Defining the relevant combinatorial space of the PKC/CARD-CC signal transduction nodes

Biological signal transduction typically display a so-called bow-tie or hour glass topology: Multiple receptors lead to multiple cellular responses but the signals all pass through a narrow waist of central signaling nodes. One such critical signaling node for several inflammatory and oncogenic signaling pathways in humans are the CARD-CC / Bcl10 / MALT1 (CBM) complexes, which get activated by upstream protein kinase C (PKC). In humans, there are four phylogenetically distinct CARD-CC family (CARD9, -10, -11 and -14) proteins and 9 true PKC isozymes ( to {iota}). At this moment, less than a handful of PKC/CARD-CC relationships are known from experimental evidence. In order to explore the biologically relevant combinatorial space out of all 36 potential permutations in this two-component signaling event, we made use of CRISPR/Cas9 genome-edited HEK293T cells to mutate CARD10 for subsequent pairwise cotransfections of all CARD-CC family members and activated mutants of all true PKCs. By quantitative reporter gene expression readout, we could define specific strong and weak PKC/CARD-CC relationships. Surprisingly as many as 21 PKC/CARD-CC combinations were found to have synergistic effects. We also discovered heterodimerization between different CARD-CC proteins, and that this can influence their PKC response profile. This information will be valuable for future studies of novel signaling pathways dependent on the CBM complex signaling nodes.