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, 457 (2), 323-34

A Robust Methodology to Subclassify Pseudokinases Based on Their Nucleotide-Binding Properties


A Robust Methodology to Subclassify Pseudokinases Based on Their Nucleotide-Binding Properties

James M Murphy et al. Biochem J.


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.


Figure 1
Figure 1. Pseudokinase domains selected for characterization of nucleotide binding
(A) Schematic cartoon of the eukaryotic protein kinase domain illustrating the amino acid motifs thought to be required for phosphoryl transfer that are usually absent from pseudokinase domains (depicted in the same style as [71]). (B) Purified pseudokinase domains (∼1 μg) were resolved by reducing SDS/PAGE and then were Coomassie Blue-stained. Molecular mass markers are indicated on the left-hand side. In each case, the predominant species present in the preparation is the pseudokinase domain. (C) Multiple sequence alignment of canonical catalytic motifs in PKA (protein kinase A), a model serine/threonine protein kinase, and JAK2(JH1), a model tyrosine kinase, with the 31 pseudokinase domains examined in the present study. Consensus sequences of the catalytic motifs and the protein kinase secondary structure are depicted as described previously ([72] and [73]). φ, hydrophobic; δ, hydrophilic; Φ, large hydrophobic; X, any amino acid. Subdomains are labelled according to the nomenclature proposed in [15]. The G-loop and the VAIK, HRD and DFG motifs of catalytically active protein kinases and their pseudokinase counterparts are boxed in red, and residues thought to be essential for robust catalytic activity are shaded in green. Details of the species of origin, domain boundaries and expression strategy are given for each protein studied, with further details presented in the Experimental section and Supplementary Table S1 (at It should be noted that equivalent results were obtained for BubR1 with the domain boundaries of residues 720–1043 (results not shown).
Figure 2
Figure 2. Thermal denaturation curves for selected Class 1 (non-binding) pseudokinase domains
Thermal denaturation curves of the Class 1 pseudokinases, BubR1 (A), wild-type RYK (B), CASK (D) and MviN (G) (upper panels) with the corresponding histograms depicting the ΔTm values in each condition (lower panels), as derived from analysis of upper panel curves. Although thermal denaturation of TFVK>VAVK RYK (C) and IRAK3 (F) was not affected by nucleotides or cations, TYK2(JH2) (E) exhibited modest, but consistent, thermal shifts between experiments in the presence of ATP–Mg2+, ATP–Mn2+ or GTP–Mg2+ . Thermal shifts for all three of these proteins were detected in the presence of the inhibitor VI16832 and/or DAP. A colour key for the curve labelling is to the right-hand side of the curves. In the histograms, bars are coloured according to the colour key when ΔTm>3C and pale blue otherwise. Thermal denaturation curves for the other Class 1 pseudokinases, Ror1, PTK7/CCK4, SgK223, SgK495, TRB2, GCN2 [also known as EIF2AK4 (eukaryotic translation initiation factor 2-α kinase 4)], VRK3, BPK1, NRBP1 and SCYL1 (SCY1-like 1), are shown in Supplementary Figure S2 (at
Figure 3
Figure 3. Thermal denaturation curves and ΔTm analyses of Class 2 (nucleotide binding) and Class 3 (cation binding) pseudokinases
Thermal denaturation data of the Class 2 pseudokinases, MLKL (A), STRADα (B), wild-type EphB6 (C) and ULK4 (E), presented as described in the legend to Figure 2. (D) The R813D mutation converts EphB6 from a Class 2 into Class 4 pseudokinase. Note that wild-type EphB6 (C) and ULK4 (E) are classified as Class 2 (nucleotide-binding) as the presence of Mn2+ and Mg2+ does not enhance ATP binding. Data for the Class 3 (cation-binding) pseudokinases ROP2 and SgK269 are presented in (F) and (G) respectively. Note that ROP2 and SgK269 are classified as Class 3 (cation-binding) pseudokinases because the presence of nucleotides does not confer further thermal stability above and beyond that of cation alone.
Figure 4
Figure 4. ΔTm analyses of Class 4 (nucleotide- and cation-binding) pseudokinases
Histograms depicting the ΔTm value in each condition derived from thermal denaturation curves presented in Supplementary Figure S3 (at for JAK1(JH2) (A), JAK2(JH2) (B), ILK (C), HER3/ErbB3 (D), SgK071 (E), CRN (F), ROP5BI (G), IRAK2 (H) and TARK1 (I). Note that some conditions are destabilizing, leading to negative ΔTm values. TARK1 (I) was categorized as Class 4 owing to the observation of ATP–Mg2+, ADP–Mg2+, AMP-PNP–Mg2+ and GTP–Mg2+ binding. Interestingly TARK1 also appears to bind Mn2+ in the absence of nucleotides, leading to thermal-stability shifts under all Mn2+ -containing conditions.

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