A Novel Mammalian Lithium-sensitive Enzyme with a Dual Enzymatic
Activity, 3'-Phosphoadenosine 5'-Phosphate Phosphatase and
Inositol-polyphosphate 1-Phosphatase*
José M.
López-Coronado,
José M.
Bellés,
Florian
Lesage
,
Ramón
Serrano, and
Pedro L.
Rodríguez§
From the Instituto de Biología Molecular y Celular de
Plantas, Universidad Politécnica de Valencia-Consejo Superior
de Investigaciones Científicas, Camino de Vera, E-46022,
Valencia, Spain and the
Institut de Pharmacologie
Moleculaire et Cellulaire, CNRS UPR411, 660 route des Lucioles,
06560, Valbonne, France
 |
ABSTRACT |
We report the molecular cloning in Rattus
norvegicus of a novel mammalian enzyme (RnPIP), which shows both
3'-phosphoadenosine 5'-phosphate (PAP) phosphatase and
inositol-polyphosphate 1-phosphatase activities. This enzyme is the
first PAP phosphatase characterized at the molecular level in mammals,
and it represents the first member of a novel family of dual
specificity enzymes. The phosphatase activity is strictly dependent on
Mg2+, and it is inhibited by Ca2+ and
Li+ ions. Lithium chloride inhibits the hydrolysis of both
PAP and inositol-1,4-bisphosphate at submillimolar concentration;
therefore, it is possible that the inhibition of the human homologue of
RnPIP by lithium ions is related to the pharmacological action of
lithium. We propose that the PAP phosphatase activity of RnPIP is
crucial for the function of enzymes sensitive to inhibition by PAP,
such as sulfotransferase and RNA processing enzymes. Finally, an
unexpected connection between PAP and inositol-1,4-bisphosphate
metabolism emerges from this work.
 |
INTRODUCTION |
The sulfation reactions in mammals affect many different
physiological processes, including deactivation and bioactivation of
xenobiotics, inactivation of hormones and catecholamines, structure and
function of macromolecules, and elimination of end products of
catabolism (1). Sulfation involves the transfer of a sulfate group from
3'-phosphoadenosine 5'-phosphosulfate
(PAPS)1 to an acceptor
molecule in a reaction that is catalyzed by a family of
sulfotransferase enzymes (2). Usually, the obligate co-substrate PAPS
donates its sulfuryl group to a functional hydroxyl group: PAPS + R-OH
PAP + R-OSO3
. The products are a sulfoconjugate
(R-OSO3
) and 3'-phosphoadenosine 5'-phosphate (PAP). PAP
is an inhibitory end product in any sulfation reaction because it acts
as a competitive inhibitor with respect to PAPS (3). For instance, the
M and P forms of human phenolsulfotransferases are potently inhibited by PAP, exhibiting a Ki value of approximately 0.1 µM (4).
In addition, a toxic effect of PAP on RNA processing enzymes (5'
3'
exoribonucleases) has been recently described in yeast (5). This effect
might be attributed to the fact that PAP mimics the monomers of a
polyribonucleotide chain and consequently, it might prevent the attack
to the phosphodiester bond of RNA processing enzymes. These 5'
3'
exoribonucleases are highly conserved in evolution (5), therefore it is
very likely that PAP accumulation in mammals has similar inhibitory
effects on RNA processing, leading to aberrant changes in the pattern
of gene expression. Consequently, a rapid removal of PAP is required
both to maintain the activity of sulfotransferase enzymes and to
prevent the accumulation of PAP to levels that could be inhibitory for
RNA processing enzymes. Thus, it is reasonable to predict the existence
in mammals of hydrolytic enzymes, such as PAP phosphatases, which could
rapidly recycle PAP to AMP and inorganic phosphate. Indeed, a
preliminary characterization of a 3',5'-bisphosphate nucleotidase
purified from guinea pig liver has been reported (6), although this enzyme showed a very poor affinity for PAP (Km 3 mM). No clear role for this enzymatic activity nor
molecular data of the corresponding gene have been reported.
In this work, we present for the first time the molecular cloning of a
mammalian PAP phosphatase. The enzyme was cloned by functional
complementation of a Saccharomyces cerevisiae hal2 mutant.
Hal2 is a 3',5'-bisphosphate nucleotidase that specifically hydrolyzes
the 3'-phosphate from PAP (7, 8), thereby preventing the accumulation
of PAP. In yeast, PAP is generated as a side product of PAPS reductase,
which is a key enzyme in the sulfate assimilation pathway that leads to
the synthesis of methionine. PAP accumulation has deleterious effects
on the cell because it inhibits PAPS reductase (9), and hence
methionine biosynthesis, and RNA processing enzymes (5). The PAP
phosphatase activity of Hal2 is crucial for the function of PAPS
reductase, which is reflected in the fact that hal2 cells
are auxotrophic for methionine (7, 10). We took advantage of this
phenotype to clone the first mammalian PAP phosphatase by functional
complementation of the auxotrophy for methionine of hal2
cells. Interestingly, the mammalian PAP phosphatase has a dual
enzymatic activity, as it is also active as inositol-polyphosphate
1-phosphatase.
 |
EXPERIMENTAL PROCEDURES |
Chemicals--
All nonradioactive organic compounds were
obtained from Sigma. Radioactive compounds were purchased from NEN Life
Science Products. PAPS (Sigma) is supplied with 4 mol of lithium/mol of PAPS. Given the instability of this compound, removal of lithium is not
recommended. [35S]PAPS (NEN Life Science Products) is
obtained as the triethylammonium salt. Therefore,
[35S]PAPS was the substrate employed to assay the
activity of RnPIP against this compound.
Yeast Strain and Growth Media--
The Saccharomyces
cerevisiae strain used in this study was JRM4 (MATa
leu2-3, 112 ura3-251,328,372 hal2::LEU2), kindly
provided by Dr. J. R. Murguia (Universidad Politécnica,
Valencia, Spain). Complementation of the auxotrophy for methionine of
the hal2 mutant was assayed in minimal synthetic glucose
medium (SD), 2% glucose, 0.7% yeast nitrogen base without amino acids
and 50 mM succinic acid adjusted to pH 5 with Tris. When
indicated, the SD medium was supplemented with 100 µg/ml methionine
to give SDM medium.
Cloning of RnPIP--
The yeast strain JRM4 was transformed with
a Rattus norvegicus heart cDNA library constructed in
the pFL61 vector (11), a yeast expression vector where expression of
the rat cDNA is driven by the constitutive phosphoglycerate kinase
(PGK) promoter. Transformants were first selected by uracil prototrophy
in SDM. 105 primary transformants were obtained and pooled
together. To select clones with the ability to complement the
auxotrophy for methionine of the hal2 mutant, the primary
transformants were spread on minimal medium lacking methionine. Four
clones (pFL61-PIP) were obtained that complemented the auxotrophy for
methionine of the hal2 mutant. The yeast shuttling plasmid
was recovered by electroporation of Escherichia coli WM1100
cells. Partial sequencing and restriction analysis of the plasmids
recovered from the E. coli transformants showed that all of
them represented the same cDNA.
Expression of RnPIP in E. coli and Protein Purification--
The
coding region of RnPIP was PCR-amplified from pFL61-PIP
using Pwo DNA polymerase (Roche Molecular Biochemicals) and
the following primers (XhoI site underlined): upstream,
5'-CCCCTCGAGATGGCTTCCAGC; downstream,
5'-CCCCTCGAGCCCCTTCAGGGAATGAG.
The PCR-amplified product was subcloned into the pT7-Blue vector
(Novagen) to give pT7-PIP. The PIP open reading frame was released with XhoI and subcloned into the XhoI
site of pGEX-KG (12), thus generating a fusion protein between
glutathione S-transferase (GST) and PIP. The pGEX-KG-PIP
construct was verified by sequencing with an ABI 377 automatic
sequencer, and it was introduced into E. coli DH5
cells.
The expression of the recombinant protein was induced with 0.1 mM isopropyl-1-thio-b-D-galactopyranoside for a
period of 1 h at 30 °C, and the GST fusion protein was affinity purified on glutathione-Sepharose 4B (Amersham Pharmacia Biotech) as
indicated by the suppliers. The pGEX-KG-PIP construct contains a
thrombin cleavage site and a poly-glycine spacer between the GST and
the RnPIP moieties (12). Thus, specific proteolysis of the fusion
protein could be achieved by thrombin cleavage. The resulting RnPIP
protein behaves identically to the GST fusion protein in the
biochemical assays we have performed.
Enzyme Assay--
PAP phosphatase and
inositol-polyphosphate 1phosphatase activities were assayed by one
of the two following methods (8). Briefly, phosphatase activity was
routinely assayed (assay 1) in a 100-µl mixture containing 50 mM Tris-HCl, pH 7.5, 1 mM magnesium chloride,
and the indicated amount of GST fusion protein and substrate. After a
30-min incubation at 30 °C, the inorganic phosphate released was
quantified by the malachite green method as described previously (13).
Under these conditions, the enzyme activity was linear with protein
quantity (up to 5 µg) and reaction time (up to 1 h). The
concentration of either Ca2+ or Li+ that
decreased the activity of the enzyme by 50% relative to a reaction
without these cations (IC50) was estimated at a substrate concentration of 0.2 mM.
Assay 2 is described next, and it involved a HPLC analysis of the
reaction products. The products were detected either by ultraviolet-absorption (8) or, in the case of radioactively labeled
substrates, by coupling the HPLC device to a RadioFlow detector LB509
(EGG Berthold).
HPLC Analysis of the Hydrolysis of PAP, [35S]PAPS,
and [3H]Inositol-1,4-bisphosphate--
The conditions
for the HPLC analysis of the hydrolysis of PAP and PAPS have been
described previously (8). To improve the resolution of the analysis of
PAPS hydrolysis, the mobile phase was 3% methanol instead of 4%.
Inositol-1,4-bisphosphate hydrolysis was assayed in a reaction mixture
containing 0.2 M potassium Bicine (pH 8.0), 1 mM Mg2+, and 10 µM
[3H]inositol-1,4-bisphosphate (10 Ci/mmol). At the
indicated time, 10 µl of reaction mixture were injected into a
4.6 × 10-mm Guard Cartridge 10-µm SAX column linked to a
4.6 × 250-mm Partisil 10-µm SAX column (pS Phase Sep),
equilibrated in Milli-Q water, and maintained at 22 °C with a flow
rate of 1 ml/min. Inositol phosphates were eluted with a linear
gradient of (NH4)H2PO4, pH 3.7, according to Jenkinson (14). [35S]PAPS,
[35S]adenosine 5'-phosphosulfate, and
[3H]inositol phosphates were detected with a RadioFlow
detector LB509 (EGG Berthold) using Optiflow as scintillation mixture
at a flow rate of 3 ml/min.
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RESULTS |
Cloning of RnPIP--
To explore the presence in animals of PAP
hydrolytic enzymes, a screening was conducted for mammalian genes that
complement the auxotrophy for methionine of the yeast hal2
mutant. A rat cDNA library was constitutively expressed in
hal2 cells, and as a result, a cDNA was identified that
complemented the auxotrophy for methionine. Sequencing of this cDNA
reveals a 927-base pair open reading frame that putatively encodes a
protein of 308-amino acid residues with a molecular mass of 33.2 kDa
(Fig. 1). The cDNA has 27 base pairs
of 5'-untranslated sequence and 961 base pairs of 3'-untranslated
region. The protein encoded by this gene, named RnPIP,
complements the auxotrophy for methionine of hal2 cells to
the same extent as Hal2, as demonstrated by the similar growth rates in
methionine-free minimal medium of hal2 cells transformed with RnPIP or ScHAL2 (data not shown).

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Fig. 1.
The nucleotide sequence and the deduced amino
acid sequence of the RnPIP cDNA. The amino
acid sequence of the putative coding region is shown below the
nucleotide sequence. Stop codons in frame with the open reading frame
are indicated with an asterisk. An in frame TGA stop codon
is found 12 nucleotides 5' to the first ATG translation initiation
codon. The three conserved regions (17) involved in the coordination of
phosphate and metal ions and nucleophilic water activation are
indicated in boldface and are underlined. The
nucleotide sequence of the RnPIP gene has been deposited at
the GenBankTM/EBI data base under the accession number
AJ000347.
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Southern and Northern Blot Analyses--
A commercially available
genomic DNA blot was used to analyze the presence of RnPIP
in the rat genome as well as in other eukaryotic species (Fig.
2A). A major EcoRI
hybridization band of approximately 4.0 kilobase pairs was detected in
the rat genome. Other bands of lower intensity were also detected,
suggesting that additional sequences related to RnPIP are
present in the rat genome. Hybridization bands were also detected in
the genome of mouse, dog, cow, rabbit, and chicken. Data base searches
in the human nucleotide data base reveal the presence of human genes with high sequence similarity to RnPIP (see below). A rat
multiple tissue Northern blot containing poly(A)+ RNA was
used to examine the transcript level of the RnPIP gene in
different tissues (Fig. 2B). The RnPIP mRNA
was detected in all the tissues examined. The transcript level was
especially high in heart, brain, and kidney. The size of the mRNA
fits well with the length of the cDNA sequence reported above.

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Fig. 2.
Southern and Northern blot analyses of the
RnPIP gene. A, a genomic DNA blot that
contains DNA from seven eukaryotic species was supplied by
CLONTECH. The blot contains approximately 4 µg of
genomic DNA digested with EcoRI from each of the following
species: rat, mouse, dog, cow, rabbit, chicken, and yeast.
Hybridization with a radiolabeled full-length cDNA was performed
under high stringency conditions (23). A 1 kilobase DNA ladder (Life
Technologies, Inc.) was used as molecular size standards. B,
a Northern blot containing approximately 2 µg of poly(A)+
RNA from eight different rat tissues was supplied by
CLONTECH. The membrane was hybridized with a
full-length cDNA probe. An RNA ladder (Life Technologies, Inc.) was
used as molecular size standards. The size of the mRNA transcript
is indicated in kilobases (kb).
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Biochemical Characterization of the RnPIP Protein--
The RnPIP
protein was expressed in E. coli as a GST fusion protein.
Following induction with
isopropyl-1-thio-b-D-galactopyranoside, the fusion protein
was purified by affinity chromatography on glutathione-Sepharose resin.
The phosphatase activity of the purified protein with respect to
different substrates is summarized in Table
I. PAP was the preferred substrate for
RnPIP. The Km value for PAP hydrolysis was too low
to be determined with the standard colorimetric assay (13) or even
using the HPLC-based method where the products are detected by
ultraviolet absorption (8). The detection limit of the HPLC analysis
for PAP hydrolysis is approximately 2 µM, therefore the
Km for PAP must be below 2 µM. PAPS
was used as substrate with a 50% efficiency as compared with PAP. No
appreciable activity was observed against AMP, 3'-AMP,
fructose-1,6-bisphosphate, or pNPP. Interestingly, inositol-1,4-bisphosphate also served as a substrate for the enzyme, although with a 45% efficiency as compared with PAP. In addition, RnPIP hydrolyzed inositol-1,3,4-trisphosphate but not
inositol-1,4,5-trisphosphate or inositol monophosphate. Therefore,
besides the preferred PAP, RnPIP hydrolyzes the typical substrates of
inositol-polyphosphate 1-phosphatase (15).
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Table I
Substrate specificity of the RnPIP enzyme
The RnPIP protein was expressed in E. coli as a GST fusion
protein and purified as described under "Experimental Procedures."
The phosphatase activity was quantified by the malachite green method
(13) as described under "Experimental Procedures." The activity
obtained with different substrates (0.2 mM)a under
conditions of maximal activity, pH 7.5 and 1 mM
Mg2+, is expressed as the percent activity observed with
3'-PAP. The specific activity with 3'-PAP of GST-PIP was 0.8 µmol
Pi × min 1 × milligram protein 1. The results
are the average of two independent experiments, each performed in
duplicate. Standard deviations were less than 5%.
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To determine the position of the phosphate liberated from PAP and
inositol-1,4-bisphosphate, we have analyzed by HPLC the reaction
products of either PAP or inositol-1,4-bisphosphate hydrolysis (Fig.
3). RnPIP converts 3'-PAP to AMP by
hydrolysis of the 3'-phosphate (Fig. 3A). The 2'-phosphate
from 2'-PAP was also hydrolyzed by the enzyme, although with a lower
efficiency. In this respect, RnPIP qualifies as a
3'(2'), 5'-bisphosphate nucleotidase. Inositol-1,4-bisphosphate was
converted to inositol-4-phosphate (Fig. 3B), therefore RnPIP catalyzes the removal of the 1'-phosphate, as inositol-polyphosphate 1-phosphatase does.

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Fig. 3.
Identification by HPLC analysis of the
reaction products of either PAP or inositol-1,4-bisphosphate hydrolysis
catalyzed by RnPIP. A, PAP hydrolysis. GST-PIP (1 µg)
was incubated for 1 h at 30 °C in a buffer containing 0.2 M potassium Bicine (pH 8.0), 1 mM
Mg2+, and 1 mM PAP as substrate (mixture of
3'-PAP and 2'-PAP). The conditions of the HPLC analysis have been
previously described (8). 1, standards of AMP, 3'-(2')PAP,
and 3'-AMP were analyzed to determine the retention time; 2,
reaction mixture at time zero; 3, reaction mixture at 1 h. B, inositol-1,4-bisphosphate hydrolysis. GST-PIP (0.1 µg) was incubated for 10 min at 30 °C in a buffer containing 0.2 M potassium Bicine (pH 8.0), 1 mM
Mg2+, and 10 µM
[3H]inositol-1,4-bisphosphate (10 Ci/mmol). The
conditions of the HPLC analysis are described in experimental
procedures. 1, standards of
[3H]inositol-1-phosphate (1),
[3H]inositol-4-phosphate (4), and
[3H]inositol-1,4-bisphosphate (1, 4) were analyzed to
determine the retention time; 2, reaction mixture at time
zero; 3, reaction mixture at 10 min.
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Determination of the Km Values for PAPS and
Inositol-1,4-bisphosphate Hydrolysis--
The sensitivity limit of the
colorimetric assay for determination of inorganic phosphate prevents
the determination of Km values in the low micromolar
range. Fortunately, the above described HPLC analysis, together with
the availability of [3H]inositol-1,4-bisphosphate and
[35S]PAPS, made it possible to determine the
Km values for these substrates (Table
II). The apparent Km
for inositol-1,4-bisphosphate was 0.2 µM ± 0.1. The
Km for PAP could not be accurately determined, as
radioactively labeled PAP is not available. [35S]PAPS is
commercially available and RnPIP hydrolyzes the 3'-phosphate of PAPS
with a 50% efficiency as compared with PAP, generating [35S]adenosine 5'-phosphosulfate (Table II). Therefore,
although PAPS is not likely a physiological substrate for animal PAP
phosphatases (16), we have also determined the Km
value for PAPS. The apparent Km for
[35S]PAPS was 1.2 µM ± 0.3.
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Table II
Km and Vmax values for PAP, PAPS, and
inositol-1,4-bisphosphate hydrolysis
Inositol-1,4-bisphosphate and PAPS hydrolysis were assayed in a
reaction mixture containing 0.2 M potassium Bicine (pH
8.0), 1 mM Mg2+, and different concentrations of
[3H]inositol-1,4-bisphosphate (10 Ci/mmol) or
[35S]PAPS (1-3 Ci/mmol), respectively. The
Km value for either inositol-1,4-bisphosphate or
PAPS hydrolysis was determined by measuring reaction rates at substrate
concentrations of 0.055, 0.166, 0.333, and 0.5 µM. The
reaction products were analysed by HPLC and detected with a RadioFlow
detector as described under "Experimental Procedures." The reaction
product of either [3H]inositol-1,4-bisphosphate or
[35S]PAPS hydrolysis is [3H]inositol-4-phosphate or
[35S]adenosine 5'-phosphosulfate, respectively. The
estimation of the Km value for PAP is described in
the text. The Vmax values are equivalent to the data
presented in Table I. The results are the average of two independent
experiments, each performed in duplicate.
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Cation Sensitivity of RnPIP--
Hal2 and inositol-polyphosphate
1-phosphatase belong to a family of magnesium-dependent,
lithium-sensitive phosphatases (17). Accordingly, the phosphatase
activity of RnPIP was strictly Mg2+-dependent,
with an optimal concentration of 1 mM (data not shown). Ca2+ inhibited the phosphatase activity of RnPIP (Fig.
4A) (20 µM CaCl2 for 50% inhibition at 0.2 mM PAP),
probably because of competition for Mg2+, as the inhibition
was abolished by increasing the Mg2+ concentration (data
not shown). RnPIP was not affected by high Na+
concentrations, whereas it was very sensitive to Li+ (0.8 mM LiCl for 50% inhibition at 0.2 mM PAP)
(Fig. 4B). The effect of Li+ on this enzyme is
unique among the group of monovalent cations (data not shown).

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Fig. 4.
Effect of Ca2+ and
Li+ on the PAP phosphatase activity of
RnPIP. A, inhibition by Ca2+. B,
inhibition by Li+. Increasing amounts of either
Ca2+ or Li+ were used in a standard phosphatase
reaction with 2 µg of GST-PIP and 0.2 mM PAP as
substrate. A similar inhibition was obtained when
inositol-1,4-bisphosphate was used as substrate. The results are
expressed as percent activity observed in the absence of cations and
are the average of at least two independent experiments, each performed
in duplicate. Standard deviations were less than 5%.
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 |
DISCUSSION |
Biochemical characterization of the RnPIP protein shows that it is
a dual enzyme that hydrolyzes both PAP and inositol-1,4-bisphosphate. The Vmax value for PAP hydrolysis was
approximately 2-fold higher than for inositol-1,4-bisphosphate
hydrolysis. Although the Km for PAP could not be
accurately determined (<2 µM), we could determine the
Km for the closely related substrate PAPS (1.2 µM) and for inositol-1,4-bisphosphate (0.2 µM), indicating a high affinity for these substrates. The
enzyme is very sensitive to Li+ and, therefore, RnPIP is
the fourth animal enzyme that is inhibited by Li+ ions in
the therapeutic range, the other three being inositol monophosphate
phosphatase (18), inositol-polyphosphate 1-phosphatase (15), and
fructose-1,6-bisphosphatase (19). RnPIP is the first PAP phosphatase
characterized at the molecular level in mammals, and a human homologue
of RnPIP can be identified in the human data base of
expressed sequence tags (ESTs) (Fig. 5).
We predict that the human homologue of RnPIP will show a similar
sensitivity to Li+ ions. Consequently, it might represent
another target in lithium therapy, and the inhibition by lithium of the
human enzyme could also contribute to the therapeutic or toxic effects
of Li+ treatment.

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Fig. 5.
Human ESTs share high similarity with
RnPIP. A schematic diagram showing the amino acid alignment of
RnPIP with human ESTs (HsEST) corresponding either to the N
or C terminus of the protein is shown. The accession numbers for the
EST1 and EST2 are H97426 and AA643182, respectively.
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In addition to PAP phosphatase, RnPIP also qualifies as
inositol-polyphosphate 1-phosphatase. A precedent for this sort of dual
enzymatic activity is found in plants (20). The SAL1 enzyme of
Arabidopsis thaliana exhibits both PAP phosphatase and
inositol-polyphosphate 1-phosphatase activities. However, the
hydrolysis of inositol-1,4-bisphosphate proceeds with a 34% efficiency
as compared with PAP. Furthermore, the Km value for
inositol-1,4-bisphosphate was much higher (90 µM) than
the Km value for PAP (2 to 10 µM). In
contrast, RnPIP hydrolyzes both PAP and inositol-1,4-bisphosphate with
very high affinities. However, some features clearly distinguish the RnPIP enzyme from the canonical inositol-polyphosphate 1-phosphatase (15). First, RnPIP hydrolyzes both PAP and inositol-1,4-bisphosphate, whereas PAP hydrolysis by inositol-polyphosphate 1-phosphatase has not
been reported. Second, bovine inositol-polyphosphate 1-phosphatase fails to complement the yeast hal2 mutant (data not shown).
Finally, amino acid sequence comparison also reflects a clear
difference between RnPIP and inositol-polyphosphate 1-phosphatase (Fig.
6). RnPIP is only 27% identical to
bovine inositol-polyphosphate 1-phosphatase, whereas the mouse
inositol-polyphosphate 1-phosphatase is 85% identical to bovine
inositol-polyphosphate 1-phosphatase (Fig. 6). On the other hand, the
sequence identity of RnPIP to non-animal PAP phosphatases is also low
(25-30%) (Fig. 6). Instead, ESTs of proteins with unidentified
function, which show high identity (60-90%) to RnPIP, are present in
mouse, pig, fruit fly, and man (Fig. 6). For instance, comparison of
human ESTs corresponding either to the N or the C terminus of RnPIP
reveals 85-90% amino acid sequence identity to the respective part of
RnPIP (Fig. 5). These data, taken together with the biochemical
characterization of the protein, indicate that RnPIP is a novel
mammalian enzyme, which probably represents the first member of an
animal gene family of dual enzymes (Fig. 6).

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Fig. 6.
Dendrogram representing the phylogenetic
relationships of the novel family of dual specificity phosphatases,
i.e. PAP-inositol-1,4-bisphosphate phosphatases
(PIPases). The family of
magnesium-dependent lithium-sensitive phosphatases
comprises fructose-1,6-bisphosphatase (FBPases),
inositol-polyphosphate 1-phosphatase (IPP1pases), inositol
monophosphatase (IMPases), and PAP phosphatase
(PAPases) enzymes (17). RnPIP is 20, 27, 30, 90, 31, 30, and
25% identical to AtFBP, BtIPP1, MmIPP, HsEST, AtSAL1, ScHal2, and
ScImp1, respectively. The PILEUP program was employed to align the
amino acid sequence of the following proteins and ESTs: AtFBP (A. thaliana, Swiss-Prot P25851), BnFBP (Brassica napus,
Swiss-Prot Q07204), BtIPP1 (Bos taurus, Swiss-Prot P21327),
HsIPP1 (Homo sapiens, Swiss-Prot P49441), MmIPP (Mus
musculus, Swiss-Prot P49442), RnPIP (R. norvegicus, EBI
AJ000347), MmEST (M. musculus, GenBankTM
AA008240), HsEST (H. sapiens, GenBankTM H97426),
SsEST (Sus scrofa, EBI Z84066), DmEST (Drosophila
melanogaster, GenBankTM AA990707), AtSAL1 (A. thaliana, GenBankTM U40433), AtSAL2 (A. thaliana, EBI Z83312), OsRHL (Oryza sativa,
GenBankTM U33283), AtAHL(A. thaliana,
GenBankTM U55205), ScHal2 (S. cerevisiae,
Swiss-Prot P32179), and EcCysQ (E. coli, Swiss-Prot P26264),
ScImp1 (S. cerevisiae, PIR S70117), ScImp2 (S. cerevisiae, Swiss-Prot P38710), AnQutG (Aspergillus
nidulans, Swiss-Prot P25416), HsIMP(H. sapiens, EBI
S38980), BtIMP (B. taurus, EBI J05394), XlIMP (Xenopus
laevis, EBI X65513), LeIMP1 (Lycopersicon esculentum,
Swiss-Prot P54926), and EcSuhB (E. coli, Swiss-Prot
P22783).
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In summary, we propose that the PAP phosphatase activity of RnPIP, by
preventing accumulation of PAP, is necessary to maintain active
sulfotransferase (6, 21) and RNA processing enzymes (5). RNA processing
is crucial in cell metabolism. The importance of sulfation in animals
is illustrated by the phenotype of brachymorphic mice, which have
abnormal hepatic detoxification, bleeding times, and postnatal growth
(22). This phenotype is because of a missense mutation in
SK2 (sulfurylase kinase 2) (22), a member of the gene family
encoding the bifunctional enzymes that synthesize the universal sulfate
donor, PAPS. In addition, the inositol-polyphosphate 1-phosphatase
activity of RnPIP could play a role in the phosphoinositide-signaling pathway. Finally, the unexpected connection between PAP and
inositol-1,4-bisphosphate metabolism remains to be investigated.
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FOOTNOTES |
*
This project was supported by Grants AIR3-CT94-1508 and
BIO4-CT96-0775 of the European Union (Brussels).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ000347.
§
To whom correspondence should be addressed. Tel.: 34 96 3877860;
Fax: 34 96 3877859; E-mail: prodriguez{at}ibmcp.upv.es.
 |
ABBREVIATIONS |
The abbreviations used are:
PAPS, 3'-phosphoadenosine 5'-phosphosulfate;
PAP, 3'-phosphoadenosine
5'-phosphate;
GST, glutathione S-transferase;
HPLC, high
performance liquid chromatography;
Bicine, N,N-bis(2-hydroxyethyl)glycine;
pNPP, p-nitrophenyl phosphate;
EST, expressed sequence tag.
 |
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