Biochemical analysis of coestimulatory signaling pathways in T cells

STEMPIN, C; HAECKER, H; REDECKE, V

St Jude Childrens Research hospital. Memphis, TN. USA

Conferencia; Infectious Disease Research Conference; 2008

Activation through the T-cell receptor (TCR) leads to the initiation of multiple signaling cascades that regulate survival, cytokine production and proliferation of T-cells. A key event upon TCR engagement is the formation of a signaling complex between CARMA1, BCL10 and MALT1, referred to as CBM complex. These three molecules interact physically and functionally. The components of this complex were found to be essential for T-cell activation, in particular by controlling the NF-kB and MAP kinase signal transduction pathways. Therefore, within the CBM complex, experimental data suggest that the adaptor protein BCL10 play a key role by providing a molecular link between CARMA1 and MALT1. It has been uncertain whether other additional molecules that exist in these signaling pathways are involved, because of the lack of appropriate techniques for the analysis of activated signaling complexes in living cells. We have developed a novel technique to identify protein interacting partners. This technique is based on the fact that many proteins, including BCL10, oligomerize upon activation (Hacker et al., 2006). We mimic stimulus-dependent dimerization by fusing bait proteins, such as BCL10, to the bacterial protein gyrase B (GyrB), which can be induced to dimerize by the bivalent antibiotic coumermycin A1 (CM) (Farrar et al., 2000). When equipped with epitope tags and stably expressed in cell lines, these constructs allow not only selective activation of BCL10, but also purification and identification of the assembled signaling components by mass spectrometry (MS) (Hacker et al., 2006). For the first time it is possible to selectively activate BCL10 and identify specific components of the CBM signaling complex from living cells. Moreover, we have successfully identified a novel NF-kB activating protein as part of the BCL10 complex by using BCL10 as bait and the semi-quantitative MS method as analysis tool.