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

Murphy, James M. and Zhang, Qingwei and Young, Samuel N. and Reese, Michael L. and Bailey, Fiona P. and Eyers, Patrick A. and Ungureanu, Daniela and Hammaren, Henrik and Silvennoinen, Olli and Varghese, Leila N. and Chen, Kelan and Tripaydonis, Anne and Jura, Natalia and Fukuda, Koichi and Qin, Jun and Nimchuk, Zachary and Mudgett, Mary Beth and Elowe, Sabine and Gee, Christine L. and Liu, Ling and Daly, Roger J. and Manning, Gerard and Babon, Jeffrey J. and Lucet, Isabelle S. (2014) A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties. Biochemical Journal, 457 (2). pp. 323-334. ISSN 0264-6021. PMCID PMC5679212. https://resolver.caltech.edu/CaltechAUTHORS:20140502-093100054

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1042/BJ20131174 DOIArticle
http://www.biochemj.org/bj/457/bj4570323.htmPublisherArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5679212PubMed CentralArticle
Additional Information:© The Authors Journal compilation © 2014 Biochemical Society. Received 30 August 2013; 8 October 2013; accepted 10 October 2013. We thank Professor Dario Alessi (University of Dundee, Dundee, U.K.) for providing the GST–STRADα expression construct; Professor Seung-Taek Lee (Yonsei University, Seoul, Korea) for the PTK7/CCK4 template DNA; Dr Alessandra Gentile (Institute for Cancer Research and Treatment, Candiolo, Italy) for the Ror1 template cDNA; Dr Elton Zeqiraj (Tanenbaum-Lunenfeld Research Institute, Toronto, Canada) for assistance with the secondary-structure cartoon in Figure 1(C); and the Monash University Protein Production Unit for access to the Corbett RT-PCR instrument used for all of the thermal-shift assays. This work was supported by the National Health and Medical Research Council (NHMRC) [grant numbers 637342 and 1011804]; the Australian Research Council (ARC) [fellowships FT100100100 and FT110100169 (to J.M.M. and J.J.B.)]; the Leukaemia Foundation and the Australian Stem Cell Centre via scholarships to L.N.V.; the National Institutes of Health (NIH) [grant numbers R01 HL58758 (to J.Q.) and AI73756 (to M.L.R.)]; the Medical Research Council of Academy of Finland, the Sigrid Juselius Foundation, the Medical Research Fund of Tampere University Hospital, the Finnish Cancer Foundation and the Tampere Tuberculosis Foundation (to O.S.); the American Heart Association [grant number 11BGIA7440051 (to N.J.)]; and the National Science Foundation [grant number IOS-0821801 (to M.B.M.)]; with additional support from the Victorian State Government Operational Infrastructure Support and the NHMRC Independent Research Institutes Infrastructure Support Scheme (IRIISS) [grant number 361646].
Funders:
Funding AgencyGrant Number
National Health and Medical Research Council (NHMRC)637342
National Health and Medical Research Council (NHMRC)1011804
Australian Research CouncilFT100100100
Australian Research Council FT110100169
Leukaemia FoundationUNSPECIFIED
Australian Stem Cell CentreUNSPECIFIED
NIHR01 HL58758
NIHAI73756
Medical Research Council (Finland)UNSPECIFIED
Sigrid Juselius FoundationUNSPECIFIED
Tampere University HospitalUNSPECIFIED
Finnish Cancer FoundationUNSPECIFIED
Tampere Tuberculosis FoundationUNSPECIFIED
American Heart Association11BGIA7440051
NSFIOS-0821801
Victorian State GovernmentUNSPECIFIED
National Health and Medical Research Council (NHMRC)361646
Subject Keywords:nucleotide binding; non-catalytic protein-interaction domain; protein kinase; pseudoenzyme; pseudokinase
Issue or Number:2
PubMed Central ID:PMC5679212
Record Number:CaltechAUTHORS:20140502-093100054
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20140502-093100054
Official Citation:Murphy, J. M., Zhang, Q., Young, S. N., Reese, M. L., Bailey, F. P., Eyers, P. A., . . . Lucet, I. S. (2014). A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties. Biochemical Journal, 457(2), 323-334. doi: 10.1042/bj20131174
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:45463
Collection:CaltechAUTHORS
Deposited By: Jason Perez
Deposited On:02 May 2014 21:59
Last Modified:03 Oct 2019 06:31

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