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

James M. Murphy, Qingwei Zhang, Samuel N. Young, Michael L. Reese, Fiona P. Bailey, Patrick A. Eyers, Daniela Ungureanu, Henrik Hammaren, Olli Silvennoinen, Leila N. Varghese, Kelan Chen, Anne Tripaydonis, Natalia Jura, Koichi Fukuda, Jun Qin, Zachary Nimchuk, Mary Beth Mudgett, Sabine Elowe, Christine L. Gee, Ling LiuRoger J. Daly, Gerard Manning, Jeffrey J. Babon, Isabelle S. Lucet

Research output: Contribution to journalArticle

124 Citations (Scopus)

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 catalysisindependent 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 thatmay 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; cationindependent 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.

Original languageEnglish (US)
Pages (from-to)323-334
Number of pages12
JournalBiochemical Journal
Volume457
Issue number2
DOIs
StatePublished - Jan 15 2014

Fingerprint

Nucleotides
Assays
Phosphotransferases
Cations
Hot Temperature
Benchmarking
Divalent Cations
Protein Kinases
Proteins
Adenosine Triphosphate

Keywords

  • Non-catalytic protein-interaction domain
  • Nucleotide binding
  • Protein kinase
  • Pseudoenzyme
  • Pseudokinase

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

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. https://doi.org/10.1042/BJ20131174

A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties. / Murphy, James M.; Zhang, Qingwei; Young, Samuel N.; Reese, Michael L.; Bailey, Fiona P.; Eyers, Patrick A.; Ungureanu, Daniela; Hammaren, Henrik; Silvennoinen, Olli; Varghese, Leila N.; Chen, Kelan; Tripaydonis, Anne; Jura, Natalia; Fukuda, Koichi; Qin, Jun; Nimchuk, Zachary; Mudgett, Mary Beth; Elowe, Sabine; Gee, Christine L.; Liu, Ling; Daly, Roger J.; Manning, Gerard; Babon, Jeffrey J.; Lucet, Isabelle S.

In: Biochemical Journal, Vol. 457, No. 2, 15.01.2014, p. 323-334.

Research output: Contribution to journalArticle

Murphy, JM, Zhang, Q, Young, SN, Reese, ML, Bailey, FP, Eyers, PA, Ungureanu, D, Hammaren, H, Silvennoinen, O, Varghese, LN, Chen, K, Tripaydonis, A, Jura, N, Fukuda, K, Qin, J, Nimchuk, Z, Mudgett, MB, Elowe, S, Gee, CL, Liu, L, Daly, RJ, Manning, G, Babon, JJ & Lucet, IS 2014, 'A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties', Biochemical Journal, vol. 457, no. 2, pp. 323-334. https://doi.org/10.1042/BJ20131174
Murphy, James M. ; Zhang, Qingwei ; Young, Samuel N. ; Reese, Michael L. ; Bailey, Fiona P. ; Eyers, Patrick A. ; Ungureanu, Daniela ; Hammaren, Henrik ; Silvennoinen, Olli ; Varghese, Leila N. ; Chen, Kelan ; Tripaydonis, Anne ; Jura, Natalia ; Fukuda, Koichi ; Qin, Jun ; Nimchuk, Zachary ; Mudgett, Mary Beth ; Elowe, Sabine ; Gee, Christine L. ; Liu, Ling ; Daly, Roger J. ; Manning, Gerard ; Babon, Jeffrey J. ; Lucet, Isabelle S. / A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties. In: Biochemical Journal. 2014 ; Vol. 457, No. 2. pp. 323-334.
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