A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties

Abstract

Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.

ATP binding to the pseudokinase domain of JAK2 is critical for pathogenic activation

“…A recent study evaluating nucleotide binding in 30 pseudokinase domains indicated that almost half of pseudokinases retain nucleotide binding, which in most cases is not associated with phosphotransfer activity (20). These and similar observations (23,(28)(29)(30)(31) have brought up the question about the functional role of ATP binding in pseudokinases.…”

“…The structures of JAK1 and TYK2 JH2 are highly similar to JAK2 JH2 (5,19), and all three JH2s bind ATP (20). The regulatory residues Ser523 and Tyr570 in JAK2 are not conserved in other JAK family members, and the catalytic function of JH2 appears to be a unique characteristic of JAK2, which is also the only JAK to function as homodimers on type I cytokine receptors.…”

“…Furthermore, some pseudokinases that are incapable of binding ATP, like Vacciniarelated kinase 3, effectively mimic the ATP-bound conformation through bulky and acidic amino acid substitutions in the active site (37). Because almost half of pseudokinases studied so far possess some form of nucleotide binding activity (20), it seems likely that future studies will find even more evidence for a functional role of ATP binding in this group of proteins.…”

RIPK1- and RIPK3-induced cell death mode is determined by target availability

“…Bacmids and baculoviruses were generated using established procedures, 48,49 before recombinant proteins were expressed and purified from Sf21 insect cells essentially as previously described. 16,48 Briefly, following cell lysis by sonication, N-terminal His 6 -tagged proteins were purified by Ni 2 þ -affinity chromatography and the tag cleaved by incubation with TEV protease for 2 h at 221C.…”

“…16,48 Briefly, following cell lysis by sonication, N-terminal His 6 -tagged proteins were purified by Ni 2 þ -affinity chromatography and the tag cleaved by incubation with TEV protease for 2 h at 221C. Detagged mMLKL (179-464) was extensively dialyzed, before further Ni 2 þ chromatography to eliminate undigested protein and TEV protease followed by a final Superdex-200 gel filtration (GE Healthcare, Rydalmere, NSW, Australia) in 200 mM NaCl and 20 mM HEPES, pH 7.5.…”