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.
Highlights
The discovery of protein phosphorylation on histidine [1] was made 52 years ago [2]
In the pre-study of commercial histones mentioned in the Introduction, the chemical phosphorylation by phosphoramidate was found to be highest for histone H1 from calf thymus
The present study shows that phosphohistidine phosphatase (PHPT1) can dephosphorylate a chemically phosphorylated histone H1 that does not contain histidine residues
Summary
The discovery of protein phosphorylation on histidine [1] was made 52 years ago [2]. Most of the work related to phosphohistidine has so far been performed on the bacterial phosphoenolpyruvate-glucose phosphotransferase system [3] and two-component systems [4]. Phosphohistidine phosphatase 1 (PHPT1), 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. 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. Mass spectrometry (MS) was used to investigate the occurrence of N-e-phospholysine residues in a phosphorylated histone H1.2 preparation, and to measure the activity of PHPT1 against free N-w-phosphoarginine. 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
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