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

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. Abbreviations: AMP-PNP, adenosine 5′-[β,γ-imido]triphosphate; BPK1, bradyzoite pseudokinase 1; CASK, calcium/calmodulin-dependent serine protein kinase; CCK4, colon carcinoma kinase 4; CH2, calponin homology 2; CRN, CORYNE; DAP, N′2′-(4-aminomethyl-phenyl)-5-fluoro-N′4′-phenyl-pyrimidine-2,4-diamine; EphB6, Ephrin type-B receptor 6; ErbB3, v-erb-b2 avian erythroblastic leukaemia viral oncogene homologue 3; HER3, human epidermal growth factor receptor 3; ILK, integrin-linked kinase; IRAK, interleukin-1-receptor-associated kinase; ITC, isothermal titration calorimetry; JAK, Janus kinase; JH, JAK homology; KSR2, kinase suppressor of Ras 2; MLKL, mixed lineage kinase domain-like; NRBP1, nuclear receptor-binding protein 1; PEAK1, pseudopodium-enriched atypical kinase 1; PTK7, protein tyrosine kinase 7; ROP, rhoptry protein kinase; Ror1, receptor tyrosine kinase-like orphan receptor; RT-PCR, real-time PCR; RYK, receptor-like tyrosine kinase; SgK, sugen kinase; STRADα, STE20-related kinase adaptor α; TARK1, tomato atypical receptor-like kinase 1; TRB2, tribbles pseudokinase 2; TYK2, tyrosine kinase 2; ULK4, unc-51 like kinase 4; VRK3, vaccinia-related kinase 3