Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death.

Necroptosis is considered to be complementary to the classical caspase-dependent programmed cell death pathway, apoptosis. The pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) is an essential effector protein in the necroptotic cell death pathway downstream of the protein kinase Receptor Interacting Protein Kinase-3 (RIPK3). How MLKL causes cell death is unclear, however RIPK3-mediated phosphorylation of the activation loop in MLKL trips a molecular switch to induce necroptotic cell death. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high-molecular-weight, membrane-localized complex and cell death. Using alanine-scanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, whereas MLKL mutations in the other cluster did not prevent membrane translocation but prevented killing; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. Finally, we identified a small molecule that binds the nucleotide binding site within the MLKL pseudokinase domain and retards MLKL translocation to membranes, thereby preventing necroptosis. This inhibitor provides a novel tool to investigate necroptosis and demonstrates the feasibility of using small molecules to target the nucleotide binding site of pseudokinases to modulate signal transduction.

The Use of JAK-specific inhibitors as chemical biology tools.

The JAK family of protein tyrosine kinases are now recognized as important participants in a wide range of pathologies, from cancer to inflammatory diseases. In the last decade, the drive to develop drugs targeting members of this family has begun to deliver a panel of small molecule inhibitors of JAK family members, with a range of potencies and specificities. A number of these compounds have already found widespread use as biochemical tools in the elucidation of JAK activity in specific signaling and disease processes; however, many of the first generation compounds are poorly characterized with suboptimal potencies and selectivities.Herein, we present the data for those small molecule JAK inhibitors that have been described in the peer-reviewed literature and the benefits and potential issues that may be associated with the use of these tool compounds.