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 micro mol.min-1.mg-1 at pH 7.5 with 7 micro 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 micro mol.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.
Ek
Pia
P
Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
Pettersson
Gunilla
G
Ek
Bo
B
Gong
Feng
F
Li
Jin-Ping
JP
Zetterqvist
Orjan
O
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001671:Biochemistry / Q000379:methods; D002899:Chromosomes, Human, Pair 9; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D008099:Liver / Q000201:enzymology; D008322:Mammals; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D012333:RNA, Messenger / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
D010748:Phosphopeptides; D012333:RNA, Messenger; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1