Identification and characterization of a mammalian 14‐kDa phosphohistidine phosphatase

Protein histidine phosphorylation in eukaryotes has been sparsely studied compared to protein serine/threonine and tyrosine phosphorylation. In an attempt to rectify this by probing porcine liver cytosol with the phosphohistidine‐containing peptide succinyl‐Ala‐His(P)‐Pro‐Phe‐ p ‐nitroanilide (phosphopeptide I), we observed a phosphatase activity that was insensitive towards okadaic acid and EDTA. This suggested the existence of a phosphohistidine phosphatase different from protein phosphatase 1, 2A and 2C. A 1000‐fold purification to apparent homogeneity gave a 14‐kDa phosphatase with a specific activity of 3 µmol·min −1 ·mg −1 at pH 7.5 with 7 µ m phosphopeptide I as substrate. Partial amino‐acid sequence determination of the purified porcine enzyme by MS revealed similarity with a human sequence representing a human chromosome 9 gene of hitherto unknown function. Molecular cloning from a human embryonic kidney cell cDNA‐library followed by expression and purification, yielded a protein with a molecular mass of 13 700 Da, and an EDTA‐insensitive phosphohistidine phosphatase activity of 9 µmol·min −1 ·mg −1 towards phosphopeptide I. No detectable activity was obtained towards a set of phosphoserine‐, phosphothreonine‐, and phosphotyrosine peptides. Northern blot analysis indicated that the human phosphohistidine phosphatase mRNA was present preferentially in heart and skeletal muscle. These results provide a new tool for studying eukaryotic histidine phosphorylation/dephosphorylation.