From the
We have isolated additional cDNA clones encoding type II
inositol polyphosphate 5-phosphatase (5-phosphatase II) resulting in a
combined cDNA of 3076 nucleotides encoding a protein of 942 amino
acids. The 5-phosphatase II hydrolyzed both
Ins
(1, 4, 5) P
The phosphatidylinositol signaling pathway influences numerous
cellular responses including calcium ion mobilization, protein
phosphorylation, and cell proliferation in response to various
extracellular agonists
(1, 2) . In one of the reactions
of this system, phospholipase C acts on phosphatidylinositol
(4, 5) -bisphosphate
(PtdIns
(4, 5) P
Hansen et al. (17) first
identified two types of soluble inositol phosphate 5-phosphatases from
rat brain. Since that time, several forms of inositol polyphosphate
5-phosphatase have been identified in a variety of tissues, and these
can be grouped according to molecular mass and affinity for
Ins
(1, 4, 5) P
There is also
5-phosphatase activity that hydrolyzes
PtdIns
(4, 5) P
The
PtdIns
(4, 5) P
Expression
vectors beginning at methionine 250 were constructed by PCR between a
sense oligonucleotide containing EcoRV, NheI, and
NcoI restriction sites and the 5-phosphatase II nucleotides
749-759 and an antisense 5-phosphatase II oligonucleotide for
nucleotides 1717-1733. This PCR product was attached to the
5-phosphatase II 3` cDNA at a SphI site at nucleotide 1156,
and the PCR portion was sequenced to confirm that the plasmid was
without mutations. Expression vectors beginning at this methionine are
designated 5-PtaseS and encode 5-phosphatase II amino acids
250-942.
For studies comparing the native and C939S mutant
5-phosphatase II enzymes, the 3` untranslated region of the cDNA was
removed by PCR between a sense oligonucleotide for 5-phosphatase II
nucleotides 2175-2191 and an antisense oligonucleotide containing
nucleotides 2810-2830 followed by an XbaI restriction
site. The mutation of cysteine 939 to serine was accomplished by
changing a single nucleotide 2817 to guanosine within the antisense PCR
oligonucleotide. These 3` PCR portions of 5-phosphatase II were
attached to the 5` portion of the clone at a NheI site at
nucleotide 2714. The native and C939S PCR portions were confirmed to be
without mutations by sequencing.
For studies on
For
[
As a second method of testing
association, Sf9 cells infected with baculovirus encoding 5PtaseS,
5PtaseL, or control MEG-01 and labeled with
[
To determine whether the presence of the C XXX motif in 5-phosphatase II results in isoprenylation of the
protein, we carried out labeling studies using
[
The 5-phosphatase type II was purified and cloned as an
enzyme that cleaves the phosphate from the 5-position of soluble
Ins
(1, 4, 5) P
The 5-phosphatase
II ends with a consensus sequence -CNPL for isoprenylation, and the
enzyme is isoprenylated on cysteine 939 based on its incorporation of
[
The
5-phosphatase II is the first enzyme in the inositol phosphate pathway
directly shown to be isoprenylated, and it is interesting to speculate
on the possible effect this modification has on the function of the
5-phosphatase. One role for isoprenylation would be to promote the
membrane association of 5-phosphatase II. Our experiments demonstrate
that at least part of the recombinant 5-phosphatase II from COS-7 cells
fractionates to the cell pellet, and, in platelets, 45% of the
PtdIns
(4, 5) P
Another possibility
is that the isoprenylation of 5-phosphatase II serves to orient or
stabilize the association of 5-phosphatase II with some other protein
on membranes. A precedent for such a function is the report that
isoprenylated Rho activates membrane phosphatidylinositol
(4) P
5-kinase, whereas nonisoprenylated Rho has no effect
(38) .
Nonisoprenylated rhodopsin kinase is 4-fold less active toward Rho in
an in vitro assay than the isoprenylated kinase
(39) .
Similarly, farnesylation and carboxylmethylation of the
Of the other proteins homologous to 5-phosphatase II,
only the 43-kDa 5-phosphatase I has a consensus sequence for
isoprenylation
(12, 13) . Isoprenylation could account
for its membrane association
(10, 12) . Another possible
5-phosphatase from mammalian sources is the gene disrupted in
Lowe's oculocerebrorenal syndrome
(33) . The predicted
amino acid sequence for this protein does not end with an
isoprenylation consensus sequence and so it may be cytosolic.
Two
other inositol polyphosphate phosphatases share the ability of the
5-phosphatase II to hydrolyze a soluble and corresponding lipid
substrate. The 3-phosphatase that degrades
Ins
(1, 3) P
Regulation of
the levels of cellular PtdIns
(4, 5) P
We thank Theodora S. Ross for assistance in the
cloning of 5-phosphatase II. We thank Richard Cerione, Patrick Casey,
and F. Anderson Norris for helpful discussions; Cecil Buchanan for
technical assistance; and J. Evan Sadler, Monita Wilson, and John D.
York for critical review.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
to
Ins
(1, 4) P
and the phospholipid
PtdIns
(4, 5) P
to PtdIns
(4) P both
in vitro and in vivo. There are two motifs highly
conserved between types I and II 5-phosphatase and several other
proteins presumed to be inositol phosphatases suggesting a possible
role in catalysis. The type II 5-phosphatase also contains homology to
several GTPase activating proteins although no such activity for
5-phosphatase II was found. The predicted protein ends with the
sequence CNPL, suggesting that it is isoprenylated as a mechanism for
membrane attachment. We found evidence for isoprenylation by
demonstrating incorporation of [
H]mevalonate into
native but not C939S mutant 5-phosphatase II expressed in Sf9 insect
cells. Furthermore, we showed that membrane localization and the
activity of 5-phosphatase II toward its lipid substrate
PtdIns
(4, 5) P
is reduced by eliminating
5-phosphatase II isoprenylation in the mutant C939S relative to the
native enzyme.
)
(
)
to
generate Ins
(1, 4, 5) P
. This
metabolite is further converted to
Ins
(1, 3, 4, 5) P
by the
action of a 3-kinase. These two soluble inositol phosphates are
involved in calcium ion mobilization. Specific 5-phosphatase enzymes
terminate calcium signaling by hydrolyzing
Ins
(1, 4, 5) P
and
Ins
(1, 3, 4, 5) P
to
Ins
(1, 4) P
and
Ins
(1, 3, 4) P
, respectively
(1, 2) .
and
Ins
(1, 3, 4, 5) P
. The first
group of 5-phosphatases of approximately 32-43 kDa have high
affinity for both inositol phosphate substrates. This type of enzyme
was first isolated from platelet cytosol and was named type I based on
its position of elution from an anion exchange resin
(3, 4, 5) . Enzymes with similar characteristics
have been recognized and cloned from a variety of tissue sources
(6, 7, 8, 9, 10, 11, 12, 13) .
Immunoprecipitation of 5-phosphatase activity from platelets with
antisera to the bovine brain and human placental isozymes suggest that
these type I activities may be the same enzyme
(10, 14) . A second group of 5-phosphatases have
molecular masses of 69-75 kDa and lower affinities for
Ins
(1, 4, 5) P
and
Ins
(1, 3, 4, 5) P
. Type II
5-phosphatase was originally isolated from human platelet cytosol
(15) . Enzymes of similar size and substrate affinity have been
observed in rat liver cytosol
(6) and human erythrocyte
membranes
(9) . A partial cDNA encoding platelet 5-phosphatase
II was obtained by screening pooled bacterial lysates from a
gt11
cDNA library for ability to bind
[
H]Ins
(1, 3, 4, 5) P
(16) . Finally, 5-phosphatases have been reported of 115
kDa from bovine brain cytosol
(7) and 160 kDa from rat brain
cytosol
(17) . These larger forms of 5-phosphatase act primarily
on Ins
(1, 4, 5) P
.
to PtdIns
(4) P.
PtdIns
(4, 5) P
5-phosphatase activity has
been identified in several tissues including chick, rat, and bovine
brain
(18, 19, 20) , rat kidney
(21) ,
human erythrocytes
(22) , and human platelets
(23) . In
most of these tissues, it is both cytosolic and membrane-associated,
and in cases in which the enzyme has been most highly purified, it
appears to be capable of hydrolyzing the 5-phosphate from both
PtdIns
(4, 5) P
and the soluble
Ins
(1, 4, 5) P
(18, 20, 23) .
5-phosphatase in platelets
has been identified as 5-phosphatase II
(23) . Polyclonal
antibodies raised against recombinant 75-kDa
Ins
(1, 4, 5) P
5-phosphatase II
deplete PtdIns
(4, 5) P
5-phosphatase
activity from the cytosolic and membrane fractions of platelets
implying that this protein catalyzes both reactions. In this report, we
have isolated additional cDNA clones encoding 5-phosphatase II and
report corrections in the previously published sequence. We find that
5-phosphatase II has 2 regions of sequence conserved with 5-phosphatase
I and several other proteins. We also find that 5-phosphatase II is
isoprenylated on the carboxyl-terminal sequence CNPL, and this
modification affects its ability to hydrolyze
PtdIns
(4, 5) P
.
Materials
[H]Mevalonolactone
was purchased from American Radiolabeled Chemicals, Inc.
[
P]PO
, horseradish peroxidase-linked
anti-rabbit IgG, and ECL Western blotting detection reagents were
purchased from Amersham Life Sciences.
PtdIns
(4, 5) P
was purchased from Boehringer
Mannheim, and Silica Gel 60 TLC plates (20
20 cm, 0.2 mm) were
from Merck. pVL1393 baculoviral expression vector and BaculoGold
transfection kit were from PharMingen. pCB6 mammalian expression vector
was from Jeffrey Milbrandt (Washington University, St. Louis).
Compactin was from Sandra Hofmann (University of Texas Southwestern
Medical Center), and mevinolin was from Alfred Alberts (Merck Research
Laboratories). Purified CDC42Hs and GST-CDC42Gap and bacteria
expressing GST-C-Ha-Ras, GST-Rac1, and GST-RhoA were from Richard
Cerione (Cornell University). Purified GST-Rab5b was from David Wilson
(Washington University, St. Louis). Most other materials were from
Sigma.
Cloning of Additional 5-Phosphatase II Sequence
A
2384-base pair clone originally isolated by Ross et al. (16) was used to obtain an additional 5` sequence of
5-phosphatase II. A human erythroleukemia cell gt11 cDNA library
(Rodger McEver, University of Oklahoma) was subdivided and amplified as
described
(16) . The 5` end of 5-phosphatase II was amplified by
PCR between an antisense oligonucleotide directed to the 5` sequence of
this clone and a primer located 16-37 base pairs 5` of the
EcoRI site of
gt11. The amplified PCR product was found
to contain an additional 656 nucleotides of 5` sequence. The original
clone was labeled by random hexamer priming and used to screen a human
fetal brain Lambda Zap II cDNA library (Stratagene). A single clone was
obtained that contained all of the original plus an additional 330
nucleotides as confirmed by partial sequencing and restriction
digestion. We used a 5`-Amplifinder RACE kit (CLONTECH) to obtain the
most 5` 39 nucleotides with an antisense 5-phosphatase II
oligonucleotide as primer to synthesize single-stranded cDNA from
placental poly(A)
RNA (CLONTECH). DNA was sequenced on
both strands by the Sanger dideoxy sequencing method (Sequenase; U. S.
Biochemical Corp.). The sequence corrected in this publication was
sequenced from at least two cDNA clones, and the 3` region was also
sequenced from a genomic clone.
Expression Vector Construction and
Mutagenesis
pBlueSKII/5ptaseL was constructed
by combining the two 5-phosphatase II cDNAs described above at a
BamHI site at 5-phosphatase II nucleotide 499. This combined
5-phosphatase II cDNA contains a sequence encoding 5-phosphatase II
amino acids 14-942 and was used to construct the pCB6 mammalian
expression vectors and, subsequently, the pVL1393 transfer vector for
expression of 5-phosphatase II in Sf9 insect cells.
Baculovirus Expression and Labeling
Recombinant
baculoviruses were constructed according to the manufacturers'
instructions using the shuttle vector pVL1393 (PharMingen) containing
either 5PtaseS, 5PtaseL, or 5PtaseS/C939S or a control construct
(protein tyrosine phosphatase MEG-01)
(24) . Sf9 insect cells
were grown in TNM-FH medium with 10% heat-inactivated fetal calf serum
and 100 µg of gentamicin/ml
(25) .
P labeling of Sf9 cell phospholipids, approximately 3
10
Sf9 insect cells were infected with baculovirus
encoding either control MEG-01
(24) or 5PtaseS. Three days
after infection, the cells were harvested from 60-mm dishes and
resuspended in 450 µl of phosphate-free Grace's insect media
containing 1 mCi of [
P]PO
at room
temperature. Samples of 100 µl were taken after various times and
added to 100 µl of 1
N HCl.
P-Labeled
phosphatidylinositols were further extracted with chloroform/methanol,
separated by TLC, and measured as described
(26) .
H]mevalonate labeling of 5-phosphatase II,
approximately 3
10
Sf9 cells infected with
baculovirus encoding either 5PtaseS or 5PtaseL were grown for 48 h in
60-mm dishes and then incubated with fresh media containing 100
µ
M compactin for 4 h.
[
H]Mevalonolactone (0.75 mCi) was added (1.4 ml
of media/dish) for an additional 18 h. Cells were pelleted and lysed by
sonication in 200 µl of buffer containing 50 m
M Mes/Tris,
pH 6.5, 3 m
M MgCl
, 2 m
M EGTA, 10
m
M 2-mercaptoethanol, 1 m
M phenylmethylsulfonyl
fluoride, 10 µg of leupeptin/ml, 10 µg of benzamidine/ml, and 1
µ
M pepstatin A. Supernatant and particulate fractions were
obtained by centrifugation for 10 min at 16,000
g at 4
°C. These fractions were resolved by SDS-PAGE and transferred to
nitrocellulose. Labeling with [
H]mevalonate was
detected by soaking the nitrocellulose in 1
M sodium
salicylate for 30 min followed by fluorography for 17 days. The
nitrocellulose was then washed and immunoblotted to identify
5-phosphatase II protein. For comparison of
[
H]mevalonate labeling of native and C939S mutant
5-phosphatase II, Sf9 cells were infected with baculovirus encoding
either 5PtaseS or 5PtaseS/C939S. They were labeled essentially as
described above except that the [
H]mevalonate
media was reused from the experiment above and the fluorography was for
20 days.
Membrane Localization of 5-Phosphatase II
COS-7
cells were maintained in Dulbecco's modified Eagle's
medium, 10% fetal calf serum, 10 units of penicillin/ml, and 10 µg
of streptomycin/ml. These cells (80-85% confluent on 60-mm
dishes) were transiently transfected using 40 µg of Lipofectin
(Life Technologies, Inc.) and 10 µg of pCB6/5PtaseL or
pCB6/5PtaseL/C939S. After 3 days, cells were incubated in fresh media
containing no addition, 100 µ
M mevinolin, 100
µ
M mevinolin, and 10 m
M mevalonic acid, or 10
m
M mevalonic acid. After 6 h of drug treatment, cells were
harvested in phosphate-buffered saline and sonicated in 150 µl of
buffer A containing 10 m
M Tris, pH 7.4, 140 m
M KCl,
1.5 m
M MgCl, 1 m
M phenylmethylsulfonyl
fluoride, 10 µg of benzamidine/ml, 1 µ
M pepstatin A,
10 µg of aprotinin/ml, and 10 µg of leupeptin/ml. Soluble and
particulate fractions were prepared by centrifugation at 16,000
g at 4 °C for 10 min. The pellets were extracted in the
same buffer with 1% Triton X-100 for 30 min with shaking, and a
detergent supernatant and pellet fraction were prepared by
centrifugation again at 16,000
g at 4 °C for 10
min. Half of each of these fractions was resolved by SDS-PAGE on 8%
polyacrylamide gels and immunoblotted for 5-phosphatase II.
Enzyme Assays and Western Blotting
Preparation of
[P]Ins
(1, 4, 5) P
and assay of 5-phosphatase activity using this substrate were as
described
(3) . Assay of 5-phosphatase activity using
[
H]PtdIns
(4, 5) P
was
as described
(23) except that
PtdIns
(4, 5) P
was at a concentration of 50
µ
M and the assay was conducted at room temperature. Under
these conditions, the assay was linear for 1 min and about 70% of
linear after 2 min. TLC was developed as described
(26) . The
enzyme concentration used in PtdIns
(4, 5) P
assays was determined by measuring the hydrolysis of
Ins
(1, 4, 5) P
(assuming that
5-phosphatase II hydrolyzes 2.5 µmol of
Ins
(1, 4, 5) P
/min/mg of protein).
For experiments testing the effect of small GTP-binding proteins on
5-phosphatase II activity, the enzyme assays described above were done
with the addition of GST-Ras, GST-Rac, or GST-Rho (at at least 1:1
G-protein:5-phosphatase) in the GDP- or GTP
S-bound state. All
immunoblotting was done with affinity-purified antibody to
5-phosphatase II (2 ng/ml) raised against a recombinant antigen
containing amino acids 233-428
(16) . Secondary antibody
was horseradish peroxidase-conjugated anti-rabbit IgG from Amersham
(1:5000 dilution), and blots were developed using ECL (Amersham).
GAP Assays and Association of 5-Phosphatase II with
GTP-binding Proteins
GAP assays were performed as described
(27) with CDC42Hs, GST-c-Ha-Ras, GST-Rac1, GST-RhoA, or
GST-Rab5b as the GTP-binding protein and Sf9 supernatants expressing
5PtaseS, 5PtaseL, or control MEG-01 or purified control CDC42GAP as the
GTPase activating protein. 5-Phosphatase II was tested for association
with GST-c-Ha-Ras, GST-Rac1, or GST-RhoA on glutathione-agarose beads
exactly as described
(28) .
S]methionine and
[
S]cysteine (0.2 mCi/ml) for 20 h were sonicated
in buffer A. GST-c-Ha-Ras, GST-Rac1, GST-RhoA or CDC42 (1 µg each)
or whole platelets (50 µg) were resolved by SDS-PAGE on a 12.5%
gel, transferred to nitrocellulose, and blocked in Tris-buffered saline
with 3 g/100 ml of milk, 0.1% Tween 20, and 100 µg of GTP
S/ml.
A supernatant (centrifugation at 16,000
g for 10 min)
containing
S-labeled proteins was used to overlay the
nitrocellulose (2 h at 4 °C), and binding was determined by
autoradiography.
cDNA Cloning and Expression
The previously
published cDNA sequence for 5-phosphatase II consisted of 2382
nucleotides and encoded amino-terminal amino acid sequence obtained
from the protein purified from platelets. However, this cDNA sequence
had an open reading frame upstream of the putative amino terminus, and
a full length transcript appears to be about 4400 nucleotides based on
Northern blot analysis. We therefore isolated additional cDNA clones to
further define the structure of 5-phosphatase II. We have obtained
clones containing 695 nucleotides 5` of the previously published
sequence and that predict an additional 232 amino acids (Fig. 1).
We have been unable to obtain cDNA clones with additional 5` sequences
despite screening several million clones from HEL cell, human
cerebellar, human fetal brain, human placenta, and MEG01 cDNA libraries
and attempting 5`-rapid amplification of cDNA end PCR on several
occasions. We also re-examined the previously published sequence and
found several sequencing errors. These corrections altered the reading
frame in two regions identified by underlined sequences in
Fig. 1
. The nucleotide sequence of 5-phosphatase II predicts an
open reading frame encoding 942 amino acids. Translation of the protein
may begin at methionine 30 which has a surrounding nucleotide consensus
sequence for initiator methionines
(29) . However, it is
possible that translation starts further 5` since there is no in-frame
upstream stop codon in the cDNA, and the full length transcript is
predicted to be 4.4 kilobases from Northern blot analysis
(16) .
The amino acid sequence obtained as an amino-terminal sequence of the
purified protein from platelets begins at amino acid 270 (Fig. 1,
arrow) and is preceded by a basic amino acid suggesting that
the purified protein may be proteolyzed from a larger precursor.
Figure 1:
Nucleotide and encoded amino acid
sequence of the human inositol polyphosphate 5-phosphatase type II
cDNA. Nucleotides are numbered on the left; amino
acids are numbered on the right. The start of amino
acid sequence obtained as the amino-terminal sequence of the purified
protein from platelets is indicated by an arrow. Amino acid
sequence altered by correction of nucleotide sequencing errors is
underlined. Consensus amino acid sequence for isoprenylation
is boxed.
The
PtdIns
(4, 5) P5-phosphatase activity in
homogenates of platelets is depleted by an antibody to recombinant
5-phosphatase II
(23) suggesting that the same enzyme
hydrolyzes both the soluble and lipid substrate. We expressed 5PtaseS
in Sf9 cells and showed directly that it is able to hydrolyze
PtdIns
(4, 5) P
to PtdIns
(4) P in
vitro (Fig. 2). The kinetics of this hydrolysis are complex.
Note that Sf9 supernatant (0.4 µg) hydrolyzed 6.3% of added
substrate (50 µ
M) in 2 min while Sf9 supernatant (1.3
µg) hydrolyzed 21.5% of added substrate. The fact that the reaction
plateaus after 2 min when there is still substrate present suggests
that much of the PtdIns
(4, 5) P
is not
available for hydrolysis by 5-phosphatase II. One explanation for this
finding is that the 5-phosphatase II binds strongly to
PtdIns
(4, 5) P
/detergent micelles
restricting substrate availability. A similar phenomenon has been found
for the inositol polyphosphate 4-phosphatase hydrolysis of
PtdIns
(3, 4) P
(26) . Consistent with
this hypothesis, we found that mixing 5-phosphatase II with unlabeled
PtdIns
(4, 5) P
or PtdIns
(4) P
containing detergent micelles prior to addition of radiolabeled
substrate eliminated subsequent hydrolysis of
H-labeled
PtdIns
(4, 5) P
. Since the extent of
substrate hydrolysis, but not the rate, is proportional to enzyme
concentration over a wide range, it is difficult to compare the ability
of 5-phosphatase II to hydrolyze the lipid- versus water-soluble substrate. However, it is clear that the lipid is a
very good substrate. Thus, using the results at 2 min, 1.3 µg of
Sf9 supernatant hydrolyzed 0.54 nmol of
PtdIns
(4, 5) P
or 0.2 µmol/min/mg of
protein compared to an activity of 0.05 µmol of
Ins
(1, 4, 5) P
hydrolyzed/min/mg of
protein.
Figure 2:
Hydrolysis of PtdIns(4,5)P by
5-phosphatase II. PtdIns(4,5)P
( PIP
)
hydrolyzed by 0.4 µg (
) or 1.3 µg (
) of supernatant
from Sf9 cells expressing 5-phosphatase II is plotted as a function of
time. Results shown are typical of three
experiments.
We also determined that 5-phosphatase II hydrolyzes
PtdIns
(4, 5) P in vivo. We used Sf9
cells infected for 3 days with baculovirus encoding 5PtaseS or encoding
an irrelevant protein MEG-01. Cells were labeled with
PO
for 1-30 min and incorporation of
isotope into inositol lipids was determined as described under
``Experimental Procedures.'' Under these non-steady state
conditions, incorporation of
PO
reflects
turnover of inositol lipids. Cells infected with MEG-01 baculovirus had
more
P incorporated into PtdInsP
than into
PtdInsP at all time points (Fig. 3) as did uninfected Sf9 cells
(data not shown). Thus, Sf9 cells continue to maintain cellular
inositol phospholipids despite infection with baculovirus. In contrast,
cells expressing 5-phosphatase II had less radioactivity incorporated
into PtdInsP
than into PtdInsP due to hydrolysis of the
5-phosphate group with the consequent increase in levels of PtdInsP.
Expression of 5-phosphatase II did not affect the incorporation of
P into total cellular phospholipid (61 cpm/nmol of
cellular phospholipid/10 min versus 66 cpm/nmol of cellular
phospholipid/10 min for 5-phosphatase II) indicating that the rate of
labeling is not affected by 5-phosphatase II expression. This result
shows that the recombinant 5-phosphatase II is able to function in
vivo as a PtdIns
(4, 5) P
5-phosphatase,
and that overexpression of the enzyme causes a measurable change in
cellular inositol phospholipid levels.
Figure 3:
Incorporation of
PO
into inositol lipids in Sf9 cells. Sf9
cells infected with baculovirus encoding 5-phosphatase II or control
MEG-01 tyrosine phosphatase were labeled with
PO
for the indicated times. Radioactivity incorporated into
PtdInsP
or PtdInsP was determined after separation on TLC.
The inset shows the ratio of radioactivity in PtdInsP
divided by that in PtdInsP in the infected cells. Results shown
are typical of five experiments.
Homology Motifs
The two known 5-phosphatase
enzymes I and II have no amino acid similarity (20.9% identity in 115
amino acids) except in two regions corresponding to 5-phosphatase II
amino acids 472-483 and 545-570 (Fig. 4). The high
degree of similarity in these regions suggests these regions may be
involved in a function common to both of these proteins such as
substrate binding or catalysis. Several other proteins show much
greater overall similarity to 5-phosphatase II including the protein
encoded by the gene mutated in Lowe's oculocerebrorenal syndrome
(51.1% identity in 744 amino acids) and predicted proteins from
Caenorhabditis elegans (25.5% identity in 255 amino acids),
Arabadopsis thaliana (46% identity in 121 amino acids), and
Saccharomyces cerevisiae (32.9% identity in 374 amino acids).
The two motifs mentioned above are also conserved in these proteins,
further suggesting similarity in function among these proteins
(Fig. 4).
Figure 4:
Two regions of amino acid similarity
between human inositol polyphosphate 5-phosphatase type II and the
amino acid sequence of related predicted proteins. The sequences
compared were: C. elegans, predicted amino acid sequence of a
protein encoded by C. elegans cosmid (GenBank accession number
L14433); 5Ptase II, human 5-phosphatase II; 5Ptase I,
human and dog 43-kDa inositol polyphosphate 5-phosphatase type I;
OCRL, the protein encoded by the gene mutated in Lowe's
oculocerebrorenal syndrome; A. thaliana, predicted amino acid
sequence of a protein encoded by A. thaliana cDNA (GenBank
accession number T04155); S. cerevisiae, predicted amino acid
sequence of a protein encoded by S. cerevisiae cosmid (GenBank
accession number Z38062). Residues showing conservation among at least
four proteins are indicated as Consensus sequence. Amino acid
sequence number is indicated in parentheses. Alignment was
performed using the program ALIGN (59).
An additional region of 5-phosphatase II amino acid
sequence is similar to several GTPase-activating proteins
(Fig. 5). This sequence similarity was noted previously, but the
corrected sequence for the 5-phosphatase II extends the region that can
be aligned to the GAP proteins
(30) . We have tested recombinant
5-phosphatase II for GTPase activating activity toward CDC42, Rac, Rho,
Ras, and Rab5b and find none (data not shown). We also find no evidence
for 5-phosphatase II binding to CDC42, GST-c-Ha-Ras, GST-Rac1, or
GST-RhoA and found no effect of these GTP-binding proteins on
5-phosphatase II hydrolyzing activity as described under
``Experimental Procedures'' (data not shown). While these
results provide no evidence for association of 5-phosphatase II with
any GTP-binding protein, the apparent homology with binding domains of
known GTPase activating proteins suggests that such an association may
yet exist.
Figure 5:
Amino acid similarity between human
5-phosphatase II and presumptive GAP domains of other proteins. The
sequences compared were: 5Ptase II, human 5-phosphatase II;
human -chimerin (RACGAP); human CDC42GAP; PI3K 85 kDa,
bovine 85-kDa
-subunit of phosphatidylinositol 3-kinase. Amino
acid sequence numbers are indicated. Boxed amino acids are
identical in at least two of the four aligned sequences. Percentage of
identical amino acids among the sequences is indicated. Alignment was
performed using the program ALIGN (59).
Protein Isoprenylation
The 5-phosphatase II
sequence ends with CNPL. This sequence fits the C XXX consensus
for modification of proteins by the addition of an isoprene group where
C is cysteine and X is any amino acid. The terminal amino acid
of the 5-phosphatase II is a lysine, further suggesting that the most
likely modification is addition of a geranylgeranyl group
(35, 36) . There are three lysines near the possible
isoprenylation signal which may promote membrane association
(31) .
H]mevalonate. Mevalonate incorporation into
cellular proteins is considered to reflect isoprenylation of these
proteins with either a farnesyl or a geranylgeranyl group
(32) .
We infected Sf9 cells with baculovirus encoding 5PtaseS which yields an
80-kDa protein and 5PtaseL which results in a prominent doublet of
100-103 kDa and a minor proteolysis product of 80 kDa. These two
5-phosphatase II species have identical enzyme activity toward soluble
and lipid substrates (data not shown). Infected cells were labeled with
[
H]mevalonate as described under
``Experimental Procedures,'' and the proteins from lysates of
the cells were separated by SDS-PAGE and transferred to nitrocellulose
membranes prior to autoradiography. [
H]Mevalonate
was incorporated into proteins of the predicted sizes
(Fig. 6 A). The
H-labeled proteins were
identified as 5-phosphatase II by Western blotting with an antibody to
5-phosphatase II (Fig. 6 A). There was insufficient isotope
incorporation to further characterize the isoprenyl group. We
constructed a 5-phosphatase II cDNA in which cysteine 939 is mutated to
serine. This eliminates the site for attachment of an isoprene moiety.
This mutant 5-phosphatase II has the same
Ins
(1, 4, 5) P
hydrolyzing activity
as the native 5-phosphatase II (data not shown). Sf9 cells expressing
this C939S mutant 5-phosphatase did not incorporate
[
H]mevalonate into 5-phosphatase II even though
equivalent amounts of recombinant protein were expressed (Fig.
6 B).
Figure 6:
[H]Mevalonate
labeling and Western blotting of 5-phosphatase II. A,
membranes from Sf9 cells infected with baculovirus encoding an 80-kDa
( a) or 103-kDa ( b) 5-phosphatase II and labeled with
[
H]mevalonate were subjected to SDS-PAGE and
transferred to nitrocellulose. The nitrocellulose was soaked in sodium
salicylate and autoradiographed for 17 days to determine
[
H] mevalonate incorporation. The
H-labeled protein was identified on the identical piece of
nitrocellulose by immunoblotting with antibody to 5-phosphatase II.
B, Sf9 cells infected with baculovirus encoding an 80-kDa wild
type 5-phosphatase II ( c) or the C939S 5-phosphatase II mutant
( d) were assayed for [
H]mevalonate
incorporation and Western blotting as described in
A.
We next determined whether isoprenylation affects the
cellular localization of the 5-phosphatase II by examining the
subcellular distribution of recombinant enzyme in COS-7 cells
expressing 5-phosphatase II or the 5-phosphatase II C939S mutant. COS-7
cells express the longer 5-phosphatase at levels similar to those
previously reported for the shorter 5-phosphatase (5ptaseL supernatant,
6.5 nmol/min/mg, versus control supernatant, 3.8 nmol/min/mg)
(16) . In COS-7 cells expressing 5-phosphatase II, there was
approximately equal distribution of recombinant 5-phosphatase II in
supernatant and pellet fractions (Fig. 7). Extraction with Triton
X-100 did not solubilize the bulk of membrane-associated 5-phosphatase
II. The C939S mutant 5-phosphatase II was not isoprenylated and was
found mostly in the supernatant fraction. Likewise, inhibition of
cellular isoprenylation with mevinolin resulted in a shift of the
native 5-phosphatase II to the supernatant. Addition of mevalonate to
the media during mevinolin treatment overcame the mevinolin inhibition
of isoprenylation and restored the equal distribution of 5-phosphatase
II between the supernatant and pellet fractions (data not shown).
Mevalonate alone had no effect on the distribution of 5-phosphatase II,
and neither mevinolin nor mevalonate affected the localization of the
C939S mutant 5-phosphatase II in the supernatant fraction. Results
similar to those shown in Fig. 7were seen in three experiments.
However, in some additional experiments, nearly all of the expressed
recombinant protein was found in the pellet fraction in both native and
C939S transfected cells, and, in those experiments, mevinolin treatment
did not alter the distribution of enzyme. It is possible that the large
amount of recombinant protein expressed transiently in COS-7 cells is
denatured in some cases precluding the expression of soluble enzyme.
Figure 7:
Subcellular fractionation of 5-phosphatase
II. COS-7 cells expressing 5-phosphatase II or the 5-phosphatase II
C939S mutant or COS-7 cells expressing 5-phosphatase II and treated for
6 h with 100 µ
M mevinolin were separated into supernatant
( s), detergent-extracted supernatant ( ds), and pellet
( p) fractions. Equivalent proportions of each fraction were
resolved by SDS-PAGE and transferred to nitrocellulose. The
5-phosphatase II was visualized by immunoblotting with antibody to
5-phosphatase II.
We next asked whether isoprenylation of 5-phosphatase II has a
functional effect on its ability to act on its soluble or lipid
substrates. We prepared native and C939S mutant 5-phosphatase II in Sf9
cells and assayed for activity using
Ins
(1, 4, 5) Pand
PtdIns
(4, 5) P
. There are inherent
difficulties in comparing the activity of an enzyme for a soluble
substrate with the activity for PtdInsP
in a
two-dimensional environment, and, while the extent of hydrolysis of
PtdInsP
is proportional to protein concentration, this
assay approaches linearity only at very short times (Fig. 2).
Thus, the hydrolysis of PtdIns
(4, 5) P
can
be expressed only as apparent specific activity with a defined length
of assay and protein concentration. Therefore, we compared the ability
of wild type and C939S mutant 5-phosphatase II to hydrolyze
PtdIns
(4, 5) P
both as a function of time
with constant amounts of Sf9 supernatant protein (,
experiments 1 and 2) and at a fixed point with varying amounts of Sf9
supernatant protein (, experiment 3). With both of these
assay conditions, the C939S mutant 5-phosphatase II showed a decreased
ability to hydrolyze PtdIns
(4, 5) P2 when compared to
the wild type enzyme. The C939S mutant 5-phosphatase II had a
29-40% decreased 5-phosphatase activity toward
PtdIns
(4, 5) P
relative to
Ins
(1, 4, 5) P
(). The
native and C939S mutant 5-phosphatase II had the same activity using
Ins
(1, 4, 5) P
as indicated by assay
of equivalent amounts of protein as determined by Western blotting
(data not shown).
. However, an antibody
to the recombinant protein depletes PtdIns
(4, 5) P
5-phosphatase activity in platelet cytosolic and membrane
fractions
(23) . We now report that recombinant 5-phosphatase II
hydrolyzes PtdIns
(4, 5) P
both in vitro and in intact Sf9 cells. Whether 5-phosphatase II is the major
enzyme that controls cellular PtdIns
(4, 5) P
levels is unknown. Larger proteins have been identified in other
tissues as PtdIns
(4, 5) P
5-phosphatases.
Palmer et al. (20) have purified two immunologically
related PtdIns
(4, 5) P
5-phosphatases of 155
and 115 kDa from bovine brain cytosol, and Roach and Palmer
(22) identified a 105-kDa PtdIns
(4, 5) P
5-phosphatase in human erythrocyte cytosol. With the additional
nucleotide sequence we report for the 5-phosphatase II in this paper,
the open reading frame of 5-phosphatase II is sufficient to encode a
protein of 107 kDa. It is possible that the full length cDNA of this
protein encodes the larger forms of the 5-phosphatases from other
tissues and that there is tissue-dependent proteolytic processing or
artifactual proteolysis to account for the different sized proteins
that have been identified. Of note we have expressed recombinant
constructs of 80 kDa and 103 kDa and find that both are equally active.
Alternatively, it is possible that the other forms of 5-phosphatase
represent distinct isozymes. It is clear from the comparison of
5-phosphatase II to sequences in GenBank that there is a family of
proteins that share at least two common motifs. One of these proteins,
the 43-kDa 5-phosphatase I, hydrolyzes
Ins
(1, 4, 5) P
but not
PtdIns
(4, 5) P
despite its membrane
localization
(10, 23) . The protein encoded by the gene
mutated in Lowe's oculocerebrorenal syndrome (OCRL) shares these
conserved motifs with the two known 5-phosphatases, but no enzyme
activity has yet been identified for this protein
(33) . The
OCRL protein is predicted to be as large as 112 kDa and thus could also
account for larger 5-phosphatases that have been identified in other
tissues. Likewise, there are homologs of the 5-phosphatase II in C.
elegans, A. thaliana, and S. cerevisiae. While
nothing is known about the 5-phosphatase activity of these organisms,
in S. cerevisiae, antibodies to
PtdIns
(4, 5) P
inhibit cell proliferation
(34) implying an importance for this lipid.
H]mevalonate in Sf9 cells. It is interesting
that the isoprenylation sequence for 5-phosphatase contains a proline.
Generally, the two central amino acids of the carboxyl-terminal
isoprenylation sequence are aliphatic amino acids, and proline in the
third position of the 5-phosphatase isoprenylation motif is unusual in
mammalian systems
(35, 36) . However, the large
hepatitis Delta antigen ends with the carboxyl-terminal isoprenylation
sequence -CRPQ and becomes modified by the addition of a geranylgeranyl
moiety. Site-specific mutagenesis studies have demonstrated that this
sequence does not tolerate mutation of the proline to an aliphatic
amino acid
(37) . This suggests the possible existence of a
novel prenyltransferase that isoprenylates the hepatitis Delta antigen
and perhaps mammalian proteins with similar sequences such as the
5-phosphatase II
(35)
(
)
.
activity fractionates with
the membrane or cytoskeleton
(23) . The three basic amino acids
preceding the isoprenylation motif may also combine with this
isoprenylation to promote the membrane location of 5-phosphatase II as
it does with K-Ras
(31) . In K-Ras, a polybasic region
containing 6 lysines is needed in addition to isoprenylation for
complete association of K-Ras with the membrane. Mutation of the
polybasic region to leave three lysines in addition to its
isoprenylation signal results in approximately equal distribution of
K-Ras between an S100 and P100 fraction
(31) . 5-Phosphatase II
which is not isoprenylated because of mutation of its isoprenylation
motif or because of inhibition of isoprenylation by mevinolin does not
localize to the cell pellet. However, it is apparent that membrane
association is not absolutely necessary for 5-phosphatase II to
hydrolyze PtdIns
(4, 5) P
. We find that the
C939S mutant 5-phosphatase II is capable of hydrolyzing
PtdIns
(4, 5) P
in an in vitro assay, although the mutant enzyme is less active relatively than
the wild type enzyme toward the lipid substrate.
subunit
of transducin leads to an increased association with membranes that can
be accounted for by an improved interaction with other proteins in the
transducin-metarhodopsin II complex
(40) . There is as yet no
direct evidence that 5-phosphatase II associates with any other
cellular protein, although it cannot be extracted from a COS-7 cell
pellet with Triton X-100, and, in platelets, 16% of 5-phosphatase
activity is associated with cytoskeleton
(23) . This raises the
possibility of a secondary association with a cytoskeletal protein
following recruitment to the membrane. The 5-phosphatase II also
contains a region of apparent homology with other proteins that serve
as GTPase activating proteins in the Rac and Rho families of
GTP-binding proteins. The 5-phosphatase II used for GTPase activation
and association experiments was produced in Sf9 cells and thus should
have been isoprenylated, but it was not presented to any GTP-binding
proteins in a lipid environment. The association of 5-phosphatase II
with a GTP-binding protein may depend on orientation of 5-phosphatase
II in a lipid bilayer. An isoprenylated and membrane-associated
5-phosphatase II would be ideally situated to terminate both GTP-Rho
activity and the increased levels of
PtdIns
(4, 5) P
that follow the 5-kinase
activation.
is the same enzyme that
hydrolyzes PtdIns
(3) P
(41) . Similarly, the inositol
polyphosphate 4-phosphatase has recently been shown to degrade
PtdIns
(3, 4) P
as well as
Ins
(1, 3, 4) P
(26) . Both of
these enzymes are found bound in part to cell membranes, and it will be
interesting to find if they are also isoprenylated.
is not
understood. It is clear that a portion of the total pool of
PtdIns
(4, 5) P
cycles continuously between
PtdIns
(4) P and PtdIns
(4, 5) P
and
that only a portion of the total cellular
PtdIns
(4, 5) P
is hydrolyzed by
phospholipase C in response to agonists
(42, 43, 44) .
PtdIns
(4, 5) P
has other functions in
addition to serving as the source of
Ins
(1, 4, 5) P
. It regulates
cellular enzymes including µ-calpain
(45) , protein kinase C
(46, 47, 48) , and ATPases
(49, 50) and binds some proteins containing pleckstrin homology
domains
(51) . PtdIns
(4, 5) P
binds
to components of the cytoskeleton to regulate actin assembly
(52, 53, 54, 55, 56) ,
associates with clathrin assembly proteins
(57) , and affects
secretion
(58) . Regulation of cellular
PtdIns
(4, 5) P
levels by 5-phosphatase II
may affect a variety of cell functions in addition to calcium
signaling.
Table:
Wild type or mutant 5-phosphatase activity using
Ins(1,4,5)Pand PtdIns (4,5)P
, phosphatidylinositol 4,5-bisphosphate;
Ins(1,4,5)P
, inositol 1,4,5-trisphosphate;
Ins(1,3,4,5)P
, inositol 1,3,4,5-tetrakisphosphate;
Ins(1,4)P
, inositol 1,4-bisphosphate;
Ins(1,3,4)P
, inositol 1,3,4-trisphosphate; GST, glutathione
S-transferase; PCR, polymerase chain reaction; Mes,
4-morpholineethanesulfonic acid; PAGE, polyacrylamide gel
electrophoresis; GTP
S, guanosine
5`- O-(3-thiotriphosphate; GAP, GTPase activating protein.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.