Phosphohistidine phosphatase 1 (PHPT1) also dephosphorylates phospholysine of chemically phosphorylated histone H1 and polylysine

Abstract

Background. Phosphohistidine phosphatase 1 (PHPT1), also named protein histidine phosphatase (PHP), is a eukaryotic enzyme dephosphorylating proteins and peptides that are phosphorylated on a histidine residue. A preliminary finding that histone H1, which lacks histidine, was phosphorylated by phosphoramidate and dephosphorylated by PHPT1 prompted the present investigation.

Methods. Histone H1 and polylysine were phosphorylated at a low concentration (3.9 mM) of phosphoramidate. Their dephosphorylation by recombinant human PHPT1 was investigated by using a DEAE-Sepharose spin column technique earlier developed by us for studies on basic phosphoproteins and phosphopeptides. Determination of protein-bound, acid-labile phosphate was performed by a malachite green method. Mass spectrometry (MS) was used to investigate the occurrence of N-ϵ-phospholysine residues in a phosphorylated histone H1.2 preparation, and to measure the activity of PHPT1 against free N-ω-phosphoarginine.

Results. Histone H1.2, which lacks histidine, was phosphorylated by phosphoramidate on several lysine residues, as shown by MS. PHPT1 was shown to dephosphorylate phosphohistone H1 at a rate similar to that previously described for the dephosphorylation of phosphohistidine-containing peptides. In addition, phosphopolylysine was an equally good substrate for PHPT1. However, no dephosphorylation of free phosphoarginine by PHPT1 could be detected.

Conclusion. The finding that PHPT1 can dephosphorylate phospholysine in chemically phosphorylated histone H1 and polylysine demonstrates a broader specificity for this enzyme than known so far.

Key words:

• Histone H1
• phosphohistidine phosphatase
• phospholysine
• phospholysine phosphatase
• PHP
• PHPT1
• protein histidine phosphatase

Acknowledgements

Gunilla Pettersson and Elvy Netzel are gratefully acknowledged for performing some of the experiments in this work.

Funding

The work was supported by the Medical Faculty of Uppsala University.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.