IDENTIFICATION OF A CONSENSUS CYCLIN-DEPENDENT KINASE PHOSPHORYLATIONSITE UNIQUE TO THE NUCLEAR FORM OF HUMAN DEOXYURIDINE TRIPHOSPHATE NUCLEOTIDOHYDROLASE

In the preceding report (Ladner, R. D., McNulty, D. E., Carr, S. A., R oberts, G. D., and Caradonna, S. J. (1996) J. Biol. Chem. 271, 7745-77 51, we identified two distinct isoforms of dUTPase in human cells. The se isoforms are individually targeted to the nucleus (DUT-N) and mitoc hondria (DUT-M). The proteins are nearly identical, differing only in a short region of their amino termini. Despite the structural differen ces between these proteins, they retain identical affinities for dUTP (preceding article). In previous work, this laboratory demonstrated th at 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 pho sphorylated, a more in depth analysis of dUTPase phosphorylation was u ndertaken. [P-32]Orthophosphate-labeled dUTPase was purified from HeLa cells, revealing that only the nuclear form of dUTPase is phosphoryla ted. Electrospray tandem mass spectrometry was used to identify the ph osphorylation site as Ser-11 in the amino-terminal tryptic peptide PCS EETPAIpSPSKR (the NH2-terminal Met is removed in the mature protein). Mutation of Ser-11 by replacement with Ala blocks phosphorylation of d UTPase in vivo. Analysis of the wild type and Ser-11 --> Ala mutant in dicates 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 pla y a role in subunit association of the nuclear form of dUTPase. The am ino acid context of this phosphorylation site corresponds to the conse nsus 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 kinas e phosphorylation in vivo.