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Identification and Characterization of a Mammalian 14-Kda

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Identification and Characterization of a Mammalian 14-Kda Eur. J. Biochem. 269, 5016–5023 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03206.x Identification and characterization of a mammalian 14-kDa phosphohistidine phosphatase Pia Ek1, Gunilla Pettersson1,BoEk2, Feng Gong1, Jin-Ping Li1 and O¨ rjan Zetterqvist1 1Department 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 cloning from a human embryonic kidney cell cDNA- sparsely studied compared to protein serine/threonine and library followed by expression and purification, yielded a tyrosine phosphorylation. In an attempt to rectify this by protein with a molecular mass of 13 700 Da, and an probing porcine liver cytosol with the phosphohistidine- EDTA-insensitive phosphohistidine phosphatase activity containing peptide succinyl-Ala-His(P)-Pro-Phe-p-nitro- of 9 lmolÆmin)1Æmg)1 towards phosphopeptide I. No anilide (phosphopeptide I), we observed a phosphatase detectable activity was obtained towards a set of phos- activity that was insensitive towards okadaic acid and phoserine-, phosphothreonine-, and phosphotyrosine pep- EDTA. This suggested the existence of a phosphohistidine tides. Northern blot analysis indicated that the human phosphatase different from protein phosphatase 1, 2A phosphohistidine phosphatase mRNA was present pre- and 2C. A 1000-fold purification to apparent homogeneity ferentially in heart and skeletal muscle. These results gave a 14-kDa phosphatase with a specific activity of 3 provide a new tool for studying eukaryotic histidine )1 )1 lmolÆmin Æmg at pH 7.5 with 7 lM phosphopeptide I phosphorylation/dephosphorylation. as substrate. Partial amino-acid sequence determination of Keywords: dephosphorylation; N-phosphorylation; phos- the purified porcine enzyme by MS revealed similarity phoamidase; phosphopeptide; protein histidine phospha- with a human sequence representing a human chromo- tase. some 9 gene of hitherto unknown function. Molecular Boyer and coworkers detected protein-bound phosphohis- particularly well-studied example. The other is the reversible tidine in rat-liver mitochondrial succinyl-CoA synthetase protein histidine phosphorylation by protein kinases and almost 40 years ago [1,2]. Despite the long time interval and phosphatases [3,11]. An important contribution to the latter the fact that phosphohistidine represents a substantial field was the purification of a yeast protein histidine kinase fraction of eukaryotic protein-bound phosphate [3], only a in 1991 [12]. Access to this enzyme also made possible the few phosphohistidine-containing proteins have been detec- preparation of 32P-labelled histone H4, which was later ted compared to the large number of eukaryotic proteins used as substrate in the search for phosphohistidine phosphorylated on serine, threonine and tyrosine residues. phosphatases. Using such an approach, the catalytic One reason for this difference may be that the N-bound subunits of the well-studied serine/threonine protein phos- phosphate of phosphohistidine easily escapes detection by phatases 1, 2A and 2C were shown to display kcat/Km values 6 7 )1 )1 common analytical procedures, due to its lability under in the order of 10 )10 M Æs for the phosphohistidine- acidic conditions, e.g. during fixation and staining of gels containing histone H4. These values were at least as high as after SDS/PAGE [4]. those for their naturally occurring phosphoserine-contain- The studies on eukaryotic protein histidine phosphory- ing substrates [13,14]. lation and dephosphorylation have dealt with essentially Notwithstanding these findings, there is still a possibility two aspects. One is the intermediary phosphorylation of that additional phosphohistidine phosphatases exist. For enzymes [5–10], of which nucleoside diphosphate kinase is a instance, a 13-kDa bovine liver phosphoamidase has been reported to possess activity also towards intermediary phosphorylated nucleoside diphosphate kinase and succi- Correspondence to J.-P. Li, Department of Medical Biochemistry nyl-CoA synthetase [15]. In a recent review [16], data in and Microbiology, Box582, SE-751 23 Uppsala, Sweden. press on a 14-kDa phosphatase from rabbit liver that Fax: + 46 18 4714209, Tel. + 46 18 4714241, dephosphorylates the phosphohistidine of autophosphory- E-mail: [email protected] lated ATP-citrate lyase is reported [17]. Abbreviations: His(P), phosphohistidine; MDEA, N-methyl-dietha- In the search for new phosphohistidine phosphatases we nolamine; phosphopeptide I, Suc-Ala-His(P)-Pro-Phe-pNA; tried a different approach. It seemed reasonable to assume pNA, p-nitroanilide. that the immediate environment of the N-phosphate of Note: The nucleotide sequence for human 14-kDa phosphohistidine protein-bound phosphohistidine can influence its sensitivity phosphatase has been submitted to the GenBank Nucleotide Sequence to a phosphatase. Of potential interest in such a context was Database under the accession number AF393504. the finding that the rate of isomerization of the histidine- (Received 9 June 2002, revised 21 August 2002, proline bond in the peptide Suc-Ala-His-Pro-Phe-pNA is accepted 27 August 2002) influenced by the degree of protonation of its imidazole ring Ó FEBS 2002 A mammalian 14-kDa phosphohistidine phosphatase (Eur. J. Biochem. 269) 5017 [18]. We therefore performed a chemical N-phosphorylation phosphopeptide was analysed by the malachite green of the imidazole of this peptide by means of phospho- method [19]. The peptide concentration was calculated by amidate, and used Suc-Ala-His(P)-Pro-Phe-pNA (phospho- using the molar absorbancy 13 600 of pNA at 315 nm. The peptide I) as a probe to search for new phosphohistidine nature of the phosphohistidine isomer in phosphopeptide I phosphatases. As the result of that screening we detected a was not determined, but it was considered to be 3-phos- 14-kDa phosphatase in porcine liver cytosol and were also phohistidine, rather than 1-phosphohistidine, due to the able to ascribe phophohistidine phosphatase activity to a 48 h phosphorylation time used [4,20]. 14-kDa protein coded for by a human chromosome 9 gene. Phosphatase assays EXPERIMENTAL PROCEDURES The standard assay of the phosphohistidine phosphatase activity was performed at 30 °C and pH 7.5 in a 150-lL Materials reaction mixture consisting of 25 mM Hepes, 15 mM Phosphoamidate was prepared according to the method of MDEA, 1 mM MgCl2 and 7 lM phosphopeptide I. The Wei and Matthews [4]. Suc-Ala-His-Pro-Phe-pNA was reaction was stopped after 10–30 min by addition of 2 M purchased from Bachem AG, Switzerland. Malachite green KOH to give pH 12.5. After 10 min incubation at 0 °C, 1 M reagent was obtained from Apoteksbolaget, So¨ dersjukhuset, HCl was added to give pH 9.0. The mixture was then Stockholm, Sweden. MDEA was bought from Riedel de applied to a 1-mL Mono Q column equilibrated in 0.085 M Hae¨ n. Diisopropyl fluorophosphate was from Fluka MDEA, pH 9.0, in 20% methanol. After washing with Chemie AG, Switzerland. Phenylmethanesulfonyl fluoride 2 mL of the equilibration buffer, elution was performed and okadaic acid were from Sigma. Trypsin of modified with a 25-mL linear gradient of MDEA from 0.085 M to )1 sequencing grade was obtained from Promega, and LysC of 0.765 M in 20% methanol at 1 mLÆmin . The absorbance Achromobacter lytii from Wako. The O-phosphorylated at 315 nm was monitored and the degree of dephosphory- phosphopeptides EQTRsLDGR-NH2, DHTGFLtEY- lation was calculated from the peak areas of phosphorylated VATRWC-NH2, DADEyL-NH2, EGDNDyIIPL-NH2, and dephosphorylated peptides. In a control experiment, KKPRyEIRW-NH2, and LIEDNEyTARQGA-NH2 the stoichiometry of the cleavage was confirmed by the (phosphorylated residues indicated by lower-case) were gifts occurrence of equal amounts of orthophosphate and from Mrs Ulla Engstro¨ m at the Ludwig Institute of Cancer unphosphorylated Suc-Ala-His-Pro-Phe-pNA. The identity Research, Uppsala. The O-phosphopeptides GRRPsLFG of the peptide product was controlled by amino acid ˚ and APDyTTPEMY-NH2 were obtained from Dr Ake analysis. Engstro¨ m at the Peptide Synthesis and Analysis Laboratory In initial phosphohistidine phosphatase assays with of the Department of Medical Biochemistry and Microbio- porcine liver cytosol, the phosphorylated and dephosphory- logy, Uppsala University. The SP6 primer 5¢-ATT TAG lated peptides were separated on a 24-mL Superose 12 GTG ACA CTA TAG-3¢ and the three oligonucleotide column in 0.10 M MDEA, pH 9.0. In this system, PCR-primers described below were from DNA Technology phosphopeptide I was eluted after 0.5 column volumes, A/S, Denmark. The T7 primer 5¢-TAA TAC GAC TCA whereas the corresponding dephosphopeptide was eluted CTA TAG GG-3¢ and protein molecular weight references after 0.9 column volumes. Both peptides were traced by the were from Amersham Biosciences. Terminator ready reac- absorbance at 315 nm. tion mix was from PE Biosystems. DEAE cellulose (DE-52) Phosphoamidase activity of the porcine liver phospho- was from Whatman, Sephadex G-200 Fine, Mono Q HR 5/5 histidine phosphatase was assayed by incubating the columns, and the gel filtration columns Sephadex G-25 phosphatase with 1 mM phosphoamidate in 25 mM Hepes, PD-10, Superose 12 HR 10/30, Superose 12 PC 3.2/30, and pH 7.5, at 30 °C for 30 min [15]. The released orthophos- Superdex 75 PC 3.2/30 were from Amersham Biosciences. phate was measured by a modification of the method of Martin and Doty [21], using toluene instead of benzene. To keep background hydrolysis of the acid-labile phospho- Preparation of the phosphohistidine-containing peptide amidate as low as possible, the sample (200 lL), isobutanol/ Suc-Ala-His(P)-Pro-Phe-pNA toluene 1 : 1 (250 lL), and 5% ammonium molybdate in The protocol used was adapted from that used for the 2 M H2SO4 (50 lL) were added in that order, and extraction synthesis and isolation of free 3-phosphohistidine by Wei was performed by immediate vortex-mixing in a capped and Matthews [4].
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