Abstract
The MLKL pseudokinase is the terminal effector in the necroptosis cell death pathway. Phosphorylation by its upstream regulator, RIPK3, triggers MLKL’s conversion from a dormant cytoplasmic protein into oligomers that translocate to, and permeabilize, the plasma membrane to kill cells. The precise mechanisms underlying these processes are incompletely understood, and were proposed to differ between mouse and human cells. Here, we examine the divergence of activation mechanisms among nine vertebrate MLKL orthologues, revealing remarkable specificity of mouse and human RIPK3 for MLKL orthologues. Pig MLKL can restore necroptotic signaling in human cells; while horse and pig, but not rat, MLKL can reconstitute the mouse pathway. This selectivity can be rationalized from the distinct conformations observed in the crystal structures of horse and rat MLKL pseudokinase domains. These studies identify important differences in necroptotic signaling between species, and suggest that, more broadly, divergent regulatory mechanisms may exist among orthologous pseudoenzymes.
Highlights
The Mixed Lineage Kinase domain-Like (MLKL) pseudokinase is the terminal effector in the necroptosis cell death pathway
Cognate pseudoenzymes are thought to have arisen from gene duplications of active enzymes, where the duplicate is relieved of selective pressures to enable evolution of new, non-catalytic functions, often within the same pathway[43]. It is typical of pseudokinases, like the TYK2 JH2 domain, KSR2, STRADα and HER3/ERBB3 to bind and regulate the activities of their cognate active kinases: the TYK2 JH1 domain, MEK1, LKB1 and EGFR, respectively[44,45,46,47]
Rather than the pseudokinase MLKL regulating the activity of RIPK3, it is RIPK3 that controls MLKL activation via engagement and phosphorylation of the MLKL pseudokinase domain[18,19,26,27,34]
Summary
The MLKL pseudokinase is the terminal effector in the necroptosis cell death pathway. Pig MLKL can restore necroptotic signaling in human cells; while horse and pig, but not rat, MLKL can reconstitute the mouse pathway This selectivity can be rationalized from the distinct conformations observed in the crystal structures of horse and rat MLKL pseudokinase domains. Revealed differences between necroptosis signaling in mouse and human cells[12,15,33,34,36] indicate that the precise mechanism of MLKL activation by RIPK3, and how they might vary across vertebrates, remains incompletely understood. The capacity of pseudokinase orthologues to complement signaling pathways in other species has not been widely examined, here our studies reveal remarkable selectivity among orthologues, with pig MLKL able to be activated by human RIPK3 and horse MLKL by mouse RIPK3. These findings raise the possibility that activation loop phosphorylation may promote activation loop mobility, and destabilise inactive MLKL conformations, to facilitate molecular switch interconversion and MLKL dissociation from RIPK3
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