Deoxyuridine triphosphate nucleotidohydrolase (dUTPase; EC 3.6.1.23) was purified from HeLa cells by immunoaffinity chromatography. Based on SDS-polyacrylamide gel electrophoresis, two distinct forms of dUTPase were evident in the purified preparation. These proteins were further characterized by a combination of NH2-terminal protein sequencing, mass spectrometry, and mass spectrometry-based protein sequencing. These analyses indicate that the two forms of dUTPase are largely identical, differing only in a short region of their amino-terminal sequences. Despite the structural difference, both forms of dUTPase exhibited identical binding characteristics for dUTP. Each form of dUTPase has a distinct cellular localization. Cellular fractionation and isopycnic density centrifugation indicate that the lower molecular weight form of dUTPase (DUT-N) is associated with the nucleus, while the higher molecular weight species (DUT-M) fractionates with the mitochondria. The DUT-N isoform is approximately 30-fold more abundant in HeLa cells than DUT-M as determined by densitometry. The NH2-terminal protein sequence of both DUT-N and DUT-M did not match previous reports of the predicted amino-terminal sequence for human dUTPase (McIntosh, E.M., Ager, D.D., Gadsden, M.H., and Haynes, R.H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8020-8024; Strahler, J.R., Zhu X., Hora, N., Wang, Y.K., Andrews, P.C., Roseman, N.A., Neel, J.V., Turka, L., and Hanash, S.M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4991-4995). A cDNA corresponding to the DUT-N isoform was isolated utilizing an oligonucleotide probe based on the determined NH2-terminal sequence. The cDNA contains a 164-amino acid open reading frame, encoding a protein of Mr 17,748. The DUT-N cDNA sequence matches the previously cloned cDNAs with the exception of a few discrepancies in the 5' end. Our data indicate a 69-base pair addition to the 5' end of the previously reported open reading frame.

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, we identified two distinct isoforms of dUTPase in human cells. These isoforms are individually targeted to the nucleus (DUT-N) and mitochondria (DUT-M). The proteins are nearly identical, differing only in a short region of their amino termini. Despite the structural differences between these proteins, they retain identical affinities for dUTP (preceding article). In previous work, this laboratory demonstrated that dUTPase is posttranslationally phosphorylated on serine residue(s) (Lirette, R., and Caradonna, S. (1990) J. Cell. Biochem. 43, 339 -353). To extend this work and determine if both isoforms of dUTPase are phosphorylated, a more in depth analysis of dUTPase phosphorylation was undertaken. [ 32 P]Orthophosphatelabeled dUTPase was purified from HeLa cells, revealing that only the nuclear form of dUTPase is phosphorylated. Electrospray tandem mass spectrometry was used to identify the phosphorylation site as Ser-11 in the amino-terminal tryptic peptide PCSEETPAIpSPSKR (the NH 2 -terminal Met is removed in the mature protein). Mutation of Ser-11 by replacement with Ala blocks phosphorylation of dUTPase in vivo. Analysis of the wild type and Ser-11 3 Ala mutant indicates that phosphorylation does not regulate the enzymatic activity of the DUT-N protein in vitro. Additionally, experiments with the Ser-11 3 Ala mutant indicate that phosphorylation does not appear to play a role in subunit association of the nuclear form of dUTPase. The amino acid context of this phosphorylation site corresponds to the consensus target sequence for the cyclin-dependent protein kinase p34 cdc2 . Recombinant DUT-N was specifically phosphorylated on Ser-11 in vitro with immunoprecipitated p34 cdc2 . Together, these data suggest that the nuclear form of dUTPase may be a target for cyclin-dependent kinase phosphorylation in vivo.Human dUTPase 1 was first purified from HeLa cells by Caradonna and Adamkiewicz (2). The enzyme was characterized as a homodimer with a monomeric molecular weight of 22,500 and a K m for dUTP of 2.5 M. The dUTPase monomers associate in the presence of divalent cations such as magnesium or manganese to form the active enzyme. In later work, we identified the human enzyme as a serine phosphoprotein (1). Studies on herpesvirus infection of HeLa cells have shown that cellular dUTPase activity decreases postinfection, while the virus-encoded dUTPase activity increases. It was postulated that the associated decrease of cellular dUTPase activity was not due to rapid degradation but rather correlated with dephosphorylation of the host dUTPase protein. These data suggest that phosphorylation may play a role in regulating the enzymatic activity of the human dUTPase protein. More recently, Strahler and co-workers demonstrated that, upon peripheral blood lymphocyte stimulation, there is a large induction of the phosphorylated form of dUTPase. This induction of dUTPase protein coincided with the onset of DNA replication, suggesting a link between dUTPase phosphorylation and the proliferation status of the cell (4).In ...

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