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

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.