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INTRODUCTION |
Xanthine dehydrogenase is the rate-limiting step in the catabolism
of purines, where it catalyzes the conversion of hypoxanthine to
xanthine and xanthine to uric acid. In this reaction,
XDH1 utilizes
NAD+ preferentially as the electron acceptor. However, when
XDH is converted to XO, the preferred electron acceptor becomes
molecular oxygen resulting in the formation of superoxide and hydrogen
peroxide. This generation of reactive oxygen species is thought to be
the basis of XDH/XO involvement in various pathological conditions such
as ischemia-reperfusion injury. Reversible conversion of XDH to XO can
occur after the oxidation of eight cysteine residues in the molecule
into four cystines by agents such as pyrimidines or oxidized
glutathione (1, 2). This conversion may be reversed upon the addition
of reducing agents such as dithiothreitol. XDH can also be converted
into XO irreversibly through proteolysis (3). Experimental proteolysis
by trypsin has allowed the identification of three different parts of
the molecule: a 20-kDa N-terminal fragment, a 40-kDa flavin-binding
fragment, and an 80-kDa molybdopterin-binding fragment, all of which
remain attached after proteolysis (3). It is believed that both
reversible and irreversible conversion of XDH to XO are due to
conformational changes in the molecule that reduce its affinity for
NAD+.
The notion that XDH/XO is a phosphoprotein has been the subject of
controversy. Initial reports of the presence of a phosphoserine in milk
xanthine oxidase concluded that, in addition to two phosphates on the
FAD and one phosphate in the molybdopterin cofactor, there was a
phosphate covalently bound to a serine residue of the protein (4, 5).
Later reports indicated that no covalently attached phosphate to milk
XDH/XO could be detected by NMR or chemical analysis (6, 7). However,
using biochemical analysis of immunoprecipitated XDH from chick embryo
hepatocytes, Schieber and Edmondson (8) demonstrated the
incorporation of labeled phosphate into XDH and estimated 3 mol of
phosphate/mol of protein. Although the latter report described the
covalent phosphorylation of serine residues in chicken xanthine
dehydrogenase (8), the phosphorylation of mammalian XDH remains to be
demonstrated conclusively.
In this report, evidence is provided that rat XDH/XO can be
phosphorylated in pulmonary microvascular endothelial cells. Moreover, the phosphorylation of XDH is greatly increased in response to hypoxia,
which is known to induce XDH/XO (9). The increase in XDH/XO
phosphorylation in hypoxia is accompanied by an increase in XDH/XO
activity. Hypoxia-induced XDH/XO phosphorylation is partially blocked
by inhibitors of p38 kinase. Furthermore, our data indicate that p38 is
significantly activated in hypoxia. Hence, p38 kinase is postulated to
be involved in the phosphorylation of XDH/XO in hypoxia.
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EXPERIMENTAL PROCEDURES |
Materials and Reagents--
RPMI 1640, phosphate-free
Dulbecco's modified Eagle's medium, fetal bovine serum, dialyzed
fetal bovine serum, penicillin G potassium, streptomycin, fungizone,
and glutamine were obtained from Life Technologies,
Inc. Protein kinase and phosphatase inhibitors were obtained
from Calbiochem. [32P] Orthophosphate was obtained from
PerkinElmer Life Sciences. EDTA, Tris, and dithiothreitol were
from Sigma. Rabbit anti-XO antibody was from LabVision, Fremont, CA,
and anti p38 antibodies were from New England Biolabs, Beverly, MA.
Horseradish peroxidase-conjugated anti-rabbit IgG and the
chemiluminescent horseradish peroxidase substrate
SuperSignal were from Pierce.
Cell Culture and Exposure to Hypoxia--
Rat pulmonary
microvascular endothelial cells (RPMEC) were a gift from Dr. Una Ryan
(Avant Immunotherapeutics, Needham, MA) and were cultured as previously
described (10). For hypoxic exposure, cells were placed in humidified
airtight incubation chambers (Billups-Rothenberg, Del Mar, CA) and
gassed with 3% O2, 5% CO2, and balance
N2. The hypoxic chambers were kept in a 37 °C incubator
for the duration of the experiment. Normoxic cells were kept in a
tissue culture incubator maintained at 5% CO2 and
37 °C.
Xanthine Oxidase Activity Measurements--
The activities of
xanthine dehydrogenase and xanthine oxidase in response to different
treatments were assayed using a slight modification of a fluorimetric
assay that measures both xanthine oxidase and xanthine dehydrogenase
activity (11). The principle of the assay involves the conversion of
pterin into the fluorescent product isoxanthopterin. The rate of
product formation with oxygen as the electron acceptor represents the
activity of xanthine oxidase, whereas the combined activities of
xanthine oxidase and xanthine dehydrogenase are measured with methylene
blue as the electron acceptor. In brief, cells were washed once in
phosphate-buffered saline and then scraped off the plate in 50 mM sodium phosphate (pH 7.4), 1.5 mg/ml dithiothreitol, and
1× protease inhibitor mixture 3 (Calbiochem). The cells were sonicated
for 5 s and centrifuged at 10,000 × g for 5 min.
The supernatant was collected and assayed immediately or stored at
80 °C overnight. For testing the effect of phosphorylation on XDH
or XO activity, lysates were prepared in 50 mM Tris-HCl (pH
7.4), 1.5 mg/ml dithiothreitol, and 1× protease inhibitor mixture 3, and aliquots were treated with calf intestine alkaline phosphatase (New
England Biolabs, Beverly, MA) before the assay.
SDS-PAGE, Western Blotting, Phosphoprotein Analysis, and
Immunoprecipitation--
Aliquots from the cell lysates prepared as
described above were assayed for protein using the Bradford protein
assay (12) and then diluted with 2× Laemmli loading buffer for
SDS-PAGE (13). Equal amounts of protein were then loaded in each well
of 4-20% Tris/glycine gels. After electrophoresis for 90 min at 125 V
constant voltage, the gel was blotted onto an Immobilon-P membrane by
electrophoretic transfer at 25 V constant voltage overnight. The
membrane was then washed, blocked with 5% milk, and probed with
antibodies against xanthine oxidase (LabVision). The immunoreactive
bands were visualized using a secondary antibody conjugated to
horseradish peroxidase and a chemiluminescent substrate according to
the manufacturer's instructions (SuperSignal, Pierce). The
intensity of the bands was quantified using a Molecular Dynamics
densitometer and ImageQuant software.
For phosphoprotein analysis RPMEC, which had been passaged the day
before, were incubated with 32PO4 at 0.5 mCi/ml
in phosphate-free medium. The cells were exposed to normoxia, hypoxia,
and/or kinase inhibitors for 1-18 h. At the end of the exposure, the
cells were washed and lysed (0.3 ml of radioimmunoprecipitation assay
lysis buffer/3.5-cm dish). XDH/XO was then immunoprecipitated with
rabbit anti-XDH/XO antibody as described (14) and run on SDS-PAGE. In
brief, cell lysates were first precleared with protein A-Sepharose.
Then they were incubated with anti-XDH/XO antibody at 4 °C
overnight. Next, protein A-Sepharose was added to the samples, which
were then rocked for 2 h at 4 °C. The complex was washed 10 times with 0.5 ml of radioimmunoprecipitation assay buffer, and the
specifically bound XDH/XO was eluted from the complex by heating in 2×
Laemmli sample buffer followed by centrifugation. The samples were then
run on SDS-PAGE as described above. The gels were dried on filter
paper or electrophoretically transferred onto Immobilon-P membranes.
Phosphorylation of XDH/XO was detected by autoradiography and
quantified by either phosphorimaging or by scanning and then
quantifying the bands on film.
Statistical Analysis--
The values plotted in all figures are
means, and the error bars reflect the standard deviation from the mean.
Statistical analysis was carried out using SigmaStat (Jandel
Scientific). Student's t test was used to determine whether
differences between normoxic and hypoxic enzyme activity were
significant in Fig. 1. When comparisons between multiple groups were
carried out, one-way analysis of variance was employed. Statistical
significance was considered at p >0.05.
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RESULTS |
Hypoxia Stimulates XDH/XO Phosphorylation and Enzymatic Activity in
RPMEC--
Previous reports have indicated that exposure of
endothelial cells to hypoxia increased XDH/XO activity (9, 15, 16) and
mRNA expression within a few hours of exposure (9, 16). An increase
in protein expression has been reported in vivo after 24 h of exposure to hypobaric hypoxia (17) and in vitro
after exposure to 48 h of hypoxia (16). The following experiments were designed to examine the effect of acute exposure (4 h) of cells to
hypoxia on posttranslational modification of the XDH/XO protein. RPMEC
were exposed to hypoxia for 4 h before XDH/XO activity was
assayed. Because the changes in XDH and XO activities always occurred
in parallel, total XDH/XO activity is reported for all experiments
(nmol of isoxanthopterin formed/min/mg of protein). A significant
increase in XDH/XO activity was observed by 4 h of exposure of
RPMEC to hypoxia (Fig. 1), a finding that
is consistent with an earlier report (9). Because the increase in the
XDH/XO enzyme activity in hypoxia was not due to a change in the amount of XDH/XO protein in hypoxia as determined by Northern blotting (data
not shown), posttranslational modification of the protein was
investigated as a mechanism of enzyme activation. Upon examining the
primary sequence of XDH/XO, potential phosphorylation sites for several
protein kinases were identified. Therefore, the possibility of XDH/XO
phosphorylation was examined in cultured cells. RPMEC were
metabolically labeled with 32P, and XDH/XO was
immunoprecipitated at various time points (see "Experimental
Procedures"). Bands that corresponded to XDH/XO incorporated a
significant amount of 32P, indicating that XDH/XO was
covalently phosphorylated (Fig. 2,
Normoxia).

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Fig. 1.
Exposing RPMEC to hypoxia for 4 h causes
a 2.3-fold increase in total (XDH/XO) activity. * indicates
p < 0.05 versus normoxia.
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Fig. 2.
XDH/XO is phosphorylated in RPMEC, and its
phosphorylation is significantly increased in hypoxia. Cells were
incubated with [32P]orthophosphate (1 mCi/ml) for 4 h. XDH/XO was immunoprecipitated with a rabbit anti-XDH/XO antibody
followed by SDS-PAGE, Western blotting, and autoradiography. Left
panel represents a 1-h film exposure showing significant
phosphorylation of XDH/XO in hypoxia. A longer film exposure (24 h) was
needed to demonstrate phosphorylation of XDH/XO from normoxic cells
(right panel).
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The possibility that the observed increase in XDH/XO activity after
4 h of exposure to hypoxia could be due to posttranslational modification of the protein, specifically phosphorylation, was examined. When RPMEC were incubated with 32P and exposed to
hypoxia for 1, 2, 4, and 24 h, there was a significant increase in
XDH/XO phosphorylation as compared with normoxia with a maximum 50-fold
increase at 4 h (Fig. 2). This increase was consistently observed
in repeated experiments. The increase in phosphorylation was not due to
an increase in the protein level because probing the same membrane with
an anti-XDH/XO antibody after decay of the 32P
radioactivity revealed no significant differences in the amount of
XDH/XO between hypoxic and normoxic samples (data not shown).
Upon demonstrating that hypoxia stimulated XDH/XO phosphorylation and
enzyme activity, the effect of dephosphorylation on enzyme activity was
investigated in normoxic and hypoxic samples. Samples were incubated
with either alkaline phosphatase or inactivated alkaline phosphatase
(control) and then assayed for XDH/XO enzyme activity. As shown in Fig.
3, alkaline phosphatase treatment reduced the activity of both normoxic and hypoxic samples. However, only the
reduction in enzyme activity in hypoxic samples (64%) was statistically significant. These results are consistent with a role for
phosphorylation in modulating XDH/XO activity.

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Fig. 3.
Treatment of normoxic and hypoxic RPMEC
samples with alkaline phosphatase reduces XDH/XO enzyme activity.
This effect was statistically significant for hypoxic samples. *
indicates p < 0.05 versus normoxic control
and versus alkaline phosphatase-treated hypoxic
samples.
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Casein Kinase II and p38 Kinase Inhibitors Block Hypoxia-induced
Phosphorylation of XDH/XO As Well As Hypoxia-stimulated XDH/XO Enzyme
Activity--
To identify the kinase(s) or phosphatase(s) involved in
XDH/XO phosphorylation, RPMEC were preincubated with various inhibitors prior to labeling and exposing to hypoxia. As shown in Table
I, inhibitors of protein kinase A
and protein kinase G reduced base-line XDH/XO phosphorylation but had
no effect on hypoxia-stimulated phosphorylation. Inhibitors of protein
kinase C had no effect on XDH/XO phosphorylation in either normoxic or
hypoxic cells. A casein kinase II (CK2) inhibitor partially blocked
hypoxia-stimulated XDH/XO phosphorylation (Fig.
4). Inhibitors of p38 kinase, a
stress-activated kinase reported to be activated in hypoxia (18, 19),
also partially blocked hypoxia-stimulated phosphorylation of XDH/XO (Fig. 4). In conclusion, these results suggest that phosphorylation of
XDH/XO in hypoxia involves p38 kinase and CK2.
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Table I
Percent of inhibition of XDH/XO phospholabeling
Inhibitors of protein kinases A, G, and C reduce base-line
phosphorylation of XDH/XO but do not affect hypoxia-stimulated
phosphorylation. Protein kinase A (PKA) inhibitor (adenosine
3',5'-cyclic monophosphorothioate, 8-bromo-, Rp
isomer) was used at 50 µM. Protein kinase G (PKG)
inhibitor (guanosine 3',5'-cyclic monophosphorothioate,
8-(4-chlorophenylthio)-, Rp isomer, triethylammonium
salt) was used at 5 µM. Protein kinase C (PKC) inhibitors
calphostin and bisindolylamide were used at 1 µM.
Inhibitors of protein kinases A, G, and C inhibited XDH/XO
phosphorylation in normoxia but had little or no effect on
hypoxia-stimulated phosphorylation.
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Fig. 4.
Inhibitors of CK2 and p38 kinase block
hypoxia-stimulated XDH/XO phosphorylation. ICK2,
inhibitor of CK2
(5,6-dichloro-1- -D-ribofuranosylbenzimidazole, 50 µM). Ip38, inhibitor of p38 MAP kinase (SB
203580, 1 µM).
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Phosphatase inhibitors, cypermethrin and cyclosporin A, which inhibit
protein phosphatase 2B or calcineurin, had no effect on XDH/XO
phosphorylation in either normoxia or hypoxia (results not shown).
Okadaic acid, which inhibits protein phosphatase 2A, was toxic to RPMEC
at a concentration of 100 nM, and therefore, its effect
could not be assessed.
After identifying inhibitors that block hypoxia-induced phosphorylation
of XDH/XO, the effect of these inhibitors on XDH/XO enzyme activity was
investigated. As shown in Fig. 5, both
p38 kinase and CK2 inhibitors blocked hypoxia-stimulated XDH/XO enzyme activity. Taken together, these results are consistent with a role for
phosphorylation by p38 kinase and CK2 in modulating XDH/XO enzymatic
activity.

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Fig. 5.
Inhibitors of CK2 and p38 kinase
block hypoxia-stimulated XDH/XO enzyme activity. CK2
inhibitor
(5,6-dichloro-1- -D-ribofuranosylbenzimidazole, 50 µM), and p38 MAP kinase inhibitor (SB 203580, 1 µM) were used. * indicates p < 0.05 versus normoxic control, hypoxic + CK2 inhibitor, and
hypoxic + p38 inhibitor samples.
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Hypoxia Stimulates p38 Kinase in Rat Pulmonary Microvascular
Endothelial Cells--
The MAP kinase p38 has been reported to be
induced in hypoxia in PC12 and other cell types (18, 19). This enzyme
is involved in stress-activated pathways responding to stressors such
as hyperosmolarity, ultraviolet radiation, and inflammatory cytokines.
Because inhibitors of p38 kinase partially prevented hypoxia-stimulated
phosphorylation of XDH/XO, the effect of hypoxia on activation of p38
kinase in pulmonary endothelial cells was examined. Because p38 kinase
becomes phosphorylated upon activation, antibodies that recognize
phosphorylated (activated) p38 were used to probe Western blots from
normoxic and hypoxic cells. To ensure that the changes in
phosphorylated p38 immunolabeling were due to increased
phosphorylation of the protein rather than total amount of p38,
duplicate blots were probed with antibodies that recognize total p38
protein (both phosphorylated and unphosphorylated). For quantification,
films were scanned with a densitometer, and the intensities of the
phosphorylated p38 kinase bands were normalized to the intensities of
the corresponding total p38 kinase bands. Hypoxia resulted in an
increase in phosphorylated p38 kinase after 30 min of hypoxia with a
peak (a 4-fold increase over normoxic cells) at 1 h (Fig.
6, A and B). By
24 h of hypoxia there was no difference in phosphorylated p38
kinase between normoxic and hypoxic cells. These results indicate that
hypoxia causes the activation of p38 kinase in pulmonary endothelial
cells, a phenomenon that chronologically precedes the peak of XDH/XO
phosphorylation observed at 4 h of hypoxia.

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Fig. 6.
Hypoxia stimulates p38 kinase in RPMEC.
A, hypoxia increases the amount of phosphorylated p38 kinase
relative to total p38 kinase. B, maximum increase in the
relative amount of phosphorylated p38 kinase normalized to total p38
kinase was observed at 1 h of exposure. The data (mean ± S.D.) are derived from three different experiments. * indicates
p < 0.05 versus normoxic control
mean.
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Hyperosmolarity and Arsenite Activate p38 Kinase but Do Not
Significantly Alter the Phosphorylation of XDH/XO--
Because
inhibitors of p38 kinase reduce the phosphorylation of XDH/XO in
hypoxia and because p38 becomes activated in RPMEC in hypoxia,
experiments were performed to determine whether p38 kinase activators
other than hypoxia stimulate XDH/XO phosphorylation. When RPMEC were
exposed to hyperosmolar sorbitol (400 mM) or sodium arsenite (0.5 mM), both of which have been reported
to activate p38 kinase in other cell types (20, 21), there was
activation of p38 kinase by both agents within 15 min (Fig.
7). However, when RPMEC were prelabeled
with 32P and then treated with sorbitol or arsenite for 30 min, no significant increase in XDH/XO phosphorylation over control
cells was observed (Fig. 8). Taken
together, these results indicate that activation of p38 kinase in
hypoxia might be necessary but not sufficient to cause the
phosphorylation of XDH/XO.

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Fig. 7.
Sorbitol (400 mM) and
sodium arsenite (0.5 mM) cause an increase in
phosphorylated p38 kinase without change in total p38
kinase.
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Fig. 8.
Sorbitol and arsenite do not increase XDH/XO
phosphorylation. Cells were labeled with 32P as in
Fig. 2 and exposed to sorbitol (400 mM), sodium arsenite
(0.5 mM), or hypoxia for 1 and 4 h.
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DISCUSSION |
This study demonstrates that XDH/XO is a phosphoprotein. To
the best of our knowledge, this is the first evidence of
phosphorylation of XDH/XO in mammalian cells. Furthermore, our results
demonstrate that the phosphorylation of XDH/XO is greatly increased in
hypoxia and is accompanied by an increase in XDH/XO enzyme activity. A causal relationship between increased phosphorylation and increased XDH/XO enzyme activity is supported by the finding that
dephosphorylating XDH/XO reduces the hypoxia-stimulated enzyme activity
and the finding that kinase inhibitors that block hypoxia-stimulated
XDH/XO protein phosphorylation also block hypoxia-stimulated enzyme
activity. These results also implicate p38 in the mechanism of XDH/XO
phosphorylation by demonstrating that p38 kinase is induced in hypoxia
and that an inhibitor of this enzyme partially blocks the
phosphorylation of XDH/XO. The possible implications of these findings
in regard to the regulation of XDH/XO by hypoxia are discussed below.
Up-regulation of XDH/XO has been reported in several diseases.
For example, XDH/XO has been implicated in the acute respiratory distress syndrome (22, 23), the multisystem organ failure (24), and
reexpansion pulmonary edema (25). The ability of XDH/XO to generate
reactive oxygen species is believed to be the basis for the role of
this enzyme in the pathogenesis of these diseases. The production of
reactive oxygen species by XDH/XO is significantly increased upon the
conversion of XDH to XO either through proteolysis or through
posttranslational modification (26, 27). Both proteolysis and oxidation
of the cysteines in XDH cause conformational changes in the molecule,
which reduce its affinity for NAD+ leaving molecular oxygen
as the electron acceptor instead (3). In animal models of lung injury,
XDH/XO has been shown to be greatly increased (400-fold) in the
bronchoalveolar lavage and serum of mice infected with the influenza
virus (28). Using an in vivo model of acute lung injury, our
laboratory has demonstrated an increase in lung XDH/XO activity and
gene expression in response to hypoxia, lipopolysaccharide, and
interleukin-1
treatment (17). Pharmacological inhibition of XDH/XO
prevents the development of pulmonary edema in this animal model
further supporting a role for this enzyme in the pathogenesis of acute
lung injury.
Hypoxia is known to trigger a series of events that has been
likened to an inflammatory reaction. Some of these events involve the
activation of transcription through the action of transcription factors
such as HIF-1 (29-31). Other events, however, are considered too rapid
to be due to transcriptional processes. One example of a
nontranscriptional hypoxic response is the mobilization of P-selectin
and its release from membranous organelles, which allows it to bind and
activate neutrophils (32). The present data suggest another
nontranscriptional endothelial response to hypoxia, namely the
phosphorylation of XDH/XO and subsequent up-regulation of its enzymatic activity.
Two kinases, CK2 and p38 kinase, were shown to be involved in
hypoxia-stimulated phosphorylation of XDH/XO. CK2, a ubiquitously expressed protein kinase, is believed to play an important role in
regulating DNA replication and transcription as well as in regulating
cell growth and metabolism. Upon examination of the primary sequence of
XDH/XO using a phosphorylation site prediction program (33), several
CK2 sites were identified. Although there is no information related to
the effect of hypoxia on CK2, hypoxia is known to increase the levels
of polyamines in the lung (34), which in turn are strong activators of
CK2 (35, 36). Our finding that the CK2 inhibitor partially blocks
hypoxia-stimulated XDH/XO phosphorylation and hypoxia-stimulated XDH/XO
activity strongly suggests a role for this kinase in mediating the
effects of hypoxia on XDH/XO.
p38 kinase was also found to be important in mediating the
phosphorylation of XDH/XO in hypoxia. This enzyme, which belongs to the
family of MAP kinases, can be activated by a variety of stresses
including hyperosmolarity and UV radiation. Recently, p38 kinase was
found to be activated by hypoxia as well (18, 19). The partial blocking
of hypoxia-induced XDH/XO phosphorylation by p38 kinase inhibitor
implicates this kinase in the XDH/XO phosphorylation pathway. The
involvement of p38 kinase in hypoxia-stimulated XDH/XO phosphorylation
is further supported by the finding that p38 is activated in hypoxic
RPMEC. However, the inability of other p38 kinase stimulators, such as
sorbitol and arsenite, to significantly increase XDH/XO phosphorylation
suggests that p38 kinase activation alone is not sufficient to cause
the XDH/XO phosphorylation and that other components are involved in
the activation pathway. Although it is yet unclear how the two kinases,
p38 kinase and CK2, interact in the XDH/XO phosphorylation pathway, a
recent report demonstrated that stress-activated p38 kinase directly interacts with CK2 causing its activation in HeLa cells (37).
The mechanisms by which phosphorylation leads to increased enzyme
activity need further investigation and are beyond the scope of the
current study. However, one might speculate that the negative charges
in the XDH/XO molecule introduced by phosphorylation might affect the
affinity of the enzyme for its substrates (for example, see Ref. 38).
Alternatively, these charges might alter protein-protein interactions
either between different XDH/XO molecules affecting their tendency to
dimerize or between XDH/XO and other unidentified proteins. An example
of the latter mechanism of enzyme regulation involves the reactive
oxygen species-producing neutrophil NADPH oxidase. This enzyme is
believed to become activated during oxidative burst through the
phosphorylation of one of its subunits, p47phox. Upon
phosphorylation p47phox is translocated from the cytosol to
the membrane where it associates with other subunits to activate the
enzyme (39). It is noteworthy that p38 kinase has been shown to cause
the phosphorylation of p47phox as well as the
redistribution of other oxidase components, such as flavocytochrome
b558, from vesicles to the plasma membrane through exocytosis resulting in activation of the enzyme (40). Another
example of phosphorylation indirectly affecting enzyme activity
involves spinach nitrate reductase, which belongs to the same family of
molybdopterin-FAD oxidoreductases that includes XDH/XO. Phosphorylation
of nitrate reductase causes it to bind the protein 14-3-3, resulting in
the inhibition of the enzyme activity (41, 42). 14-3-3 is a family of
proteins known to be involved in protein-protein interactions with
various signal transduction components (for review, see Ref. 43).
Alternatively, the phosphorylation of XDH/XO might alter XDH/XO
subcellular localization by modulating its interaction with other
proteins. Further studies are required to decipher the consequences of
XDH/XO phosphorylation at the molecular level such as its potential
role in the interaction of the enzyme with other proteins and in
modulating its subcellular localization.
In conclusion, the findings that XDH/XO becomes phosphorylated and
activated in hypoxia and that this phosphorylation is mediated by
kinases known to be involved in signal transduction processes in
inflammation and hypoxia implicate XDH/XO as an important component of
the inflammatory reaction. Furthermore, the findings point to kinases
as novel potential targets in regulating XDH/XO physiology. For
instance, the p38 kinase inhibitor SB 203580 has been shown to
reduce inflammation and lipopolysaccharide-induced mortality in animals
(44). One possible action of this inhibitor might include modulating
XDH/XO phosphorylation.