From the
Inositol polyphosphate 4-phosphatase, an enzyme of the inositol
phosphate signaling pathway, catalyzes the hydrolysis of the 4-position
phosphate of inositol 3,4-bisphosphate, inositol 1,3,4-trisphosphate,
and phosphatidylinositol 3,4-bisphosphate. The amino acid sequences of
tryptic and CNBr peptides of the enzyme isolated from rat brain were
determined. Degenerate oligonucleotide primers based on this sequence
were used to amplify a 74-base pair polymerase chain reaction product.
This product was used to isolate a 5607-base pair composite cDNA, which
had an open reading frame encoding a protein with 939 amino acids with
a predicted molecular mass of 105,588 Da. The rat brain polymerase
chain reaction product was used as a probe to isolate a human brain
cDNA that predicts a protein with 938 amino acids and a molecular mass
of 105,710 Da. Remarkably, the human and rat proteins were 97%
identical. Recombinant rat protein expressed in Escherichia coli catalyzed the hydrolysis of all three substrates of the
4-phosphatase. Northern blot hybridization indicates that the
4-phosphatase is widely expressed in rat tissues with the highest
levels of expression occurring in brain, heart, and skeletal muscle.
Polyclonal antiserum directed against the carboxyl terminus of the
4-phosphatase immunoprecipitated >95% of the 4-phosphatase activity
in crude homogenates of rat brain, heart, skeletal muscle, and spleen,
suggesting that this enzyme accounts for the 4-phosphate activ-ity
present in rat tissues. This antiserum also immunoprecipitated the
4-phosphatase from human platelet sonicates.
Inositol polyphosphate 4-phosphatase is a
Mg
Phosphatidylinositols
phosphorylated at the D-3 position are formed in response to
extracellular agonists. The D-3 phosphate-containing lipids,
We now report the isolation of
cDNAs encoding rat and human brain 4-phosphatases that are 97%
identical at the amino acid level. We demonstrate the expression of
active recombinant enzyme in Escherichia coli and have
determined the tissue distribution of expression by Northern blot
analysis. In addition, an antibody directed against the 4-phosphatase
is shown to immunoprecipitate >95% of the observed 4-phosphatase
activity from the supernatant of crude extracts of several rat tissues
and human platelets.
We have isolated cDNA molecules encoding human and rat brain
4-phosphatase. This conclusion is based on the following criteria: 1)
the cDNA encodes a protein of the appropriate molecular weight that
contains all of the peptide sequence obtained from the native enzyme,
2) bacterially expressed recombinant protein catalyzes the hydrolysis
of the three substrates of 4-phosphatase, and 3) antisera raised
against the carboxyl-terminal peptide immunoprecipitates >95% of the
4-phosphatase activity from supernatants of human platelets and rat
brain, heart, skeletal muscle, and spleen. This suggests that this
enzyme may be the only 4-phosphatase expressed in these tissues. In
addition, Northern blot analysis indicates that the expression of mRNA
in various rat tissues correlates with the enzyme activity levels
previously reported for bovine tissues, with brain having the highest
activity(16) .
The amino acid sequence predicted by the rat
brain 4-phosphatase cDNA is 97% identical to that of human
4-phosphatase. The majority of differences between the two sequences
were conservative substitutions except for deletion of one amino acid
from the human sequence. A TBLASTN search (17) of the Genbank
data base disclosed that the 4-phosphatase amino acid sequence is 25%
identical over 300 amino acids to a predicted gene found in Caenorhabditis elegans cosmid R01H10, suggesting that it may
encode a related protein. This remarkable conservation between species
suggests that 4-phosphatase has an essential function. The amino acid
sequence of 4-phosphatase has no significant similarity to any other
inositol phosphate metabolizing enzyme that has been cloned to date.
Most notably, inositol polyphosphate 1-phosphatase has no similarity to
4-phosphatase even though it hydrolyzes one of the same substrates
(Ins(1,3,4)P
Several features of the 4-phosphatase sequence suggest
that its expression may be regulated. The 3`-UTR contains eight ATTTA
motifs that have been shown to be responsible for rapid degradation of
mRNA (15). The amino acid sequence of the 4-phosphatase contains two
potential PEST sequences. PEST sequences are proline, glutamic acid,
aspartic acid, serine, and threonine-rich domains that have been shown
to be common in proteins that are rapidly degraded by the
calcium-dependent thiol protease, calpain(20) . Potential PEST
sequences were evaluated using the computer program PEST-FIND (21).
PEST scores greater than 0 are considered strong PEST regions, and
those between 0 and -5 are considered weak PEST
regions(20) . The 4-phosphatase has two PEST domains. These are
KASPTSTEEEQVMLR (Fig. 2, amino acids 486-500) and
RLHGEGGEDVFPCSSTCSSK (Fig. 2, amino acids 555-574) with
PEST scores of 2.1 and 0.5, respectively. Proteolysis by calpain may
explain the instability of 4-phosphatase in the absence of
EDTA(1) . 4-phosphatase also contains several potential protein
kinase phosphorylation sites. Motifs for protein kinase C,
multifunctional calmodulin kinase II, myosin-I heavy chain kinase, S6
kinase, proline-dependent kinase, and casein kinase I and II are all
present(22) .
The cloning of the 4-phosphatase will allow its
role in the inositol phosphate signaling pathway to be studied further.
The recent observation that PtdIns(3,4)P
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank
-independent enzyme that catalyzes the hydrolysis
of the 4-position phosphate of inositol 3,4-bisphosphate
(Ins(3,4)P
)
(
)and inositol
1,3,4-trisphosphate (Ins(1,3,4)P
)(1) . Recently, the
4-phosphatase was shown to catalyze the hydrolysis the 4-position
phosphate of phosphatidylinositol 3,4-bisphosphate
(PtdIns(3,4)P
) with a first order rate constant 120- and
900-fold greater than that of inositol 3,4-bisphosphate and inositol
1,3,4-trisphosphate, respectively(2) . This suggests that the
4-phosphatase is an enzyme involved in the D-3 phosphate
containing phosphatidylinositol pathway.
PtdIns(3)P
, PtdIns(3,4)P
, and
PtdIns(3,4,5)P
, are believed to be formed chiefly by the
PtdIns-3 kinase that has been shown to physically associate with a
variety of activated tyrosine kinases(3, 4) . Activation
of the PtdIns 3-kinase has been implicated in the signal transduction
of platelet-derived growth factor receptor-mediated
mitogenesis(5) , the oxidative burst of neutrophils (6), and
translocation of the glucose transporter to the plasma
membrane(7) . The D-3 phosphate-containing
phosphatidylinositols are not hydrolyzed by phospholipase C enzymes (8, 9) and thus may act directly as signaling molecules
in these processes. The targets for these potential second messengers
are yet to be defined, although PtdIns(3,4)P
and
PtdIns(3,4,5)P
have recently been shown to activate
isoenzymes of protein kinase C in
vitro(10, 11) .
Materials
Frozen unstripped rat brains
were purchased from Pel-Freeze. Unlabeled Ins(1,3,4)P and
leupeptin were purchased from Calbiochem.
[
H]Ins(3,4)P
, unlabeled
Ins(3,4)P
, and [
P]PtdIns(3,4)P
were prepared as described(2) . GeneAmp
dNTP,
10
PCR buffer, and AmpliTaq
DNA polymerase were
purchased from Perkin-Elmer Corp. Oligonucleotides were synthesized by
the Washington University Protein and Nucleic Acid Laboratory. The
Colony/Plaque Screen
hybridization filters,
Aquassure
scintillation mixture,
[
P]dATP, [
P]ATP, and
[
H]Ins(1,3,4)P
were purchased from
DuPont NEN. The sequencing grade trypsin (modified), T4 polynucleotide
kinase, and the random primed labeling kit were purchased from
Boehringer Mannheim. phenylmethylsulfonyl fluoride, keyhole limpet
hemocyanin, bromoacetic acid N-hydroxysuccinimide ester,
protein A-Sepharose, and CNBr were from Sigma. A Uni-ZAP
rat brain cDNA library (oligo(dT) primed), a
Zap II human
fetal brain cDNA library (oligo(dT) and random primed), and E. coli strain XL1-Blue were purchased from Stratagene. A rat brain
5`-STRETCH
library (random primed), rat brain
QUICK-clone
cDNA, and rat brain multi-tissue Northern blot
were purchased from Clontech. The Sequenase
(version 2.0)
DNA sequencing kit was purchased from U. S. Biochemical Corp.
[thio-
S]dATP was purchased from
Amersham Corp. The Wizard
miniprep kit was purchased from
Promega. The LongRanger
gel solution was from Fisher. The
TA Cloning
kit was from Invitrogen. The QIAexpress PQE-32
expression vector and Ni-NTA agarose were purchased from Qiagen.
Protein Sequencing
The 4-phosphatase was
partially purified from rat brain as previously described(2) .
The purified protein was run on a 6% preparative SDS-polyacrylamide gel
electrophoresis gel, and the 110-kDa protein (35 µg) band was
excised from the gel and electroeluted, acetone precipitated, and then
either treated with trypsin or CNBr; the resulting peptides were then
separated by high pressure liquid chromatography and sequenced as
previously described(12) .
cDNA Cloning
The degenerate
oligonucleotides 5`-GAATTCCCNGTNCTNTTYAAYGT-3` (sense) and
5`-GAGCTCTTRTCNCCRAANCKYTC-3` (antisense) were derived from the amino
acid sequence of a CNBr peptide RVQPVLFNVGINEQQTLAERFGDTSLQ (Fig. 2, amino acids 744-770) with the addition of a
5`-EcoRI and 5`-XhoI restriction site to the sense
and antisense oligonucleotides, respectively. These oligonucleotides
(350 ng each) were used as primers in a 20-µl PCR containing 0.6
units of AmpliTaq DNA polymerase, 0.25 mM
GeneAmp
dNTP, 1
PCR buffer, and 1 ng of rat brain
QUICK-clone
cDNA (Clontech). The reaction was carried out
for 30 cycles with a 1-min denaturation step at 94 °C, a 1.5-min
annealing step at 50 °C, and a 2-min extension step at 72 °C.
PCR amplification was then repeated using 1 µl of the first
reaction as template using the same primers and cycling program. The
resulting 74-bp PCR product (PCR product A) was subcloned using a TA
Cloning
kit (version 1.3) (Invitrogen) and sequenced to
verify that the PCR product predicted the correct amino acid sequence
of the CNBr peptide. The product was end labeled with
[
P]ATP using T4 polynucleotide kinase
(Boehringer Mannheim) and used to probe a rat brain Uni-ZAP
cDNA library (Stratagene). Clones were plaque purified and
rescued into Bluescript
SK± plasmid using the R408
helper phage following the manufacturer's protocol (Stratagene).
One of the clones isolated, clone 4, was used as template to generate
PCR product B. The primers 5`-AGTCTGTGAGTTGGAAGAGT-3` (sense) and
5`-GAGCCGTTCTGCATAGTAGC-3` (antisense) derived from the 5`-sequence of
clone 4 were used for PCR under the conditions described above. The
resulting 480-bp product was labeled with
P using a random
primed labeling kit (Boehringer Mannheim) and then used to screen the
Uni-ZAP
cDNA library (Stratagene) and the random primed
rat brain 5`-STRETCH
cDNA library (Clontech). cDNA
libraries were screened by probing plaques bound to duplicate
Colony/Plaque Screen hybridization filters (Dupont NEN) following the
manufacturer's protocol for aqueous hybridization. DNA sequencing
reactions were performed using the Sequenase
(version 2)
kit (U. S. Biochemical Corp.) except for a short C-rich segment of the
3`-UTR (bp 3480-3540), which was sequenced with Taq polymerase using the manufacturer's protocol (Perkin Elmer).
Both strands of the open reading frame were sequenced. The human cDNA
was isolated by the same protocol from the fetal brain cDNA library
mentioned above using
P end-labeled PCR product A as
probe.
Figure 2:
Nucleotide and predicted amino acid
sequence of the rat 4-phosphatase cDNA. The numbering of the
nucleotides (leftside) and the amino acids (rightside) begin at the start methionine. Peptide
sequence obtained from purified 4-phosphatase are underlined; solidlines indicate tryptic peptides, and dashedlines indicate CNBr peptides. ATTTA sequences implicated
in mRNA instability are also underlined. The polyadenylation
signal is boxed. There is also a box at amino acid
574 indicating the 11 amino acid deletion found in clone
13.
Bacterial Expression of Inositol Polyphosphate
4-Phosphatase
The 689-bp AvaI-BamHI
fragment of clone 6b and the 2678-bp BamHI-HindIII
fragment of clone 4 (Fig. 1) were subcloned into Bluescript
SK± cut with AvaI and HindIII. The 3379-bp KpnI-HindIII fragment of this construct was then
subcloned into the PQE-32 expression vector (Qiagen). The resulting
construct expressed an amino-terminal six histidine tag and the
4-phosphatase minus amino acid residues 1-13. The recombinant
protein was expressed in XL1-blue strain of E. coli (Stratagene). Bacteria were grown at 37 °C in LB broth
containing 100 µg of ampicillin/ml until an A of 0.6 OD was reached.
Isopropyl-1-thio-
-D-galactopyranoside (1 mM) was
then added, and culture growth was continued for 3 h at room
temperature. Bacteria were harvested by centrifugation at 4000
g for 20 min, and the pellet was resuspended in a buffer
containing 50 mM potassium phosphate, 10 mM Tris, pH
8.0 (buffer A), with 1 µg of lysozyme/ml (2 ml/g, wet weight) and
incubated on ice for 30 min. The suspension was then sonicated on ice
using a probe sonicator at 200 watts for 30-s intervals until the cells
were lysed (approximately 2 min). The lysate was centrifuged at 20,000
g for 20 min. The supernatant was then mixed with
Ni-NTA-agarose (1 ml of 50% slurry/ml of supernatant) for 30 min at 4
°C. The Ni-NTA-agarose was transferred to a 1-cm diameter column
and washed with five column volumes of buffer A. The column was then
washed with four column volumes of buffer A containing 20 mM imidazole, pH 8.0, and four column volumes of buffer A containing
250 mM imidazole, pH 8.0. Fractions containing 4-phosphatase
activity were pooled and concentrated using a centriprep 30 spin
concentrator (Amicon).
Figure 1:
Independent clones isolated from cDNA
libraries. The relationship between the composite 4-phosphatase clone
and the isolated cDNA clones and probes used for screening are shown.
The thinbars indicate the location of the sequence
of each independent cDNA clone: clones 4, 7, 13, and 15 (isolated from
a Stratagene library), clones 2B, 6B, and 10A (isolated from Clontech
library), and human clones 12A and 12C (isolated from a Stratagene
library). A partial restriction map of the composite cDNA is given for ApaI (A), BamHI (B), EcoRI (E), and HindIII (H). The relative location
of PCR products A and B are also indicated. Note that clone 13 is
missing 11 amino acids (at 574) present in other rat and human
clones.
Northern Blot Analysis
PCR product B was P-labeled using the random hexamer procedure described
above. A rat multi-tissue Northern blot was then probed following the
protocol recommended by the manufacturer (Clontech). The blot was
stripped of probe by boiling in 1% SDS for 10 min and reprobed with
P-labeled
-actin control probe.
Production of Rabbit Polyclonal Peptide
Antiserum
The peptide CPPEGTYGKVET corresponding to the
carboxyl-terminal tryptic peptide was synthesized by Washington
University Protein and Nucleic acid Laboratory and coupled to keyhole
limpet hemocyanin with bromoacetic acid N-hydroxysuccinimide
ester(13) . Rabbit polyclonal serum was raised to antigen by
Pocono Rabbit Farms and Laboratory (Cacadensis, PA).
Immunoprecipitation of Inositol Polyphosphate
4-Phosphatase
Rat brain homogenate was prepared by
homogenizing 10 minced unstripped rat brains in a polytron using 20
mM HEPES, pH 7.5, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg of leupeptin/ml
(homogenization buffer). The supernatant was prepared by centrifuging
the homogenate for 30 min at 20,000 g.
Immunoprecipitations were performed by adding 5 µl of a mixture of
preimmune rabbit serum and some quantity of immune serum (0-0.2
µl) to 100 µl of supernatant and incubating at 4 °C for 4
h. Then, 90 µl of a 50% slurry of a protein A-Sepharose CL-4B in
homogenization buffer was added and mixed overnight at 4 °C. The
amount of 4-phosphatase remaining in the supernatant was then assayed
using both a soluble and lipid substrate as previously
described(2) , except that 0.1% Triton X-100 was substituted for
the octylglucoside in the lipid assay buffer.
Sequence and Structural Features of 4-Phosphatase
cDNA
The amino acid sequence was determined for tryptic and
CNBr peptides derived from rat brain inositol polyphosphate
4-phosphatase. A 74-base pair PCR product was obtained using degenerate
sense and antisense oligonucleotides derived from the amino acid
sequence of a CNBr peptide as described under ``Experimental
Procedures.'' This PCR product was labeled and used to screen an
oligo(dT) primed Stratagene Unizap rat brain library. Three overlapping
clones obtained from this screen were sequenced (Fig. 1). A
480-base pair PCR product derived from the 5`-end of clone 4 was then
used to screen both a Stratagene Unizap library and a Clontech random
primed rat brain 5`-stretch library. One additional clone obtained from
the Stratagene library and three clones from the Clontech library were
sequenced. The composite cDNA contained an open reading frame that
predicted a protein of 939 amino acids of 105,588 Da. This corresponds
closely to the molecular mass of 110 kDa determined for the bovine
4-phosphatase by SDS-polyacrylamide gel electrophoresis and gel
filtration chromatography(1) . The open reading frame that
contains an ATG that conforms to the Kozak consensus (14) and an
in-frame stop TGA occurs at nucleotide -39 (Fig. 2). The
predicted amino acid sequence of the open reading frame contains the
sequences of seven tryptic and two CNBr peptides that were obtained
from the native enzyme (Fig. 2). The 3`-UTR contains eight ATTTA
sequences that correspond to the AUUUA motifs shown to be present in
mRNAs that are rapidly degraded(15) . The 3`-UTR terminates in a
poly(A) tail preceded by a AAUAAA polyadenylation signal (Fig. 2). One of the clones isolated (clone 13) lacked 33
nucleotides corresponding to amino acids 574-585 ( Fig. 1and Fig. 2). This may indicate an alternatively
spliced form of 4-phosphatase. The PCR product A was used to isolate a
cDNA encoding human inositol polyphosphate 4-phosphatase from a human
fetal brain cDNA library. Two clones were isolated and sequenced, one
of which (clone 12A) contained the entire open reading frame of human
4-phosphatase (Fig. 1). Remarkably, the human and rat proteins
are 97% identical with most of the amino acid substitutions being
conservative (Fig. 3). The rat 4-phosphatase is one amino acid
longer than the human 4-phosphatase as a result of a serine inserted at
position 489. The nucleotide sequences of the human and rat clones were
90% identical throughout the coding region but only 60% identical in
the 5`-UTR.
Figure 3:
Alignment
of the amino acid sequences of rat and human 4-phosphatases. The amino
acid sequence of rat (top) and human (bottom) were
aligned using the Genetics Computer Group program GAP (23). Identical
amino acids are indicated by verticallines. A doubledot indicates a conservative substitution, and
a singledot indicates a change caused by a single
nucleotide change.
Northern Blot Analysis
PCR product B was
end labeled and hybridized to a multi-tissue Northern blot to determine
the tissue distribution of expression (Fig. 4). The results
indicate that 4-phosphatase is widely expressed in rat tissues, the
relative expression level being brain > heart = skeletal
muscle > spleen = kidney > lung = testis. The size
of the 4-phosphatase mRNA transcript was estimated to be 6.2 kilobases,
indicating that the composite cDNA clone lacks approximately 0.6
kilobases of 5`-UTR. The nature of the hybridizing 12-kilobase
transcript found primarily in brain is unknown.
Figure 4:
Northern blot of rat mRNA isolated from
various tissues. Lanes1-8 are mRNA isolated,
respectively, from heart, brain, spleen, lung, liver, skeletal muscle,
kidney, and testes screened with 4-phosphatase PCR B probe (upperpanel) or -actin control probe (lowerpanel).
Heterologous Expression of
4-Phosphatase
A construct that contains a histidine-tagged
version of 4-phosphatase was expressed in E. coli. This
construct contains 6 histidine residues at the amino terminus and lacks
the first 13 amino acids of the predicted protein. Lysates of bacteria
expressing this construct were found to contain 4-phosphatase activity,
whereas extracts from bacteria-expressing vector only had no detectable
activity (data not shown). The soluble activity was partially purified
on a Ni-NTA affinity column as described under ``Experimen-tal
Procedures'' and was shown to hydrolyze Ins(3,4)P,
Ins(1,3,4)P
, and PtdIns(3,4)P
, the three
substrates of 4-phosphatase (Fig. 5). There was no hydrolysis of
Ins(1,4)P
, an inositol phosphate that is not a substrate of
the 4-phosphatase, by this preparation (data not shown). These
experiments indicate that the recombinant enzyme has the same substrate
specificity as the native enzyme. We estimated the concentration of
recombinant 4-phosphatase in the partially purified extracts by
comparing immunoblots of varying amounts of bacterial extract versus native 4-phosphatase. This estimation indicated that
the recombinant protein was only 16% as active as the native enzyme (4
µmol of Ins(3,4)P
hydrolyzed/min/mg of protein versus 25 µmol of Ins(3,4)P
hydrolyzed/min/mg
of protein). Whether this results from the addition of the histidine
tag to the amino terminus, deletion of the first 13 amino acids, or
inactivation during purification is unknown.
Figure 5:
Time course of the hydrolysis of
4-phosphatase substrates by recombinant enzyme. 0.6 µg () or
1.2 (
) µg of partially purified histidine-tagged recombinant
enzyme was incubated with (A) 10 µM
Ins(3,4)P
or 10 µM (B)
Ins(1,3,4)P
. C, TLC plate of PtdIns(3,4)P
hydrolyzed by 0.6 µg of recombinant
enzyme.
Immunoprecipitation of 4-Phosphatase
A
polyclonal antiserum directed against a carboxyl-terminal peptide of
the 4-phosphatase was capable of immunoprecipitating >95% enzyme
activity present in crude rat brain supernatant (Fig. 6). The
fraction of activity remaining in the supernatant after
immunoprecipitation with increasing amounts of antiserum was identical
for Ins(3,4)P and PtdIns(3,4)P
, indicating that
the same enzyme was responsible for catalyzing the hydrolysis of both
substrates (Fig. 6). In addition, approximately 95% of the
activity could be removed from supernatants of human platelets and rat
heart, skeletal muscle, and spleen using this antiserum, indicating
that this enzyme is the primary 4-phosphatase activity found these
tissues (data not shown).
Figure 6:
Immunoprecipitation of 4-phosphatase
activity from crude rat brain supernatant. The activity of
4-phosphatase remaining in the supernatant after immunoprecipitation
with increasing amounts of rabbit polyclonal antiserum directed against
a COOH-terminal peptide was determined for the substrate
Ins(3,4)P (
) or PtdIns(3,4)P
(
).
(12) . However, the 4-phosphatase is the
first Mg
-independent inositol phosphate phosphatase
to be cloned, and sequence similarity with other members of this
category such as the inositol polyphosphate 3-phosphatase (18) and polyphosphoinositide phosphatase (19) may yet be
observed.
is also a
substrate for this enzyme (11) suggests that it may have a role
in controlling the levels of this potential messenger. Expression of
the 4-phosphatase in eukaryotic cells will allow us to study the role
of the enzyme in metabolizing PtdIns(3,4)P
and may prove to
be a powerful tool for studying the signaling function of
PtdIns(3,4)P
in vivo.
/EMBL Data Bank with accession
number(s) U26397,
U26398.
, inositol
3,4-bisphosphate; Ins(1,4)P
, inositol 1,4-bisphosphate;
Ins(1,3,4)P
, inositol 1,3,4-trisphosphate; PtdIns(3)P,
phosphatidylinositol 3-phosphate; PtdIns(3,4)P
,
phosphatidylinositol 3,4-bisphosphate; PtdIns(3,4,5)P
,
phosphatidylinositol 3,4,5-trisphosphate; PCR, polymerase chain
reaction; UTR, untranslated region; bp, base pair(s).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.