Regulation of Malt1 and Bcl10 function by the ubiquitin and SUMO systems: Implications for NF-κB signaling in lymphocytes.

Since T cells play a crucial role in orchestrating the immune response, a detailed knowledge of TCR signaling is pivotal for understanding aberrant T or B cell responses in autoimmunity or cancer. The transcription factor NF-kappaB triggers the expression of many target genes involved in antigen-specific proliferation, differentiation and survival of T cells, thus representing a central mediator of T cell activation. At a molecular level, the IkB kinase (IKK) complex represents the gatekeeper of NF-kappaB activation in many pathways. In T cells, formation of a Carma1/Bcl10/Malt1 (CBM) complex was shown to be essential for IKK activation. However, the molecular mechanisms, which connect CBM complex formation to IKK activation, had remained elusive. In the presented work Malt1 was identified as a novel target for regulatory ubiquitination in the course of TCR signaling. The ubiquitin ligase TRAF6 (TNF receptor associated factor 6) was shown to associate with Malt1 and to mediate Malt1 ubiquitination in vitro and in vivo by attachment of lysine 63-linked ubiquitin chains to multiple lysine residues in the Malt1 C-terminus. Through RNAi evidence and the finding that the TRAF6 binding sites in Malt1 are required for NF-kappaB and T cell activation, the presented work strongly supports a critical role for TRAF6 in TCR signaling to NF-kappaB. By reconstitution experiments using Malt1 deficient primary T cells, a crucial function for C-terminal Malt1 ubiquitination in NF-kappaB and T cell activation could be demonstrated. Finally, Malt1-attached ubiquitin chains were shown to provide the interaction surface for recruitment of the regulatory IKK complex component IKKgamma through its ubiquitin-binding domain. Furthermore, binding of ubiquitin chains by IKKgamma was demonstrated to be essential for TCR triggered NF-kB activation. Based on the presented findings, a new model for CBM dependent IKK activation can be suggested: Upon CBM complex formation, TRAF6 associates with the C-terminus of Malt1 and mediates ubiquitination of multiple lysines in the vicinity. IKKgamma is then recruited to ubiquitin-chains attached to Malt1, what could either induce IKK complex activation by induced proximity or by recruitment of an IKK activating kinase. Both mechanisms are not mutually exclusive. Thus, Malt1 ubiquitination represents the missing link between IKK and CBM complexes, thereby directing TCR proximal signals to IKK activation. In addition, a second, ubiquitin-like protein modification was investigated. It was demonstrated that Bcl10 can be a target of sumoylation and that attachment of SUMO occurs specifically at lysine 110. The presented data suggest an interplay between Bcl10 sumoylation and subcellular localization. Sumoylation is enhanced upon nuclear localization of Bcl10 and SUMO fusion to Bcl10 leads to its nuclear import and retention. Furthermore, Bcl10-SUMO fusion results in an enhanced NF-kappaB activation potential compared to wildtype Bcl10. Aberrant nuclear localization of Bcl10 is observed in many MALT lymphomas, which critically depend on constitutive NF-kappaB activation, and correlates with advanced stages. The molecular mechanisms underlying this phenomenon are poorly understood. Thus, sumoylation could represent a novel mechanism to mediate nuclear localization of Bcl10. Additionally, the data suggest that sumoylation of nuclear Bcl10 could influence NF-kappaB activity by a yet uncharacterized mechanism