Histidine phosphorylation in human cells; a needle or phantom in the haystack?

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.