From the Department of Biochemistry, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Here, we identify a novel rat
phosphatidylinositol-5-phosphate 4-kinase,
phosphatidylinositol-phosphate kinase II (PIPKII
). PIPKII
comprises 420 amino acids with a molecular mass of 47,048 Da, showing
greater homology to the type II
and II
isoforms (61.1 and 63.7%
amino acid identities, respectively) of phosphatidylinositol-phosphate kinase than to the type I isoforms. It is predominantly expressed in
kidney, with low expression in almost all other tissues. PIPKII
was
found to have phosphatidylinositol-5-phosphate 4-kinase activity as
demonstrated in other type II kinases such as PIPKII
. The PIPKII
that is present endogenously in rat fibroblasts, PC12 cells, and rat
whole brain lysate or that is exogenously overexpressed in COS-7 cells
shows a doublet migrating pattern on SDS-polyacrylamide gel
electrophoresis. Alkaline phosphatase treatment and metabolic labeling
in [32P]orthophosphate experiments revealed that
PIPKII
is phosphorylated in vivo, resulting in a shift
in its electrophoretic mobility. Phosphorylation is induced by
treatment of mitogens such as serum and epidermal growth factor.
Immunostaining experiments and subcellular fractionation revealed that
PIPKII
localizes dominantly in the endoplasmic reticulum (ER).
Phosphorylation also occurs in the ER. Thus, PIPKII
may have an
important role in the synthesis of phosphatidylinositol bisphosphate in
the ER.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phosphatidylinositol 4,5-bisphosphate
(PI(4,5)P2)1 is a
phospholipid with a variety of functions in vivo including
not only the production of second messengers such as diacylglycerol and inositol 1,4,5-trisphosphate, but also the regulation of actin regulatory proteins and the activation of phospholipase D and ADP-ribosylation factor. It has also been reported that
PI(4,5)P2 synthesis is potentiated by various stimuli
including GTPS (1-3), phorbol esters (4), tyrosine kinases (5), and
integrins (6). The variations in its function and the regulation of its synthesis indicate that enzymes responsible for the production of
PIP2, such as PI kinase and PIPK, also show large
diversities. Among PIPKs, two major subtypes (types I and II), each
comprising two isoforms (I
, I
, II
, and II
), have been
identified to date (13, 16-18), and it is thought that the role for
each subtype in vivo is different. The type I isozyme has
been reported to be activated by phosphatidic acid (7), to bind
physically to the small GTPases Rho (8) and Rac (9), and to be involved in Ca2+-dependent exocytosis in PC12 cells
(10). Human PIPKI
has been shown to be identical to the
STM7 gene, the putative gene responsible for Friedreich's
ataxia, suggesting that this isozyme plays roles in vesicular
trafficking such as neurotransmitter release (11). On the other hand,
type II isozymes have also been reported to have several functions
in vivo. In platelets, PIPKII
was shown to translocate to
the cytoskeletal fraction after stimulation by thrombin (12). PIPKII
was identified by its specific interaction with a cytoplasmic region of
the p55 tumor necrosis factor-
receptor, and a role for PIPK in
tumor necrosis factor-
signaling has been suggested (13).
Here, we identify a novel PIPKII isozyme (PIPKII) by a reverse
transcription-PCR method using degenerate primers designed from highly
conserved primary sequences in PIPK family members. PIPKII
is
phosphorylated on serine residues in vivo, resulting in a
mobility shift on SDS-polyacrylamide gel electrophoresis. Mitogenic
stimulation, such as by serum, EGF, or PDGF treatment, results in
phosphorylation of PIPKII
. The results of immunofluorescence experiments and subcellular fractionation suggest that PIPKII
has
important roles in the production of PIP2 in the ER.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials--
PIPs were purified by neomycin column
chromatography from crude phospholipids extracted from bovine spinal
cord as described (14). [-32P]dCTP,
[
-32P]ATP, [32P]orthophosphate, and
[3H]PI(4,5)P2 were from NEN Life Science
Products. The Colony/ PlaqueScreen used to screen the cDNA
library was from NEN Life Science Products. The polyvinylidene
difluoride membranes used for Western blot analysis were from Nihon
Eido (Tokyo, Japan). Ni2+-nitrilotriacetic acid-agarose was
from QIAGEN Inc. (Chatsworth, CA). The Partisphere SAX column was from
Whatman International Ltd. (Maidstone, United Kingdom). The thin-layer
chromatography silica plates and the cellulose plate used to separate
phospholipids and phosphoamino acids, respectively, were from Merck
(Darmstadt, Germany). Monoclonal anti-Myc antibody was purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-BiP antibody was from Stressgen Biotech Corp. Monoclonal anti-
-tubulin antibody was from Chemicon International, Inc. (Temecula, CA). Rhodamine- and
fluorescein-conjugated anti-rabbit IgG antibodies and
fluorescein-conjugated anti-mouse IgG antibody were from Organon
Teknika Corp. (West Chester, PA). Rhodamine-conjugated wheat germ
agglutinin was from Molecular Probes, Inc. (Eugene, OR).
Cell Culture-- COS-7 and 3Y1 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. PC12 cells were grown in Dulbecco's modified Eagle's medium containing 10% horse serum and 5% fetal bovine serum.
Reverse Transcription-Polymerase Chain Reaction--
Total RNA
isolated from rat brain was reverse-transcribed into cDNA by murine
leukemia virus reverse transcriptase and used as a template for PCR
using degenerate primers (5'-GAITAYTGYCCIRWIGTITTYMG-3', 5'-ATICYIABIAIIARRCTRTARTCCAT-3', and 5'-ATICYIABIAIIARIGARTARTCCAT-3') corresponding to two highly conserved sequences in mammalian and yeast
PIPKs ((D/E)YCPXVFR and MDYSLLLG(I/M)). The polymerase chain reaction was carried out as follows: 95 °C for 1 min, 43 °C for 1 min, and 72 °C for 2 min, for 40 cycles. The PCR product, ~500 base pairs long, was subcloned into the SmaI site of the
pBluescript SK() vector and sequenced.
cDNA Cloning of PIPKII--
The PCR product encoding a
novel sequence was cut out from the vector with EcoRI and
BamHI, labeled with [
-32P]dCTP, and used as
a probe for screening a rat brain cDNA library. The longest clone
obtained (~2.4 kilobases) encoded an open reading frame as long as
~400 amino acids, but did not include a potent start codon. On the
other hand, another partial clone was obtained that included a potent
start codon preceded by a sequence consistent with a Kozak consensus
sequence (15), but did not include a stop codon. From the sequences of
these two clones, we could determine the complete sequence for this
novel PIPK.
Northern Blot Analysis-- A partial fragment corresponding to 418-1500 base pairs of cDNA was labeled and used as a probe for Northern blot analysis. Hybridization was carried out on mouse multiple tissue Northern blot membrane (MTNTM, CLONTECH).
Production of Polyclonal Antibody-- A partial fragment encoding amino acids 130-420 was ligated into the PstI-HindIII site of a pQE32 His tag expression vector (QIAGEN Inc.). The His-tagged protein was expressed in Escherichia coli and purified on Ni2+-nitrilotriacetic acid-agarose as described by the manufacturer. The purified protein was injected as an antigen into rabbits to raise polyclonal antiserum. The resulting antibody was affinity-purified with the antigen protein transferred onto a polyvinylidene difluoride membrane or immobilized on a Hi-Trap NHS-activated column (Amersham Pharmacia Biotech).
Transfection into COS-7 Cells--
The full-length cDNAs of
mouse PIPKI and rat PIPKII
and PIPKII
were ligated into the
SalI-BamHI site of pCMV-Myc or the XhoI-BamHI site of pSR
XEBNeo mammalian
expression vectors. Twenty micrograms of each plasmid was mixed with
1 × 107 cells, and the mixtures were subjected to
electroporation with a Gene Pulser (Bio-Rad). The cells were cultured
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum.
Measurement of PIPK Activity--
Forty-eight hours after
electroporation, the expression vector-transfected COS-7 cells were
lysed with lysis buffer (20 mM Hepes, pH 7.2, 50 mM NaCl, 30 mM sodium pyrophosphate, 1%
Nonidet P-40, 1 mM EGTA, 25 mM NaF, 0.1 mM sodium vanadate, and 1 mM
phenylmethylsulfonyl fluoride). The expressed enzyme was
immunoprecipitated with monoclonal anti-Myc antibody and washed three
times with lysis buffer and once with reaction buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, and 1 mM EGTA). The reaction was started by adding 50 µM PIP, 50 µM ATP, and 10 µCi of
[-32P]ATP in 50 µl. After incubating for 10 min at
room temperature, the lipids were extracted with 1 N HCl
and chloroform/methanol (2:1, by volume) and spotted on TLC plates. The
plates were developed in chloroform/methanol/ammonia/water (14:20:3:5,
by volume), and the products were observed by autoradiography or
quantified by a Fuji BAS2000 image analyzer.
Analysis of Phosphoinositides by SAX HPLC-- Phosphoinositides separated by TLC were scraped out, deacylated, and analyzed by SAX HPLC as described (19).
Dephosphorylation of Phosphoinositides by SHIP-- A partial fragment corresponding to 1084-3947 base pairs of the cDNA of human Src homology domain-containing inositol-polyphosphate phosphatase (SHIP) was cut out with SalI and BamHI and ligated into the SalI-BamHI site of pCMV-Myc. The resulting expression vector was transfected into COS-7 cells as described above. Myc-SHIP was immunoprecipitated, and the dephosphorylation of lipids was carried out in 50 mM Tris-HCl, pH 7.5, and 10 mM MgCl2 at 37 °C for 60 min. The lipids were extracted and separated by TLC (chloroform/methanol/acetic acid/water, 43:38:5:7, by volume).
Dephosphorylation of PIPKII by Alkaline
Phosphatase--
Myc-tagged PIPKII
was immunoprecipitated from the
lysate of overexpressing COS-7 cells. The immunoprecipitates were
washed first with lysis buffer and then with alkaline phosphatase
buffer (50 mM Tris-HCl, pH 8.2, 50 mM NaCl, 1 mM MgCl2, 1 mM dithiothreitol, and
1 mM phenylmethylsulfonyl fluoride), after which 2 units of calf intestine alkaline phosphatase (Takara Shuzo Co., Ltd.) or storage
buffer for calf intestine alkaline phosphatase (10 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 50 mM
KCl, 0.1 mM ZnCl2, and 50% glycerol) was
added. The reaction was carried out at 30 °C for 60 min.
Metabolic 32P Labeling of PC12 Cells and Phosphoamino
Acid Analysis--
The culture medium was changed to phosphate-free
Dulbecco's modified Eagle's medium, and the PC12 cells were cultured
for 30 min. [32P]Orthophosphate (0.2 mCi/ml) was then
added, and the cells were incubated for 24 h. Labeled cells were
lysed in lysis buffer, and PIPKII was immunoprecipitated with
anti-PIPKII
antibody and transferred to a polyvinylidene difluoride
membrane. The band corresponding to PIPKII
was cut out and
hydrolyzed in 6 N HCl for 1 h at 110 °C. The
resulting amino acids, together with standard phosphoamino acids, were
spotted on TLC plates and separated by electrophoresis in pH 3.5 buffer
(5% acetic acid and 0.5% pyridine). The labeled phosphoamino acids
were detected by autoradiography. The positions of the standard
phosphoamino acids were detected by ninhydrin staining.
Immunofluorescence of PIPKII--
Cells growing on glass
coverslips were fixed with 3.7% formaldehyde in phosphate-buffered
saline for 15 min and permeabilized with 0.2% Triton X-100 in
phosphate-buffered saline for 5 min. Incubation with the first antibody
(polyclonal anti-PIPKII
and monoclonal anti-BiP) was carried out for
1 h, and incubation with the second antibody or
rhodamine-conjugated wheat germ agglutinin for 30 min. The cells were
observed with a confocal fluorescence microscope (Bio-Rad).
Subcellular Fractionation-- The subcellular fractionation was performed as described (20) with some modifications. Rat liver or 3Y1 fibroblasts were homogenized in 0.25 M sucrose, 50 mM triethanolamine HCl, pH 7.5, 50 mM potassium acetate, 6 mM magnesium acetate, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin. After centrifugation at 800 × g for 10 min and 10,000 × g for 10 min to devoid nuclei and mitochondria, respectively, the "post-mitochondrial" supernatant was obtained. The supernatant was layered over a cushion of 1.3 M sucrose in the same buffer and centrifuged at 202,000 × g for 2.5 h to yield three distinct fractions: the "post-microsomal" supernatant (representing the cytosol), interfacial "smooth microsomes" (representing the smooth ER and the Golgi apparatus), and the "rough microsomal" pellet (representing the rough ER).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identification of a Novel PIPK-- To identify novel PIPKs, we applied a reverse transcription-PCR method using degenerate primers corresponding to amino acid sequences highly conserved among mammalian PIPKs and their putative yeast homologs, Mss4p and Fab1p. Total RNA isolated from rat brain was reverse-transcribed and used as a template for further PCR. The PCR product was subcloned into the pBluescript vector and sequenced. Among several sequences corresponding to known PIPKs, one novel sequence homologous to the type II isoform of PIPK was obtained. We then tried to isolate a full-length cDNA using this fragment as a probe. By screening a rat brain cDNA library including random-primed clones, we obtained two clones encoding overlapping sequences. Between these clones, we found an open reading frame of 1260 base pairs encoding 420 amino acids (Fig. 1A). The calculated molecular mass is 47,048 Da.
|
The Novel PIPK Belongs to the Type II Subfamily--
The whole
amino acid sequence of the novel PIPK was revealed to be homologous to
the type II and II
isoforms of PIPK rather than to the type I
isoforms (61.1 and 63.7% identities to types II
and II
,
respectively, for the entire amino acid sequence, compared with 33.0%
identity to the type I isoforms for the kinase domain), indicating that
this PIPK is a third member of the type II isoform subgroup (Fig.
1B). Thus, we propose that this novel PIPK be called type
II
(PIPKII
). Alignment of the amino acid sequences between type
II PIPKs revealed that PIPKII
has a highly conserved kinase homology
domain separated by an insert domain showing no similarity to other
PIPK family members (18).
Tissue Distribution of PIPKII--
To study the tissue
distribution of PIPKII
, Northern hybridization was carried out on
mRNA from various mouse tissues. An mRNA of ~3.5 kilobases
was detected in almost all tissues, with the most abundant expression
in kidney (Fig. 2). The pattern of distribution is different from that of any type I
isoform or any other type II isoform (13, 16-18), suggesting specific
functions for this isoform.
|
PIPKII Is a Phosphatidylinositol-5-phosphate 4-Kinase--
We
transfected a Myc-tagged version of the full-length cDNA of
PIPKII
(Myc-PIPKII
) into COS-7 cells. The protein expressed in
the whole cell lysate and the anti-Myc immunoprecipitate was detected
in a doublet form by Western blotting with anti-Myc antibody (Fig.
3A and discussed further
below). Myc-PIPKII
was immunoprecipitated with anti-Myc antibody,
and the PIPK activity was measured. The immunoprecipitate
phosphorylated PIP purified from bovine spinal cord (see
"Experimental Procedures"), whereas anti-Myc immunoprecipitates from cells transfected with vector alone failed to do so (Fig. 3B). By using SAX HPLC, the resulting PIP2 was
confirmed to be PI(4,5)P2 (Fig. 3C).
|
PIPKII Is a Phosphoprotein--
A polyclonal antibody was
produced with a partial His-tagged protein expressed in E. coli as an antigen. With this polyclonal antibody, endogenous
PIPKII
was detected as doublet bands at 47 kDa in lysates from rat
brain, PC12 cells, and 3Y1 fibroblasts by Western blotting (Fig.
4A). When the full-length
cDNA (without the Myc tag) was transfected into COS-7 cells, the
same doublet band was detected (Fig. 4A), suggesting that
this doublet corresponds to some modification of PIPKII
such as
proteolysis or phosphorylation and is not due to cross-reactivity of
the antibody to another protein. To examine the possibility that these
doublet bands correspond to phosphorylated PIPKII
, we treated the
Myc-tagged version of PIPKII
with alkaline phosphatase. The
Myc-PIPKII
that was immunoprecipitated from overexpressing COS-7
cells showed a doublet banding pattern with the upper band predominant.
When the immunoprecipitated Myc-PIPKII
was incubated with calf
intestine alkaline phosphatase, the upper band disappeared completely
(Fig. 4B), whereas the lower band increased in intensity.
This indicates that the doublet migrating pattern is due to the
phosphorylation of PIPKII
. To confirm this conclusion, we next
labeled PC12 cells metabolically with
[32P]orthophosphate. After labeling, the ells
were lysed, and PIPKII
was immunoprecipitated, showing that
PIPKII
was phosphorylated (Fig. 4C). Together with the
results of Western blotting, it was confirmed that this phosphorylated
protein corresponds to the upper band of PIPKII
(Fig.
4C). Next, the phosphorylated band was cut out from
membrane, and phosphoamino acid analysis was carried out. The results
show that PIPKII
phosphorylation occurs predominantly on serine
residues (Fig. 4D). To determine whether the enzymatic
activity of PIPKII
is affected by its phosphorylation, we measured
the activity of Myc-PIPKII
after alkaline phosphatase treatment.
Myc-PIPKII
retained considerable activity even after alkaline
phosphatase treatment (Fig. 4E), indicating that the phosphorylation of PIPKII
does not affect its enzymatic
activity.
|
PIPKII Is Phosphorylated in Response to Extracellular
Stimuli--
In response to extracellular stimuli such as growth
factors or hormones, intracellular protein kinases are activated and
phosphorylate their physiological substrates. Since PIPKII
was found
to be phosphorylated on serine residues in vivo, we examined
whether the level of PIPKII
phosphorylation is potentiated by
extracellular stimuli. First, we treated rat 3Y1 fibroblasts with 10%
serum for various periods. The upper band of PIPKII
increased in a time-dependent manner, suggesting that PIPKII
is
phosphorylated in response to serum (Fig.
5A). We then examined other
extracellular stimuli including EGF, PDGF, bradykinin, and
lysophosphatidic acid for their abilities to induce the phosphorylation
of PIPKII
. Among them, EGF and PDGF enhanced the phosphorylation as
well as serum (Fig. 5, B and C). Lysophosphatidic
acid and bradykinin also induced phosphorylation to a lesser extent.
Fig. 4D clearly shows that PIPKII
phosphorylation does
not take place on tyrosine residues. Moreover, PIPKII
was not
recognized by an anti-phosphotyrosine antibody, PY20 (data not shown).
Therefore, it seems likely that the phosphorylation is mediated by a
serine/threonine kinase downstream of mitogenic signals mediated by
receptor tyrosine kinases. Protein kinase C does not seem to be
involved since phorbol 12-myristate 13-acetate did not potentiate
phosphorylation (Fig. 5, B and C). In addition, a
specific protein kinase C inhibitor, H-7, did not suppress
phosphorylation in 3Y1 cells (data not shown).
|
Intracellular Localization of PIPKII--
Using a polyclonal
antibody, we next examined the intracellular localization of PIPKII
.
The polyclonal antibody used was confirmed to recognize
specifically the doublet band corresponding to PIPKII
in 3Y1 cell
lysates by Western blotting (Fig. 4A). When rat 3Y1
fibroblasts were stained, PIPKII
was seen to predominate in the
perinuclear regions, suggesting that it is localized in microsomal
organelles such as the ER. To confirm this possibility, we
double-stained rat 3Y1 fibroblasts with anti-PIPKII
antibody and
with anti-BiP antibody, an ER-retaining protein. Both staining patterns
(Fig. 6A) clearly indicate the
localization of this enzyme in the ER. This staining pattern does not
overlap with that of wheat germ agglutinin, a trans-Golgi
staining reagent (Fig. 6A).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Purification and cDNA cloning of the 53-kDa
PIPKII from erythrocytes revealed that the lipid kinase belongs to a
distinct kinase family different from those of PI 3- and PI 4-kinases
and protein kinases (16). This family also seems to include yeast homologs such as Mss4p and Fab1p. Furthermore, cDNA cloning of types I
and I
, members of another subtype of mammalian PIPK, also
showed them to belong to this same distinct lipid kinase family (17,
18). Members of this novel lipid kinase family have several conserved
regions within their primary sequences. Using a reverse
transcription-PCR method involving degenerate primers corresponding to
these highly conserved sequences, we succeeded in identifying a novel
PIPK isoform and named it PIPKII
.
Although PIPKII seems to belong to the type II subtype, the
similarity between PIPKII
and other members of the type II PIPK family is not very high (61.1% for II
and 63.3% for II
)
compared with the homology between PIPKII
and PIPKII
(76.7%).
This, together with the difference in its expression pattern from that
of other PIPKs, suggests that PIPKII
has some distinct functions
in vivo.
PIPKII was detected as a doublet migrating protein by Western
blotting with a specific polyclonal antibody not only in rat brain
lysates, but also in 3Y1 fibroblasts and PC12 cells. The same doublet
patterns were also observed when PIPKII
was overexpressed in COS-7
cells. The evidence presented in this study shows that PIPKII
is
phosphorylated in vivo and that the upper band represents the phosphorylated form. Furthermore, phosphoamino acid analysis revealed that phosphorylation occurs predominantly on serine residues. We also observed that mitogens such as serum and growth factors immediately induced phosphorylation of PIPKII
. The total cellular amount of PIP2 and the PIPK activity have been reported to
increase in response to various extracellular stimuli, including EGF
(5), formyl-methionyl-leucyl-phenylalanine, platelet-activating factor (1), thrombin (22), phorbol ester (4), and adhesion to fibronectin (6).
Some of these extracellular stimuli have been reported to increase PIPK
activity, especially in the cytoskeleton. In addition, the involvement
of G-proteins, including small GTPases such as Rac and Rho, has also
been suggested by data showing that the PIPK activity is potentiated by
non-hydrolyzable GTP or is associated with recombinant Rho and Rac
proteins. Despite the above observations, the exact molecular mechanism
by which PIPK is regulated has not been made clear. Here, we provide
evidence for the phosphorylation of PIPKII
. It is possible that PIPK
is regulated by a protein kinase downstream of extracellular stimuli. At present, we do not know what kinase is responsible for the phosphorylation. The phosphorylation was found to be enhanced by
tyrosine kinase activators such as EGF and PDGF rather than activators
related to triplet G-protein-coupled signalings, such as bradykinin and
lysophosphatidic acid. Moreover, dibutyryl cAMP (data not shown) and
phorbol 12-myristate 13-acetate did not increase the phosphorylation
markedly. Considering that phosphorylation occurs on serine residues
rather than on tyrosine residues, a serine kinase, other than protein
kinase A or C, downstream of a tyrosine kinase must phosphorylate
PIPKII
. Although the exact roles of the phosphorylation remain
unclear, it is possible that the phosphorylation of PIPKII
regulates
its localization. Hinchliffe et al. (12) reported that the
translocation of PIPKII
to the cytoskeletal fraction of platelets in
response to thrombin is inhibited by okadaic acid treatment, suggesting
the importance of dephosphorylation for translocation. Although they
also showed that the activity of PIPKII
is regulated by its
phosphorylation state (23), we did not observe any change in the
activity of PIPKII
after phosphorylation by mitogenic stimulation or
dephosphorylation by alkaline phosphatase (Fig. 4E and
data not shown).
In this study, we demonstrated that PIPKII is specifically localized
in the ER in rat 3Y1 fibroblasts. Although most PIPK activity is found
in the plasma membrane and cytosol, Helms et al. (24)
reported that PI(4,5)P2 synthesis occurs in the ER. Several
phosphoinositide-metabolizing enzymes have been reported to be
localized in the microsomal fraction. Wong et al. (25) reported that PI 4-kinase
is localized in the ER, whereas PI 4-kinase
is localized in the Golgi apparatus in HeLa cells. Most PI
synthase activity is also detected in the ER (24, 26, 27). It is
conceivable that PI(4,5)P2 synthesis occurs efficiently in
microsomes because of the relay of substrates between PI synthase, PI
kinase, and PIPK. In addition, PI5P, the preferential substrate for
type II isozymes in PI(4,5)P2 synthesis, is rare in NIH3T3 cells (21) compared with PI4P, which exits abundantly in the cell. It
may be important for this minor phosphoinositide to be localized in a
restricted area such as in microsomes with its metabolizing enzyme,
PI5P 4-kinase, for efficient PI(4,5)P2 synthesis. Many of
the characteristics of PI5P have yet to be elucidated, including its
synthetic pathway as well as the identity of PI 5-kinase and its exact
intracellular localization. However, together with the observation that
PIPKII
is localized in the ER after phosphorylation by mitogenic
signals, our results suggest that PIPKII
is involved in the
synthesis of PI(4,5)P2 in the ER.
Shibasaki et al. (28) reported that the type I PI4P 5-kinase
overexpressed in COS-7 cells by an adenovirus expression system is
localized mainly in plasma membranes and the cytosol. They further
reported that type I PIPKs induce a pine needle-like structure of the
actin cytoskeleton downstream from Rho. In contrast, we observed no
change in the actin cytoskeleton when type II and II
isozymes
were transiently overexpressed in COS-7 cells (data not shown). From
these results, it is possible to conclude that each subfamily of PIPK
has a distinct localization and function and is also responsible for
the synthesis of distinct intracellular PIP2 sources.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Hisamaru Hirai for the
gift of human SHIP cDNA and Dr. Hisamitsu Ishihara for mouse
PIPKI cDNA.
![]() |
FOOTNOTES |
---|
* 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) AF030558 and AF033355.
To whom correspondence should be addressed. Tel.: 81-3-5449-5510;
Fax: 81-3-5449-5417; E-mail: takenawa{at}ims.u-tokyo.ac.jp.
The abbreviations used are:
PI(4, 5)P2, phosphatidylinositol
4,5-bisphosphateGTPS, guanosine
5'-O-(3-thiotriphosphate)PIP2, phosphatidylinositol bisphosphatePI, phosphatidylinositolPIPK, phosphatidylinositol-phosphate kinasePIP, phosphatidylinositol
phosphatePCR, polymerase chain reactionEGF, epidermal growth
factorPDGF, platelet-derived growth factorER, endoplasmic
reticulumSAX, strong anion exchangeHPLC, high pressure liquid
chromatographySHIP, Src homology domain-containing
inositol-polyphosphate phosphatasePI5P, phosphatidylinositol
5-phosphatePI4P, phosphatidylinositol 4-phosphate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|