IDENTIFICATION OF A CONSENSUS CYCLIN-DEPENDENT KINASE PHOSPHORYLATIONSITE UNIQUE TO THE NUCLEAR FORM OF HUMAN DEOXYURIDINE TRIPHOSPHATE NUCLEOTIDOHYDROLASE
Rd. Ladner et al., IDENTIFICATION OF A CONSENSUS CYCLIN-DEPENDENT KINASE PHOSPHORYLATIONSITE UNIQUE TO THE NUCLEAR FORM OF HUMAN DEOXYURIDINE TRIPHOSPHATE NUCLEOTIDOHYDROLASE, The Journal of biological chemistry, 271(13), 1996, pp. 7752-7757
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.