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

Protein kinase-like domains that lack conserved residues known to catalyze phosphoryl transfer, termed pseudokinases, have emerged as important signaling domains across the kingdoms of life. Although predicted to function principally as catalysis-independent protein interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amidst controversy. We established a thermal stability 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 attributions 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; or nucleotide binding enhanced by cations. While 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 signaling 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 kinas...

Source: BJ Signal - October 10, 2013 Category: Biochemistry Authors: J M Murphy, Q Zhang, S N Young, M L Reese, F P Bailey, P A Eyers, D Ungureanu, H Hammarén, O Silvennoinen, L N Varghese, K Chen, A Tripaydonis, N Jura, K Fukuda, J Qin, Z Nimchuk, M Mudgett, S Elowe, C Lesley Gee, L Liu, R J Daly, G Manning, J J Babon, I Tags: BJ Signal Source Type: research