In the past 10 years have pseudokinases come to prominence as crucial regulators of intracellular signaling pathways. Pseudokinases, unlike catalytically-active protein kinases, typically function as protein interaction domains to nucleate the formation of signaling complexes or modulate the activities of other protein domains, such as conventional protein kinases.

One such pseudokinase, Mixed lineage kinase domain-like (MLKL), is the most-terminal (known) protein effector in the necroptotic cell death pathway. In 2013, we proposed that a “molecular switch” mechanism underlies activation of MLKL, where phosphorylation of MLKL’s pseudokinase domain by the upstream protein kinase, RIPK3, triggers MLKL to induce cell death [1]. Consistent with this idea, we [2] and others showed that deletion of the pseudokinase domain from MLKL derepresses the N-terminal four-helix bundle (4HB) domain to drive cell death. These data point to the pseudokinase domain functioning as a brake to prevent 4HB domain-mediated cell death, although the precise mechanism by which the 4HB domain kills cells remains highly contentious. In support of this model, we have identified a small molecule ATP-mimetic that binds the pseudokinase domain of MLKL and can jam the molecular switch to inhibit necroptosis in cellular assays [2], providing proof-of-principle that pseudokinase functions are amenable to therapeutic modulation by small molecules.

To probe the molecular switch mechanism, we have characterised the necroptotic functions of MLKL orthologs. The MLKL domain architecture is peculiar to MLKL orthologs in chordates, but somewhat unexpectedly, we have identified remote orthologs of MLKL in plants and fungi. Studies of orthologs in cells lacking mouse Mlkl suggest a convergent mechanism for MLKL ortholog-mediated cell death and indicate, surprisingly, that the evolutionary origins of necroptosis lie in single-celled eukaryotes, rather than having evolved as a backup to the better-characterised cell death pathway, apoptosis.