From the Institut Claude de Préval, INSERM
Unité 326, Hôpital Purpan, 31059 Toulouse Cedex, the
§ INSERM Unité 469, CCIPE, 34094 Montpellier
Cedex 5, and the ¶ Institut Louis Bugnard, INSERM Unité
466, Hôpital Rangueil, 31043 Toulouse Cedex, France
Received for publication, December 21, 2000, and in revised form, March 20, 2001
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ABSTRACT |
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Recent studies highlight the
existence of an autonomous nuclear polyphosphoinositide metabolism
related to cellular proliferation and differentiation. However, only
few data document the nuclear production of the putative second
messengers, the 3-phosphorylated phosphoinositides, by the
phosphoinositide 3-kinase (PI3K). In the present paper, we examine
whether GTP-binding proteins can directly modulate 3-phosphorylated
phosphoinositide metabolism in membrane-free nuclei isolated from pig
aorta smooth muscle cells (VSMCs). In vitro PI3K assays
performed without the addition of any exogenous substrates revealed
that guanosine 5'-( Vascular smooth muscle cells
(VSMCs)1 play a central role
in the fibroproliferative response during the development of
atherosclerosis and of restenosis after angioplasty (1, 2). Recently,
we have demonstrated that phosphoinositide 3-kinase (PI3K) was
essential for the progression of VSMCs throughout the G1
phase of the cell cycle (3), implying that a better understanding of
the PI3K signaling pathway might be of pathophysiological relevance.
PI3K phosphorylates the D3 position of the inositol ring in
phosphoinositides (PI) to generate the putative second messengers
PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (4). In
addition to proliferation, the PI3K products have also been involved in
cell transformation, apoptosis, vesicle trafficking, and cytoskeleton
organization (5, 6).
Three distinct classes of PI3Ks have now been identified on the basis
of their in vitro substrate specificity, structure, and mode
of regulation (7, 8). The most studied are class I PI3Ks, which
in vitro phosphorylate PtdIns, PtdIns(4)P, and PtdIns(4,5)P2, and display a preference for
PtdIns(4,5)P2 in vivo. Class I PI3Ks form a
heterodimeric complex and are subdivided according to the adaptor
protein associated with the catalytic subunit. Class IA PI3Ks consist
of a 110-kDa catalytic subunit (p110 Moreover, there is now considerable evidence that a nuclear PI cycle,
apart from that occurring in the plasma membrane, is involved in the
regulation of nuclear functions (14). Indeed, it has been demonstrated
that nuclei contain almost all the enzymes involved in the classical PI
cycle, including kinases required for the synthesis of
PtdIns(4,5)P2, phospholipases C, and diacylglycerol kinase
(15, 16). Furthermore, specific changes in the nuclear levels of PI
have been implicated in both cell growth and differentiation (17-19).
To date, information concerning the role and the regulation of nuclear
PI3K are still very limited. Immunocytochemical and biochemical
analyses demonstrate the presence of the p85 Chemicals and Antibodies--
All culture reagents were obtained
from Life Technologies Inc. U73122, wortmannin, and LY294002 were
obtained from Biomol (Plymouth Meeting, PA). GTP Cell Culture and Isolation of VSMC Nuclei--
VSMCs were
prepared from 6-week-old pig thoracic aorta using the explant technique
(26) and cultured in Dulbecco's modified Eagle's medium containing
10% fetal calf serum, as previously described (27). For all the
experiments, VSMCs were used from the third to the sixth passage.
Growing VSMCs were washed twice with ice-cold calcium- and
magnesium-free PBS and once with a hypotonic buffer containing 5 mM Tris-HCl, 1.5 mM KCl, 2.5 mM
MgCl2, pH 7.4. All subsequent procedures were carried out
at 4 °C. Medium was then switched to hypotonic buffer supplemented
with 200 µM Na3VO4, 1 mM NaF, 1 mM EDTA, 1 mM EGTA, 100 µM phenylmethylsulfonylfluoride, 10 µg/ml each
aprotinin, benzamidin, and leupeptin for 1 min, and VSMCs were lyzed by
the addition of 1% Nonidet-P40 and 1% deoxycholic acid. Cells were
allowed to swell for 1 min and were sheared by three passages through a
25-gauge needle. The cell lysate was layered over a 0.3 M/2
M sucrose discontinuous gradient and was centrifuged at
500 × g for 15 min in polypropylene tubes pretreated with a siliconizing agent (Sigmacote) in a further attempt to reduce
nuclei adsorption. Then, nuclei were recovered at the interface of 0.3 M/2 M sucrose and washed once with the assay
buffer containing 1 mg/ml fatty-acid free bovine serum albumin, 40 mM Hepes, pH 7.5, 2 mM EGTA, 2 mM
EDTA, 1 mM dithiothreitol, 50 mM NaCl, 4 mM MgCl2, 200 µM
Na3VO4, 1 mM NaF, and proteases
inhibitors, as above. As to the yield of the nuclear isolation, an
average of 0.5 × 106 nuclei were obtained from 1 × 106 cells. 1 × 106 cells and 1 × 106 nuclei contained 300 µg and 30 µg of proteins, respectively.
Lipid Kinase Assay--
Nuclei were disrupted by sonication (10 kHz for 3 × 1 s) using an ultrasonic cell disrupter (Branson
Sonifier 250) and treated for 1 h at 4 °C with 10 units/1.5 × 106 nuclei RNase-free DNase I. All
assays were conducted in a final volume of 100 µl of assay buffer
containing 5 × 106 nuclei, 1 µM
thapsigargin (ATPase inhibitor), and 5 µM U73122 (phospholipase C inhibitor). For experiments in the presence of PI3K
inhibitors and a Gi/G0 inhibitor, nuclei were
preincubated with 20 nM wortmannin or 10 µM
LY294002 and 5 ng/ml pertussis toxin, respectively, for 15 min on ice.
When indicated, 100 µM GTP Gel Electrophoresis and Immunoblotting--
Proteins from whole
cells or purified nuclei were resuspended in electrophoresis sample
buffer, incubated for 1 h at room temperature with 10 units/105 cells RNase-free DNase I, boiled for 10 min, separated by SDS-PAGE using 7.5 and 10% polyacrylamide gel,
transferred onto nitrocellulose membrane (Schleicher & Schuell), and
immunoblotted as previously described (30). Immunodetection was
achieved using the relevant antibody, anti-p85 Immunofluorescence--
VSMCs were plated on glass coverslips,
stimulated for 3 days with 10% fetal calf serum, and fixed in 70%
ethanol for 30 min at 4 °C. Cells were then permeabilized, and the
DNA was denaturated for 30 min in a solution of 2 N HCl,
0.5% Triton X-100, 0.5% Tween 20 in PBS. Cells were washed
extensively with PBS and were blocked for 1 h in PBS containing
0.5% bovine serum albumin (w/v) and 0.5% Tween 20. Coverslips were
incubated with the anti-p85 Electron Microscopy--
The purified nuclei were immediately
fixed in 3% glutaraldehyde in PBS, postfixed in osmium tetroxide,
dehydrated in graded ethanol series, and embedded in Epon 812. Ultrathin sections were then cut (Reichert ultratome), placed on
300 mesh copper grids, counterstained with uranyl acetate and lead
citrate, and examined in a Hitachi 300 transmission electron microscope.
Purity of VSMC Nuclear Preparations--
Membrane-depleted nuclei
from pig aorta VSMCs were isolated by hypotonic shock combined with
detergents. The purity of nuclear preparations was evaluated by
biochemical and immunochemical analyses. Lactate dehydrogenase and
5'-nucleotidase activities, recognized as markers for cytoplasm and
plasma membrane were, respectively, found to be 0.29 ± 0.12%
(n = 3) and 0.18 ± 0.08% (n = 3)
of the activity in the total homogenate. Furthermore, Western blot
analysis using anti-tubulin antibody showed the absence of
immunoreactivity to the cytoskeletal proteins in the purified nuclei
(Fig. 1A). In addition, the
nuclear fraction was highly enriched with nucleoporin p62, a protein of
nuclear pore complex (Fig. 1B). Finally, electron microscopy
analysis confirmed that the isolation procedure yielded nuclei of high
purity (Fig. 1C). No appreciable morphological change of the
nuclei was noted during the isolation procedure, the nucleolar
structure was maintained, and the lysis procedure completely removed
the nuclear envelope to leave the naked laminar layer and nuclear pore
remnants (Fig. 1C, right panel).
VSMC Nuclei Contain a GTP-dependent PI3K
Activity--
Previous studies suggest the existence of a nuclear PI3K
pathway (20-24), but the intranuclear location and regulation of a PI3K activity has not clearly been demonstrated. Therefore, we first
investigated whether membrane-free nuclei were able to produce 3-PI
from endogenous precursors. We assessed nuclear PI3K activity in
vitro by phosphate incorporation from [
As shown in Fig. 2A
(upper panel), HPLC analyses revealed a peak of
[32P]phosphatidic acid, suggesting a residual PLC
activity, but we never detected radiolabeled 3-PI. To look for the
possible presence of a G protein-regulated PI3K (11-13), we next
tested whether 100 µM GTP Substrate Specificity of the Nuclear GTP-dependent
PI3K--
The above data suggest either that the
GTP-dependent PI3K is selective for nuclear
PtdIns(4,5)P2 or that PtdIns and PtdIns(4)P present in the
nuclear membrane were removed during nuclei isolation. To address this
question, vesicles containing PtdIns/phosphatidylserine (100 µM/200 µM, final concentrations) were added
to nuclear PI3K assays, and the formation of 32P-labeled
lipids derived from PtdIns was analyzed by HPLC (Fig. 4). Incubation of nuclei with exogenous
PtdIns in the absence of GTP A 117-kDa PI3K
Having shown that a nuclear PI3K phosphorylates exogenous PtdIns in the
absence of GTP The Nuclear GTP-dependent PI3K Could Be Regulated by G
Proteins--
We next sought to determine whether nuclear
heterotrimeric Gi/G0 proteins could be
responsible for the nuclear PI3K activation by using PTX. Pretreatment
of isolated nuclei with 5 ng/ml PTX inhibited about 50% of the
GTP In the present study, we provide the first evidence that a
GTP-dependent PI3K generates the second messenger
PtdIns(3,4,5)P3 from a pre-existing nuclear pool of
PtdIns(4,5)P2, directly within VSMC nucleus.
Furthermore, we showed that this PI3K activity, which could be related
to a PI3K PtdIns(3,4,5)P3 Synthesis in VSMC Nuclei--
We
prepared highly purified VSMC nuclei stripped of their nuclear
envelope. Our data demonstrated that the GTP
The PtdIns(3,4,5)P3-dependent signaling
pathways in the nucleus are still unknown. However,
PtdIns(3,4,5)P3 synthesis was associated with activation of
the serine/threonine kinases, protein kinase C Nuclear GTP-dependent and -independent PI3Ks--
In
response to GTP
We further showed that VSMC nuclei contain a PI3K activity able to
generate PtdIns(3)P from exogenous PtdIns in the absence of GTP
Our experiments with bacterial toxin and the nuclear identification of
G
Clearly, further investigations will be required to answer important
points, in particular to clarify the role of the nuclear heterotrimeric
G protein-activated PI3K in VSMC pathophysiology. It will be of special
interest to study the regulation of this PI3K throughout the cell
cycle. In this regard, the recent demonstration that serum-induced VSMC
proliferation is mediated primarily via G-thio)triphosphate (GTP
S) specifically
stimulated the nuclear synthesis of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), whereas guanosine
5'-(
-thio)diphosphate was ineffective. PI3K inhibitors wortmannin
and LY294002 prevented GTP
S-induced PtdIns(3,4,5)P3
synthesis. Moreover, pertussis toxin inhibited partially
PtdIns(3,4,5)P3 accumulation, suggesting that nuclear
Gi/G0 proteins are involved in the activation
of PI3K. Immunoblot experiments showed the presence of
G
0 proteins in VSMC nuclei. In contrast with previous
reports, immunoblots and indirect immunofluorescence failed to detect
the p85
subunit of the heterodimeric PI3K within VSMC nuclei. By
contrast, we have detected the presence of a 117-kDa protein
immunologically related to the PI3K
. These results indicate the
existence of a G protein-activated PI3K inside VSMC nucleus that might
be involved in the contol of VSMC proliferation and in the
pathogenesis of vascular proliferative disorders.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
,
) (9) and a 85-kDa
adaptor protein (p85
,
) (10) containing Src homology 2 (SH2)
domains that link them to tyrosine kinase signaling. In contrast, class
IB PI3K or PI3K
defines a G protein-coupled receptor-regulated PI3K
(11). It is made of a p110
catalytic subunit and a p101 regulatory
subunit unrelated to p85 (12). The p110
can be activated in
vitro by both the
and
subunits of heterotrimeric G
proteins (11-13). This stimulation is considerably enhanced by the
p101 adaptor (12).
regulatory subunit in
the nuclei of rat and human cells (20-22) and the growth factor-dependent nuclear translocation of the p110
catalytic subunit in osteoblast-like cells (23), suggesting that class IA PI3Ks exist in the nucleus. Recently, a study based on the immunolocalization of epitope-tagged p110
in HepG2 cells reported that PI3K
translocates to the nucleus after serum stimulation (24).
Since a nuclear G protein-regulated PI3K activity has not yet been
demonstrated, we investigated whether GTP-binding proteins directly
modulate 3-phosphorylated phosphoinositide (3-PI) metabolism in
membrane-free nuclei isolated from pig aorta VSMCs. Our data provide
the first evidence for the existence of a pertussis toxin
(PTX)-sensitive PI3K inside VSMC nucleus. This enzyme, which might be
related to PI3K
, phosphorylates an intranuclear pool of
PtdIns(4,5)P2, suggesting that nuclear
PtdIns(3,4,5)P3 may regulate protein kinases involved in
vascular proliferative disorders.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S, GDP
S, and
RNase-free DNase I were from Roche Molecular Biochemicals.
[
-32P]ATP (3000 Ci/mmol) was purchased from
PerkinElmer Life Sciences. Monoclonal anti-p110
, fluorescein
isothiocyanate-conjugated anti-rabbit antibodies, and the enhanced
chemiluminescence (ECL) system were purchased from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA). Polyclonal anti-p110
was kindly
provided by S. Roche. Specific polyclonal anti-G
0
antibodies directed against the last 10 amino acids of the common
carboxyl-terminal sequence of the G
01 and G
02 (anti-G
0(C-ter) or against the amino
acid 291-302 of the
subunit of G01
(anti-G
01) were obtained and characterized as previously
described (25). Horseradish peroxidase-conjugated anti-rabbit/mouse
antibodies and polyclonal anti-p85
antibody were, respectively,
supplied from New England Biolabs Inc. (Saint Quentin, France) and
Upstate Biotechnology Inc. (Lake Placid, NY). Monoclonal
anti-nucleoporin p62 antibody was from BD Transduction Laboratories
(San Diego, CA). Lactate dehydrogenase and 5'-nucleotidase kits,
polyclonal anti-tubulin antibodies, and all other reagents were
obtained from Sigma. COS-7 cells overexpressing human p110
or
p110
were a generous gift of A. Yart and P. Raynal.
S or 100 µM
GDP
S was added for another 15 min. The assays were then started by
the addition of 1 mM ATP (10 µl) containing 65 µCi of
[
-32P]ATP, and the 32P incorporation was
allowed for 15 min at 30 °C under shaking. For exogenous lipid
phosphorylation, 30 µl of lipid vesicles containing 100 µM PtdIns and 200 µM phosphatidylserine
were added 5 min before starting the assay. Reactions were stopped by
the addition of 1360 µl of chloroform/methanol (1:1, v/v), 300 µl
of 2 N HCl, and 280 µl of 200 mM EDTA. Lipids
were immediately extracted after the modified procedure of Bligh and
Dyer (28). Lipids were then analyzed either on oxalate-coated
thin-layer chromatography plates (Silica Gel 60, Merck) developed in
isopropanolol:acetic acid:H2O (65:1:34) or by HPLC on a
Partisphere SAX column (Whatman International Ltd, U. K.) after
deacylation, as previously described (29). The synthesis of radioactive
standard PtdIns(3)P, PtdIns(3,4)P2, and
PtdIns(3,4,5)P3 was performed from specific anti-p85
immunoprecipitates, essentially as described in Payrastre et
al. (30).
(1/1000) for 1 h
at room temperature and anti-tubulin (1/1000), anti-nucleoporin p62
(1/1000), monoclonal and polyclonal anti-PI3K
(1/100 and 1/1500,
respectively), anti-G
0(C-ter) and anti-G
01 (1/500) overnight at 4 °C. Horseradish
peroxidase-conjugated secondary antibodies (1/2500) were incubated of
1 h at room temperature, and immunoreactive proteins were
visualized with ECL reagents according to the manufacturer's instructions.
antibody (1/20, overnight at 4 °C)
then with fluorescein isothiocyanate-conjugated anti-rabbit antibody
(1/100, 1 h), mounted in Mowiol, and examined under epifluorescent
illumination using a Zeiss microscope coupled to a Micromax camera
(Princeton Instruments Inc.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Purity of VSMC nuclear preparations.
Membrane-depleted nuclei were obtained by hypotonic shock combined with
detergents, separated from lysate, and washed once time as detailed
under "Experimental Procedures." A and B,
detection of markers by Western blotting analyses. 15 µg of proteins
from 5 × 104 cells (lane 1) and 5 × 105 purified nuclei (lane 2) were fractionated
on SDS-PAGE, transferred and probed with anti-tubulin antibody
(A). 3 µg of proteins from 1 × 104 cells
(lane 1) and 1 × 105 purified nuclei
(lane 2) were probed with anti-nucleoporin p62 antibody
(B). The immunoblots shown are representative of three
independent experiments. C, transmission electron
micrographs of purified nuclei. Nuclei were fixed and embedded, and
ultrathin sections were examined at different magnifications
(left panel, ×10,000; middle panel, ×12,000;
right panel, ×20,000). Note the presence of the lamina
layer (L) and nuclear pore remnants (P) and the
absence of the nuclear envelope. The black bar represents
the width of 1 µm.
-32P]ATP
into inositol lipids. All assays were conducted in the presence of 2 mM EGTA and 5 µM U73122, a phospholipase C
(PLC) inhibitor, so that nuclear PLC activity would not interfere with
PI3K activity.
S, a nonhydrolyzable GTP
analogue, could trigger the production of 3-PI in isolated nuclei (Fig.
2A, middle panel). Under this condition, we detected the
synthesis of only one PI3K product, the PtdIns(3,4,5)P3,
and the incorporation into
[32P]PtdIns(3,4,5)P3 was 49073 ± 2000 cpm/107 nuclei (n = 3) (Fig.
3A). This peak coincided with
the HPLC profile of pure [32P]PtdIns(3,4,5)P3
used as a control (Fig. 2B). In addition, the [32P]phosphatidic acid peak was always present, and we
never observed the production of any other inositol lipids, especially
PtdIns(3)P and PtdIns(3,4)P2. No further stimulation of
PtdIns(3,4,5)P3 was observed with 200 µM
GTP
S (data not shown). Moreover, 100 µM GDP
S was
unable to activate PtdIns(3,4,5)P3 synthesis, as presented in Fig. 2A (lower panel). These results were
confirmed by TLC showing the specific activation of only
PtdIns(3,4,5)P3 synthesis by GTP
S in isolated nuclei
(Fig. 3B, lanes 1 and 3). A
weak wortmannin-insensitive production of PtdIns(3,4,5)P3
was observed in the absence of GTP
S (Fig. 3B, lanes
1 and 2), but in general, the PI3K activity was too small to be detectable. These data strongly suggest that a nuclear
GTP-dependent PI3K phosphorylates a pre-existing
intranuclear pool of PtdIns(4,5)P2 to produce
PtdIns(3,4,5)P3. We next investigated the effects of two
specific PI3K inhibitors on GTP
S-induced nuclear PI3K activation.
Both HPLC (Fig. 3A) and TLC studies (Fig. 3B) showed that accumulation of PtdIns(3,4,5)P3 was reduced by
60-80% after preincubation of nuclei with 20 nM
wortmannin or 10 µM LY294002, demonstrating that the
nuclear GTP-dependent PI3K is sensitive to PI3K inhibitors
in a classical range of concentrations.
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Fig. 2.
GTP S induces a
PtdIns(3,4,5)P3 synthesis in VSMC nuclei. A,
membrane-free nuclei were obtained as indicated in Fig. 1. Nuclear
lipid kinase activity was assayed with 5 × 106 nuclei
(N) without adding exogenous lipids. Before starting the
reaction, nuclei were preincubated for 15 min in the absence
(upper panel) or in the presence of 100 µM
GTP
S (middle panel) or with 100 µM GDP
S
(lower panel). The assays were started by the addition of
[
-32P]ATP, and incorporation was achieved for 15 min
at 30 °C with shaking. Then radioactive lipids were extracted and
analyzed by HPLC after deacylation. The data shown are representative
of three different experiments. B, HPLC profile of purified
[32P]PtdIns(3,4,5)P3 control after
deacylation. PA, phosphatidic acid; Pi,
inorganic phosphate; PIP3,
PtdIns(3,4,5)P3.
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Fig. 3.
Effects of PI3K inhibitors on
GTP S-induced nuclear
PtdIns(3,4,5)P3 synthesis. Membrane-depleted nuclei
were isolated, and nuclear lipid kinase activity was assayed as
described in Fig. 2. Nuclei were pretreated for 15 min with 20 nM wortmannin or 10 µM LY294002 before
incubation with 100 µM GTP
S. A, lipids were
extracted and analyzed by HPLC. Quantification of
[32P]PtdIns(3,4,5)P3 synthesis is expressed
as cpm/107 nuclei. Data for GTP
S alone are the mean ± S.E. from three independent experiments. Data for PI3K inhibitors
represent the mean of two independent experiments. B, lipids
were extracted and analyzed by TLC. The data shown are representative
of two different experiments. The position of standard radioactive 3-PI
is shown as the control. PI3P, PtdIns(3)P;
PI3,4P2, PtdIns(3,4)P2;
PIP3, PtdIns(3,4,5)P3.
S resulted in the synthesis of
[32P]PtdIns(4)P, as already described (15), and also of
[32P]PtdIns(3)P (36,963 ± 4105 cpm/107
nuclei, n = 3) but not of any other 3-PI (Fig.
4A, upper panel, and Fig. 4B). This
result suggests that membrane-free VSMC nuclei also contain a
GTP-independent PI3K phosphorylating in vitro exogenous PtdIns to PtdIns(3)P but unable to phosphorylate the intranuclear pool
of PtdIns(4,5)P2. In the presence of GTP
S,
[32P]PtdIns(3)P synthesis was increased by 40%
(57114 ± 572 cpm/107 nuclei, n = 3, p < 0.01), and under that condition, we also measured PtdIns(3,4,5)P3 synthesis (Fig. 4A, lower
panel). These results indicate that the nuclear
GTP-dependent PI3K can phosphorylate both endogenous
nuclear PtdIns(4,5)P2 and exogenous PtdIns.
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Fig. 4.
Exogenous PtdIns phosphorylation by nuclear
GTP-dependent PI3K. Membrane-depleted nuclei were
isolated, and nuclear lipid kinase activity was assayed in the presence
of exogenous lipids. Nuclei (N) were preincubated in the
absence (A, upper panel, and B) or in
the presence (A, lower panel, and B)
of 100 µM GTP S. Vesicles of PtdIns/phosphatidylserine
at the final concentrations 100 µM/200 µM
were added 5 min before starting the reaction. A,
representative HPLC profiles of three different experiments.
B, quantification of [32P]PtdIns(3)P synthesis
is expressed as cpm/107 nuclei. Data are the mean ± S.E. from three independent experiments. The asterisk
indicates significant difference using the Student's t test
(p < 0.01). PA, phosphatidic acid;
Pi, inorganic phosphate; PI3P,
PtdIns(3)P; PI4P, PtdIns(4)P; PIP3,
PtdIns(3,4,5)P3.
but Not p85
Is Expressed Inside VSMC
Nuclei--
We next performed immunoblot experiments to identify the
two PI3K isoforms expressed in isolated nuclei (Figs.
5 and 6).
The existence of a nuclear PI3K that phosphorylates both PtdIns and PtdIns(4,5)P2 in a GTP-dependent manner
suggests that it could be a class IB PI3K. Indeed, we detected the
presence of the catalytic subunit of the G protein-regulated PI3K by
using two different anti-PI3K
antibodies (Fig. 5). The analysis with
a monoclonal antibody against the amino-terminal region of human
p110
revealed a 117-kDa protein in both the total cell homogenate
(Fig. 5A, lane 1) and the nuclear fraction (Fig.
5A, lane 2), assayed here in similar amounts in
terms of nuclei number. A positive control with human p110
overexpressed in COS-7 cells also showed a single band at 117 kDa (Fig.
5A, lane 3). Moreover, the p110
detection was
specific, as the monoclonal antibody did not cross-react with human
p110
overexpressed in COS-7 and as no signal was detected in VSMC
nuclei when the primary antibody was omitted (Fig. 5A, lanes 4 and 5). The polyclonal anti-p110
antibody also confirmed the presence of a 117-kDa protein in VSMC
nuclei (Fig. 5B). Unfortunately, as previously noted in
HepG2 cells (24), all available antibodies were unable to detect
endogenous PI3K
by immunofluorescence in VSMCs.
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Fig. 5.
Expression of PI3K
in VSMCs. A, proteins from 1 × 105 whole VSMCs (lane 1) and 1 × 105 purified nuclei (lane 2) were fractionated
on 7.5% SDS-PAGE, transferred, and probed with monoclonal anti-p110
antibody. As a positive control, proteins from 1 × 105 COS-7 cells overexpressing human p110
were also
probed (lane 3). The specificity was tested by probing
proteins from 1 × 105 COS-7 cells overexpressing
human p110
(lane 4) and proteins from 1 × 105 VSMC nuclei without the primary antibody incubation
(lane 5). B, proteins from VSMC purified nuclei
(1 × 105 to 5 × 105 nuclei) were
immunoblotted with polyclonal anti-p110
antibody. The data are from
a single experiment representative of at least three others.
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Fig. 6.
Expression of p85 in
VSMCs. A, Western blotting analysis of p85
. 15 µg
of proteins from 5 × 104 cells and 5 × 105 nuclei were fractionated on 7.5% SDS-PAGE,
transferred, and immunoblotted with polyclonal anti-p85
antibody.
The data are from a single experiment representative of at least three
others. B, p85
subcellular localization by
immunofluorescence. Cells were plated on glass coverslips, fixed,
permeabilized, and incubated with polyclonal anti-p85
antibody. The
bar represents the width of 10 µm.
S and considering that p85
has been reported in rat
liver nuclei (20), we checked whether class IA PI3Ks would also be
present in VSMC nuclei (Fig. 6). We used an anti-p85
antibody to
detect this adaptor protein and loaded the same amount of proteins (15 µg) from cell homogenate or from purified nuclei (Fig.
6A). In contrast to previous reports, p85
was
undetectable in VSMC nuclei when compared with total cell homogenates,
although 10 times as much nuclei were assayed. The absence of nuclear
p85
expression was further confirmed by indirect immunofluorescence
microscopy. As shown in Fig. 6B, the p85
labeling was
evident as a ring at the perinuclear region and as fluorescent dots in
the cytoplasm and the plasma membrane, whereas the nuclear interior
remained unstained. These results strongly suggest that the
heterodimeric PI3K p85
/p110 is absent from our nuclear preparations but could be present in the nuclear envelope, whereas a 117-kDa PI3K
-like kinase is, significantly, located inside VSMC nuclei.
S-induced PI3K activity (Fig.
7A). Moreover, to identify
PTX-sensitive G proteins inside VSMC nuclei, we performed immunoblots
using specific antibodies directed against both
G
01/
02 subunits
(anti-G
0(C-ter)) or against the
subunit of
G01 (anti-G
01). As shown in Fig.
7B, a protein of 42-43 kDa is recognized by both antibodies
in our nuclear preparations. These results suggest that PTX-sensitive G
proteins are present in VSMC nuclei and could be involved in nuclear
PI3K activation.
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Fig. 7.
Effects of PTX on
GTP S-induced nuclear
PtdIns(3,4,5)P3 synthesis and expression of
G
0 proteins in VSMCs.
Membrane-depleted nuclei were isolated, and nuclear lipid kinase
activity was assayed as described in Fig. 2. A, nuclei were
pretreated for 15 min with 5 ng/ml pertussis toxin before incubation
with 100 µM GTP
S. Lipids were extracted and analyzed
by HPLC. Quantification of
[32P]PtdIns(3,4,5)P3 synthesis is expressed
as cpm/107 nuclei. Data for GTP
S alone are the mean ± S.E. from three independent experiments. Data for PTX represent the
mean of two independent experiments. B, Western blotting
analysis of heterotrimeric G
0 protein. Protein from
1 × 105 whole cells or 5 × 105
purified nuclei were fractionated on 10% SDS-PAGE, transferred, and
probed with polyclonal anti-G
0(C-ter) or
anti-G
01 antibodies. Experiments illustrated are
representative of at least three distinct experiments.
PIP3, PtdIns(3,4,5)P3.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, is coupled to nuclear Gi/G0 heterotrimeric G proteins.
S-responsive PI3K and
the PtdIns(4,5)P2 resist treatment with non-ionic
detergents, suggesting that this enzyme and its substrate are tightly
associated with non-membrane nuclear structures. In agreement with
these results, PtdIns(4,5)P2 was localized in
nucleolus-associated heterochromatin in rat pancreas (31), and
picomole-sensitive mass assays have revealed that 35% of nuclear
PtdIns(4,5)P2 (about 30 pmol/mg of protein) remained in rat
liver nuclei treated with Triton X-100 (32). In addition, recent data
from Boronenkov et al. (33) provide new insights about the
localization of PtdIns(4,5)P2 within the nucleus. They
demonstrated that PtdIns(4,5)P2 is spatially organized to
"nuclear speckles" in mammalian cells. Interestingly, nuclear
speckles are separated from known membrane structures and contain
pre-mRNA processing factors, and their morphology is tightly linked
to the status of mRNA transcription. These observations suggest
that speckles might function as centers for PI-signaling pathways in
nuclei. However, PI3K activity was not addressed in these studies. In
this respect, our finding of a nuclear G protein-regulated PI3K is of
special interest, and we can speculate that phosphorylation of
PtdIns(4,5)P2 into PtdIns(3,4,5)P3 might
modulate speckle functions.
, and Akt/protein
kinase B (34, 35), and the nuclear translocation of these two
PtdIns(3,4,5)P3 effectors was demonstrated upon mitogenic
stimulation (36, 37). More recently, Neri et al. (38) show
that nuclear PtdIns(3,4,5)P3 production correlates both
with p85
/p110 PI3K and protein kinase C
translocations to the
nucleus of nerve growth factor-treated PC12 cells. Thus, the G
protein-activated PI3K could produce PtdIns(3,4,5)P3 inside VSMC nuclei to recruit and/or activate downstream effectors. In this
respect, we also observed the presence of protein kinase C
and
active Akt/protein kinase B in VSMC nuclei (data not shown). The
nuclear targets of protein kinase C
and Akt/protein kinase B only
begin to be described. Indeed, a major component of the nucleolus,
nucleolin, has been shown to be phosphorylated by protein kinase C
(36), and Akt/protein kinase B has been reported to promote
phosphorylation of the nuclear transcription factors CREB (cAMP-response element-binding protein) (39) and FKHR1 (forkhead in rhabdomyosarcoma 1) (40). Finally, evidence has been provided that
PIP3BP, a PtdIns(3,4,5)P3-binding protein, is exported out of the nucleus by the expression of constitutively activated PI3K (41).
S, we never detected nuclear PtdIns(3)P or
PtdIns(3,4)P2 synthesis from endogenous substrates,
suggesting that PtdIns and PtdIns(4)P, which have been reported located
in the nuclear membrane (14, 32), were totally removed during nuclei
isolation. On the other hand, the GTP-dependent PI3K might be PtdIns(4,5)P2-selective. To address this question, we
added exogenous PtdIns in assays. We found that GTP
S stimulated a
PtdIns(3)P synthesis, demonstrating that the nuclear
GTP-dependent PI3K uses PtdIns as a substrate in
vitro. This result suggests that GTP-binding proteins activate a
class IB PI3K in VSMC nucleus. Accordingly, immunoblot analysis
revealed the presence of a 117-kDa catalytic subunit of G
protein-regulated PI3K
inside VSMC nuclei, suggesting that the
nuclear GTP-dependent PI3K could be PI3K
or a
PI3K
-like kinase. In this respect, Stephens et al. (12)
purified two G protein-activated PI3Ks from pig neutrophils. Both were
heterodimers composed of the 101-kDa regulatory protein and either a
120-kDa or a 117 kDa catalytic subunit. Only, the p120 cDNA was
cloned and was shown to be highly related to PI3K
. Furthermore, two recent reports demonstrated the presence of a PI3K
-mediating ion
channel stimulation in smooth muscle cells (42, 43). However, the
mechanism governing the nuclear targeting of the
GTP-dependent PI3K is unclear. Nevertheless, Metjian
et al. (24) showed that serum induces translocation of
tagged p110
into HepG2 nuclei and suggested that p101 could regulate
this relocation. In contrast with this observation, preliminary data
from our laboratory seem to indicate that serum does not modify nuclear
PI3K
-like kinase expression.2
S.
Immunoblots and immunofluorescence analyses failed to detect the
adaptor protein p85
inside the VSMC nuclei, suggesting that the
GTP-independent PI3K is different from p85
/p110 PI3Ks (class IA).
This conflicts with the data from Lu et al. (20) and
Marchisio et al. (44), who found p85
in the nuclear
matrix of rat liver and of differentiated HL60 cells, respectively.
Such a discrepancy might be due to differences in cell type or in
nuclei isolation. However, p85
/p110 could be expressed in the
nuclear envelope of VSMCs. We also cannot exclude the presence of a
class IA PI3K containing the p85
adaptor protein or class II/III
PI3K inside VSMC nuclei. These hypotheses are currently under
investigation. However, it is noteworthy that this GTP-independent PI3K
was unable to phosphorylate the pool of PtdIns(4,5)P2
within the nucleus.
01 allowed us to propose that PTX-dependent
G proteins are present in the nucleus and participate in the activation
of the GTP-dependent PI3K. In support of this hypothesis,
the association between G
0 and the mitotic spindle was
observed in certain cancer cell lines (45). It has also been shown that
growth factor-induced cell division is paralleled by the translocation
of
i to the nucleus (46). Moreover, G
subunits
might also play an important role in GTP-dependent PI3K
activation, since G
directly stimulates PI3K
activity in
vitro (47), and a
subunit-like activity has been reported
in rat liver nuclei (48). Interestingly, PI3K
was implicated in both
G
i- and
-mediated survival pathways elicited by G
protein-coupled receptors in COS-7 cells (49).
in vitro and
that targeted inhibition of G
reduces intimal hyperplasia and
limits restenosis in vivo (50) appears essential.
![]() |
ACKNOWLEDGEMENTS |
---|
We appreciate the expert technical assistance
of J. C. Thiers for the electron microscopy studies. We thank S. Roche for providing the polyclonal antibody to p110, A. Yart and P. Raynal for COS-7 cells transfected with plasmid encoding human p110
or p110
. We are grateful to B. Payrastre and C. Racaud-Sultan for
helpful discussions and critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Association pour la Recherche contre le Cancer, the Ligue Nationale contre le Cancer, and the Fondation pour la Recherche Médicale.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 all correspondence should be addressed. Tel.:
33-5-61-77-94-15; Fax: 33-5-61-77-94-01; E-mail:
monique.douillon@purpan. inserm.fr.
Published, JBC Papers in Press, April 12, 2001, DOI 10.1074/jbc.M011572200
2 D. Bacqueville, P. Déléris, and M. Breton-Douillon, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
VSMC, vascular
smooth muscle cell;
PI, phosphoinositides;
PI3K, PI 3-kinase;
3-PI, 3-phosphorylated phosphoinositides;
PtdIns, phosphatidylinositol;
PtdIns(4)P, Ptd 4-monophosphate;
PtdIns(3)P, Ptd 3-monophosphate;
PtdIns(4, 5)P2, Ptd 4,5-bisphosphate;
PtdIns(3, 4)P2, Ptd 3,4-bisphosphate;
PtdIns(3, 4,5)P3, Ptd 3,4,5-trisphosphate;
PLC, phospholipase C;
PAGE, polyacrylamide gel electrophoresis;
GTPS, guanosine 5'-(
-thio)triphosphate;
GDP
S, guanosine
5'-(
-thio)diphosphate;
LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one;
PTX, pertussis toxin;
PBS, phosphate-buffered saline;
HPLC, high performance
liquid chromatography.
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