From the Department of Biochemistry, The Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639 Japan
Received for publication, February 20, 2001, and in revised form, March 23, 2001
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ABSTRACT |
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We have characterized a novel Sac
domain-containing inositol phosphatase, hSac2. It was ubiquitously
expressed but especially abundant in the brain, heart, skeletal muscle,
and kidney. Unlike other Sac domain-containing proteins, hSac2 protein
exhibited 5-phosphatase activity specific for phosphatidylinositol
4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate.
This is the first time that the Sac domain has been reported to possess
5-phosphatase activity. Its 5-phosphatase activity for
phosphatidylinositol 4,5-bisphosphate (Km = 14.3 µM) was comparable with those of Type II
5-phosphatases. These results imply that hSac2 functions as an inositol
polyphosphate 5-phosphatase.
Phosphatidylinositol
(PI)1 phosphates, trace
amounts of phospholipids in eukaryotic cells, exist as seven different
molecules that are produced as a result of the phosphorylation of
single or multiple sites of the inositol head group of
phosphatidylinositol, including PI(4)P, PI(3)P, PI(5)P,
PI(4,5)P2, PI(3,4)P2, PI(3,5)P2 and
PI(3,4,5)P3. These lipids are now widely recognized as
important regulators in a variety of cellular functions such as
membrane trafficking, cytoskeletal reorganization, cell survival and
cell proliferation (1-4).
A variety of phosphoinositides are hydrolyzed by phosphatases with
different substrate specificities. They are classified into four groups
according to the position of the phosphate that they hydrolyze, 1-, 3-, 4-, or 5-phosphatase. Among many phosphoinositide phosphatases,
5-phosphatase forms a fairly large family. According to the substrate
specificity, it is classified into four types. Type I 5-phosphatases
hydrolyze only water-soluble substrates such as
Ins(1,4,5)P3 and Ins(1,3,4,5)P4 (5).
Type II enzymes hydrolyze not only the water-soluble inositol
phosphates but also lipid substrates such as PI(4,5)P2 and
PI(3,4,5)P3. Type II 5-phosphatases are further divided
into four subgroups. All type II 5-phosphatases have two conserved
motifs, WXGDXNXR and
KXRXPAW(T/C)DR(I/V)LW(R/)K. The first
subgroup includes a 75-kDa protein isolated from human platelets and is
now called 5-phosphatase II or GIP (GAP domain-containing inositol
5-phosphatase) (6). This subgroup also includes Lowe's
oculocerebrorenal syndrome (OCRL) protein. This protein
hydrolyzes the phospholipid substrates 10-30-fold more than type I
5-phosphatase (7). In the kidney proximal tubule cells from a Lowe's
syndrome patient, PI(4,5)P2 was found to accumulate, even
though other inositol polyphosphate 5-phosphatases were present,
suggesting that OCRL protein regulates the PI(4,5)P2 levels
in the cells (8). Synaptojanin 1 and 2 are also subjected to group II
enzymes that participate in synaptic vesicle trafficking. Synaptojanin
forms complexes with dynamin, amphiphysin, and Grb2 to promote synaptic
vesicle recycling (9, 10). Synaptojanins contain two phosphatase
domains. One is a phosphatase domain at the C terminus, which is common
to all type II 5-phosphatases. This domain can hydrolyze
PI(4,5)P2 to PI(4)P but cannot use PI(4)P, PI(3)P, or
PI(3,5)P2 as a substrate (11). In addition to this
5-phosphatase, synaptojanins contain Sac domains in the N
terminus, which are homologous to yeast Sac1. Recently, the
synaptojanin Sac domain and yeast Sac1 protein were found to
dephosphorylate PI(4)P, PI(3)P, and PI(3,5)P2 but not
PI(4,5)P2 (12). Thus synaptojanins have both a
PI(4,5)P2 5-phosphatase activity and a phosphoinositide
phosphatase activity.
Proline-rich inositol polyphosphate 5-phosphatase (PIPP) is a recently
identified type II 5-phosphatase that localizes at membrane ruffling
areas (13). This phosphatase hydrolyzes phosphate at the 5-position of
Ins(1,4,5)P3, Ins(1,3,4,5)P4, and
PI(4,5)P2. 5-Phosphatase that induces arbolization
(Pharbin) and skeletal muscle- and kidney-enriched inositol phosphatase
(SKIP) also seem to be members of the type II 5-phosphatases (14,
15). Pharbin has a CaaX motif at the C terminus
and localizes at membranes. It hydrolyzes Ins(1,4,5)P3 and
Ins(1,3,4,5)P4 more effectively than PI(4,5)P2.
On the other hand, SKIP preferentially hydrolyzes phosphoinositides,
such as PI(4,5)P2 and PI(3,4,5)P3 to
water-soluble inositol polyphosphates.
Type III enzymes hydrolyze phosphate at the 5-position of
phosphoinositide and inositol polyphosphate, which have a 3-position phosphate group. There are two such enzymes designated as SHIP 1 and 2 SH2-containing inositol phosphatase (16, 17). SHIPs contain
an SH2 domain at the N terminus and form complexes with intracellular signaling molecules such as Grb2 and Shc (18). Because
these enzymes hydrolyze PI 3-kinase products such as
PI(3,5)P2 and PI(3,4,5)P3, SHIPs are thought to
have a negative function and to terminate signals from PI 3-kinase.
Type IV 5-phosphatase was originally reported to hydrolyze only
PI(3,5)P2 and PI(3,4,5)P3, but it has now been
found that it also hydrolyzes PI(4,5)P2 (19). However, this
group of 5-phosphatases does not use water-soluble inositol phosphates
as substrates.
The Sac domain was originally found in yeast Sac1p (20, 21). The
SAC1 mutation suppressed the phenotypes seen in Sec14 mutant yeast. Sec14p is the yeast PI/phosphatidylcholine transfer protein. Several of the Sac domain-containing proteins have been identified in yeast. Fig4p protein, which was composed only of the Sac
domain, was identified from the yeast mutant that causes mating defects
(22). Inp51p, Inp52p, and Inp53p are 5-phosphatases that contain
the Sac domain in the N terminus-like synaptojanin. Recently, Guo
et al. (12) found that these Sac domains, except Inp51p,
possess phosphoinositide phosphatase activity. The Sac domain is
~400 amino acids in length and consists of seven conserved motifs
that appear to define the catalytic regions of enzyme activity (see
Fig. 3). The sequence RXNCLDCLDRTN within the sixth motif is
the most particular. The CX5R(T/S) motif found
within this sequence appears to define the catalytic region of the
phosphatase and is also seen in a variety of metal-independent protein
phosphatases (12). In addition to synaptojanins, three additional Sac
domain-containing proteins (KIAA0851, KIAA0966, and KIAA0274) exist in
humans. Among these proteins, only the rat homolog of KIAA0851 (rSac1),
which is the most closely related to yeast Sac1p, was characterized (23). This enzyme showed the same spectrum for the substrate specificity of yeast Sac1p and hydrolyzed PI(3)P > PI(4)P > PI(3,5)P2. However, KIAA0966 (hSac2) and
KIAA0274 (hSac3) still have not been characterized.
We studied the characterization of hSac2 and determined the lipid
specificity. This enzyme has a different substrate specificity than rSac1, in that it can dephosphorylate the 5-position of
phosphoinositide, PI(4,5)P2, and PI(3,4,5)P3.
It hydrolyzes PI(4,5)P2 most effectively, and its substrate
specificity for PI(4,5)P2 is comparable with those of Type
II phosphatases.
Materials--
Phosphoinositides (PI(3)P, PI(4)P,
PI(3,4)P2, PI(3,5)P2, PI(4,5)P2,
and PI(3,4,5)P3) and phosphatidylinositol
(Ins(1,4,5)P3 and Ins(1,3,4,5)P4) were
purchased from Cell Signals, Inc. [ Molecular Cloning of hSac2--
Full-length human cDNA clone
of human Sac2 (GenBankTM accession number Hj06369) was
obtained from Kazusa DNA Research Institute. hSac2 is a 4924-bp
cDNA with 17-bp poly(A)+ stretch, which was inserted at the
SalI-NotI site of pBluescript II SK+ vector.
Northern Blot Analysis--
A membrane containing mRNA (2 µg of poly(A) was contained in each lane) (human 12-lane multiple
tissue Northern blot) was purchased from CLONTECH.
The N-terminal 666-base pair cDNA were 32P-labeled
using a random primer labeling kit (Takara Shuzo) following the
manufacturer's protocol.
Baculovirus Expression of Recombinant hSac2--
The expression
constructs of glutathione S-transferase (GST) or GST-tagged
full-length hSac2 (GST·hSac2) were constructed as
follows. GST construct was produced by ligating
BglII-BamHI site polymerase chain reaction
products encoding full-length GST amplified from pGEX-2T bacterial
expression vector into the BamHI site of pFASTBAC1 (Life
Technologies, Inc.). Then BamHI-NotI site full-length hSac2 fragments amplified by polymerase chain reaction were
introduced into the BamHI site of the GST construct
(GST·hSac2). The GST and GST·hSac2 proteins were expressed in
Sf9 cells by infecting them with 1 ml of 1 × 109 units/ml recombinant virus and cultured in Sf 900 medium containing 5% fetal calf serum at 28 °C for 48 h.
Phosphatase Activity Assay--
Baculovirus-expressing
Sf9 cells were centrifuged and lysed in a cold lysis buffer (40 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). The cells were briefly sonicated and centrifuged at
10,000 × g at 4 °C for 30 min. The supernatant was
applied to 400 µl of glutathione-Sepharose 4B beads (Amersham
Pharmacia Biotech), then washed five times with a wash buffer (40 mM Tris-HCl, pH 7.5, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100) and three times with assay
buffers for phosphoinositide phosphatase (50 mM Tris
maleate, pH 6.0, 5 mM MgCl2, 25 mM
KCl, 0.25%
The 5-phosphatase activity for PI(4,5)P2 was measured as
follows. The PI(5)P 4-kinase was used to produce
[32P]PI(4,5)P2. Myc-tagged full-length human
PI(5)P 4-kinase
An investigation of the effect of cations or chelate, and pH on
enzyme activity was carried out under the same conditions described
above. Phosphatase activities were examined under various conditions of
cations or chelate and pH values in Tris maleate or Tris/HCl buffer in
the presence of 50 µM PI(4,5)P2 as substrate were determined.
Identification of Novel Sac Domain-containing Protein--
The Sac
domain is a recently identified novel inositol polyphosphate
phosphatase, and several family proteins have been identified in yeast.
Nemoto et al. (23) identified a rat homologue of yeast Sac1p, which was a prototypic member of the Sac domain-containing protein. Sac domain-containing proteins possess conserved amino acid
motifs that are essential for inositol polyphosphate phosphatase activities. In an attempt to identify another member of the Sac domain-containing proteins, we searched the cDNA data base based on
the amino acid sequence of these motifs and identified two novel
clones. One is KIAA0274, (GenBankTM accession number
NM014845) which is homologous to yeast Fig4p protein (22). The other is
KIAA0966 (GenBankTM accession number XM005971), a
4923-bp clone, with an estimated open reading frame of 3396-bp that
encodes a protein with a molecular mass of 120 kDa (Fig.
1). Unlike other Sac domain-containing
proteins, KIAA0966 has no yeast counterparts (Fig.
2). We named this hSac2 (human Sac2).
hSac2 shares 34.2% amino acid identity with Sac1p and possesses seven
motifs conserved in the Sac domain-containing proteins. As shown in
Fig. 3, an active site motif
CX5R(T/S) is found within the sixth motif, which
also exists in metal-independent protein phosphatases and inositol
polyphosphate phosphatases. Some dissimilar amino acids in the fifth
motif (Val417 to Asp427) were also observed
(Fig. 3).
Tissue Distribution of hSac2--
The expression of hSac2 was
analyzed by Northern blot analysis. The 4.9-kilobase transcript was
detected in every tissue tested but was especially high in the brain,
heart, skeletal muscle, kidney, and placenta (Fig.
4).
Enzymatic Properties of hSac2--
The Sac-containing proteins
studied so far exhibited intrinsic inositol phosphatase activities.
Yeast Sac1 and rat Sac1 exhibited phosphatase activities for PI(3)P,
PI(4)P, and PI(3,5)P2 but not PI(4,5)P2
(22). We expressed GST-conjugated full-length hSac-2 protein in
insect cells and purified the protein. The intrinsic phosphatase
activities of the purified recombinant protein for the
phosphoinositides were analyzed. Unexpectedly, hSac2 only hydrolyzed PI(4,5)P2 and PI(3,4,5)P3, as
synaptojanin 1 or SKIP did (Fig.
5a). It did not hydrolyze
PI(3)P, PI(4)P or PI(3,5)P2, as rat Sac1 did. We also
analyzed its phosphatase activities for inositol phosphates,
Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Neither of
them was hydrolyzed, and this revealed that hSac2 is a
phosphoinositide-specific 5-phosphatase (Fig. 5a). The
mutant in which asparagine at amino acid 460 was replaced with alanine
lost its phosphatase activity, suggesting that the Sac domain is the
only domain responsible for phosphatase activity (data not shown). The
apparent Km value of the PI(4,5)P2
5-phosphatase activity determined by a Lineweaver-Burk plot analysis
was 14.3 µM, which was comparable with those of Type II
5-phosphatase, SKIP and synaptojanin 1 (Fig. 5, b and c) (15, 25).
To determine whether hSac2 is a 4-phosphatase or 5-phosphatase, we
generated 32P-labeled PI(4,5)P2 from PI(5)P
using PI(5)P 4-kinase
To further characterize the enzymatic activities, we analyzed the
optimal pH for the phosphatase activities of hSac2. High phosphatase
activity was maintained over a wide range between pH 5.5 and 8.0 and
was optimal at pH 6.0 (Fig.
7a). Next we examined the
effect of cations on the catalytic activities. Guo et al. (12) reported that inositol phosphatase activity of the Sac domain of
yeast Inp53p was inhibited partly by Mg2+. However, in the
presence of Mg2+ or K+, phosphatase activities
were slightly up-regulated, and addition of 5 mM
Mg2+ in the presence of 25 mM K+
increased the PI(4,5)P2 activity 4-fold (Fig.
7b). Addition of other cations, such as Mn2+ or
Ca2+, or EDTA did not affect PI(4,5)P2
5-phosphatase activity of hSac2.
In this study, we found that hSac2 is 5-phosphatase for
PI(4,5)P2 and PI(3,4,5)P3. This is the first
report to demonstrate that the Sac domain has phosphoinositide
5-phosphatase activity, whereas the Sac domain of yeast Sac1p and rat
Sac1 has already been shown to dephosphorylate PI(3)P, PI(4)P, and
PI(3,5)P2. As shown in Fig. 3, the motifs of the catalytic
site of the Sac domain in hSac2 are conserved, but some
alternations in other parts of the Sac domain are present, which
may contribute to the substrate specificity.
Thus far phosphoinositide and inositol polyphosphate 5-phosphatases
have been classified into five types. In these types, all
phosphatases have two conserved motifs that are essential for
5-phosphatase activity. However, hSac2 does not contain such motifs,
and phosphatase activity seems to exist in the Sac domain, suggesting a
new type of phosphoinositide 5-phosphatase. We report here that the
hSac2 protein is a 5-phosphatase of which the substrate specificity is
different from that of the rat Sac1, suggesting that each Sac domain
may have different substrate specificity. In mammalians, at least three
Sac-containing proteins (Sac1, Sac2, and Sac3) are present in addition
to synaptojanins. Thus it remains to be determined whether these Sac
proteins have different substrate specificities and functions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]ATP was
purchased from PerkinElmer Life Science Products. Tetramethylrhodamine B isothiocyanate-conjugated wheat germ agglutinin and rhodamine-labeled phalloidin were each purchased from Sigma and Molecular Probes. Polyclonal antibody against c-Myc was purchased from Santa Cruz Biotechnology.
-D-octyl glucoside, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). The recombinant proteins were eluted with
three bed volumes of elution buffer (10 mM glutathione in 50 mM Tris-maleate, pH 6.0). Phosphoinositide phosphatase
and inositol 5-phosphatase activity assays were carried out as
described by Maehama et al. (24) using 50 µM
PI(3)P, PI(4)P, PI(5)P, PI(3,4)P2, PI(3,5)P2,
PI(4,5)P2, PI(3,4,5)P3,
Ins(1,4,5)P3, and Ins(1,3,4,5)P4 as substrates
and purified baculovirus-expressed recombinant proteins as enzymes.
Recombinant hSac2 protein was added, the mixture was further
incubated at 37 °C for 15 min, and the phosphate release was
quantified using a malachite green-based colorimetric assay for
inorganic phosphate. (24).
was expressed in COS-7 cells and purified by
imunoprecipitation with anti-c-Myc monoclonal antibody. The
PI(5)P/PI(4,5)P2 mixture was extracted with
chloroform/methanol (2:1, v/v) and then dried with nitrogen gas. A
phosphatase activity assay was performed under the same conditions as
described above. The lipids were extracted and separated by TLC using
n-propyl alcohol, 2 M acetic acid (2:1, v/v) and
visualized by autoradiography.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide and corresponding amino acid
sequence of hSac2 cDNA. The amino acid sequence of the Sac
domain is underlined.
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Fig. 2.
Comparison of the domain structures of the
various Sac domain-containing proteins. hSac2, yeast Sac1p, rat
Sac1p, Inp52p, and Inp53p share the homology sequence at the C terminus
of the Sac domain (white box). Inp51p, Inp52p, and Inp53p
have a type II 5-phosphatase catalytic domain and a proline-rich region
at the C terminus of the protein. However, hSac2 has no putative domain
besides the Sac domain.
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Fig. 3.
Comparison of highly conserved sequences of
the various Sac domains with that of hSac2. The Sac domains of
hSac2, Sac1p, rat Sac1p, Inp52p, and Inp53p contain the conserved amino
acids that encode the putative catalytic sequence
(CX5R) in motif 6. However, those of Inp51p are
replaced with other amino acids, which are the reason for the lack of
phosphatase activity seen in this protein. Highly conserved residues
are highlighted as white on black.
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Fig. 4.
Northern blot analysis of expression of
hSac2. Northern blot analysis of human hSac2 was carried out using
N-terminal 666-base pair hSac2 cDNA as a probe. 2 µg of human
mRNA was used in each lane. The numbers on the
left are the sizes of the markers. Lane 1, brain;
lane 2, heart; lane 3, skeletal muscle;
lane 4, colon (no mucosa); lane 5, thymus;
lane 6, spleen; lane 7, kidney; lane
8, liver; lane 9, small intestine; lane 10,
placenta; lane 11, lung; lane 12, peripheral
blood leukocyte. kbp, kilobase pair.
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Fig. 5.
Enzymatic properties of the recombinant hSac2
protein. a, phosphatase activity analysis of the
recombinant GST·hSac2 protein for phosphoinositides and inositol
phosphates in vitro. 50 µM of various
phosphoinositide and inositol phosphate substrates were incubated with
25 ng of GST·hSac2 at 37 °C for 15 min. The reaction product was
measured by the malachite green method. b, kinetic
properties of hSac2. PI(4,5)P2 (5-100 µM) was
incubated with 25 ng of GST·hSac2 at 37 °C for 15 min. The
reaction product was measured by the malachite green method.
Km values were calculated by Lineweaver-Burk
analysis.
(26). Then 32P-labeled
PI(4,5)P2 was incubated with the recombinant hSac2 protein. If hSac2 is a 5-phosphatase, labeled PI(4)P will be formed, and if it
is a 4-phosphatase, labeled phosphate is hydrolyzed and only unlabeled
PI(5)P will be generated. As shown in Fig.
6, labeled phosphatidylinositol
monophosphate was formed by the incubation with hSac2 protein. This
result confirmed that hSac2 is a 5-phosphatase.
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Fig. 6.
hSac2 is a 5-phosphatase.
[4-32P]PI(4,5)P2 were incubated with
25 ng of GST or GST·hSac2 at 37 °C for 15 min. The product was
analyzed by TLC. Generation of [4-32P]PI(4)P was caused
by GST·hSac2. The positions of phospholipids are indicated on the
right.
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Fig. 7.
Catalytic properties of hSac2. The
effects of pH and cations on PI(4,5)P2 phosphatase activity
of the hSac2 were analyzed. a, PI(4,5)P2 (50 µM) was incubated with 25 ng of GST·hSac2 at 37 °
for 20 min in a buffer between pH 5.0 and 9.0. b,
PI(4,5)P2 phosphatase activities were measured in a maleate
buffer containing 2.5-25 mM EDTA (open
squares), KCl (open triangles), MnCl2
(open circles), MgCl2 (closed
circles), MgCl2 in the presence of 25 mM
KCl (closed squares), and CaCl2 (closed
triangles).
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ACKNOWLEDGEMENTS |
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We thank Dr. Takahiro Nagase (Kazusa DNA Research Institute, Kisarazu, Japan) for kindly donating a cDNA clone of KIAA0966 (hSac2) and Dr. Takeshi Endo (Chiba University, Chiba, Japan) for much helpful advice.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
The Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639 Japan. Tel: 81-3-5449-5510; Fax: 81-3-5449-5417; E-mail: takenawa@ims.u-tokyo.ac.jp.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M101579200
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ABBREVIATIONS |
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The abbreviations used are: PI, phosphatidylinositol; PI(3)P, phosphatidylinositol (3)-monophosphate; PI(4)P, phosphatidylinositol (4)-monophosphate; PI(5)P, phosphatidylinositol (5)-monophosphate; PI(4, 5)P2, phosphatidylinositol (4,5)-bisphosphate; PI(3, 4,5)P3, phosphatidylinositol (3,4,5)-trisphosphate; Ins(1, 4,5)P3, inositol (1,4,5)-trisphosphate; Ins(1, 3,4,5)P4, inositol (1,3,4,5)-tetrakisphosphate; GST, glutathione S-transferase; hSac2, human Sac2; OCRL, Lowe's oculocerebrorenal syndrome; SHIP, SH2-containing inositol phosphatase; GIP, GAP domain containing inositol 5-phosphatase; pharbin, 5-phosphatase that induces arbolization; PIPP, proline-rich inositol polyphosphate 5-phosphatase; SKIP, skeletal muscle- and kidney-enriched inositol phosphatase.
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