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A Quantitative Method for the Measurement of Protein Histidine Phosphorylation

  • Paul V. AttwoodEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2077)

Abstract

The method described in this chapter provides a quantitative means of assaying for protein histidine phosphorylation and thus protein histidine kinase activity, even in the presence of other protein kinases, for example, serine/threonine or tyrosine kinases. The method involves the measurement of 32P, derived from [γ32P]ATP, incorporation into phosphohistidine in a protein substrate. The method makes use of the differential stabilities of phosphohistidine and the common phosphohydroxyamino acids to alkali and acid treatments to measure phosphohistidine incorporation. Phosphoserine and phosphothreonine are depleted by alkali treatment, while phosphohistidine, which is alkali-stable, is removed by acid treatment. Phosphotyrosine is stable to both alkali and acid treatments. The method is filter-based and allows for rapid assay of multiple protein histidine kinase samples, for example, screening for histidine kinase activity, allowing for the calculation of specific activity. In addition, quantitative time-course assays can also be performed to allow for kinetic analysis of histidine kinase activity.

Key words

Phosphohistidine Histidine kinase Quantitative assay Phosphotyrosine Cherenkov Radioactive phosphate 

References

  1. 1.
    Attwood PV (2013) Histidine kinases from bacteria to humans. Biochem Soc Trans 41:1023–1028CrossRefGoogle Scholar
  2. 2.
    Fuhs SR, Hunter T (2017) pHisphorylation: the emergence of histidine phosphorylation as a reversible regulatory modification. Curr Opin Cell Biol 45:8–16CrossRefGoogle Scholar
  3. 3.
    Attwood PV, Wieland T (2015) Nucleoside diphosphate kinase as protein histidine kinase. Naunyn Schmiedeberg's Arch Pharmacol 388:153–160CrossRefGoogle Scholar
  4. 4.
    Fuhs SR, Meisenhelder J, Aslanian A, Ma L, Zagorska A, Stankova A et al (2015) Monoclonal 1- and 3-phosphohistidine antibodies: new tools to study histidine phosphorylation. Cell 162:198–210CrossRefGoogle Scholar
  5. 5.
    Kee JM, Oslund RC, Perlman DH, Muir TW (2013) A pan-specific antibody for direct detection of protein histidine phosphorylation. Nat Chem Biol 9:416–421CrossRefGoogle Scholar
  6. 6.
    Hohenester UM, Ludwig K, Krieglstein J, Konig S (2010) Stepchild phosphohistidine: acid-labile phosphorylation becomes accessible by functional proteomics. Anal Bioanal Chem 397:3209–3212CrossRefGoogle Scholar
  7. 7.
    Oslund RC, Kee JM, Couvillon AD, Bhatia VN, Perlman DH, Muir TW (2014) A phosphohistidine proteomics strategy based on elucidation of a unique gas-phase phosphopeptide fragmentation mechanism. J Am Chem Soc 136:12899–12911CrossRefGoogle Scholar
  8. 8.
    Potel CM, Lin MH, Heck AJR, Lemeer S (2018) Widespread bacterial protein histidine phosphorylation revealed by mass spectrometry-based proteomics. Nat Methods 15:187–190CrossRefGoogle Scholar
  9. 9.
    Wei YF, Matthews HR (1990) A filter-based protein kinase assay selective for alkali-stable protein phosphorylation and suitable for acid-labile protein phosphorylation. Anal Biochem 190:188–192CrossRefGoogle Scholar
  10. 10.
    Attwood PV, Piggott MJ, Zu XL, Besant PG (2007) Focus on phosphohistidine. Amino Acids 32:145–156CrossRefGoogle Scholar
  11. 11.
    Tan E, Lin Zu X, Yeoh GC, Besant PG, Attwood PV (2003) Detection of histidine kinases via a filter-based assay and reverse-phase thin-layer chromatographic phosphoamino acid analysis. Anal Biochem 323:122–126CrossRefGoogle Scholar
  12. 12.
    Huang JM, Wei YF, Kim YH, Osterberg L, Matthews HR (1991) Purification of a protein histidine kinase from the yeast Saccharomyces cerevisiae. The first member of this class of protein kinases. J Biol Chem 266:9023–9031PubMedGoogle Scholar
  13. 13.
    Besant PG, Attwood PV, Piggott MJ (2009) Focus on phosphoarginine and phospholysine. Curr Protein Pept Sci 10:536–550CrossRefGoogle Scholar
  14. 14.
    Besant PG, Lasker MV, Bui CD, Turck CW (2000) Phosphohistidine analysis using reversed-phase thin-layer chromatography. Anal Biochem 282:149–153CrossRefGoogle Scholar
  15. 15.
    Wei YF, Matthews HR (1991) Identification of phosphohistidine in proteins and purification of protein-histidine kinases. Methods Enzymol 200:388–414CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.School of Molecular SciencesThe University of Western AustraliaCrawleyAustralia

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