A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties
Details
Publication Year 2014-01-15,Volume 457,Issue #2,Page 323-34
Journal Title
The Biochemical journal
Publication Type
Journal Article
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.
Publisher
PORTLAND PRESS
Keywords
nucleotide binding, non-catalytic protein-interaction domain, protein kinase, pseudoenzyme, pseudokinase
Research Division(s)
Cancer And Haematology; Cell Signalling And Cell Death
Terms of Use/Rights Notice
© The Authors Journal compilation © 2014 Biochemical Society


Creation Date: 2014-01-24 12:02:23
Last Modified: 2015-04-02 11:33:07
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