Identification of sequence determinants of human nuclear dUTPase isoform localization.
dUTP nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate and is the central regulator of cellular dUTP pools. Nuclear (DUT-N) and mitochondrial (DUT-M) isoforms of the protein have been identified in humans and arise from the same gene by the alternative use of 5' exons. Recently, it has been shown that these isoforms are aberrantly expressed in some cancers and overexpression of dUTPase in the nucleus is associated with resistance to chemotherapeutic agents that target thymidylate biosynthesis. In this study, we have examined the signals necessary for dUTPase isoform localization using green fluorescent protein fusion constructs. We report that the N-terminal 23 amino acids of DUT-N are required but not sufficient for complete nuclear localization. Within this region, we identified a small cluster of basic residues (K(14)R(15)R(17)) that resemble a classic monopartite nuclear localization signal (NLS). Mutation of these residues completely abolishes nuclear localization. In addition, phosphorylation of Ser11 near the putative NLS has no affect on DUT-N nuclear localization. Through deletion analysis we show improved sorting of DUT-N to the nucleus when most of the protein sequence is present. Therefore, we conclude that DUT-N may contain a complex NLS that is located throughout the entire protein.
Tinkelenberg
Beverly A
BA
Department of Molecular Biology, School of Osteopathic Medicine, University of Medicine and Dentistry of New Jersey, 2 Medical Center Drive, Stratford, NJ 08084, USA.
Fazzone
William
W
Lynch
Frank J
FJ
Ladner
Robert D
RD
D016475:3T3 Cells; D017398:Alternative Splicing / Q000235:genetics; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002451:Cell Compartmentation / Q000235:genetics; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005057:Eukaryotic Cells / Q000187:drug effects / Q000201:enzymology*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D019073:HT29 Cells; D006801:Humans; D051379:Mice; D009154:Mutation / Q000235:genetics; D009369:Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics; D011755:Pyrophosphatases / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D012694:Serine / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D013940:Thymidylate Synthase / Q000378:metabolism; D014544:Uridine Triphosphate / Q000378:metabolism*
D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D012694:Serine; D013940:Thymidylate Synthase; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase; D014544:Uridine Triphosphate