Characterization of a novel mammalian phosphatase having sequence similarity to Schizosaccharomyces pombe PHO2 and Saccharomyces cerevisiae PHO13.
p34, a specific p-nitrophenyl phosphatase (pNPPase) was identified and purified from the murine cell line EL4 in a screen for the intracellular molecular targets of the antiinflammatory natural product parthenolide. A BLAST search analysis revealed that it has a high degree of sequence similarity to two yeast alkaline phosphatases. We have cloned, sequenced, and expressed p34 as a GST-tagged fusion protein in Escherichia coli and an EE-epitope-tagged fusion protein in mammalian cells. Using p-nitrophenyl phosphate (pNPP) as a substrate, p34 is optimally active at pH 7.6 with a K(m) of 1.36 mM and K(cat) of 0.052 min(-1). Addition of 1 mM Mg(2+) to the reaction mixture increases its activity by 14-fold. Other divalent metal ions such as Co(2+) and Mn(2+) also stimulated the activity of the enzyme, while Zn(2+), Fe(2+), and Cu(2+) had no effect. Furthermore, both NaCl and KCl enhanced the activity of the enzyme, having maximal effect at 50 and 75 mM, respectively. The enzyme is inhibited by sodium orthovanadate but not by sodium fluoride or okadaic acid. Mutational analysis data suggest that p34 belongs to the group of phosphatases characterized by the sequence motif DXDX(T/V).
Ndubuisil
MacKevin I
MI
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA.
Kwok
Benjamin H B
BH
Vervoort
Jonathan
J
Koh
Brian D
BD
Elofsson
Mikael
M
Crews
Craig M
CM
D009597:4-Nitrophenylphosphatase / Q000737:chemistry*; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism / Q000494:pharmacology; D001710:Biotin / Q000494:pharmacology; D002413:Cations, Divalent / Q000737:chemistry; D002414:Cations, Monovalent / Q000737:chemistry; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005656:Fungal Proteins / Q000737:chemistry*; D006367:HeLa Cells; D018398:Homeodomain Proteins; D006801:Humans; D008670:Metals / Q000737:chemistry; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism / Q000502:physiology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D012717:Sesquiterpenes / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D015534:Trans-Activators / Q000737:chemistry*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D000894:Anti-Inflammatory Agents, Non-Steroidal; D002413:Cations, Divalent; D002414:Cations, Monovalent; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D018398:Homeodomain Proteins; D008670:Metals; D016328:NF-kappa B; C051611:PHO2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D012717:Sesquiterpenes; D015534:Trans-Activators; D014409:Tumor Necrosis Factor-alpha; C002669:parthenolide; D014638:Vanadates; D001710:Biotin; D010749:Phosphoprotein Phosphatases; D009597:4-Nitrophenylphosphatase