Identification of a Consensus Cyclin-dependent Kinase Phosphorylation Site Unique to the Nuclear Form of Human Deoxyuridine Triphosphate Nucleotidohydrolase (∗)
In the preceding report (Ladner, R. D., McNulty, D. E., Carr, S. A., Roberts, G. D., and Caradonna, S. J. (1996) J. Biol. Chem. 271, 7745-7751, 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...
Saved in:
Published in | The Journal of biological chemistry Vol. 271; no. 13; pp. 7752 - 7757 |
---|---|
Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Elsevier Inc
29.03.1996
American Society for Biochemistry and Molecular Biology |
Subjects | |
Online Access | Get full text |
Cover
Loading…
Summary: | In the preceding report (Ladner, R. D., McNulty, D. E., Carr, S. A., Roberts, G. D., and Caradonna, S. J. (1996) J. Biol. Chem. 271, 7745-7751, 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. [32P]Orthophosphate-labeled 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 NH2-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 → Ala mutant indicates that phosphorylation does not regulate the enzymatic activity of the DUT-N protein in vitro. Additionally, experiments with the Ser-11 → 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 p34cdc2. Recombinant DUT-N was specifically phosphorylated on Ser-11 in vitro with immunoprecipitated p34cdc2. Together, these data suggest that the nuclear form of dUTPase may be a target for cyclin-dependent kinase phosphorylation in vivo. |
---|---|
Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.271.13.7752 |