(Received for publication, October 16, 1995; and in revised form, December 12, 1995)
From the
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.
[P]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 NH
-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 p34
.
Recombinant DUT-N was specifically phosphorylated on Ser-11 in
vitro with immunoprecipitated p34
.
Together, these data suggest that the nuclear form of dUTPase may be a
target for cyclin-dependent kinase phosphorylation in vivo.
Human dUTPase ()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
for dUTP of 2.5 µM. 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 this report we extend previous work involving
dUTPase phosphorylation. A single site of phosphorylation correlating
to Ser-11 of the nuclear isoform of dUTPase was identified. Although
both the DUT-N and DUT-M contain the identical site, phosphorylation of
Ser-11 is unique to the nuclear isoform. This site correlates with the
consensus sequence for cyclin-dependent kinase phosphorylation and is
specifically phosphorylated by p34in
vitro, suggesting a link to the cyclin signaling pathway. Studies
with a Ser-11
Ala mutant of DUT-N suggest that this modification
is unrelated to both enzymatic activity and subunit association.
Antibodies against the carboxyl termini of
p34 (CDC2 (Ab-1)) were purchased from Oncogene Science
and used as per the manufacturer's recommendations. The peptide
to which the p34
monoclonal antibody was generated
(CDNQIKKM; CDC2 (peptide 1)) was also purchased from Oncogene Science.
CDC2 (peptide 1) effectively blocks CDC2 (Ab-1).
Peptide competition
studies were carried out by preincubating the p34 antisera (CDC2 (Ab-1)) with a 5-fold molar excess of blocking
peptide (CDC2 (peptide 1)) for 2 h at 4 °C. The immunoprecipitation
was then carried out as above.
Figure 1:
The
nuclear form of dUTPase is phosphorylated. HeLa S3 cells were labeled
with [P]orthophosphate and dUTPase was
immunoprecipitated, utilizing dUTPase-specific monoclonal antibodies,
from total cell extracts. The immunoprecipitates were fractionated by
SDS-PAGE and transferred to nitrocellulose. dUTPase protein was
detected by immunoblot analysis utilizing affinity-purified,
dUTPase-specific polyclonal antibody. The protein bands were visualized
using a chemiluminescent detection system. Lane 1, immunoblot
analysis demonstrates the presence of the major nuclear form of dUTPase
(DUT-N) and the larger, less abundant mitochondrial isoform (DUT-M).
The chemiluminescent detection reaction was quenched by exposing to
visible light, and the membrane was subjected to autoradiography to
detect the
P labeling. Lane 2, autoradiograph
demonstrating the phosphorylation of DUT-N. There is no detectable
phosphorylation associated with the DUT-M
isoform.
The number of phosphate
groups and their sequence locations in the phosphopeptide present in
the major radioactive fraction was established by electrospray mass
spectrometry(5, 6, 7) . The major peptide
signal observed had a determined M of 1638.0.
Tandem mass spectrometry ( (7) and references therein) of the
(M + 2H)
parent ion of the phosphorylated
peptide provided the partial sequence PCSEETPAXpSP-, where pS
is phosphoserine, Cys is carboxamidomethylated, and X is
either Leu or Ile, which cannot be distinguished in this experiment (Fig. 2; see legend for an explanation of the fragmentation).
This subsequence corresponds to residues 2-12 of the mature
protein (Fig. 4). The molecular weight calculated for the
tryptic peptide Pro-2-Arg-15 is 1637.7. This is in close
agreement with the predicted molecular weight for this peptide. These
data establish the identity of this tryptic peptide as residues
2-15 of the protein in which Ser-11 is phosphorylated.
Figure 2:
Mass spectrometry analysis of the DUT-N
phosphopeptide. Electrospray tandem mass spectrometry of the (M +
2H) (m/z 820) of approximately 1
pmol of the phosphopeptide. Fragment nomenclature is according to
Biemann and Roepstorff(20, 21) . The numbering
above the single-letter code sequence refers to y
ions formed by cleavage of the peptide bond of the nth amino acid from the COOH terminus with H-rearrangement to
form a charged, COOH-terminal peptide fragment
(NH
-CHR
-CO . . .
NH-CHR1-CO
H + H)
. The numbering
below the single-letter code sequence refers to b
ions formed by charge retention on the
NH
-terminal acylium fragments (NH
-CHR1-CO . . .
NHCHR
CO
); loss of CO from the
b
ions yields the a
ion
series. The mass increment between the y
and y
ions (167 Da), and the presence of satellite peaks formed by
the loss of either H
PO
(-98 Da) on all of
the y
ions that are formed by cleavage
COOH-terminal to the Pro-11 indicate that the phosphate is located on
Ser-10.
Figure 4:
Sequence comparison of known dUTPases.
Alignment of the amino acid sequences of dUTPases from human (DUT-N and
DUT-M), Saccharomyces cerevisiae (Yeast), E.
coli, simian retrovirus 1 (SRV1), mouse mammary tumor
virus (MMTV), visna virus, equine infectious anemia virus (EIAV), orf virus, vaccinia virus, herpes simplex virus type 1 (HSV-1), and varicella-zoster virus (VZV). The boxed regions (labeled I-V) correspond to the
five conserved domains common to all known dUTPases(8) . The asterisk above Ser-11 denotes the site of DUT-N
phosphorylation. The underlined region of the DUT-N sequence
indicates the consensus sequence for p34phosphorylation. Brackets in the HSV1 sequence represent
residues 118-156, which are not shown. Brackets in the VZV
sequence indicate residues 117-150, which are not
shown.
Figure 3:
In vivo verification of
phosphorylation on Ser-11 by site-directed mutagenesis. A,
Ser-11 was changed to Ala by site-directed mutagenesis. The dUTPase
native and mutant open reading frames were cloned into the eukaryotic
expression vector pEUK-C1. COS-7 cells were transfected with these
constructs and control vector (with no insert) using Lipofectin. Cells
were harvested after 60 h. Immunoblot analysis utilizing
affinity-purified polyclonal antibodies demonstrate the transient
expression of native and mutant dUTPases. Lane 1, pEUK-C1
alone; lane 2, pEUK-C1/native dUTPase; lane 3,
pEUK-C1/Ser-11 to Ala mutant dUTPase. B, COS-7 were
transfected as above, and cells were labeled with
[P]orthophosphate between 50 and 60 h
posttransfection. At 60 h cells were harvested, and dUTPase was
immunoprecipitated from total cell extracts. Immunoprecipitates were
washed and fractionated by SDS-PAGE. The dried gel was exposed to x-ray
film for 12 h at -80 °C. Lane 1, pEUK-C1, no insert; lane 2, pEUK-C1/native dUTPase; lane 3,
pEUK-C1/Ser-11
Ala mutant. Both the monoclonal and polyclonal
antibodies to human dUTPase do not cross-react with COS-7 derived
dUTPase.
To further
confirm that Ser-11 is the site of DUT-N phosphorylation, transfected
cells were labeled with [P]orthophosphate for 10
h at 50-60 h posttransfection. At 60 h, cells were harvested and
extracts were prepared. Each sample was subjected to
immunoprecipitation analysis using a human dUTPase-specific monoclonal
antibody. The immunoprecipitates were resolved by 15% SDS-PAGE and
visualized by autoradiography. The wild type DUT-N protein is readily
phosphorylated in COS-7 cells (Fig. 3B, lane
2). Phosphorylation of the mutant DUT-N however, is blocked by the
Ser-11
Ala mutation (Fig. 3B, lane 3).
The monoclonal antibody used in this experiment was determined to
quantitatively immunoprecipitate both the wild type and mutant forms of
dUTPase (data not shown). These data further indicate that Ser-11 is
the sole phosphorylation site of the nuclear form of human dUTPase in vivo.
Although DUT-N
and DUT-M differ in their NH termini, the site of Ser
phosphorylation is retained in both isoforms (Fig. 4). Despite
this conservation, only the DUT-N isoform is phosphorylated at any
detectable levels. Mass spectrometry analysis of the analogous tryptic
peptide derived from DUT-M shows no change in mass indicative of a
phosphorylated residue(22) .
To examine the
authenticity of the consensus CDK phosphorylation site, in vitro kinase assays were performed to determine if DUT-N could be
phosphorylated specifically on Ser-11 by p34. Polyclonal
antibodies generated against the carboxyl terminus of human
p34
were used to immunoprecipitate protein kinase
activity from HeLa S3 cells. The immunoprecipitates were washed, and
recombinant DUT-N protein (baculovirus-expressed) was added to in
vitro kinase assays. The samples were subsequently fractionated by
SDS-PAGE, and protein phosphorylation was detected by autoradiography. Fig. 5, lane 1, illustrates the in vitro phosphorylation of recombinant dUTPase by the immunoprecipitated
p34
protein kinase. Fig. 5, lane 2,
demonstrates that dUTPase phosphorylation can be specifically blocked
by the addition of the competing peptide to the immunoprecipitation
reaction, verifying the identity of the immunoprecipitated kinase
utilized in the in vitro assays. To confirm the specificity of
phosphorylation in vitro, a recombinant Ser-11
Ala
mutant was utilized in an identical kinase reaction. Fig. 5, lane 3, illustrates that the Ser-11
Ala mutant
specifically prevents phosphorylation by p34
,
documenting the site of DUT-N phosphorylation by p34
in vitro. Together, these experiments demonstrate that the
recombinant DUT-N protein is phosphorylated by p34
specifically on Ser-11 in vitro.
Figure 5:
Phosphorylation of dUTPase by
p34in vitro. Recombinant DUT-N
protein was phosphorylated in vitro with immunoprecipitated
p34
from HeLa cells as described under
``Experimental Procedures.'' DUT-N was subsequently
immunoprecipitated with a monoclonal antibody and fractionated by
SDS-PAGE. The dried gel was exposed to x-ray film for 12 h. Lane
1, recombinant DUT-N protein; lane 2, DUT-N protein,
competition experiment with p34
peptide; lane 3, Ser-11
Ala mutant. All reactions were carried
out under identical conditions.
We previously
hypothesized that phosphorylation may regulate subunit
association(1) . Both the wild type and Ser-11 Ala
mutant were assayed for the ability to undergo magnesium-dependent
multimerization as described previously(2) . Both the wild type
and mutant forms of the DUT-N protein formed higher molecular weight
complexes, demonstrating that monomer association is independent of the
phosphorylation state of the protein (data not shown).
The amino acid context of the DUT-N
phosphorylation site corresponds to the consensus target sequence of
the cyclin-dependent kinase p34(9) . In order to
determine that the Ser-11 phosphorylation site was authentic, we
performed in vitro experiments demonstrating that dUTPase is
specifically phosphorylated on Ser-11 by immunoprecipitated
p34
from HeLa S3 cells (Fig. 5). However, these
experiments do not confirm that p34
directly
phosphorylates dUTPase in vivo. There is a family of related
kinases that share extensive homology with p34
(e.g. CDK2 through CDK5)(12) . It is possible that DUT-N is a
target for one or several of these p34
-related kinases in vivo.
The role of dUTPase
phosphorylation remains to be elucidated. In a previous report, we
postulated that dUTPase phosphorylation may regulate enzymatic
activity(1) . Several lines of evidence, presented in this
work, suggest that phosphorylation does not regulate the enzymatic
activity of DUT-N. Mutagenesis of Ser-11 Ala prevents
phosphorylation; however, experiments with the mutant protein
demonstrate that enzymatic activity is not significantly altered in
vitro. Second, a recombinant human dUTPase protein has been
expressed that lacks the first 22 amino acids present in the nuclear
form of dUTPase. This recombinant protein does not contain the
phosphorylation site yet still retains full enzymatic
activity(18) . A third line of evidence arguing against the
regulation of dUTPase activity by phosphorylation comes from a
comparison of the enzymatic activities of the nuclear and mitochondrial
isoforms. DUT-M contains the identical site for phosphorylation as
DUT-N but is not phosphorylated in vivo. Although DUT-M is not
phosphorylated, it exhibits identical kinetic characteristics (K
= 2.5 µM) to the
phosphorylated nuclear form. Taken together, these observations suggest
that the phosphorylation of dUTPase does not significantly govern
enzymatic activity under the assay conditions utilized in
vitro.
Another possible role of phosphorylation is the
formation of the dUTPase multimer. Caradonna and Adamkiewicz (2) first described human dUTPase as a homodimer, although
molecular modeling of human dUTPase based on the E. coli dUTPase crystal structure (19) suggests that the human
protein is a homotrimer. ()Experiments utilizing both the
DUT-N recombinant and the Ser-11
Ala mutant demonstrate that
multimerization is independent of Ser-11 phosphorylation. Additional
evidence supporting this again comes from a truncated recombinant form
of dUTPase lacking 22 amino-terminal residues. This protein was shown
to trimerize independent of the Ser-11 phosphorylation
site(18) .
DUT-N phosphorylation may also regulate its
intracellular localization. The DUT-N and DUT-M isoforms differ
exclusively in their amino termini. This distinction appears to confer
the ability of DUT-M to localize in the mitochondria. It is conceivable
that the exclusive phosphorylation of DUT-N may play a role in nuclear
targeting of this protein. Taken a step further, Ser-11 may confer the
ability of DUT-N to localize in specific regions of the nucleus where
the dUTPase function is required. The Ser-11 Ala mutant should
aid in the testing of these hypotheses.
In summary, we have continued our investigation of the detailed biochemistry of dUTPase in human cells, uncovering an additional layer of detail. Elucidation of two distinct isoforms localized to the mitochondria and nucleus, respectively, as well as identification of a CDK phosphorylation site specific to the nuclear isoform, all suggest that this enzyme function is highly regulated within the cell.