tailieunhanh - Báo cáo Y học: 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-nitro-anilide (phosphopeptide I), we observed a phosphatase activity that was insensitive towards okadaic acid and EDTA. | Eur. J. Biochem. 269 5016-5023 2002 FEBS 2002 doi Identification and characterization of a mammalian 14-kDa phosphohistidine phosphatase Pia Ek1 Gunilla Pettersson1 Bo Ek2 Feng Gong1 Jin-Ping Li1 and Orjan Zetterqvist1 1 Department of Medical Biochemistry and Microbiology Uppsala University Uppsala Sweden 2Department of Plant Biology The Swedish University of Agricultural Sciences Uppsala Sweden 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-nitro-anilide 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 Limol-min-1 mg-1 at pH with 7 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 Ltmol-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. Keywords dephosphorylation N-phosphorylation phosphoamidase .

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