impairs TCR-induced IKK activation. Furthermore, the CSN is required for maintaining the stability of Bcl10 in response to T-cell activation. Taken together, our data provide evidence for a functional link between the evolutionarily conserved CSN and the adaptive immunoregulatory CBM complex in T cells.