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Updated: Sep 7, 2025

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay
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Histidine phosphorylation in human cells; a needle or phantom in the haystack?

Niels M Leijten1,2, Albert J R Heck1,2, Simone Lemeer3,4

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Histidine phosphorylation in human cells is rare, with most initially detected sites being false positives. This study found no evidence that histidine phosphorylation plays a significant role in mammalian cell signaling.

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Signaling

Background:

  • Histidine phosphorylation was proposed to be as common as serine, threonine, and tyrosine phosphorylation in mammalian cells.
  • This modification was hypothesized to be crucial for mammalian cell signaling pathways.

Purpose of the Study:

  • To investigate the presence and role of histidine phosphorylation in human cell lines.
  • To determine if histidine phosphorylation is a significant signaling mechanism in mammals.

Main Methods:

  • Applied a validated workflow for detecting histidine phosphorylation, previously successful in bacteria.
  • Analyzed four human cell lines using mass spectrometry-based proteomics.
  • Conducted rigorous control experiments to validate identified phosphorylation sites.

Main Results:

  • Initially identified numerous potential histidine phosphorylation sites across all cell lines.
  • Demonstrated that over 99% of these initial findings were artifacts, mislocalized to serine/threonine residues.
  • Confirmed only a few genuine histidine phosphorylation sites, corresponding to known enzymatic intermediates.

Conclusions:

  • Protein histidine phosphorylation is not widespread in mammalian cells as previously suggested.
  • The study found no evidence to support a role for histidine phosphorylation in mammalian cell signaling.
  • Existing methods can detect genuine histidine phosphorylation, but it appears to be a rare event in mammals.