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J Biol Chem, Vol. 274, Issue 31, 22019-22024, July 30, 1999
,,,
From the § Third Department of Internal Medicine,
Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113, Japan, the Institute for Adult Disease, Asahi
Life Foundation, 1-9-14, Nishishinjuku, Shinjuku-ku, Tokyo 160, Japan, and the ¶ Third Department of Internal Medicine, Yamaguchi
University School of Medicine, 1144 Kogushi, Ube,
Yamaguchi 755, Japan
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Activation of p85/p110-type phosphatidylinositol
(PI) kinase has been implicated in various cellular activities. This PI
kinase phosphorylates the D-4 position with a similar or higher
efficiency than the D-3 position when trichloroacetic acid-treated cell
membrane is used as a substrate, although it phosphorylates almost
exclusively the D-3 position of the inositol ring in phosphoinositides
when purified PI is used as a substrate. Furthermore, the lipid kinase activities of p110 for both the D-3 and D-4 positions were completely abolished by introducing kinase-dead point mutations in their lipid
kinase domains ( A variety of growth factors and hormones exert their cellular
effects via interactions with specific receptors that possess protein
kinase activities. The interaction of most of these ligands with their
receptors induces tyrosine kinase activation and phosphorylation of the
receptor and/or intracellular substrates. The tyrosine-phosphorylated protein serves as a docking protein for several cytoplasmic substrates with SH2 domains (1-3). p85/p110-type phosphatidylinositol
(PI)1 kinase has been
identified through its ability to associate with these
tyrosine-phosphorylated substrates (4, 5) and has been thought to be an
enzyme that phosphorylates the D-3 position of the inositol ring in
phosphoinositides, resulting in formation of PI-3-P,
PI-3,4-P2, and PI-3,4,5-P3 (6), based on
experiments using purified phosphatidylinositol as a substrate to
determine the lipid substrate specificity.
However, our findings that the lipid products of p85/p110-type PI
kinase in vivo differ markedly from those produced by a conventional in vitro method used to determine the substrate
specificity of PI kinase are of great interest. The results of this
study show that p110 Materials--
PI and dexamethasone were purchased from Sigma.
3-Isobutyl-1-methylxanthine and 2-deoxy-D-glucose were from
Wako Bioproducts, and the enhanced chemiluminescence (ECL) detection
system was from Amersham Pharmacia Biotech. [ Antibodies--
Anti-p85 antiserum and anti-phosphotyrosine
antibodies (4G10) were purchased from Upstate Biotechnology, Inc.
Anti-hemagglutinin antibodies (12CA5) were purchased from Roche
Molecular Biochemicals. Anti-p110 Cell Culture--
Sf9 cells were maintained in TC-100
medium (Life Technologies, Inc.) supplemented with 10% fetal bovine
serum (Life Technologies, Inc.) at 27 °C. Cells were harvested 2 days post-baculovirus infection. 3T3-L1 fibroblasts were maintained in
Dulbecco's modified Eagle's medium (DMEM) containing 10% donor calf
serum (Life Technologies, Inc.) in an atmosphere of 10%
CO2 at 37 °C. Two days after the fibroblasts had reached
confluence, differentiation was induced by treating the cells with DMEM
containing 0.5 mM 3-isobutyl-1-methylxanthine, 4 mg/ml
dexamethasone, and 10% fetal bovine serum for 48 h. Cells were
re-fed with DMEM supplemented with 10% fetal bovine serum every other
day. Infection with the indicated adenoviruses was carried out on day 3 post-differentiation induction, and the experiments were conducted on
day 5, at which point >90% of the cells expressed the adipocyte phenotype.
Cloning of cDNA--
Cloning and construct-reverse
transcription-polymerase chain reaction were performed to amplify
full-length p110 Gene Transduction--
The entire coding regions of p110 Immunoprecipitation and Western Blotting--
Cells were lysed
in PBS containing 1% Triton, 0.35 mg/ml phenylmethylsulfonyl fluoride,
and 100 mM sodium vanadate. Cell lysates were centrifuged
at 15,000 × g for 10 min at 4 °C to remove
insoluble materials. The supernatants were incubated with the indicated antibodies, followed by the addition of protein A-Sepharose (Amersham Pharmacia Biotech). Alternatively, in some experiments, lysates were
immunoprecipitated with anti-hemagglutinin antibodies, followed by the
addition of protein G-Sepharose (Amersham Pharmacia Biotech). The
immune complexes were collected by centrifugation, washed with PBS
containing 1% Triton X-100, boiled in Laemmli sample buffer containing
100 mM dithiothreitol, and then subjected to SDS-polyacrylamide gel electrophoresis. Immunoblotting was performed with the ECL system according to the manufacturer's instructions. In
some experiments, the band intensities were quantitated with a Model
GS-525 molecular imager (Bio-Rad).
In Vitro Generation of 32P-Labeled Phosphoinositides
(PI 3-Kinase Assay)--
Sf9 cells infected with baculovirus
were lysed with PBS containing 1% Nonidet P-40 and 0.35 mg/ml
phenylmethylsulfonyl fluoride and then immunoprecipitated with
anti-C-terminal GLUT2 tag antibodies and protein A-Sepharose. 3T3-L1
adipocytes (in 24-well culture dishes) were serum-starved for 3 h
in DMEM containing 0.2% bovine serum albumin. The cells were incubated
with 10
To exclude the possibility that D-3-phosphorylated phosphoinositides
may activate some unknown PI 4-kinase in the membrane fraction,
resulting in the synthesis of D-4-phosphorylated phosphoinositides, purified D-3-phosphorylated phosphoinositides were prepared by incubating purified PI, PI-4-P, or PI-4,5-P2 with
nonradioactive ATP and p110 In Vivo Generation of 32P-Labeled
Phosphoinositides--
3T3-L1 adipocytes infected with an adenovirus
containing p110
A similar procedure was performed in the experiments using Sf9
cells. Sf9 cells infected with the indicated baculovirus were also phosphate-starved for 18 h and then labeled with
[32P]orthophosphate (0.1 mCi/ml) for 2 h.
HPLC Analysis of Phosphoinositides--
The extracted lipid was
deacylated and subjected to anion-exchange HPLC using a Partisphere
strong anion-exchange column (Whatman) as described previously (14).
The radioactivity was detected with an on-line radiochemical detector.
Deacylated [3H]PI-4-P and
[3H]PI-4,5-P2 were used as internal
standards. 32P-Labeled PI-3-P, PI-3,4-P2, and
PI-3,4,5-P3 generated in vitro as described
above were also deacylated and used as internal standards. Each sample
and standard were co-injected with the nonradioactive nucleotides ADP
and ATP for each HPLC analysis. Deacylated PI-4-P and
PI-4,5-P2 were successfully separated from deacylated
PI-5-P and PI-3,5-P2, as reported previously (15).
p85/p110-type PI kinase has been identified through its ability to
associate with many tyrosine-phosphorylated substrates and has been
considered to phosphorylate the D-3 position of the inositol ring in
phosphoinositides (6, 16-18). However, of great interest are our
findings that the lipid products of p85/p110-type PI kinase in
vivo differ markedly from those produced by a conventional in vitro method used to determine the substrate specificity
of each PI kinase.
First, recombinant baculoviruses that express either p110
Kin
and Kin
, respectively). In addition, both PI 3- and PI 4-kinase activities of p110 and p110
immunoprecipitates were similarly inhibited by either wortmannin or
LY294002, specific inhibitors of p110. Insulin induced phosphorylation
of not only the D-3 position, but also the D-4 position. Indeed,
overexpression of p110 in Sf9 or 3T3-L1 cells induced marked
phosphorylation of the D-4 position to a level comparable to or much
greater than that of D-3, whereas inhibition of endogenous
p85/p110-type PI kinase via overexpression of dominant-negative p85
(p85) in 3T3-L1 adipocytes abolished insulin-induced synthesis of
both. Thus, p85/p110-type PI kinase phosphorylates the D-4
position of phosphoinositides more efficiently than the D-3 position
in vivo, and each of the D-3- or D-4-phosphorylated
phosphoinositides may transmit signals downstream.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and p110 phosphorylate the D-4 position of
phosphoinositides more efficiently than the D-3 position in
vivo. Thus, p85/p110-type PI kinase may activate downstream
targets by increasing not only D-3-phosphorylated (PI-3-P,
PI-3,4-P2, and PI-3,4,5-P3), but also D-4-phosphorylated (PI-4-P and PI-4,5-P2) phosphoinositides.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]ATP
and [32P]orthophosphate were obtained from ICN. All other
reagents from commercial sources were of analytical grade.
antibodies were raised against
synthetic peptides corresponding to residues 1048-1068 of p110.
Antibodies against the C-terminal GLUT2 tag were prepared as described
previously (7, 8).
from human embryonic heart cDNA, based on its
reported sequence (9). The entire coding region of p110 cDNA was
obtained as described in our previous report (7). The cDNAs coding
for the kinase-dead point mutants of p110 (Kin) and p110
(Kin), which lack amino acids 917-950 of p110 and amino acids
921-954 of p110, were designed as reported previously (10, 11). A
portion of human GLUT2 cDNA corresponding to residues 510-524 was
ligated to each cDNA to generate catalytic subunits of PI 3-kinase
tagged at their C termini.
,
p110, and their kinase-dead point mutants, all of which have an
epitope GLUT2 tag at their C termini, were cloned into transfer vectors
(either pBacPAK8 or pBacPAK9). The entire coding region of LacZ from
Escherichia coli was also cloned into pBacPAK8. Recombinant
baculoviruses were obtained by homologous recombination between the
recombinant transfer vectors and their parental virus genome according
to the manufacturer's instructions (CLONTECH). To obtain recombinant adenoviruses, the expression cosmid cassette pAdexCAwt was ligated with
each of the cDNAs coding for either LacZ from E. coli or epitope-tagged p110, followed by homologous recombination between the recombinant cosmid cassette and its parental virus genome, as
described previously (7). Recombinant adenovirus expressing p85
was prepared as described previously (12).
6 M insulin for 5 min; washed with
ice-cold PBS; and then lysed with PBS containing 1% Nonidet P-40, 0.35 mg/ml phenylmethylsulfonyl fluoride, and 100 mM sodium
vanadate. Cell lysates were precleared of insoluble materials by
centrifugation (15,000 × g, 4 °C, 10 min) and were
subjected to immunoprecipitation with anti-phosphotyrosine antibodies
and protein A-Sepharose. The PI 3-kinase activity in the
immunoprecipitates was measured as described previously (7). When
indicated, the membrane fraction of Sf9 cells, prepared as described previously (13), was used as the substrate instead of
purified PI from a commercial source. The reaction products were
deacylated and analyzed by HPLC. In some experiments, wortmannin or
LY294002 at the indicated concentrations was added to the reaction mixture of the in vitro PI kinase assay, and the inhibitory
effects on 32P-labeled phosphoinositide synthesis were investigated.
immunoprecipitates in vitro.
Phosphoinositides labeled with unlabeled phosphate groups on their D-3
positions (PI-3-P, PI-3,4-P2, and PI-3,4,5-P3)
were separated by TLC, followed by chloroform extraction. Each of them
was dried and mixed with Sf9 cell membrane fraction in the
presence of [-32P]ATP.
, p85, or control LacZ DNA were
phosphate-starved overnight in phosphate-free DMEM (Life Technologies,
Inc.), followed by serum starvation for 3 h.
[32P]Orthophosphate (0.1 mCi/ml) was added, and the cells
were cultured for an additional 2 h. Following the labeling
period, cells were incubated with or without 106
M insulin for 15 min. The reaction was then terminated with
an ice-cold PBS wash, followed by the addition of methanol and 1 N HCl (1:1) and finally lipid extraction with chloroform.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
or p110
with a tagged epitope at their COOH terminus were prepared. Sf9
cells were infected with either p110 or p110 recombinant virus or
control LacZ virus, and similar expression levels were obtained among
them (Fig. 1A).
Immunoprecipitates with antibodies against the tag were subjected to
in vitro lipid kinase assays using purified PI as
substrates, followed by HPLC analysis (Fig. 1, B and
C). The major product was PI-3-P with p110
immunoprecipitates, although small amounts of PI-4-P and
PI-3,4-P2 were also detected. Based on the amounts of
PI-3-P, PI-4-P, and PI-3,4-P2 produced, the in
vitro lipid kinase activity of p110 immunoprecipitates at the
D-4 position was estimated to be 4% of that at the D-3 position. On
the contrary, immunoprecipitates of p110, another isoform of the
catalytic subunit, produced <1% of the PI-3-P produced by p110
immunoprecipitates, and no generation of PI-4-P or
PI-3,4-P2 was detected. These results indicate that the
catalytic subunits of p85/p110-type PI kinase, p110 and p110, are
indeed PI kinases for the D-3 position.
View larger version (25K):
[in a new window]
Fig. 1.
In vitro generation of
phosphoinositides from purified PI by p110 and
p110 expressed in Sf9 cells.
Sf9 cells were infected with recombinant baculoviruses
containing control LacZ (Cont.), p110, or p110
(A). These cells were lysed and immunoprecipitated with
anti-C-terminal GLUT2 tag antibodies. The washed immunoprecipitates
were then separated by SDS-polyacrylamide gel electrophoresis and
immunoblotted with anti-C-terminal GLUT2 tag antibodies.
Anti-C-terminal GLUT2 tag immunoprecipitates from Sf9 cells
overexpressing LacZ (B, upper panel), p110
(middle panel), or p110 (lower
panel) were subjected to PI kinase assay. Purified PI and
[-32P]ATP were mixed with the immunoprecipitates as
described under "Experimental Procedures." The
32P-labeled phosphoinositides generated were separated by
HPLC (B), yielding three peaks corresponding to PI-3-P,
PI-4-P, and PI-3,4-P2, which were quantified with an
on-line radiochemical detector (C). Three other separate
experiments yielded similar results.
Quite different results were obtained when trichloroacetic acid-treated
cell membranes were used as substrates for p110 or p110, instead
of purified PI. Treating the cell membranes with 10% trichloroacetic
acid apparently denatures the protein contained in the cell membrane
fraction, and trichloroacetic acid-treated cell membrane did not
significantly incorporate 32P into the lipid (data not
shown). In the assay using trichloroacetic acid-treated membrane from
Sf9 cells as a substrate (Fig.
2B), p110
immunoprecipitates produced PI-4-P in an amount comparable to that of
PI-3-P. Similarly, PI-4-P and PI-3-P were detected in p110
immunoprecipitates, and the level of the former was ~5.4 times the
level of the latter. The amount of PI-3-P produced in p110
immunoprecipitates was 19% of that produced in p110
immunoprecipitates, whereas nearly the same amount of PI-4-P was
produced in p110 and p110 immunoprecipitates.
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To rule out the possible contamination of PI 4-kinase in the p110
immunoprecipitates and to confirm that p110 itself possesses PI
4-kinase activity, we performed two experiments using kinase-dead point
mutants of p110 (Fig. 2, A and B) and lipid
kinase inhibitors (Fig. 2, C and D). First,
p110, p110, and their kinase-dead point mutants (Kin and
Kin, respectively) were expressed at a similar level in
Sf9 cells (Fig. 2A). It was shown that the lipid
kinase activities of the p110 proteins for both the D-3 and D-4
positions were completely abolished by introducing kinase-dead point
mutations in their lipid kinase domains (Fig. 2B). In
addition, both PI 3- and PI 4-kinase activities of p110 or p110
immunoprecipitates were inhibited similarly by either wortmannin or
LY294002 (Fig. 2, C and D). Wortmannin and
LY294002 are recognized as specific inhibitors of p110, with much
higher concentrations being necessary for the inhibition of other lipid
kinases (19, 20). Subsequently, to exclude the possibility that
D-3-phosphorylated phosphoinositides may activate some unknown PI
4-kinase in the membrane fraction, resulting in the synthesis of
D-4-phosphorylated phosphoinositides, purified nonradioactive
D-3-phosphorylated phosphoinositides (PI-3-P, PI-3,4-P2, or
PI-3,4,5-P3) were added to a mixture of trichloroacetic acid-treated cell fraction and [-32P]ATP. As a result,
no D-4-phosphorylated phosphoinositide labeled with 32P was
observed (Fig. 2B, last column), which excludes
the possibility mentioned above. In addition, when purified PI-3-P,
PI-3,4-P2, or PI-3,4,5-P3 labeled with
32P at the D-3 position was incubated with trichloroacetic
acid-treated cell membrane, no phosphoinositide labeled with
32P at the D-4 position was synthesized (data not shown).
This indicates that transfer of 32P from the D-3 position
to the D-4 position is quite unlikely. Taken together, it is very
likely that the PI 4-kinase activities observed in the p110
immunoprecipitate are attributable to p110 itself.
Subsequently, to demonstrate that p85/p110-type PI kinase actually
exhibits PI 4-kinase activity in vivo, either p110 or p110 was first overexpressed in Sf9 cells, followed by
[32P]orthophosphate labeling. Sf9 cells
overexpressing either p110 or p110 at a similar level
significantly accumulated each phosphoinositide, as compared with
control cells, the exception being PI-3,4,5-P3 in
p110-overexpressing cells (Fig. 3).
The increases in PI-4-P and PI-4,5-P2 were comparable to
the increase in PI-3-P induced by p110 overexpression and even
greater than the increases in PI-3,4-P2 and
PI-3,4,5-P3, which suggests that p110 overexpression induces accumulation of not only D-3-phosphorylated, but also D-4-phosphorylated phosphoinositides in vivo. Essentially
the same results were obtained with the overexpression of both p110 and
its regulatory subunit, p85 (data not shown). p110 overexpression induced accumulation of D-4-phosphorylated phosphoinositides at a much
higher level than that of D-3 in Sf9 cells, which suggests that
p110 may function as a PI 4-kinase predominantly in intact cells.
Further study should be addressed to determine the mechanism that
causes the difference in substrate specificity between p110 and
p110.
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Finally, to demonstrate that endogenous p85/p110 produces not only
D-3-phosphorylated, but also D-4-phosphorylated phosphoinositides, and
to examine the effect of insulin on the level of phosphorylation of
phosphoinositides, either p110 or dominant-negative p85 was overexpressed in 3T3-L1 adipocytes, and the cellular level of each
phosphoinositide in the absence or presence of insulin was investigated. p110 was overexpressed in 3T3-L1 adipocytes using an
adenovirus expression system (Fig.
4A, upper panel) at
a level approximately five times that expressed endogenously in 3T3-L1 cells (lower panel). The cellular ATP level was essentially
unaltered by p110 overexpression (data not shown). The amount of
overexpressed p110 with the adenovirus expression system is much
smaller than that with the baculovirus expression system. Thus,
increases in the amounts of PI-3,4-P2,
PI-4,5-P2, and PI-3,4,5-P3 accumulated in
3T3-L1 adipocytes (Fig. 4B) were smaller than those in
Sf9 cells (Fig. 3), but such increases were still obvious.
Interestingly, the increase in PI-4-P in 3T3-L1 adipocytes
overexpressing p110 was much larger than that in PI-3-P, although
they were almost comparable in Sf9 cells overexpressing p110.
The reason for these differences according to cell type remains
unclear, but the conformation of p110 may differ somewhat by an unknown
modification, such as serine/threonine phosphorylation, according to
whether p110 is expressed in Sf9 cells or in 3T3-L1
adipocytes.
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Insulin showed no additive effect on the amount of each
phosphoinositide in p110-overexpressing cells, which also agrees well with our previous report that no additive effect of insulin and
p110 overexpression on GLUT4 translocation to the cell surface was
observed (7). In addition, very clear results were obtained in the
experiment using p85 with a hemagglutinin tag at its C terminus
(Fig. 4C, upper panel). By overexpressing
p85, which inhibits the normal activation of endogenous p110, we
were able to investigate the function of endogenous p85/p110. In this
experiment, the expression level of p85 was ~10 times that of
endogenous p85 (Fig. 4C, middle panel), which
almost completely inhibited the insulin-induced activation of
endogenous p110 (lower panel). To determine precisely the
effect of insulin on phosphoinositide phosphorylation, we calculated
the amount of freshly labeled phosphoinositide during 15 min of insulin
stimulation by subtracting the radioactivity from that labeled with
32P during the 2-h incubation before insulin treatment. As
indicated in Fig. 4D (by the numbers outside the
parentheses), in the control cells, insulin raised
32P-labeled PI-3-P, PI-4-P, and PI-4,5-P2 to
7.3, 35.0, and 3.4 times the respective levels of their freshly labeled
counterparts without stimuli. By overexpressing p85,
insulin-stimulated production of PI-3,4-P2,
PI-4,5-P2, and PI-3,4,5-P3 decreased to nearly
undetectable levels and that of PI-3-P and PI-4-P decreased by ~70
and 98%, respectively (Fig. 4D). These results clearly
indicate that insulin induces accumulation of both D-3- and
D-4-phosphorylated phosphoinositides via the activation of endogenous
p85/p110-type PI kinase. In addition, it should be noted that
p85/p110-type PI kinase, but none of the other PI-4 kinases, appears to
be critical for the insulin-induced synthesis of D-4-phosphorylated
phosphoinositides in 3T3-L1 cells.
As yet, there have been no reports of p85/p110-type PI kinase with PI
4-kinase activities or that growth factors/hormones stimulate PI
4-kinases. There are two possible explanations for these going
unnoticed. One is that researchers have assumed that the in
vitro PI kinase assay using purified PI as a substrate reflects
the physiological reaction that takes place intracellularly. However,
phosphoinositides are a rather minor component of phospholipids, usually composing <10%. The remaining phospholipids are
phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine.
Without these components around phosphoinositides, the conformation of
lipid kinases might be altered. This may explain the exhibition by
p85/p110-type PI kinase of a substrate specificity different from that
observed with purified PI, as was reported for some isoforms of protein kinase C (21). In addition, recently, the substrate specificity of PI
4-P-5-kinases, which has been reported based on in vitro assay, was also corrected (15). Thus, it is understandable that results
obtained from the artificial in vitro assay do not reflect the actual intracellular situation. The other possible reason is that
many investigators employed stimulation with epidermal growth factor,
nerve growth factor, or platelet-derived growth factor to analyze the
function of p85/p110-type PI kinase (22-24). Stimulation with these
growth factors does activate p85/p110-type PI kinase, but phospholipase
C, which hydrolyzes PI-4,5-P2 (25), is also activated.
Thus, as reported previously, epidermal growth factor, nerve growth
factor, or platelet-derived growth factor does not increase the amount
of PI-4,5-P2, despite the possibly generation by activated
p85/p110-type PI kinase of PI-4,5-P2 probably via PI-4-P
production. Because of this complicated alteration in the amount of
D-4-phosphorylated phosphoinositide, it seems that earlier studies did
not pay attention to the effect of p85/p110-type PI kinase on the
amount of D-4-phosphorylated phosphoinositide. In contrast, insulin
stimulation does not lead to phospholipase C activation (26). Thus,
the generation of D-4-phosphorylated phosphoinositides (PI-4-P and
PI-4,5-P2) by p85/p110-type PI kinase became as apparent as
that of D-3-phosphorylated phosphoinositides (PI-3-P,
PI-3,4-P2, and PI-3,4,5-P3). However, even in
the case of insulin stimulation, since the cell has significant basal
PI 4-kinase activity, if cells are prelabeled with 32P for
too long a period (12 h or longer), the effect of insulin for a very
short time would be difficult to detect. This is the reason we labeled
the cells for 2 h. In the living body, cells are always stimulated
by many factors in the serum. It is thus reasonable to consider the PI
4-kinase activity of p85/p110 to be important for the regulation of
D-4-phosphorylated phosphoinositides since p85/p110 is likely to be the
only enzyme that phosphorylates the D-4 position in response to insulin.
The importance of D-4-phosphorylated phosphoinositides has been
established by several investigators. PI-4,5-P2 regulates the function of several actin-binding proteins (27-31) and is likely to mediate certain processes of protein sorting or secretion (32, 33),
membrane ruffling (34), and the formation of focal adhesions (28), some
of which reportedly occur in response to growth factor/hormone stimuli
in parallel with p85/p110-type PI kinase activation. Thus, some of
these cellular events might be attributable to D-4-phosphorylated phosphoinositides produced by p85/p110-type PI kinase. In addition, PI-4,5-P2 plays an important role as a precursor for the
second messengers diacylglycerol, inositol 1,4,5-P3 (25),
and PI-3,4,5-P3 (22). In this respect, the cellular content
of PI-4,5-P2 might affect the signal intensity of
phospholipase C. Our surprising findings also raise the possibility
that the substrate specificity of various lipid kinases and
phosphatases, when incubated with purified phosphoinositide in an
in vitro assay, might be different from the true,
i.e. cellular, substrate specificity.
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ACKNOWLEDGEMENTS |
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We thank Drs. I. Saito and Y. Kanegae for helpful advice and the generous donation of recombinant pAdex1CAlacZ, the expression cosmid cassette, and the parental adenovirus DNA-terminal protein complex. We thank Professor T. Takenawa, Dr. K. Fukami, and Dr. F. Shibasaki for valuable discussions.
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FOOTNOTES |
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* This work was supported by Grant-in-aid 09470214 for Scientific Research (to T. A.) from the Ministry of Education, Science, and Culture of Japan.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. Tel.:
81-3-3815-5411 (ext. 3133); Fax: 81-3-5803-1874 or 81-3-3344-6275;
E-mail: asano- tky@umin.ac.jp.
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ABBREVIATIONS |
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The abbreviations used are: PI, phosphatidylinositol; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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