Identification of a Consensus Cyclin-dependent Kinase Phosphorylation Site Unique to the Nuclear Form of Human Deoxyuridine Triphosphate Nucleotidohydrolase

Abstract

, 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 ...

Intracellular Location of Thymidylate Synthase and Its State of Phosphorylation

“…It is possible that the mitochondrial location of TS is due to the nonspecific adherence of TS to the surface of the mitochondria, but the extensive washing procedure employed would appear to mitigate against this possibility, as well as our recent finding that the enzyme can be released on hypotonic lysis of the mitochondria. 3 Preliminary results reveal that the H35 TS is labeled on a serine, as has been found also for dUTPase (33). Because of these common properties of TS and dUTPase, it would not be unreasonable to assume that the two enzymes reside in the same regions of the cell, possibly in the proposed replitase complex (12).…”

“…Alternatively, the enzyme might be chaperoned into the nucleus by a protein similar to that described recently for the Id family of helix-loop-helix proteins (32). It is of interest to note that dUTPase, like TS, although associated with the nucleus, does not contain a characteristic nuclear localization signal either (15,33).…”

“…This does not appear to be an isolated case as more and more instances are being reported on the influence of protein phosphorylation on the cellular location of various proteins (34,36,37), particularly those associated with cell division. It is interesting to note that only the nuclear form of dUTPase is also phosphorylated (33). Whether a similar case can be made for TS remains to be determined.…”

The Human dUTPase Gene Encodes both Nuclear and Mitochondrial Isoforms

“…To determine the structural differences between these isoforms, a combination of NH 2 -terminal protein sequencing and mass spectrometry was utilized to characterize each form in detail, demonstrating that the two isoforms are largely identical, differing only in a short region of their amino termini. Analysis of dUTPase phosphorylation demonstrated that DUT-N is serinephosphorylated at a consensus cyclin-dependent protein kinase phosphorylation site (Ser 11 of DUT-N), suggesting a link to cell cycle regulation (15). While DUT-M contains the identical site, it does not undergo phosphorylation in HeLa cells.…”

“…While DUT-M contains the identical site, it does not undergo phosphorylation in HeLa cells. Site-directed mutagenesis analysis demonstrates that serine phosphorylation of DUT-N does not regulate enzymatic activity, and the biological significance of DUT-N phosphorylation remains unclear (15). Kinetic analysis of each isoform demonstrates that they retain identical affinities for dUTP despite their structural differences.…”