Identification and characterization of a mammalian 14-kDa phosphohistidine phosphatase

• Protein histidine phosphorylation in eukaryotes has beensparsely studied compared to protein serine/threonine andtyrosine phosphorylation. In an attempt to rectify this byprobing porcine liver cytosol with the phosphohistidinecontainingpeptide succinyl-Ala-His(P)-Pro-Phe-p-nitroanilide(phosphopeptide I), we observed a phosphataseactivity that was insensitive towards okadaic acid andEDTA. This suggested the existence of a phosphohistidinephosphatase different from protein phosphatase 1, 2Aand 2C. A 1000-fold purification to apparent homogeneitygave a 14-kDa phosphatase with a specific activity of 3lmolÆmin)1Æmg)1 at pH 7.5 with 7 lM phosphopeptide Ias substrate. Partial amino-acid sequence determination ofthe purified porcine enzyme by MS revealed similaritywith a human sequence representing a human chromosome9 gene of hitherto unknown function. Molecularcloning from a human embryonic kidney cell cDNAlibraryfollowed by expression and purification, yielded aprotein with a molecular mass of 13 700 Da, and anEDTA-insensitive phosphohistidine phosphatase activityof 9 lmolÆmin)1Æmg)1 towards phosphopeptide I. Nodetectable activity was obtained towards a set of phosphoserine-,phosphothreonine-, and phosphotyrosine peptides.Northern blot analysis indicated that the humanphosphohistidine phosphatase mRNA was present preferentiallyin heart and skeletal muscle. These resultsprovide a new tool for studying eukaryotic histidinephosphorylation/dephosphorylation.

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinsk bioteknologi -- Medicinsk bioteknologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Medical Biotechnology -- Medical Biotechnology (hsv//eng)

Nyckelord

dephosphorylation

N-phosphorylation

phosphoamidase

phosphopeptide

protein histidine phosphatase

Biochemistry

Biokemi

Biokemi

Biochemistry

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)