From the Department of Biochemistry, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108 and CREST, Japan Science and Technology Corporation, Tokyo 108, Japan
Received for publication, November 8, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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Phosphatidylinositol 4-phosphate 5-kinase (PIPK)
catalyzes a final step in the synthesis of phosphatidylinositol
4,5-bisphosphate (PIP2), a lipid signaling
molecule. Strict regulation of PIPK activity is thought to be
essential in intact cells. Here we show that type I enzymes of PIPK
(PIPKI) are phosphorylated by cyclic AMP-dependent protein
kinase (PKA), and phosphorylation of PIPKI suppresses its activity.
Serine 214 was found to be a major phosphorylation site of PIPK type
I Phosphatidylinositol 4,5-bisphosphate
(PIP2)1 is a
signal-generating phospholipid with crucial roles in various cellular
processes. PIP2 is the best substrate for
phosphoinositide-specific phospholipase C, and PIP2
hydrolysis generates two second messengers, 1,2-diacylglycerol (DG) and
inositol 1,4,5-trisphosphate. Inositol 1,4,5-trisphosphate binds to
specific receptors and induces release of calcium from intracellular
stores (1), whereas DG activates protein kinase C (PKC) (2). In
mammalian cells, PIP2 can be further phosphorylated by
phosphoinositide 3-kinase for generation of phosphatidylinositol 3,4,5-trisphosphate, a mediator of cell growth and survival (3, 4). In
addition, PIP2 directly modulates the activity of numerous enzymes and proteins involved in diverse cellular processes including exocytosis (5), cytoskeletal re-organization (6-8), and membrane trafficking (9).
Consistent with this important role played by PIP2 in
cellular signaling, intracellular PIP2 levels are strictly
regulated (10, 11). In response to various extracellular stimuli, a hydrolysis of PIP2 has taken place, and its levels are
rapidly decreased, resulting in the shortage of PIP2.
However, since compensatory synthesis of PIP2 is rapidly
induced, PIP2 levels are constantly maintained, and the
subsequent generation of second messengers inositol 1,4,5-trisphosphate
and DG is renewed (12).
PIP2 is synthesized from phosphatidylinositol (PI) by two
lipid kinases, phosphatidylinositol kinase (PIK) and PIPK. At least two
immunologically distinct PIPK subtypes, PIPK type I and PIPK type II
(13), exist in mammalian cells and are composed of three isoforms A variety of stimuli, including GTP Considering this, we hypothesized that PIPKI itself is a substrate for
some protein kinase in response to extracellular stimuli, which allows
it to regulate the enzymatic activity. In particular, this is
reasonable, since PIPKI from plasma membrane of
Schizosaccharomyces pombe is phosphorylated by casein kinase
I and is thereby inactivated (33), and activation of PKC by phorbol
ester PMA treatment, which leads to phosphorylation of cellular
proteins, stimulates PIP2 synthesis (22, 23). Moreover,
tyrosine phosphoproteins associated with EGF receptor have PI4K and
PIPK activity (24, 28). Despite these data, there is still no direct
evidence for regulation of PIPK activity by phosphorylation in
mammalian cells. Thus, in this study, we examine whether there is a
kinase that catalyzes PIPK phosphorylation and thereby regulates the activity.
Here we show that PIPKI is phosphorylated and inactivated by protein
kinase A (PKA) both in vitro and in vivo, and we
identify the PKA phosphorylation site of PIPKI Materials--
PI4P was purified by neomycin column
chromatography from crude phospholipids extracted from bovine spinal
cord (34). [ Cell Culture--
NIH 3T3 cells were cultured in Dulbecco's
modified Eagle's medium supplemented with 10% bovine calf serum.
Serum starvation was performed by culturing cells in Dulbecco's
modified Eagle's medium without serum for 12-24 h.
Transient Expression and Stimulation of NIH 3T3
Cells--
Full-length cDNAs encoding mouse PIPKI Measurement of PIPK Activity--
Expression plasmid-transfected
NIH 3T3 cells were lysed with lysis buffer (20 mM Tris-HCl,
pH 7.5, 50 mM NaCl, 30 mM sodium pyrophosphate,
1% Nonidet P-40, 1 mM EGTA, 1% Triton X-100, 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 then 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 PI4P, 50 µM ATP, and 1 µCi of
[
All assays were performed in the linear range with regard to the
protein amounts and the incubation time in each assay system.
In Vitro Phosphorylation of PIPKI by PKA--
Phosphorylation of
PIPKI by PKA was carried out as described previously (36). 1 µg of
GST-PIPKI protein was immobilized on 20 µl of glutathione-Sepharose
beads and then incubated in 50 µl of 25 mM Tris-HCl, pH
7.5, containing 5 mM MgCl2, 2 mM
EGTA, 1 µCi of [ Dephosphorylation of PIPKI by Alkaline
Phosphatase--
Myc-tagged PIPKI was immunoprecipitated from lysates
of transfected NIH 3T3 cells. The immunoprecipitates were washed twice with lysis buffer and then once 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). Immunoprecipitates
were subsequently resuspended in 2 units of calf intestine alkaline phosphatase (Takara Shuzo Co., Ltd., Tokyo, Japan) in 10 mM
Tris-HCl, pH 8.0, 1 mM MgCl2, 50 mM
KCl, 0.1 mM ZnCl2, and 50% glycerol and
incubated at 30 °C for 60 min.
Dephosphorylation of PIPKI Metabolic 32P Labeling and Phosphoamino Acid
Analysis--
NIH 3T3 cells were transfected with Myc-tagged PIPKI Tryptic Peptide Mapping--
Tryptic peptide mapping was
performed as described previously (37). 32P-Labeled PIPKI
was digested twice with 10 µg of tosylsulfonylphenylalanyl chloromethyl ketone-treated trypsin. Released phosphopeptides were
spotted onto on cellulose thin layer plates and separated first by
electrophoresis at 1,000 V in pH 1.9 buffer for 27 min and then by
ascending chromatography in n-butyl
alcohol/pyridine/acetic acid/water (75:50:15:60).
PIPKI Is a Phosphoprotein--
Anti-Myc antibody
precipitates of Myc-tagged PIPKI expressed in NIH 3T3 cells appeared as
broad bands on Western blots. Thus, we first examined whether these
electrophoretic mobility shifts are caused by phosphorylation of PIPKI.
After NIH 3T3 cells expressing Myc-PIPKI Phosphorylation of PIPKI by PKA in Vivo Suppresses PIPKI
Activity--
To identify the kinase chiefly responsible for
phosphorylation of PIPKI in NIH 3T3 cells, the effects of various
protein kinase inhibitors, the PKC inhibitor H7, the PKA inhibitor H89,
the calmodulin dependent kinase inhibitor KN-62, and tyrosine kinase
inhibitor genistein on PIPKI
Next, to determine whether the enzymatic activity of PIPKI is affected
by its phosphorylation, the activity of Myc-PIPKI PKA Catalyzes the Phosphorylation and Suppresses the Activity of
PIPK in Vitro--
The effect of PKA-catalyzed phosphorylation of
PIPKI
In addition, treatment of Myc-PIPKI
To determine directly the effect of phosphorylation on the activity of
PIPKI, activity of PIPKI PKA Catalyzes Phosphorylation of PIPKI
To confirm this, peptide-map analysis was performed on tryptic cleavage
fragments of wild type and mutant proteins phosphorylated by PKA
in vitro. Three major phosphopeptides were detected in two-dimensional maps of wild type PIPKI Mechanism of LPA-induced Activation of PIPKI
To assess the requirement of Ca2+ in this regulation, we
next used a combination of EGTA and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, antagonist of Ca2+ mobilization and
Ca2+ entry. Interestingly, LPA-induced dephosphorylation
and activation of PIPKI
Furthermore, we examined whether major serine/threonine phosphates such
as protein phosphatase type 1 (PP1) and protein phosphatase type 2 (PP2A) are involved in the LPA-induced dephosphorylation of PIPKI PP1 Dephosphorylates and Activates PIPKI In the present study, we show that PIPKI is a phosphoprotein, and
the PIPKI phosphorylation state is dynamically regulated by the
opposing actions of a PIPKI kinase and a PIPKI phosphatase in response
to external stimuli (Fig. 9). Most of the
PIPKI (PIPKI
) that is catalyzed by PKA. In contrast, lysophosphatidic
acid-induced protein kinase C activation increased PIPKI
activity.
Activation of PIPKI
was induced by dephosphorylation, which was
catalyzed by an okadaic acid-sensitive phosphatase, protein phosphatase
1 (PP1). In vitro dephosphorylation of PIPKI
with PP1
increased PIPK activity, indicating that PP1 plays a role in
lysophosphatidic acid-induced dephosphorylation of PIPKI
. These
results strongly suggest that activity of PIPKI
in NIH 3T3 cells is
regulated by the reversible balance between PKA-dependent phosphorylation and PP1-dependent dephosphorylation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
(13-18). Most PIP2 synthesis is catalyzed by
type I enzymes, which phosphorylate the D-5 position of the inositol
ring of PI 4-phosphate (PI4P). Recently, type II enzymes have been
reported to be a PI 5-phosphate 4-kinase (19), which suggests the
presence of an alternative pathway of PIP2 synthesis catalyzing phosphorylation of PI 5-phosphate (PI5P).
S (20, 21), phorbol esters (22,
23), tyrosine phosphatase inhibitors (24), integrin (25), and EGF
(26-28), have been reported to regulate PIP2 synthesis. Type I enzymes were shown to be activated by acid phospholipids, especially phosphatidic acid (29), and to be regulated by GTP-binding proteins, Rho (30, 31) and Rac (32) in a manner dependent on GTP
S.
In EGF-induced membrane ruffling, PIPKI
is physiologically downstream of the small G protein ADP-ribosylation factor, ARF6 (32).
However, a lot of parts are not yet clear to explain how PIPK activity
is regulated by extracellular stimulation during a few minutes in cells.
. Furthermore, we
demonstrate that activation of PIPKI
in response to LPA treatment is
induced by dephosphorylation of PIPKI
. Our findings suggest that
phosphorylation and dephosphorylation of PIPKI
are important for
regulation of PIP2 biosynthesis and phospholipid signaling
in cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP and
[32P]orthophosphate were from PerkinElmer Life Sciences.
Polyvinylidene difluoride membranes for Western blot analysis were from
Nihon Eido (Tokyo, Japan). Thin layer chromatography silica plates and cellulose plates for separation of phospholipids, phosphoamino acids,
and phosphopeptides were from Merck. Monoclonal and polyclonal anti-Myc
antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). Protein kinase inhibitors H7, H89, KN-62, and genistein were
purchased from Seikagaku Co. (Tokyo, Japan). Okadaic acid and
calphostin C were from Wako Life Science Reagents (Tokyo, Japan). LPA
and PMA were from Sigma. PKA catalytic subunit was from Promega
(Madison, WI).
, PIPKI
,
and PIPKI
were ligated into the SalI-BamHI
site of pCMV-Myc or the BamHI site of pEF-BOS-Myc mammalian
expression vectors. NIH 3T3 cells were seeded into 60-mm dishes at a
density of 2 × 105 cells per dish and cultured
overnight. 15 µg of expression plasmid was transfected into cells by
the Ca2+ phosphate method (35). After incubation for 4 h, cells were washed with fresh medium and cultured for an additional
12 h. In stimulation experiments, the culture medium was aspirated
and replaced with fresh serum-free medium at least 12 h prior to
stimulation. In protein kinase or phosphatase inhibitor experiments,
various inhibitors were added throughout the preincubation for times
indicated prior to addition of growth factors.
-32P]ATP in 50 µl. After incubation for 10 min at
room temperature, the lipids were extracted with 1 N HCl
and chloroform/methanol (2:1, by volume) and spotted onto TLC plates.
The plates were developed in chloroform/methanol/acetone/acetic
acid/water (40:13:15:12:7), and the products were observed by
autoradiography or quantified with a Fuji BAS 2000 image analyzer.
-32P]ATP, and 0.1 µg of PKA
catalytic subunit for 30 min at room temperature. The reaction was
stopped by addition of SDS sample buffer or ice-cold phosphate-buffered
saline (PBS). The reaction mixture was centrifuged, and the beads were
subsequently washed three times with PBS and then subjected to PIPK
activity assay or SDS-polyacrylamide gel electrophoresis and Western
blot analysis.
by Protein Phosphatase--
1 µg
of GST-PIPKI
immobilized on beads was phosphorylated with PKA and
subsequently washed first with PBS and then dephosphorylation buffer
(50 mM MOPS, pH 7.5, 1 mM MnCl2,
150 mM NaCl, and 2 mM EGTA). The resulting
beads were incubated in 50 µl of dephosphorylation buffer containing
0.5 units of PP1 or PP2A (Upstate Biotechnology Inc.) at 30 °C
for 30 min.
and placed in fresh serum-free medium for at least 12 h prior to
initiation of metabolic 32P labeling experiments. Cells
were subsequently incubated in phosphate-free Dulbecco's modified
Eagle's medium for 2 h and then labeled in the same medium
containing [32P]orthophosphate (0.2 mCi/ml) for 6 h.
After Bt2cAMP treatment, cells were lysed in lysis
buffer, and PIPKI
was immunoprecipitated with anti-Myc antibody.
Immune complexes were washed once in lysis buffer and then subjected to
SDS-polyacrylamide gel electrophoresis and autoradiography. The
radioactive band corresponding to PIPKI
was cut out of the gel and
subjected to phosphoamino acid and tryptic peptide mapping.
Phosphorylated proteins excised from gel bands were hydrolyzed in 6 N HCl for 3 h at 110 °C. The resulting amino acids,
together with standard phosphoamino acids, were spotted onto cellulose
thin layer plates and were separated by two-dimensional electrophoresis
in 2.5% formic acid and 7.8% acetic acid, pH 1.9, and then in 5%
acetic acid and 0.5% pyridine, pH 3.5. Labeled phosphoamino acids were
detected by autoradiography. Positions of the standard phosphoamino
acids were detected by ninhydrin staining.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, or
were labeled
with [32P]orthophosphate, the cell lysates were
immunoprecipitated with anti-Myc antibody and subjected to
SDS-polyacrylamide gel electrophoresis and autoradiography. As shown in
Fig. 1, phosphorylation of all type I
enzymes was evident in intact cells. These results show that the
mobility shift of Myc-PIPKI was caused by phosphorylation.
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Fig. 1.
PIPKI is a phosphoprotein. Western blot
and [32P]orthophosphate labeling of PIPKI are shown. NIH
3T3 cells were expressed with Myc-tagged PIPKI three isoforms (I ,
I
, and I
) and then labeled with [32P]orthophosphate
(0.2 mCi/ml) for 6 h. Expressed PIPKI proteins were
immunoprecipitated with anti-Myc antibody and subjected to
SDS-polyacrylamide gel electrophoresis. The results were detected by
Western blot analysis with anti-Myc antibody and autoradiography.
I.B., immunoblot.
phosphorylation were tested.
Surprisingly, H89 at 50 µM severely suppressed the shift
in mobility of Myc-PIPKI
, suggesting that phosphorylation of
PIPKI
occurred in a PKA-dependent manner. In contrast,
no significant shift in PIPKI
mobility was observed in response to
treatment of NIH 3T3 cells with any of the others inhibitors (Fig.
2A).
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Fig. 2.
PIPKI is phosphorylated and suppressed by PKA
in vivo. A, specific inhibition of
PIPKI phosphorylation by H89. NIH 3T3 cells were expressed with
Myc-PIPKI
and then treated with 50 µM H7, H89, KN62,
or genistein (Gen.) for 30 min, respectively.
Immunoprecipitated PIPKI
was subjected to Western blot analysis with
anti-Myc antibody or PIPK activity assay. The kinase reaction was
carried out in the presence of 50 µM PI4P for 10 min at
room temperature. cont., control. B,
phosphorylation by PKA is conserved in all PIPKI isoforms. NIH 3T3
cells expressing Myc-PIPKI three isoforms were treated with 50 µM H89 for 30 min. Western blot analysis with anti-Myc
antibody and PIPK activity assay were performed.
immunoprecipitated
from NIH 3T3 cells treated with or without H89 was measured. PIP kinase
activity was clearly elevated in cells treated with H89, amounting to
about 150% of that in cells not treated or treated with other kinase
inhibitors (Fig. 2A). Moreover, H89 inhibited
phosphorylation of all PIPKI isoenzymes (Fig. 2B). Thus,
type I PIPK enzymes appear to be phosphorylated and inactivated by
PKA.
, PIPKI
, and PIPKI
on their activities was tested
in vitro with the corresponding GST-PIPKI fusion proteins
expressed in and purified from Escherichia coli cells.
Incubation of PIPKI proteins with the catalytic subunit of PKA and
[
-32P]ATP resulted in phosphorylation and 50%
inhibition of all PIPKI subtypes (Fig.
3A).
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Fig. 3.
Phosphorylation of PIPKI
by PKA in vitro suppresses PIP kinase
activity. A, in vitro phosphorylation of
PIPKI by PKA. GST fusion proteins of PIPKI three isoforms were
expressed in E. coli cells. Purified GST proteins were
immobilized on glutathione-Sepharose beads and then incubated with (+)
or without (
) 0.1 µg of PKA catalytic subunit and 1 µCi of
[
-32P]ATP for 30 min at room temperature. The reaction
was stopped by addition of SDS sample buffer or PBS. The resulting
mixture or beads were subjected to SDS-polyacrylamide gel
electrophoresis or PIPK activity assay. B,
immunoprecipitated Myc-PIPKI
was incubated in the presence of 2 units of calf intestine alkaline phosphatase (CIAP) for 60 min at 30 °C or 0.1 µg of PKA for 30 min at room temperature.
cont., control.
immunoprecipitated from NIH 3T3
cell lysates with alkaline phosphatase clearly decreased the
electrophoretic mobility of Myc-PIPKI
and increased its activity by
about 170% (Fig. 3B). In contrast, incubation of the
Myc-PIPKI
immunoprecipitates with PKA catalytic subunit did not
significantly decrease PIPK activity, despite additional mobility
shifts. The shift in mobility of PIPKI
by in vitro PKA
phosphorylation was likely due to phosphorylation of physiologically
irrelevant sites that are not phosphorylated in vivo and do
not affect PIPK activity.
was examined in a
time-dependent manner during phosphorylation by PKA. After
[
-32P]ATP addition, the level of GST-PIPKI
phosphorylation was maximal in 20 min and sustained for at least 1 h. The inhibition time course of PIPKI
activity, (maximally
inhibited to 50% of the control) correlated closely with the
phosphorylation time course by PKA, showing that activity of PIPKI
is regulated by PKA-dependent phosphorylation (Fig.
4).
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Fig. 4.
Time course of PIPKI
inhibition by PKA phosphorylation. 1 µg of GST-PIPKI
immobilized on beads was phosphorylated with PKA and
[
-32P]ATP for the times indicated at room temperature.
The resulting mixtures or beads were subjected to SDS-polyacrylamide
gel electrophoresis and autoradiography or PIPK activity assay. Data
are representative of three similar experiments.
, PIP kinase
activity;
, phosphorylation of PIPKI.
Ser-214--
we next
tried to characterize the phosphorylation sites of PIPKI
by PKA.
Phosphoamino acid analysis of [32P]GST-PIPK I
, which
was produced in vitro by incubation with PKA and
[
-32P]ATP, revealed predominant phosphorylation of
only serine residues (Fig. 5). No
phosphotyrosine was detected by Western blot analysis with PY-20
anti-phosphotyrosine antibody (data not shown). To identify further the
PKA phosphorylation sites of PIPKI
, wild type and several C-terminal
deletion mutants of PIPKI
were constructed and designated I
full,
I
-(1-392), I
-(1-260), and I
-(1-175), respectively.
The wild type and mutants were expressed in E. coli cells to
produce GST fusion proteins. Purified GST proteins were incubated with
the PKA catalytic subunit and [
-32P]ATP, and fusion
protein phosphorylation was quantified. As shown in Fig.
6A, deletion mutants
I
-(1-392) and I
-(1-260) showed similar phosphorylation level to
that of I
full. In contrast, the I
-(1-175) mutant, which
lacks 365 C-terminal residues of PIPKI
, resulted in dramatically
reduced phosphorylation, by only 3.5% of I
full. These results
suggest that the PKA phosphorylation sites exist within a central
region, approximately 85 amino acids between 175 and 260 of PIPKI
.
By interrogating the primary sequence of this region, we found that
there are PKA phosphorylation site consensus sequence (RRXS)
and serine residues conserved in all three PIPKI as shown in the
results of Figs. 1, 2B, and 3A. Based on these
results, serine-to-alanine point mutations were performed in PIPKI
that corresponded to putative PKA phosphorylation sites containing
serine 207 or 214. Purified mutant and wild type PIPKI
proteins were
incubated with PKA and [
-32P]ATP, and the reaction
products were subjected to SDS-polyacrylamide gel electrophoresis and
autoradiography. No significant change in the level of phosphorylation
was observed in the S207A mutant. In contrast, the phosphorylation
level of S214A mutant was 60% less than that of wild type PIPKI
(Fig. 6B). Ser-214 was likely to be a major PKA
phosphorylation site in PIPKI
.
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Fig. 5.
Phosphoamino acid analysis of
PIPKI . 1 µg of GST-PIPKI
was
phosphorylated with PKA and [
-32P]ATP and then
subjected to SDS-polyacrylamide gel electrophoresis and
autoradiography. The radioactive band corresponding to PIPKI
was
excised and hydrolyzed in 6 N HCl for 3 h at
110 °C. The resulting amino acids were separated by two-dimensional
electrophoresis and detected by autoradiography. Positions of standard
phosphoamino acids (PS, phosphoserine; PT,
phosphothreonine; PY, phosphotyrosine) and free
orthophosphate are also indicated.
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Fig. 6.
Determination of PKA phosphorylation site of
PIPKI . A, phosphorylation of
C-terminal deletion mutants. C-terminal deletion mutants of
GST-PIPKI
were generated with endogenous restriction enzymes,
EcoRI, HindIII, and SphI and
designated I
-(1-392), I
-(1-260), and I
-(1-175),
respectively. Wild type, I
full, and deletion mutant proteins were
phosphorylated with PKA and [
-32P]ATP. B,
amino acids between 175 and 260 of PIPKI are aligned. PKA
phosphorylation site consensus sequences (RRXS) are
shaded, and serine residues conserved in all three PIPKI
isoforms are in black. Phosphorylation levels of point
mutant proteins of GST-PIPKI
were tested. C, tryptic
peptide mapping of PIPKI
. NIH 3T3 cells expressing Myc-PIPKI
were
labeled with [32P]orthophosphate. Myc-PIPKI
immunoprecipitated from the labeled cell lysate and GST-PIPKI
phosphorylated with PKA in vitro were digested with trypsin.
Released phosphopeptides were separated first by electrophoresis in pH
1.9 buffer and then by ascending chromatography. Sample origin is
marked by arrow. Main phosphopeptides are numbered.
WT, wild type.
tryptic fragments
(spots 1-3 in Fig. 6C). However, in maps of
S214A mutant, spots 2 and 3 had disappeared. These two spots appear to
be derived from incomplete digestion products by trypsin, whereas spot
1 was not changed. However, surprisingly, tryptic peptide maps of
PIPKI
expressed in NIH 3T3 cells resulted in only two
phosphopeptides, which have disappeared in maps of S214A mutant. In
addition, treatment of the cells with Bt2cAMP neither
induced phosphorylation of additional sites nor significantly increased
the phosphorylation level of Ser-214 (data not shown). Therefore, we
conclude that PIPKI
exists as the Ser-214 phosphorylated form in
most NIH 3T3 cells. Western blot analysis using lysates of NIH 3T3
cells expressing S214A mutant showed that this mutated PIPKI
protein
had migrated to the lowest mobile band (data not shown).
--
It is well
known that PIPK activity is increased in response to various
extracellular stimuli. However, little is known about the signal
pathway that leads to PIPKI
activation. Thus, we first examined how
phosphorylation of PIPKI is changed in response to extracellular
stimuli. As shown in Fig. 7A,
stimulation of NIH 3T3 cells with LPA induced a transient but clear
suppression of PIPKI
phosphorylation, which peaked at 3 min and then
rapidly declined. Good correlation was observed between PIPKI
dephosphorylation and activation. Since LPA activates pathways
involving PKC and/or Ca2+ signaling in a variety of
cells (43-46), NIH 3T3 cells were treated with the phorbol ester
PMA, an activator of PKC. This treatment induced a strong
dephosphorylation, and PIPKI
activity increased by about 150% of
the untreated control at 30 min (Fig. 7B). However, as shown
in Fig. 7C, treatment of NIH 3T3 cells with calphostin C, a
selective PKC inhibitor, completely blocked PIPKI
dephosphorylation in response to LPA.
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Fig. 7.
LPA-induced PKC activation causes
dephosphorylation of PIPKI . A
and B, dephosphorylation of PIPKI
in response to LPA and
PMA treatment. NIH 3T3 cells expressing Myc-PIPKI
were serum-starved
and then stimulated with 50 µM LPA or 100 nM
PMA for the times indicated. Immunoprecipitated PIPKI
was subjected
to Western blot analysis with anti-Myc antibody or PIPK activity assay.
C, inhibition of dephosphorylation of PIPKI
by calphostin
C (CC) or OA. Serum-starved NIH 3T3 cells were pretreated
with 1 µM OA or 100 nM calphostin C for 30 min and then subsequently stimulated with 50 µM LPA.
cont., control.
was not different (data not shown).
Ionophore, A23187, treatment also had no effect (data not shown). Taken
together, these results suggest that a PKC-dependent
pathway, not a Ca2+-dependent pathway, played a
key role in the dephosphorylation and activation of PIPKI
in
response to LPA.
.
Pretreatment of NIH 3T3 cells with okadaic acid (OA), a potent
inhibitor of PP1 and PP2A, also completely blocked dephosphorylation and activation by LPA (Fig. 7C), suggesting that an
OA-sensitive phosphatase, PP1 or PP2A, presumably regulates
dephosphorylation and activation of PIPKI
in response to LPA stimulation.
--
To examine
whether OA-sensitive phosphatase directly participates in
dephosphorylation and activation of PIPKI
, dephosphorylation assays
were performed with 32P-labeled GST-PIPKI
, which was
produced by incubation with PKA and [
-32P]ATP in
vitro. As shown in Fig. 8, PP1
clearly dephosphorylated and activated PIPKI
.
32P-Labeled GST-PIPKI
treated with PP2A was also found
to be dephosphorylated; however, its activity remained unchanged (data
not shown). Taking these results together, we conclude that activation
via dephosphorylation of PIPKI
is catalyzed by PP1.
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Fig. 8.
PP1 dephosphorylates and activates
PIPKI . In vitro
dephosphorylation of PIPKI
by PP1. 1 µg of GST-PIPKI
immobilized on beads was first phosphorylated with PKA and
[
-32P]ATP. The resulting precipitate was washed
extensively and subjected to dephosphorylation. The dephosphorylation
was carried out in the presence of 0.5 units of PP1 for 30 min at
30 °C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
expressed in NIH 3T3 cells existed in fully phosphorylated
form by PKA at serine 214 even in the resting cells, which was likely
to allow PIPKI
to be in an inactive state. In fact, when NIH 3T3
cells expressing PIPKI
were stimulated with Bt2cAMP, we
saw no significant mobility shift (data not shown). However, potential
stimuli such as LPA led to dephosphorylation and subsequent activation
of PIPKI
. PP1 was identified as a primary candidate for LPA-mediated
PKC-dependent phosphatase. In addition, from the results of
Fig. 3B and in vitro dephosphorylation with PP2A,
PP2A is likely to function at another phosphorylation site, not
Ser-214, that is phosphorylated by PKA only in vitro.
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Fig. 9.
Model of PIPKI
activation in response to extracellular stimuli. Activation
of PIPKI
is catalyzed by dephosphorylation in response to LPA
stimulation.
Ser-214 is a major phosphorylation site of PIPKI by PKA in
vitro and in vivo. This site is conserved in both type
I (14) and type II isoenzymes (18). However, type II enzymes do not have perfect PKA phosphorylation site consensus sequences, and we did
not detect any mobility shift of type II
by Western blot analysis
after treatment with either H89 or Bt2cAMP. This is
consistent with results of earlier studies showing that PIPKII
is
phosphorylated by a serine kinase but not by PKA or PKC (18). Thus, in
NIH 3T3 cells, activity regulation via phosphorylation of PIPK type I
is more likely to be important for maintaining intracellular total
PIP2 levels because PI4P, the major substrate for PIPKI, is
extremely abundant than PI5P, the substrate for PIPK type II (19).
Several groups reported that PIP2 synthesis is regulated by
extracellular stimuli. Treatment of HEK-293 cells with tyrosine kinase
inhibitor alters PIP2 levels (38), and Rho protein and phospholipase D activation appear to be involved in this
process. However, involvement of PIPK was also remained unclear, and in the present study, we did not detect any change in phosphorylation or
activation of PIPKI in NIH 3T3 cells in response to treatment with
the tyrosine kinase inhibitor, genistein. Tyrosine-phosphorylated proteins immunoprecipitated with anti-EGF antibody from EGF
receptor-transfected mouse cells or A431 cells, an epidermoid carcinoid
cell line overexpressing EGF receptors, showed PIK activity and PIPK
activity (24, 28). However, in any case, tyrosine phosphorylation by
itself did not alter the activity of PIPK. Thus, it seems likely that
some other tyrosine kinase or phosphotyrosyl protein interacts with
both EGF receptor and PIK or PIPK.
Although involvement of PKC in the inositol phospholipid metabolism has been also investigated in many reports, the exact role PKC plays is not well understood. PMA treatment increased PIP2 levels 1.5-2.5-fold in lymphocytes (23) and in human platelets (22) but did not stimulate PIP2 hydrolysis by phospholipase C. Thus, direct effect on inositol lipid kinase or phosphatase through activation of PKC was suggested (22). Our results in this study provided evidence suggesting that PKC, which is usually activated by the PIP2 hydrolysis product DG, contributes to maintenance of PIP2 level through a feedback regulation of PIPKI in response to receptor activation.
PIP2 synthesis is also regulated by GTP-binding proteins.
Phosphatidic acid (PA)-stimulated PIPK activity was found to be associated with Rac in a GTPS-dependent manner in liver
and Swiss 3T3 cells (39), and interaction between Rho and PIPK activity was demonstrated in mouse fibroblast cell lines (30, 31). In addition,
the ADP-ribosylation factor 1 (ARF1) interacts with PIPK. Godi et
al. (40) and Jones et al. (41) recently showed that
PIPKI and PI4K
are direct effectors of ARF1. ARF1 activated by
GTP
S recruits PIPKI and PI4K
to the Golgi complex, resulting in a
potent stimulation of PIP2 synthesis in Golgi membrane, and these effects were independent of the known activities of ARF on a coat
proteins and phospholipase D. At the Golgi membranes, PIP2 stimulates not only guanine nucleotide exchange factor
for ARF1 but also a GTPase-activating factor for ARF1 in a
PA-dependent manner. Thus, PIP2 up-regulates
and down-regulates ARF activity and, consequently, vesicular
trafficking in Golgi membrane. Therefore, control of PIP2
levels at this cellular compartment is essential. Phosphorylation of
PIPKI by PKA may reduce PIP2 resynthesis in Golgi membrane
and participate in feedback regulation of ARF1 activity. In fact, when
we tested the effect of PA on the activity of PIPK, inhibition of PIPKI
activity by phosphorylation was not affected by PA (data not shown).
Recently, Martin et al. (42) reported that activation of PKA
induces binding of ARF1 to Golgi membranes, and this binding is
mediated by an unknown target protein, which is phosphorylated by PKA
and dephosphorylated by OA-sensitive phosphatase. In addition, this
step is not dependent on guanine nucleotides. Therefore, we speculate
that the target protein that is phosphorylated by PKA is PIPKI and that
PIP2 formed by PIPKI promotes stable association of ARF1
with the Golgi membrane. Instead of guanine nucleotide factors,
PKA-dependent phosphorylation of PIPKI is likely to
regulate activity of ARF1 and thereby control its association with
Golgi membrane. That is, PIPKI first increases recruitment of ARF1 in Golgi membrane through PIP2 generation, and then
inactivation of PIPKI by PKA-dependent phosphorylation
would likely promote the generation or extraction of trafficking
vesicles, thereby subsequently stimulating the ARF recruitment cycle to
Golgi membrane. We expect that further studies with antibodies that
specifically recognize PIPKI phosphorylated at Ser-214 will help
address this speculation.
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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,
Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108, Japan. Tel.: 81-3-5449-5510; Fax: 81-3-5449-5417; E-mail: takenawa@ims.u-tokyo.ac.jp.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M010177200
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ABBREVIATIONS |
---|
The abbreviations used are:
PIP2, phosphatidylinositol 4,5-bisphosphate;
PI, phosphatidylinositol;
PIP, phosphatidylinositol phosphate;
PIK, phosphatidylinositol kinase;
PIPK, phosphatidylinositol phosphate kinase;
PKA, cAMP-dependent
protein kinase;
PKC, protein kinase C;
LPA, lysophosphatidic acid;
OA, okadaic acid;
PP1, protein phosphatase 1;
PP2A, protein phosphatase 2A;
PIPKI, phosphatidylinositol 4-phosphate 5-kinase type I
;
Bt2cAMP, dibutyryl cyclic AMP;
GST, glutathione
S-transferase;
PBS, phosphate-buffered saline;
ARF, ADP-ribosylation factor;
DG, diacylglycerol;
EGF, epidermal growth
factor;
PI4P, PI 4-phosphate;
GTP
S, guanosine
5'-3-O-(thio)triphosphate;
PMA, phorbol 12-myristate
13-acetate;
MOPS, 4-morpholinepropanesulfonic acid;
PA, phosphatidic
acid.
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