Necroptotic cell death is mediated by the most terminal known effector of the pathway, MLKL. Precisely how phosphorylation of the MLKL pseudokinase domain activation loop by the upstream kinase, RIPK3, induces unmasking of the N-terminal executioner four-helix bundle (4HB) domain of MLKL, higher-order assemblies, and permeabilization of plasma membranes remains poorly understood. Here, we reveal the existence of a basal monomeric MLKL conformer present in human cells prior to exposure to a necroptotic stimulus. Following activation, toggling within the MLKL pseudokinase domain promotes 4HB domain disengagement from the pseudokinase domain αC helix and pseudocatalytic loop, to enable formation of a necroptosis-inducing tetramer. In contrast to mouse MLKL, substitution of RIPK3 substrate sites in the human MLKL pseudokinase domain completely abrogated necroptotic signaling. Therefore, while the pseudokinase domains of mouse and human MLKL function as molecular switches to control MLKL activation, the underlying mechanism differs between species.

Eph receptors, the largest subfamily of receptor tyrosine kinases, are linked with proliferative disease, such as cancer, as a result of their deregulated expression or mutation. Unlike other tyrosine kinases that have been clinically targeted, the development of therapeutics against Eph receptors remains at a relatively early stage. The major reason is the limited understanding on the Eph receptor regulatory mechanisms at a molecular level. The complexity in understanding Eph signalling in cells arises due to following reasons: (1) Eph receptors comprise 14 members, two of which are pseudokinases, EphA10 and EphB6, with relatively uncharacterised function; (2) activation of Eph receptors results in dimerisation, oligomerisation and formation of clustered signalling centres at the plasma membrane, which can comprise different combinations of Eph receptors, leading to diverse downstream signalling outputs; (3) the non-catalytic functions of Eph receptors have been overlooked. This review provides a structural perspective of the intricate molecular mechanisms that drive Eph receptor signalling, and investigates the contribution of intra-and inter-molecular interactions between Eph receptors intracellular domains and their major binding partners. We focus on the non-catalytic functions of Eph receptors with relevance to cancer, which are further substantiated by exploring the role of the two pseudokinase Eph receptors, EphA10 and EphB6. Throughout this review, we carefully analyse and reconcile the existing/conflicting data in the field, to allow researchers to further the current understanding of Eph receptor signalling. Eph receptors and ephrin ligands: an overview Receptor tyrosine kinases (RTKs) are a major type of membrane receptors, which govern cell proliferation, differentiation and mobility [1]. Deregulation of RTK signalling pathways leads to many diseases, such as cancers and developmental disorders [2]. The erythropoietin-producing hepatoma (Eph) receptor subfamily is the largest amongst the RTKs with 14 members classified into type A and type B. Compared with other RTKs, Eph receptors share

Necroptotic cell death has been implicated in many human pathologies and is thought to have evolved as an innate immunity mechanism. The pathway relies on two key effectors: the kinase receptor-interacting protein kinase 3 (RIPK3) and the terminal effector, the pseudokinase mixed-lineage kinasedomain-like (MLKL). We identify proteins with high sequence similarity to the pseudokinase domain of MLKL in poxvirus genomes. Expression of these proteins from the BeAn 58058 and Cotia poxviruses, but not swinepox, in human and mouse cells blocks cellular MLKL activation and necroptotic cell death. We show that viral MLKL-like proteins function as dominant-negative mimics of host MLKL, which inhibit necroptosis by sequestering RIPK3 via its kinase domain to thwart MLKL engagement and phosphorylation. These data support an ancestral role for necroptosis in defense against pathogens. Furthermore, mimicry of a cellular pseudokinase by a pathogen adds to the growing repertoire of functions performed by pseudokinases in signal transduction.