![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 56, Issue 3, 562-569, September 1999
Division of Clinical Virology, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden
 |
Summary |
|---|
 Top
 Summary
 Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Phosphorylation of deoxycytidine analogs by cellular enzymes is
a prerequisite for the activity of these compounds. We have investigated the kinetic parameters for the phosphorylation of 1-
-D-arabinofuranosylcytosine (araC) and
2',2'-difluorodeoxycytidine (dFdC) to their diphosphate forms catalyzed
by human UMP-CMP kinase. We cloned the cDNA of this enzyme to enable
characterization of the recombinant protein, determine its expression
in different tissues, and determine the chromosome location of the
gene. We showed that the recombinant UMP-CMP kinase phosphorylated CMP, dCMP, and UMP with highest efficiency and dUMP, AMP, and dAMP with lower efficiency. The monophosphates of araC and dFdC were shown
to be phosphorylated with similar efficiency as dCMP and CMP. We
further showed, in a combined enzymatic assay, that human deoxycytidine
kinase and UMP-CMP kinase together phosphorylated araC, dFdC, and
2',3'-dideoxycytidine to their diphosphate forms. Northern blot
analysis showed that the UMP-CMP kinase mRNA was ubiquitously present
in human tissues as a 3.9-kb transcript with highest levels in
pancreas, skeletal muscle, and liver. The human UMP-CMP kinase gene was
localized to chromosome 1p34.1-1p33 by radiation hybrid analysis. We
further expressed the UMP-CMP kinase as a fusion protein to the green
fluorescent protein in Chinese hamster ovary cells, and showed that the
fusion protein was located in the cytosol and nucleus.
| |
Introduction |
|---|
|
Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Deoxycytidine
(dCyd) analogs that are used in chemotherapy of cancer and virus
infections require phosphorylation by cellular enzymes to become
active. The phosphorylation of dCyd analogs by deoxycytidine kinase
(dCK) is regarded to be the rate-limiting step in the activation of the
compounds; the further phosphorylation to the di- and triphosphates has
not been as thoroughly studied as the initial phosphorylation step.
However, it is important to identify all enzymes involved in the
activation of nucleoside analogs for a rational use of these compounds
as single drugs or in combination therapy. Among the monophosphate
kinases, only thymidylate kinase has been carefully studied. The
anti-HIV nucleoside analog 3'-azido-2',3'-dideoxythymidine (AZT) is a
poor substrate for thymidylate kinase, which results in
AZT-monophosphate accumulation in cells (Furman et al., 1986
;
Lavie et al., 1997). AZT-monophosphate is inactive in inhibiting
retroviral replication and may cause adverse effects (Tornevik et al.,
1995).
There are two pyrimidine nucleoside monophosphate kinases identified in
mammalian cells: the thymidylate kinase and the UMP-CMP kinase.
Thymidylate kinase phosphorylates dTMP and dUMP, whereas UMP-CMP
kinase phosphorylates CMP and UMP as well as dCMP and dUMP (Arima et
al., 1977). Both thymidylate kinase and UMP-CMP kinase are known to
phosphorylate structural analogs of pyrimidine nucleosides that are
used in the treatment of cancer and virus infections. Similar to AZT,
there are cytidine and uridine nucleoside analogs that accumulate at
the monophosphate level (Vilpo and Vilpo, 1993; Schinazi et al., 1994),
and the UMP-CMP kinase may accordingly be limiting for the
phosphorylation of pharmacologically important nucleoside analogs.
UMP-CMP kinase further phosphorylates (S)-1-(3-hydroxy-2-phosphonomethoxypropyl)cytosine
(cidofovir), an acyclic nucleotide analog used in treatment of
cytomegalovirus retinitis (Cihlar and Chen, 1996). The formation of the
diphosphate form may be the rate-limiting step in the phosphorylation
of this compound.
The majority of studies on mammalian UMP-CMP kinase have been performed
on partially purified enzymes from different cells and tissues (Maness
and Orengo, 1975; Arima et al., 1977; Hande and Chabner, 1978; Scott
and Wright, 1979; Seagrave and Reyes, 1987). In recent years UMP-CMP
kinases have been cloned from the yeast Saccharomyces
cerevisae and the slime mold Dictyostelium discoideum
as well as from several prokaryotes (Liljelund and Lacroute, 1986;
Wiesmüller et al., 1990; Jong et al., 1993; Bucurenci et al.,
1996). Recently, the first cloning of a mammalian UMP-CMP kinase, from
pig, was reported (Okajima et al., 1995). The pig UMP-CMP kinase was
recombinantly expressed, but no thorough characterization of the enzyme
in regard to nucleotide and nucleotide analog phosphorylation was
performed. We have in the present study cloned the cDNA of the human
homolog of UMP-CMP kinase, expressed and purified the recombinant
protein, and studied phosphorylation of both natural as well as
structurally modified nucleotides. Our study includes the first
characterization of the enzymatic conversion of the clinically
important dCyd analog 2',2'-difluorodeoxycytidine (dFdC) to its
diphosphate form.
| |
Experimental Procedures |
|---|
|
Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Cloning and Expression of Human UMP-CMP Kinase cDNA. We searched the expressed sequence tag library of the GenBank database at the National Institute for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) with the Basic Local Alignment Search Tool (BLAST) to identify a human homolog of the pig UMP-CMP kinase. The expressed sequence tag cDNA clones were obtained from Research Genetics Inc. The DNA sequences of the plasmids were determined with the automatic laser fluorescent (A.L.F.) sequencer (Amersham Pharmacia Biotech, Uppsala, Sweden).
We expressed the cDNA encoded protein in Escherichia coli as a fusion protein to glutathione-S-transferase (GST). Two oligonucleotide primers that flanked the open reading frame of the cDNA were designed with BamHI and SalI restriction enzyme sites (5'-GGGGATCCGTATGCTGAGCCGCTGCCGCA and 5'-ATGTCGACTTAGCCTTCCTTGTCAAAAATCTG). The oligonucleotides were used in a polymerase chain reaction (PCR) and the amplified DNA fragment was cloned in the BamHI to SalI sites of the pGEX-5X-1 plasmid vector (Amersham Pharmacia Biotech). The plasmid was transformed into the Escherichia coli strain BL21(DE3)pLysS (Stratagene, Inc., La Jolla, CA). Protein expression was induced at 27°C with 1 mM isopropyl-D-thiogalactoside for 12 h and
purification was performed as described (Johansson and Karlsson,
1997a). The purified recombinant protein was eluted in 50 mM Tris pH
8.0 supplemented with 10 mM reduced glutathione (Sigma Chemical Co.,
St. Louis, MO). The size and purity of the recombinant protein was
determined by SDS-polyacrylamide gel electrophoresis (Phast
system; Amersham Pharmacia Biotech). The protein concentration was
determined with Bradford Protein Assay (Bio-Rad, Hercules, CA)
and BSA was used as the concentration standard.
Enzyme Assays.
The nucleoside monophosphates and
triphosphates were obtained from Sigma.
[
-32P]ATP (3000 Ci/mmol) was obtained from
Amersham Pharmacia Biotech. All assays were performed in 50 mM Tris-HCl
pH 8.0, 5 mM MgCl2, and indicated concentrations
of nucleotides and enzyme. The enzymatic assays with radiolabeled ATP
were performed in 10 µl with 1 mM ribonucleoside monophosphate or
deoxyribonucleoside monophosphate and 2.5 µCi/µl
[-32P]ATP. The reaction mixtures were
incubated 30 min at 37°C. The reaction products were separated by
thin-layer chromatography (TLC) on poly(ethyleneimine)-cellulose
F chromatography sheets (Merck & Co., Inc., Whitehouse Station,
NJ) in 0.5 M ammonium formate pH 3.5 (Lust and Sahud, 1972). The
TLC sheets were soaked in methanol before use. The TLC sheets were
autoradiographed using phosphorimaging plates (BAS 1000; Fuji Photo
Film Co., Ltd., Tokyo, Japan).
; Uesugi et al., 1997).
Chromatography was performed on Gilson 506C HPLC system with 1 ml/min flow rate and detection of nucleotides at 254 nm. The assays for
kinetic determinations were performed with 1 mM ATP and different
concentrations of the nucleoside monophosphates ranging from 0.008 to
10 mM. The reactions mixes were incubated at 37°C for 30 min, and
stopped by boiling for 5 min before HPLC analysis.
Assays of Nucleoside Analog Phosphorylation.
Human dCK cDNA
(Chottiner et al., 1991) was cloned into the pGEX-5X-1 vector and
recombinant dCK-GST was expressed as described (Johansson and Karlsson,
1997a). One microgram of dCK, 1 µg UMP-CMP kinase, 1 µCi/µl
[-32P]ATP, 1 mM unlabeled ATP, and 1 mM
either dCyd (Sigma), 1--D-arabinofuranosylcytosine (araC; Sigma), 2',3'-dideoxycytidine (ddC; Sigma), or dFdC (Eli-Lilly) were used in the assays. The reaction mixes were incubated at 37°C for 2 h, and the reaction products were separated by TLC as
described above. Kinetic determinations for araC-MP and dFdC-MP were
performed with the HPLC method described above. araC-MP was obtained
from Sigma and dFdC-MP was enzymatically produced with recombinant
Drosophila melanogaster deoxyribonucleoside kinase (M.J.,
A.R.V.R., B. Degrève, J. Balzarini and A.K., unpublished data).
Green Fluorescent Protein (GFP) Fusion Protein.
We used the
pEGFP-N1 vector (Clontech, Palo Alto, CA) to express the enzyme as a
fusion protein with the GFP. Oligonucleotides containing
HindIII and SalI restriction enzyme sites
(5'-CGAAGCTTGTGTATGCTGAGCCGCTGCC-GCA and
5'-ATGTCGACCAGCCTTCCTTGTCAAAAATCT) were used to clone the open reading
frame of UMP-CMP kinase cDNA into the
HindIII-SalI sites of the pEGFP-N1 vector. The
Chinese hamster ovary cell line was obtained from American Type Culture
Collection. The cytosolic and nuclear dCK-GFP constructs, cell culture,
transfection, and fluorescence microscopy were performed as described
(Johansson et al., 1997b).
Northern Blot.
A human multiple tissue Northern blot was
purchased from Clontech. The Northern blot contains approximately 2 µg of poly A+ RNA per lane from eight different
human tissues. The complete UMP-CMP kinase cDNA was labeled with
[
-32P]dCTP (6000 Ci/mmol; QuickPrime,
Amersham Pharmacia Biotech) and the probe was hybridized using the
ExpressHyb hybridization solution (Clontech) as described in the
manufacturer's protocol. As a quality control the Northern blot was
afterward probed with radioactively labeled human -actin cDNA
control probe.
Chromosome Mapping.
The GeneBridge 4 (Gyapay et al.,
1996) human/hamster radiation hybrid panel was obtained from Research
Genetics Inc. (Huntsville, AL). Two oligonucleotide primers located in
the untranslated 3' region of the UMP-CMP kinase cDNA (bp 2593-2803)
were designed (5'-GGCAACATTGAGTTTTGGAGTTGATT-GA and
5'-AGGCCACAGCACAAAATTTATTGAGA). PCR amplifications were performed as
described (Stewart et al., 1997), and the PCR products were analyzed by
agarose gel electrophoresis. The results of the GeneBridge 4 radiation
hybrid panels were analyzed via Internet at
http://www-genome.wi.mit.edu. The cytogenetic location was calculated
using MapView software at http://www. gdb.org/.
| |
Results |
|---|
|
Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
cDNA Cloning and Expression of the Human UMP-CMP Kinase.
Human
expressed sequence tag cDNA clones homologous to the cDNA of pig
UMP-CMP kinase were identified in GenBank. The identified clones were
deposited by the Integrated Molecular Analysis of Genomes and their
Expression (I.M.A.G.E.) consortium (Lennon et al., 1996) (I.M.A.G.E.
clone ID 83173, 545355, 140570, 595781, 171759, and 813072). The
complete 2.9-kilobase pair cDNA sequence was determined (Fig.
1). The longest open reading frame
encoded a 228-amino-acid residue protein with a predicted molecular
mass of 26 kDa. The open reading frame of the human UMP-CMP kinase was
93% identical with the pig UMP-CMP kinase sequence at the nucleotide
level and 98% identical at the amino acid level. The open reading
frame of the pig UMP-CMP kinase lacked the first 32 amino acids present
in the open reading frame of the human enzyme. N-terminal peptide
sequences of native pig and human UMP-CMP kinase suggest, however, that
the second methionine in the human cDNA sequence is the translation
start (Hughes et al., 1993; Okajima et al., 1995). The function of
these first 32 amino acids in the human UMP-CMP kinase are not known.
Alignment of the human enzyme with UMP-CMP kinase cloned from other
species showed that the enzyme was 43% similar to yeast
Saccharomyces cerevisiae UMP kinase, 50% similar to
Dictyostelium discoideum UMP-CMP kinase, and 15% similar to
the Escherichia coli CMP kinase (Fig.
2). The UMP-CMP kinases are also related
to the adenylate kinase enzyme family and the human UMP-CMP kinase was
21 to 39% similar to the mammalian adenylate kinases. Three functional
domains have been described in the primary structure of nucleoside
monophosphate kinases: 1) the nucleoside triphosphate binding glycine
rich region, 2) the nucleoside monophosphate binding site, and 3) the
lid domain that closes over the substrate upon binding (Scheffzek et
al., 1996). The alignment showed a highly conserved glycine-rich region at amino acid residues 42 to 50 corresponding to the binding site of
the phosphate donor. Amino acid residues 65 to 95 are similar to the
nucleoside monophosphate binding site. The lid domain contains many of
the catalytically important residues and the corresponding domain was
present in human UMP-CMP kinase at amino acid residues 166 to 175.
|
|
50 kDa was detected after purification, which corresponded to the
52-kDa molecular mass of GST and UMP-CMP kinase.
Enzymatic Properties of the Recombinant Enzyme.
To verify
enzymatic activity of the recombinant enzyme, we tested the naturally
occurring ribo- and deoxyribonucleoside monophosphates for activity
(Fig. 3). We used ATP as phosphate donor
in the assays because studies on UMP-CMP kinase purified from rat liver
indicate that the preferred donors are ATP and dATP although the enzyme can use several other phosphate donors (Maness and Orengo, 1975). The
ribonucleotides UMP and CMP were both efficiently phosphorylated by the
enzyme. However, lower levels of activity was also detected for dCMP,
dUMP, AMP, and dAMP. No phosphorylation of GMP, IMP, dGMP, dIMP, or
dTMP was detected. The relative activity of UMP and CMP phosphorylation
was similar. dCMP phosphorylation was 12% and dUMP, AMP, and dAMP were
3 to 5%, compared with the activity of UMP and CMP.
|
,b), and we therefore
determined kinetic properties of the recombinant enzyme both in the
presence and absence of 10 mM DTT. CMP exhibited the lowest
Km of 0.5 mM whereas UMP and dCMP had
3-fold higher Km values and dUMP showed a
12-fold higher Km. In the absence of DTT,
the Vmax of CMP and UMP were similar, 2.1 and 1.6 µmol/mg/min, whereas the Vmax of dCMP and dUMP both were 7-fold lower. However, addition of DTT to the
assay buffer increased the Vmax of dCMP
21-fold, whereas there were no major changes in velocity for UMP, CMP,
or dUMP phosphorylation. In spite of the large increase in dCMP
Vmax in the presence of DTT, the
Km remained unaffected (data not shown). In
summary, the efficiency of the different substrates, calculated as
Vmax/Km, showed
that CMP was the best enzyme substrate. Compared with CMP, the
efficiency of dCMP phosphorylation was 2-fold lower, UMP
phosphorylation was 8-fold lower, and dUMP phosphorylation was 420-fold
lower.
|
Two-Step Phosphorylation of Nucleoside Analogs In Vitro.
We
decided to test if we could study phosphorylation of the pyrimidine
nucleoside analogs to their diphosphate derivative in vitro by
combining the nucleoside kinase dCK with UMP-CMP kinase. dFdC, araC,
and ddC were all phosphorylated to their diphosphate derivative
in the presence of dCK and UMP-CMP kinase (Fig.
4).
|
Subcellular Location.
The mammalian adenylate kinases are
located in either the cytosol or the mitochondria. Sequence analysis of
the UMP-CMP kinase primary structure did not reveal any known signal
for targeting the protein to any subcellular compartment. To verify
that UMP-CMP kinase was located in the cytosol, we decided to express
the enzyme as a fusion protein to GFP and visualize the protein in
cultured cells. The cells transfected with GFP alone showed as expected green fluorescence both in the cytosol and nucleus (Fig.
5). Although GFP lacks a nuclear import
signal, the small size of the protein allows it to passively enter the
nucleus through the nuclear envelope pores. As controls, we transfected
the cells with a wild-type nuclear dCK-GFP and a mutant cytosolic
dCK-GFP construct (Johansson et al., 1997b). Nuclear dCK showed strong
fluorescence in the nucleus and weak fluorescence in the cytosol,
whereas the cytosolic mutant of dCK showed strong fluorescence in the
cytosol but no fluorescence in the nucleus. The cells transfected with
the plasmids encoding the UMP-CMP kinase-GFP fusion showed fluorescence
in both the cytosol and nucleus, similar to the cells transfected with
wild-type GFP. We conclude that the UMP-CMP kinase-GFP fusion lacks
subcellular targeting signals and that the relative small molecular
mass of the fusion protein allows it to passively diffuse between the
cytosol and nucleus.
|
Northern Blot Analysis.
We used a multiple tissue Northern
blot with mRNA from eight different human tissues to study the
expression pattern of UMP-CMP kinase mRNA. The Northern blot analysis
showed that the mRNA transcript of UMP-CMP kinase was detected as a
3.9-kilobase band ubiquitously expressed in human tissues
(Fig. 6). Among the eight investigated tissues, the mRNA was present in all investigated tissues with the
highest level of expression in pancreas, skeletal muscle, and liver.
The -actin cDNA showed the presence of a single 2.0-kb band in all
lanes and a second band in the heart (1.8-kb). The highest level of
-actin was in skeletal muscle, lung, and heart. Expression of
-actin mRNA varies between different tissues, and the mRNA level of
actin in skeletal muscle is higher than in other tissues. We therefore
cannot draw any conclusions with regard to the level of expression of
the UMP-CMP kinase in skeletal muscle. We are presently generating
antibodies against the recombinant UMP-CMP kinase protein to study the
tissue distribution in detail.
|
Chromosome Mapping. We used the GeneBridge4 human-hamster radiation hybrid panel to determine the chromosome location of the UMP-CMP kinase gene. The cell hybrid clones were screened for the presence or absence of a PCR amplifiable marker for the UMP-CMP kinase locus. The radiation hybrid analysis indicated that the UMP-CMP kinase gene was located 3.77 centiRay distal to marker D1S2134 (GeneBridge 4 positive clones: B2, C11, D7, E2, E6, F13, I1, I4, K9, K12, P11, Q4, R3, S6, S12, V7, Y4, Y8). This marker is located at chromosome 1p34.1-1p33.
| |
Discussion |
|---|
|
Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
We study the enzymes involved in the activation of nucleoside
analogs in an attempt to reconstitute their pathways of activation. In
the present investigation we have focused on the phosphorylation of
clinically important dCyd analogs to their corresponding diphosphates. We have cloned, expressed, and characterized the human UMP-CMP kinase.
Because this is the first report on recombinant human UMP-CMP kinase we
performed a careful characterization of the enzyme using natural
substrates, in addition to the nucleoside analogs. Our data showed that
CMP, dCMP, and UMP were the best substrates for the enzyme, whereas
dUMP was poorly phosphorylated. The physiological basis of this
discrepancy is probably that dUTP is harmful to the cells and low
efficiency of dUMP phosphorylation by UMP-CMP kinase may contribute to
keeping the cellular dUTP levels low. The pattern of substrate
specificity of the recombinant UMP-CMP kinase is generally in agreement
to that reported for the tissue-purified mammalian enzymes (Maness and
Orengo, 1975; Arima et al., 1977; Scott and Wright, 1979; Okajima et
al., 1995). However, previous reports on the kinetic parameters of
UMP-CMP kinases are partially conflicting. The
Km for UMP and CMP of purified enzyme from
mammalian tissues is in most studies reported to be 10-fold lower
than the Km values of the recombinant
enzyme (Sugino et al., 1966; Maness and Orengo, 1975, 1976a; Teng et
al., 1976; Hande and Chabner, 1978). However, there are studies on
tissue-purified UMP-CMP kinase that report similar affinity of UMP and
CMP as for the recombinant enzyme (Scott and Wright, 1979; Seagrave and Reyes, 1987). The reasons for these discrepancies are not yet clear. In
regard to the affinity to dCMP and dUMP, most reports are in agreement
with the kinetic data of the recombinant enzyme (Sugino et al., 1966;
Maness and Orengo, 1975, 1976a; Arima et al., 1977; Hande and Chabner,
1978). The majority of studies on mammalian UMP-CMP kinases have been
performed on partially purified enzyme and it is therefore difficult to
compare the specific enzymatic activity of different enzyme
preparations, but a study on highly purified rat UMP-CMP kinase shows a
Vmax in the same range as for the
recombinant human enzyme (Maness and Orengo, 1976a). The intracellular
levels of UMP and CMP are in most tissues 10-fold lower than the
Km of UMP-CMP kinase and the corresponding
deoxyribonucleotide levels are more than 1000-fold lower than the
Km determined for the recombinant UMP-CMP
kinase (Traut, 1994). These data, together with the kinetic parameters
of the recombinant enzyme, indicate that UMP-CMP kinase would
preferentially phosphorylate ribonucleotides in vivo.
UMP-CMP kinase activity requires the presence of reducing agents and
dCMP phosphorylation shows the highest sensitivity (Sugino et al.,
1966, Maness and Orengo, 1975, 1976a). Inactive oxidized UMP-CMP kinase
can regain full activity by incubation with DTT (Sugino et al., 1966;
Maness and Orengo, 1976a,b). The mechanism of activation involves
cleavage of inter- and intramolecular disulfide bonds that cause the
enzyme to change its quaternary structure (Maness and Orengo,
1976b). It is currently not known which amino acids in the enzyme are
involved in formation of the disulfide bonds. The recombinant human
enzyme was active both in the presence and absence of DTT. However,
dCMP phosphorylation was markedly increased by addition of DTT, whereas
phosphorylation of other substrates were not affected. It is possible
that the fusion of the recombinant enzyme to GST affects the enzyme's
ability to form disulfide bonds and thereby make the enzyme less
dependent of a reducing environment. It is possible that dCMP
phosphorylation catalyzed by the enzyme in vivo is redox-regulated.
However, there is at present time no experimental support of the hypothesis.
Although UMP-CMP kinases from yeast and D. discoideum are
closely sequence-related to the mammalian kinases, these enzymes exhibit different patterns of substrate specificity. The yeast enzyme
is predominantly a UMP kinase and it phosphorylates CMP poorly (Jong et
al., 1993), whereas the D. discoideum enzyme phosphorylates CMP better than UMP (Wiesmüller et al., 1990). Bacterial enzymes, such as the CMP kinase of E. coli, phosphorylates CMP and
dCMP equally efficiently whereas UMP is a poor substrate for the enzyme (Bucurenci et al., 1996). Recently, structural studies of both prokaryotic as well as eukaryotic UMP-CMP kinases have been reported, and these studies will contribute to the understanding of the difference in substrate recognition between the enzymes (Scheffzek et
al., 1996; Briozzo et al., 1998).
In addition to phosphorylation of the pyrimidine nucleoside
monophosphates, we also detected low levels of AMP and dAMP
phosphorylation catalyzed by the recombinant enzyme. UMP-CMP kinases
from other species, such as yeast, have been reported to phosphorylate
other nucleotides, such as AMP and dTMP, but the activity is generally low (Jong et al., 1993). However, complementation studies performed in
thymidylate or adenylate kinase-deficient yeast cells show that the
yeast UMP-CMP kinase is a dominant suppressor of these deficiencies
(Choi et al., 1989; Schricker et al., 1992). These experiments suggest,
at least in the absence of other nucleoside monophosphate kinases, that
the yeast UMP-CMP kinase can be important for dTMP and AMP
phosphorylation. However, the physiological role of the human UMP-CMP
kinase for these alternative substrates remains to be shown.
Biochemical studies on cell extracts suggest that human cells contain a
single UMP-CMP kinase (Sugino et al., 1966; Arima et al., 1977).
Subcellular fractionation has further shown that the major UMP-CMP
kinase activity is present in the cytosol (Shiosaka et al., 1975). The
cloned human enzyme did not appear to contain a signal for transport to
another subcellular compartment and the absence of a subcellular
targeting sequence was further supported by the predominantly cytosolic
location of the UMP-CMP kinase-GFP fusion protein. However, cellular
nucleotide metabolism is separated into two compartments: the
cytosolic/nuclear and the mitochondrial. The mitochondria inner
membrane constitutes a barrier that prevents free nucleotide transport
between the cytosol and the mitochondria. The mitochondria contains
several nucleoside and nucleotide kinases that phosphorylate
pyrimidines, but no mitochondrial pyrimidine nucleoside monophosphate
kinase has yet been identified. Recently, a mitochondrial dCTP import
system was identified, and it may explain the apparent absence of a CMP
kinase in the mitochondria (Bridges et al., 1999).
The chromosome location of a human UMP-CMP kinase has been determined
to 1p32 using low-resolution somatic cell hybrid analysis and family
studies (Cook and Hamerton, 1979). The basis for these studies is the
identification of three different UMP-CMP kinase alleles that can be
distinguished by their different biochemical properties. The 1p32
location is adjacent to 1p34.1-1p33, where the gene of the cloned
UMP-CMP kinase was located. Accordingly, we conclude that the cloned
UMP-CMP kinase gene is the same as the one previously mapped to 1p32.
In a study on the incidence of bacterial infection, the UMP-CMP kinase
allele 2 was linked to prolonged respiratory infections
(Giblett et al., 1974) and patients homozygous for UMP-CMP kinase
allele 3 showed increased incidence of invasive Hemophilus
influenzae type B infections (Petersen et al., 1985). These
studies included, however, few patients, and the potential involvement
of UMP-CMP kinases in an immunodeficiency disorder is still not
clarified. Several types of solid tumors frequently exhibit 1p
deletions and the UMP-CMP kinase gene has been suggested to be included
in these deletions. However, there is no evidence that these tumors
have changed levels of UMP-CMP kinase enzyme activity (Bravard et al.,
1991).
Phosphorylation of nucleoside analogs is a prerequisite for pharmacological activity of these compounds. Although a few nucleoside analogs exert their pharmacological effects in the monophosphate form, the majority of compounds require phosphorylation to di- and triphosphates. Pyrimidine nucleoside analogs, such as araC and dFdC, are widely used in chemotherapy of malignancies. araC is used in the treatment of several hematological malignancies and dFdC is used in the treatment of solid tumors such as pancreatic cancer and nonsmall cell lung cancer. We showed that the monophosphate derivatives of araC and dFdC were efficient substrates of human UMP-CMP kinase. We also showed that the anti-HIV nucleoside analog ddC, at the monophosphate level, is a substrate of the human UMP-CMP kinase. Northern blot analysis showed that UMP-CMP kinase mRNA is present in all investigated tissues with the highest levels in pancreas, liver, and skeletal muscle. There are, however, no data suggesting that these tissues would be more sensitive to cytotoxic pyrimidine nucleoside analogs phosphorylated by UMP-CMP kinase.
We have further shown that nucleoside analogs incubated with both the nucleoside kinase dCK and the UMP-CMP kinase can be phosphorylated to their diphosphate derivatives in vitro. Assays of nucleoside analog phosphorylation using multiple kinases to reconstitute the pathways involved in the activation of the compounds will be a useful method for the screening of novel nucleoside analogs.
| |
Footnotes |
|---|
Received March 12, 1999; Accepted June 21, 1999
1 The DNA sequence reported in this paper has been deposited in the GenBank database (accession no. AF070416).
This work was supported by grants from the Swedish Medical Research Council, the Swedish Cancer Foundation, and the European Community.
Send reprint requests to: Dr. Anna Karlsson, Division of Clinical Virology, Karolinska Institute, Huddinge University Hospital, S-141 86 Stockholm, Sweden. E-mail: anna.karlsson{at}mbb.ki.se
| |
Abbreviations |
|---|
dCyd, deoxycytidine;
dCK, deoxycytidine kinase;
GST, glutathione-S-transferase;
GFP, green fluorescent
protein;
araC, 1--D-arabinofuranosylcytosine;
dFdC, 2',2'-difluorodeoxycytidine;
ddC, 2',3'-dideoxycytidine;
AZT, 3'-azido-2',3'-dideoxythymidine;
PCR, polymerase chain reaction;
TLC, thin-layer chromatography;
dAMP, deoxyadenosine monophosphate;
dCMP, deoxycytidine monophosphate;
dUMP, deoxyuridine monophosphate;
dTMP, deoxythymidine monophosphate;
MP, monophosphate.
| |
References |
|---|
|
Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
This article has been cited by other articles:
|
|
 |
|
  N. A. Kocabas, P. Aksoy, L. L. Pelleymounter, I. Moon, J.-S. Ryu, J. A. Gilbert, O. E. Salavaggione, B. W. Eckloff, E. D. Wieben, V. Yee, et al. Gemcitabine Pharmacogenomics: Deoxycytidine Kinase and Cytidylate Kinase Gene Resequencing and Functional Genomics Drug Metab. Dispos., September 1, 2008; 36(9): 1951 - 1959. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-L. Chen, D.-W. Lin, and Z.-F. Chang Identification of a putative human mitochondrial thymidine monophosphate kinase associated with monocytic/macrophage terminal differentiation. Genes Cells, July 1, 2008; 13(7): 679 - 689. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Xu, M. Johansson, and A. Karlsson Human UMP-CMP Kinase 2, a Novel Nucleoside Monophosphate Kinase Localized in Mitochondria J. Biol. Chem., January 18, 2008; 283(3): 1563 - 1571. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Wang, G. J. S. Lohman, and J. Stubbe Enhanced subunit interactions with gemcitabine-5'-diphosphate inhibit ribonucleotide reductases PNAS, September 4, 2007; 104(36): 14324 - 14329. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-H. Hsu, J.-Y. Liou, G. E. Dutschman, and Y.-C. Cheng Phosphorylation of Cytidine, Deoxycytidine, and Their Analog Monophosphates by Human UMP/CMP Kinase Is Differentially Regulated by ATP and Magnesium Mol. Pharmacol., March 1, 2005; 67(3): 806 - 814. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Segura-Pena, N. Sekulic, S. Ort, M. Konrad, and A. Lavie Substrate-induced Conformational Changes in Human UMP/CMP Kinase J. Biol. Chem., August 6, 2004; 279(32): 33882 - 33889. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Krishnan, E. A. Gullen, W. Lam, G. E. Dutschman, S. P. Grill, and Y.-c. Cheng Novel Role of 3-Phosphoglycerate Kinase, a Glycolytic Enzyme, in the Activation of L-Nucleoside Analogs, a New Class of Anticancer and Antiviral Agents J. Biol. Chem., September 19, 2003; 278(38): 36726 - 36732. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-Y. Liou, G. E. Dutschman, W. Lam, Z. Jiang, and Y.-C. Cheng Characterization of Human UMP/CMP Kinase and Its Phosphorylation of D- and L-Form Deoxycytidine Analogue Monophosphates Cancer Res., March 1, 2002; 62(6): 1624 - 1631. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. R. Van Rompay, A. Norda, K. Lindén, M. Johansson, and A. Karlsson Phosphorylation of Uridine and Cytidine Nucleoside Analogs by Two Human Uridine-Cytidine Kinases Mol. Pharmacol., April 16, 2001; 59(5): 1181 - 1186. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
