Human Biliverdin Reductase Is Autophosphorylated, and
Phosphorylation Is Required for Bilirubin Formation*
Mohammad
Salim,
Brigette A.
Brown-Kipphut, and
Mahin D.
Maines
From the Department of Biochemistry/Biophysics, University of
Rochester School of Medicine, Rochester, New York 14642
Received for publication, November 29, 2000
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ABSTRACT |
Biliverdin reductase (BVR) reduces heme oxygenase
(HO) activity product, biliverdin, to bilirubin. BVR is unique in
having dual pH/dual cofactor requirements. Using Escherichia
coli-expressed human BVR and COS cells, we show that BVR is
autophosphorylated and that phosphorylation is required for its
activity. An "in blot" autophosphorylation assay showed that BVR is
a renaturable phosphoprotein. Controls for the experiments were HO-1
and HO-2; both are phosphoproteins but are not autophosphorylated.
Autophosphorylation was pH-dependent, with activity at pH
8.7 being most prominent. In addition,
2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate fluorescence titration of BVR gave a lower Kd at pH
8.7 than at pH 7.4 (15.5 versus 28.0 µM).
Mn2+ was required for binding of the ATP analogue and for
autophosphorylation; the autokinase activity was lost when treated at
60 °C for 10 min. The loss of transferred phosphates by alkaline
treatment suggested that BVR is a serine/threonine kinase. Potato acid
phosphatase treatment reversibly inactivated the enzyme. The enzyme was
also inactivated by treatment with the serine/threonine
phosphatase, protein phosphatase 2A; okadaic acid attenuated the
inhibition. Titration of protein phosphatase 2A-released phosphates
indicated a 1:6 molar ratio of BVR to phosphate. The BVR
immunoprecipitated from COS cell lysates was a phosphoprotein, and its
activity and phosphorylation levels increased in response to
H2O2. The results define a previously unknown
mechanism for regulation of BVR activity and are discussed in the
context of their relevance to heme metabolism.
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INTRODUCTION |
In the cell, the negative feedback inhibition of heme oxygenase
(HO)1 activity by biliverdin
is a mechanism by which oxidation of heme is regulated (1, 2).
Biliverdin is the open tetrapyrrole oxidation product of heme
(iron-protoporphyrin IX, hemin, heme b) degradation by the HO system,
also called the HSP32 family of proteins (1, 3-5). In various species
including mammals and certain fish, biliverdin undergoes reduction by
the oxidoreductase biliverdin reductase (BVR) (6-11); the reductase
catalyzes the reduction of the
-meso bridge of biliverdin
to bilirubin. Synthesis of biliverdin is a prominent dimension of HO
system function in cellular defense mechanisms (12).
In plants, biliverdin analogues function in a photoregulatory capacity
(13-16), and in cyanobacteria BVR-catalyzed conversion of biliverdin
to bilirubin is important for normal phycobiliprotein synthesis (17).
In mammals, bilirubin has antioxidant and immunomodulatory properties
(18-28), induces Aryl hydrocarbon receptor-mediated activation
of cytochrome P450 1a1 expression, and inhibits protein phosphorylation
(29-31). Bilirubin inhibits responses of human lymphocytes, including
phytohemagglutinin-induced proliferation, IL-2 production, and
antibody-dependent and -independent cell-mediated cytotoxicity (21-28). Interestingly, there is a report suggesting that
higher serum bilirubin levels are associated with decreased risk for
early familial coronary artery disease (24).
BVR is unique among all enzymes characterized to date; previous studies
have identified the reductase as the only dual pH/dual adenine
nucleotide cofactor-requiring enzyme (9, 32-35). The reductase, which
is highly conserved in its primary structure and molecular properties,
uses NADH in the acidic range (peak activity at pH ~6.7), whereas
NADPH is utilized in the basic range (peak activity at pH ~8.7). BVR
is encoded by a single copy gene (36) and is posttranslationally
modified (32).
This study identifies, for the first time, BVR as a phosphoprotein and
finds that phosphorylation is essential to the activity of BVR
to reduce biliverdin to bilirubin. Furthermore, it is found that the
enzyme is autophosphorylated and that phosphorylation is reversible;
the latter criterion defines a kinase. Based on the findings, we
suggest that reversible phosphorylation of BVR is a previously unknown
mechanism by which biological activities of heme degradation products
are regulated in the cell.
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MATERIALS AND METHODS |
Chemicals--
Cofactors,
-casein, and biliverdin-HCl were
purchased from Sigma or Porphyrin Products (Logan, UT). Mixtures
of anti-phosphoserine and anti-phosphothreonine or of
anti-phosphoserine, anti-phosphothreonine, and anti-phosphotyrosine
were obtained from Promega. The sources of other reagents are noted in
connection with the appropriate experiments. All chemicals used were of
the highest purity commercially available.
Purified Enzyme Preparations--
BVR was purified from human
liver to homogeneity as before (32, 37) and was used for preparation of
a polyclonal antibody in New Zealand rabbits (9). Human HO-1 and HO-2
were expressed in Escherichia coli and purified as before
(38).
Purification of E. coli-expressed Human BVR and Measurement of
Activity--
To generate glutathione S-transferase
(GST)-BVR fusion protein, wild type human BVR (34) plasmid was used as
a template for polymerase chain reaction using the primers
GGTCGACGAATGCAGAGCCCGAGAG and
GGGCGAATTCGTCGACTTACTTCCTTG, representing
nucleotides
3 to +22 and the reverse complement of nucleotides
+881-991 plus vector sequences (italicized), respectively, and
containing SalI linkers (underlined). The product was
digested with SalI and cloned into the vector pGEX 4T-2.
Orientation was determined by restriction analysis and confirmed by
sequencing. The ligation places the BVR coding region in-frame with the
GST protein of the vector. GST-BVR fusion protein was purified from
bacterial clones containing the plasmid and grown overnight, using a
glutathione-Sepharose 4B column (Amersham Pharmacia Biotech).
The reductase portion of the fusion protein was released by thrombin
protease treatment. Activity was determined as previously described (9,
32). The rate of activity was measured as the increase in 450 nm
absorbance at 25 °C. Specific activity is expressed as nmol of
bilirubin/min/mg of protein.
Phosphotransferase Activity--
The phosphotransferase activity
of the reductase was examined using two different methods: "in
blot" and "in solution" kinase assays. The in blot kinase
assay was based on the procedure described by Ferrell and Martin (39).
Following electrophoresis, on 10% SDS-polyacrylamide gel
electrophoresis plus thioglycolate (0.002%), the protein was
transferred to a polyvinylidene difluoride membrane (Millipore). The
blot was denatured for 1 h at room temperature in 50 mM Tris-HCl, pH 8.3, 7 M guanidine-HCl, 50 mM dithiothreitol, and 2 mM EDTA. The bound
protein was allowed to renature in 10 mM Tris-HCl, pH 7.4, 140 mM NaCl, 2 mM EDTA, 1% bovine serum
albumin, 0.1% Nonidet P-40 overnight at 4 °C. The blot was treated
with 5% bovine serum albumin in 30 mM Tris-HCl at
pH 7.4 at room temperature for 1 h. It was then equilibrated in 30 mM Tris-HCl of appropriate pH and subjected to the
[
-32P]ATP labeling mix, consisting of 30 mM Tris (of appropriate pH), 10 mM
MgCl2, 2 mM MnCl2, 30 µM ATP, and ~400 µCi/ml [
-32P]ATP
for 40 min at room temperature. The blot was then washed with 250 ml of
the following: three times in 30 mM Tris buffer indicated
in appropriate experiments, once in the same buffer containing 0.5%
Nonidet P-40, two times in buffer without Nonidet P-40, once in 1 M KOH, and two times with Tris buffer only. The membrane
was dried and exposed for autoradiography.
The in solution kinase assay protocol of Brown et al. (40)
was used at pH values indicated in appropriate experiments. The reaction mix contained 30 mM Tris-HCl at the appropriate
pH, 0.5 mM dithiothreitol, 10 mM
MgCl2, 30 µM ATP (Sigma), and 5 µCi of [
-32P]ATP/50 µl. The metal dependence of BVR kinase
activity was measured in the presence of 0, 1, and 2 mM
concentrations of Ca2+ or Mn2+. The reaction
contained 5 µg of purified BVR. When used, the reaction also
contained 10 µg of dephosphorylated
-casein (Sigma) or myelin
basic protein (MBP, Sigma). The assay mix was incubated at 37 °C for
1 h and was terminated by adding 10 µl of 1% SDS. Unincorporated ATP was removed by gel filtration through a Sephadex G-50 column (Amersham Pharmacia Biotech), and the eluant was loaded onto a 0.75-mm, 15% SDS-polyacrylamide gel (41). The gel was stained using Coomassie Blue and dried under a vacuum. The same gel was
then exposed to Hyperfilm ECL (Amersham Pharmacia Biotech) and developed.
To determine whether BVR serine/threonine residues are a target of
autophosphorylation, the following experiment based on the method
described by Kamps and Sefton (42) was carried out. BVR (20 µg) was
subjected to in solution phosphorylation. After removal of
unbound ATP and treatment with SDS at 100 °C, samples were applied
to gel and transferred to an Immobilon P membrane. The membrane was
briefly exposed for autoradiography. The same membrane was subsequently
cut in two. One section was treated with 1 N KOH for 2 h at
55 °C to hydrolyze phosphate groups from phosphoserine and
phosphothreonine residues. The second section was treated similarly but
at pH 7.4. The membrane was again autoradiographed.
ATP Binding--
The pH dependence of BVR affinity for ATP was
determined by measuring the increase in fluorescence after addition of
increasing amounts of the ATP analogue TNP-ATP to BVR as described by
Senior and colleagues (43, 44). Fluorescence measurements were made in
a SPEX Fluorolog 2 fluorometer. 2-ml stirred cuvettes at room temperature containing 30 mM Tris-HCl (pH 6.7, 7.4, and
8.7), 2 mM Mn2+, 10 mM
Mg2+, and 1.0 µM BVR were used for TNP-ATP binding
experiments. The excitation wavelength was 408 nm, and the emission
spectra from 500 to 600 nm were recorded. A control cuvette
contained all components except BVR. Fluorescence spectra were taken 1 min after the addition of TNP-ATP. The TNP-ATP concentration was
determined using an extinction coefficient of 26,400 M
1 cm
1
at 408 nm.
Dephosphorylation of BVR--
The enzyme was dephosphorylated
using two different enzymes: potato acid phosphatase and protein
phosphatase 2A (PP2A). For the former, E. coli containing
BVR plasmid construct was grown overnight in LB medium. The cells were
sonicated in phosphate-buffered saline (10 mM phosphate, pH
7.4, 140 mM NaCl) and centrifuged at 10,000 × g for 15 min. GST-BVR fusion protein in the cell extract was
then bound to glutathione-Sepharose beads (Amersham Pharmacia Biotech)
at 4 °C for 1 h. Beads were washed four times with
phosphate-buffered saline and then four times with TES buffer (100 mM TES, pH 7.0, 100 mM NaCl). Beads were
incubated at 37 °C for 2 h with 15 units of potato acid
phosphatase (Sigma) in TES buffer to remove phosphate. After
incubation, beads were washed six times with TES buffer to remove
phosphatase. BVR was then cleaved off GST fusion by incubation with 5 units of thrombin protease at 4 °C overnight. The protein solution
was collected and measured by a Bradford assay. To rephosphorylate BVR,
the assay conditions described above for the in solution kinase assay
were used.
That phosphorylation is required for its reductase activity was
also examined, and the number of phosphates bound to BVR was determined
after PP2A (Upstate Biochem) treatment. BVR (8 µg) was treated with
PP2A (1.0 unit) and incubated for 30 min at 30 °C. To test for
specificity of phosphatase activity, PP2A was inhibited by okadaic acid
(1 µM) by incubating 30 °C for 5 min with prior to
addition of BVR. Thereafter, BVR activity was measured at pH 6.7 with NADH as the cofactor. To measure the stoichiometry of
BVR:phosphate after treatment with PP2A, the released phosphate was quantitated based on a phosphate standard titration curve constructed using EnzChek phosphate assay kit (E-6646, Molecular Probes).
Cell Culture--
COS cells were grown to 80% confluence in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum.
The cells were suspended in prewarmed serum-free Dulbecco's modified
Eagle's medium at 37 °C. Cell suspension was divided into
aliquots. H2O2 (1 mM) was added to
the cell suspensions, and they were incubated at 37 °C. Control
samples were not treated. After centrifugation at 2000 rpm for 5 min,
cells were lysed in 50 µl of lysis buffer A (50 mM
Tris-HCl, pH 7.4, 75 mM NaCl, 1% Nonidet P-40, 0.2 mM NaVO4, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml each leupeptin,
pepstatin, and aprotinin, 2 mM EDTA, 2 mM EGTA) on ice for 15 min. After a brief sonication, the lysates were centrifuged first at 1000 rpm for 5 min, and then the supernatant fraction was centrifuged at 45,000 rpm for 60 min. Protein
concentration was measured by Bradford assay (Bio-Rad).
For BVR immunoprecipitation, cells were lysed in lysis buffer as above.
Anti-BVR antibody coupled to protein A was added to the supernatant
fraction. For coupling, 10 µl of antibody was incubated with 40 µl
of protein A-Sepharose, which had been blocked with bovine serum
albumin for 3 h at 4 °C. 100 µg of protein of cell lysate was
added to beads, and the mixture was incubated for a minimum of 3 h
at 4 °C. The immunoprecipitate was recovered by centrifugation,
washed three times, and used for SDS-polyacrylamide gel electrophoresis
and immunoblotting. Duplicate blots were probed with anti-BVR and
antiphosphomix consisting of a mix of anti-serine/anti-threonine phospho-amino acids.
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RESULTS |
Previous studies have suggested that BVR is posttranslationally
modified (32). In this study, experiments were carried out to determine
whether BVR is a phosphoprotein and, if so, whether phosphorylation of
BVR is a reflection of autophosphorylation activity.
Autophosphorylation activity was analyzed by two different methods: the
in blot and in solution kinase assays. The results of the in blot
kinase assay are shown in Fig.
1A. Autophosphorylation activity was assessed, after denaturation of BVR with
guanidine-HCl, from phosphate incorporation into the renatured protein
in the presence of [
-32P]ATP. Incorporation of
[32P]PO4 upon renaturation was detected by
autoradiography. As shown when renatured in the presence of ATP,
phosphate was transferred to BVR. HO-1 and HO-2, which are
phosphoproteins, as indicated by immunoreactivity toward a mixture of
anti-serine/anti-threonine/anti-tyrosine (Fig. 1B),
were used as controls for autophosphorylation. As noted in Fig.
1A, neither of the HO isozymes is reversibly
phosphorylated under the same conditions that showed incorporation of
the
phosphate of ATP into BVR. The finding suggested that the
labeled phosphate signal produced by renatured BVR was not due to
nonspecific protein binding of [32P]ATP.

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Fig. 1.
Biliverdin reductase is autophosphorylated.
A, the in blot kinase assay. 20-µg aliquots of purified
human or E. coli-expressed human BVR, HO-1, or HO-2
preparations, after SDS-gel electrophoresis, were transferred to a
polyvinylidene difluoride membrane and examined for renaturable
phosphorylation at pH 7.4. The protein was denatured, renatured, and
incubated in the presence of [ -32P]ATP as detailed
under "Materials and Methods." B, 4 µg each of the
same enzyme preparations were subjected to Western blot analysis using
anti-phospho mix as the primary antibody. The ECL method of detection
was used. C, the in solution kinase assay. The kinase assay
was carried out in the presence of 2 mM Mn2+
and 10 µg of dephosphorylated -casein. 5 µg of purified E. coli-expressed BVR was used for each assay (50 µl).
Phosphotransferase activity was carried out at the indicated pH values.
The gel was processed as detailed under "Materials and Methods."
D, the same gel stained with Coomassie Blue before
autoradiography.
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Because of the pH dependence of BVR, the effect of pH on
phosphotransferase activity was measured at pH 8.7 and 6.7. In
addition, experiments were performed to examine whether the
32P signal observed in Fig. 1A is an
authentic reflection of its incorporation in BVR in the process of
renaturation, rather than a reflection of nonspecific protein binding.
For these experiments, the in solution kinase assay was used. The
results are shown in Fig. 1C. Casein, which is not a
phosphoprotein and is a commonly used acceptor of phosphate, was used
in these experiments as a negative control for autophosphorylation. The
stained gel of the same samples for control of sample loading is shown
in Fig. 1D. As noted in Fig. 1C, BVR
autophosphorylation is pH-dependent, with the activity
being prominent at pH 8.7 and essentially no phosphotransferase
activity occurring at pH 6.7. Moreover, only in lanes that contained
BVR was the 32P signal observed. In lanes that contained
casein alone, a signal was not observed under the pH conditions. This
observation suggested that the autophosphorylation of BVR in this
kinase system also is not a reflection of nonspecific ATP binding to
the protein. Moreover, it appeared that at pH 8.7 BVR transferred
phosphate to casein.
To confirm that BVR transfers phosphate to casein in the in
solution kinase assay was carried out in the presence of increasing concentrations of casein (1-20 µg) and a constant concentration of
BVR (5 µg). The data suggest that BVR does transfer phosphate to
casein, albeit not at an impressive rate, as indicated by the kinetics
of the reaction (Km = 1.1 µM;
Vmax = 2.8 pmol/mg/min). It is noted that the
pattern of casein separation on SDS gel reveals that the commercially
obtained casein separates on the gel as multiples of protein bands.
When compared with Fig. 1C, it is evident that not all
components that are present in the casein preparation are
phosphorylated in the presence of BVR.
The phosphotransferase activity of BVR specifically required
Mn2+ (Fig. 2).
Ca2+ used at the same concentrations as Mn2+
(1.0 and 2.0 mM) did not support phosphotransferase
activity. As shown in Fig. 2, A versus
B, at pH 8.7 in the absence of Mn2+, no
incorporation of labeled phosphate in BVR takes place (panel A, lane 1 versus panel B, lane
3). The Mn2+ requirement for BVR phosphotransferase
activity was further examined in an assay system containing MBP. As
detected on the autoradiograph (Fig. 2A, lane 2),
MBP, a phosphate acceptor, when present with BVR plus Mn2+,
also gave three distinct 32P signals. In the absence of
Mn2+ the signals were quite subdued (Fig. 2B,
lane 2). MBP is known to nonspecifically bind phosphate in
the absence of a kinase, and this was also observed in our hands (Fig.
2A, lane 3); as noted in panel B,
lane 1, this interaction can take place in the absence of
Mn2+. Commercially available MBP, as is the case with
casein, separates in several bands on SDS gel. Because the objective of
this experiment was not to characterize MBP phosphorylation, the kinase
activity of BVR with MBP was not further examined. The
autophosphorylation activity of BVR was thermolabile and was lost by
exposure to 60 °C for 10 min prior to use in the kinase assay (Fig.
2C, lane 1).

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Fig. 2.
Characterization of phosphotransferase
activity of human biliverdin reductase. Purified E. coli-expressed BVR was analyzed by an in solution kinase assay in
the presence (A) or absence (B) of 2 mM MnCl2 at pH 8.7 as described under
"Materials and Methods." 5 µg of BVR was used in each assay.
Assays were also carried out in the presence of MBP (10 µ g).
A, lanes: 1, BVR; 2, BVR plus MBP;
3, MBP. B, lanes: 1, MBP;
2, BVR plus MBP; 3, BVR. C, lanes:
1, BVR heated for 10 min at 60 °C; 2, the same
preparation without heating. Top panel, in solution kinase
activity at pH 8.7; bottom panel, the same gel stained with
Coomassie Blue.
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Next, the effect of pH on ATP binding was examined using the
fluorescent ATP analogue TNP-ATP. The increase in fluorescence of
TNP-ATP as the result of binding to BVR was measured. The binding was
titrated as an increase in BVR fluorescence at 550 nm as a function of
TNP-ATP concentration. As shown in Fig.
3, at pH 7.4 and 8.7 there is a
TNP-ATP-dependent increase in BVR fluorescence, with
Kd values of 15.5 µM at pH 8.7 and
28.0 µM at pH 7.4. In the absence of Mn2+ in
the assay system, the increase in fluorescence at 550 nm was not
detectable. In addition, when measured at pH 6.7 in the presence of
Mn2+ and the same concentrations of TNP-ATP, an increase in
BVR fluorescence was not detected. The base-line fluorescence of
TNP-ATP was not pH-dependent.

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Fig. 3.
Binding of TNP-ATP to human BVR.
Purified E. coli-expressed BVR was titrated with TNP-ATP in
an assay system containing 30 mM Tris buffer at pH 7.4 or
8.7, 10 mM Mg2+, 2 mM
Mn2+, and the indicated concentrations of TNP-ATP. The BVR
concentration in the assay system was 1 µM. The increase
in fluorescence was measured at 550 nm.   , pH 8.7;   , pH
7.4.
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The most common amino acid acceptors of phosphate are serine,
threonine, and tyrosine, with the latter being the least common among
the three. Because in the experiment shown in Fig. 1B we used a mixture of antibodies to all three phospho amino acids, we
sought to identify the amino acids that are acceptors of phosphate. For
these experiments, the alkaline treatment, which hydrolyses serine/threonine-bound phosphate (42) was used. BVR transfers phosphates to serine/threonine residues, as suggested by results of the
experiments shown in Fig. 4. Subsequent to autophosphorylation and
autoradiography (Fig. 4A) (control for protein
loading), the membrane was divided in two parts; one part was treated
with 1 NKOH (2 h at 55 °C) and again autoradiographed.
As shown in Fig. 4B, the
intensity of the 32P signal is reduced to a minimum
subsequent to alkaline hydrolysis (lane 1 versus
lanes 2 and 3). Fig. 4A shows that the
decrease in intensity is not due to a differential loading of the lanes (lane 1 versus lanes 2 and
3).

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Fig. 4.
Biliverdin reductase is autophosphorylated on
serine/threonine residues. Purified E. coli-expressed
BVR was subjected to in solution autophosphorylation and
electrophoresis on a 12% SDS-polyacrylamide gel and was then
transferred to an Immobilon membrane. The membrane was autoradiographed
for control of loading. Subsequently, the membrane was cut in two, and
one section was subjected to alkaline hydrolysis (1 N KOH)
before autoradiography. Experimental details are provided under
"Materials and Methods". A, lane 1,
32P signal before hydrolysis; B, lane
1, 32P signal after hydrolysis. A and
B, lanes 2 and 3, control.
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The significance of the phosphorylation state to reductase activity was
examined by investigating the effect of treatment with phosphatases on
BVR activity. The first experiment used potato acid phosphatase. For
this experiment, a protocol was designed to ensure the removal of the
added phosphatase from the BVR preparation, and also only
NADH-dependent activity was measured, to ensure that NADPH
dephosphorylation by phosphatase did not contribute to the observation.
The results are shown in Table I. As
noted, treatment with potato acid phosphatase caused a dramatic
decrease in BVR activity. The loss of activity was, however, recovered when the phosphatase-treated preparation was incubated with ATP at pH
8.7 under the conditions used for the in solution autophosphorylation assay. It was also noted that in the presence of ATP the reductase activity of the control BVR, as well as of phosphatase-treated samples,
was increased over that in the absence of ATP. This observation was
reproducible, and we cannot explain it at this time.
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Table I
Effect of phosphorylation state on human biliverdin reductase activity
E. coli-expressed GST-tagged human BVR, as linked to a
glutathione-Sepharose 4B column, was treated with potato acid
phosphatase for 2 h at 37 °C and then purified as described
under "Materials and Methods," and the GST tag was removed. The
control sample was exposed to the same treatment but minus the
phosphatase. The purified preparations were treated with 30 µM unlabeled ATP for 1 h at pH 8.7 under the same
conditions used for in solution phosphotransferase activity described
under "Materials and Methods." The activity of the control
preparation was 2830 nmol of bilirubin/min/mg. After removal of excess
ATP by passing through a gel filtration column, samples were utilized
for measuring BVR activity at pH 6.7 with NADH as the cofactor.
Experimental procedures are described in the text.
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The next series of experiments using PP2A confirmed that
phosphorylation is essential for BVR activity and that BVR is
phosphorylated on serine/threonine residues. The results are shown in
Fig. 5. Treatment of BVR with the
serine/threonine phosphatase PP2A inhibited the reductase activity. The
presence of okadaic acid, the inhibitor of PP2A, for the most part
blocked inhibition of BVR. Me2SO, the solvent for
PP2A, proved to be an inhibitor of BVR; this may well account for the
absence of full recovery of BVR activity in the presence of okadaic
acid. The stoichiometry of BVR:phosphate was determined by
measuring the number of phosphates released by PP2A treatment of BVR
and a phosphate standard curve. The results indicated a 1:6
stoichiometry of BVR:phosphate. The BVR sample after PP2A treatment, when analyzed by Western blotting using anti-phosphoserine and anti-phosphothreonine, showed a loss of phosphate signal.

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Fig. 5.
Effect of protein phosphatase 2A and okadaic
acid treatment on BVR activity. Purified human BVR (8 µg) was
treated with PP2A (1.0 unit) in Me2SO (DMSO) or
with PP2A plus okadaic acid (1 µM, dissolved in
Me2SO) at 30 °C for 30 min. Activity was measured from
the rate of bilirubin formation at pH 6.7 with NADH. Control BVR was
treated with the same concentration of Me2SO as was present
in PP2A-treated samples.
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Next, we examined whether phosphorylation of BVR also occurs in the
cell and whether it has a bearing on BVR activity. For these
experiments BVR was immunoprecipitated from COS cells and analyzed for phosphorylation using a mixture of anti-phosphoserine and
anti-phosphothreonine antibodies. The results are shown in Fig.
6. As noted in Fig. 6A
(top panel), BVR is phosphorylated in vivo
(lane 1), and its phosphorylation is increased in a
time-dependent manner in response to
H2O2 treatment. BVR phosphorylation did not
increase further when assessed after 1 h of
H2O2 treatment. The increased phosphorylation
was not a reflection of an increase in cellular BVR protein levels, as
suggested by the Western blot analysis using anti-BVR antibody (Fig.
6A, bottom panel). The reductase activity was
also increased in cells treated with H2O2 (Fig.
6B). As shown, a time-dependent increase in
activity was detected in the presence of H2O2,
with the rate of bilirubin formation measuring 3-5-fold that of the
control when determined at 15 and 60 min, respectively, after treatment
with H2O2. The early onset of increasing
activity suggests that activation of the protein, rather than increased
BVR protein, is responsible for the observation.

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Fig. 6.
Phosphorylation of biliverdin reductase in
COS cells and response to H2O2. A,
COS cells were treated with 1 mM
H2O2. Control cells were not treated. At the
indicated times, cells were lysed, and BVR was immunoprecipitated and
subjected to electrophoresis and blot transfer. For one blot, a mix of
anti-phosphoserine and anti-phosphothreonine was used as the primary
antibody (top panel). Anti-BVR antibody was used for the
second blot (bottom panel). B, COS cells were
treated with 1 mM H2O2 for the
indicated periods. After lysis, the cell extract was prepared, and the
reductase activity was measured at pH 8.7 as described under
"Materials and Methods." The activity analysis was repeated using
two separate preparations of COS cells.
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DISCUSSION |
This report has identified phosphotransfer and reduction of
biliverdin as two types of activities of human BVR and has shown that
the reductase activity of the enzyme requires phosphorylation of the
protein. Moreover, as with reductase activity, the enzyme shows pH
dependence for autophosphorylation. The enzyme appears to be best
suited for both autokinase (Fig. 1 and 2) and reductase activity (9) in
an alkaline pH range (7.4 and 8.7). pH 6.7 corresponds to the pH value
for peak reductase activity in the acidic range, whereas at this pH,
phosphotransferase activity was nearly undetectable (Fig.
1C). A plausible explanation for the observation is a
decreased affinity of BVR for adenine nucleotides in the acidic range.
This possibility is consistent with BVR activity at pH 6.7 requiring a
10-fold higher concentration of cofactor (9, 33, 34) and with
the near doubling of the Kd value for
TNP-ATP at pH 7.4 versus pH 8.7 (Fig. 3). The base-line fluorescence of TNP-ATP is prohibitive of using a great excess of the
agent in the assay system. The fact that at pH 8.7 the enzyme is an
NADPH-dependent reductase and is also most effective in
autophosphorylation would suggest that at this pH the electronic environment of the phosphate-interacting domains and/or the structure that BVR assumes at this pH allow for high affinity binding of adenine nucleotides. The biological significance of why BVR is a more
effective reductase and autokinase at pH 8.7 than at pH 6.7 is not understood.
Hydrolysis of BVR-bound phosphates under alkaline conditions suggests
that BVR is a serine/threonine phosphoprotein. Phosphorylated tyrosine
residues are resistant to alkaline/acid hydrolysis (42). Findings with
PP2A, which is a serine/threonine phosphatase, supports this
conclusion. PP2A studies also suggest a 1:6 molar ratio of BVR:phosphate. At this time it is not known how many of the phosphates are transferred to BVR by autophosphorylation. Also not known is which
serine/threonine residues are phosphorylated. Human BVR has 24 serine
and 7 threonine residues (34). These inquiries are the subject of our
future studies.
The finding with casein phosphorylation is also consistent with the
possibility that BVR is a serine/threonine kinase. Casein, as well as
MBP, is phosphorylated on serine/threonine residues. It is noteworthy
that a predicted BVR sequence,
Gly148-Ser-Leu-Arg-Phe-Thr-Ala-Ser-Pro, closely resembles
the serine/threonine kinase consensus motif
Gly-(Thr/Ser)-X-X-(Tyr/Phe)-X-Ala-Pro-Glu (45). It is relevant to note that mutation of the Ser149
residue in BVR inactivates the enzyme (data not shown). This finding is
consistent with the likelihood that the
Gly148-Ser-Leu-Arg-Phe-Thr-Ala-Ser-Pro motif in BVR is the
serine/threonine kinase domain and that phosphorylation of
Ser149 is essential for BVR activity. The data further
suggest that the other five titratable phosphates released by PP2A
treatment are bound to serine/threonine residues, which are not
acceptors of BVR phosphotransferase activity, and that they may not be
critical for BVR activity. In addition, present in BVR is the Walker
homology A motif,
Gly15-X-Gly-X-X-Gly, which
is conserved in the cyclic nucleotide-regulated protein kinase family.
Mutation of the Gly17 residue in this motif also
inactivates the reductase (data not shown).
The presently described in vitro phosphotransferase activity
of BVR, together with the finding with COS cells, although suggestive of BVR having phosphotransferase activity in vivo, does not
prove this activity. However, the assay systems used for determining autophosphorylation of E. coli-expressed BVR convincingly
suggest that in vitro transfer of ATP phosphate to the
reductase is carried out by BVR itself. In the mammalian cell, this may
not be the case, and other kinases could partake in phosphorylation of
BVR. Accordingly, the finding that in COS cells there is an increase in
the phosphorylation state of BVR in response to
H2O2 does not necessarily implicate
autophosphorylation activity; however, an increase in BVR reductase
activity is consistent with the conclusion that phosphorylation is
required for BVR reductase activity. Moreover, potato acid
phosphatase inactivation of the reductase activity (Table I) and
reversible activation of the phosphatase-treated BVR further indicate
that phosphorylation is important to BVR enzyme activity. Furthermore,
it is reasonable to speculate that in the cell, the phosphotransferase
activity of BVR may not be directed only toward itself but also
toward other substrates.
Enzyme phosphorylation plays a regulatory role in intermediary
metabolism, and many enzymes are phosphoproteins; of these a small
fraction are reversibly autophosphorylated. The biological significance
of BVR phosphotransferase activity and the dependence of the activity
on phosphorylation may be related to the central role of the HO system
in cellular defense mechanisms, cell signaling (12, 46), and the
complex interactions that exist between the enzymes of the heme
degradation pathway and products of heme degradation activity. To
elaborate, as noted above, the BVR catalytic activity product bilirubin
inhibits protein phosphorylation (29); bilirubin is also a feedback
inhibitor of reductase (9). On the other hand, biliverdin is an
inhibitor of HO activity (2). In fact, with the exception of
biliverdin, to date, no other heme pathway products or normal cell
constituents have been found to inhibit HO activity in vivo.
In addition, as noted above, induction of HO-1 and generation of
bilirubin is suspected to be a significant component of the cellular
antioxidant defense mechanisms. The finding that
H2O2, which is a potent oxidant and inducer of
HO-1 (47), causes rapid increases in the activity of the reductase and
in its phosphorylation state could be particularly relevant to the
control of cellular levels of heme degradation products. In this
scenario, the rapid conversion of biliverdin to bilirubin would allow
for the derepression of HO activity. Product inhibition of BVR activity
then would allow for an increase in effective biliverdin levels and
hence permit a return to normal of the HO activity. The finding that
the reductase is reversibly phosphorylated is further supportive of the
potential significance of BVR phosphorylation to cellular defense
mechanisms as demand arises.
 |
ACKNOWLEDGEMENTS |
We thank Suzanne Bono for preparation of the
manuscript, William McCoubrey for generation of the BVR expression
system, Parvanah Rafiee for assistance with phosphorylation assays, and
XiaoJun Wang for cell culture maintenance. We are grateful to Professor Alan Senior and Dr. Joachim Weber for helpful assistance with ATP
binding experiments.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants ES04066 and ES04391.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 716-275-5383;
Fax: 716-275-6007; E-mail: mahin maines@urmc.rochester.edu.
Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.M010753200
 |
ABBREVIATIONS |
The abbreviations used are:
HO, heme oxygenase;
BVR, biliverdin reductase;
GST, glutathione S-transferase;
MBP, myelin basic protein;
TNP-ATP, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate;
PP2A, protein phosphatase 2A;
TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic
acid.
 |
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