(Received for publication, September 24, 1996, and in revised form, December 16, 1996)
From the Department of Medicine, Phosphoinositide 3-kinase has been implicated as
an activator of cell motility in a variety of recent studies, yet the
role of its lipid product, phosphatidylinositol 1,4,5-trisphosphate (PtdIns-3,4,5-P3), has yet to be elucidated. In this study,
three independent preparations of PtdIns-3,4,5-P3 were
found to increase the motility of NIH 3T3 cells when examined utilizing
a microchemotaxis chamber. Dipalmitoyl
L- Initiation of cellular motility has been demonstrated with
multiple growth factors, including platelet-derived growth factor (PDGF)1 (1), hepatocyte growth factor (HGF)
(2), and insulin (3). The mechanisms whereby cells undergo chemotaxis
(directional cell movement) and chemokinesis (random cell movement) are
complex, requiring dissolution of cell-cell contacts (such as tight
junctions in epithelial cells) and cell-surface contacts, formation of
lamellipodia, actin filament severing and nucleation, and finally
contraction of the actin filament network leading to movement of the
cell body (4). An understanding of the signaling pathways required to
orchestrate these cellular events should provide critical new insights
into numerous biological events such as cell migration and organization
during organ development and wound healing, tumor cell metastasis, and
progression of arterial atherosclerotic plaques.
Mutations in the PDGF receptor that eliminate binding of
phosphoinositide 3-kinase PI 3-kinase-impair PDGF-dependent
chemotaxis (1, 5, 6), and selective activation of the PI 3-kinase is
sufficient to initiate motility (7). The lipid products of PI 3-kinase,
PtdIns-3,4-P2, and PtdIns-3,4,5-P3 are elevated acutely in response to PDGF (8) and are thought to act as second messengers (9-11). Although the in vivo function of these
lipids has not been demonstrated, they activate calcium-independent
protein kinase C family members in a stereospecific manner (12-16).
Thus, we investigated the possibility that PtdIns-3,4,5-P3
stimulates cell motility via activation of a PKC family member.
The majority of experiments were
performed with NIH 3T3 fibroblasts, using PDGF as the positive control.
Selected experiments were repeated with mIMCD-3 cells, a murine renal
tubular epithelial cell line that expresses the c-met
receptor and exhibits striking chemotaxis to a gradient of HGF
(17-19). All cells were cultured in Dulbecco's modified Eagle's
medium with 5% fetal calf serum using standard techniques. PDGF
(Upstate Biotechnology, Inc., Lake Placid, NY) and HGF (Institute of
Immunology, Tokyo, Japan) were used in concentrations of 10 and 40 ng/ml, respectively, based on previous dose response curves for maximal
chemotaxis (19).
PtdIns-4,5-P2 was obtained from Upstate Biologicals, and
phosphatidylserine (PtdSer) was from Avanti Polar Lipids.
Diacylglycerol (DAG) and horseradish anti-mouse conjugate were
purchased from Boehringer Mannheim.
12-O-Tetradecanoylphorbol-13-acetate (TPA) was obtained from
Life Technologies, Inc., and wortmannin was from
Sigma. Calphostin C was obtained from Calbiochem, and
P81 phosphocellulose paper was purchased from Whatman. Thin layer chromatography plates (Silica Gel 60) were obtained from EM
Separations.
Phosphoinositide 3-kinase was purified
from rat liver cytosol as described previously (20) and used
immediately for the preparation of PtdIns-3,4,5-P3. Lipid
substrates were prepared by drying under a stream of nitrogen. PtdSer
(10 mg/ml) was added to the PtdIns-4,5-P2 (2 mg/ml) as a
carrier, and the mixture was sonicated in 10 mM Hepes, pH
7.0, 1 mM EGTA for 10 min using a bath sonicator. This
mixture was then incubated with phosphoinositide 3-kinase at 37 °C
in the presence of 50 µM [ Dipalmitoyl
L- Chemotaxis was evaluated using a modified
Boyden chamber assay with a 48-well microchemotaxis chamber as
described previously (Neuro Probe Inc., Cabin John, MD) (19, 23).
Lipids were dried in a stream of nitrogen and then sonicated for 5 min
in serum-free media. The lower section of the Boyden chamber was filled
with media alone or media containing either PDGF (10 ng/ml) or
PtdSer/PtdIns-4,5-P2 (100 µM/25
µM) or
PtdSer/PtdIns-4,5-P2/PtdIns-3,4,5-P3 (100 µM/25 µM/5 µM). A
polycarbonate filter (Nucleopore Corp., Pleasanton, CA) coated with rat
tail collagen type I (Collaborative Biomedical, Bedford, MA) was placed
over the lower compartment, and 1.5 × 104 cells were
added to the upper compartment. In some experiments, wortmannin was
diluted 1:1000 in serum-free media immediately prior to use and added
at the appropriate concentration to both the upper and lower chambers
at time 0. Control experiments were performed with Me2SO
vehicle alone. After 4 h of incubation at 37 °C, filters were
removed, the cells were fixed and stained with Diff-Quik (Baxter
Healthcare Corp., Miami, FL), and the upper surface was wiped with a
cotton applicator to remove nonchemotaxing cells. For each well, cells
that had passed through the pores were counted, and the mean value of
cells/mm2 was calculated.
Confluent plates of
cells were serum-starved overnight in the presence of either 0.3%
Me2SO or 300 nM TPA followed by a wash with
phosphate-buffered saline and lysis in ice-cold lysis buffer (137 mM NaCl, 20 mM Tris, 1 mM
MgCl2, 1 mM CaCl2, 10% glycerol, 1% Nonidet P-40, 2 mM sodium vanadate, 1 mM
phenylmethylsulfonyl fluoride, pH 7.5). The suspension was centrifuged
for 10 min at 12,000 × g. Equal aliquots of
supernatant determined by protein assay (Bio-Rad) were resolved by
SDS-polyacrylamide gel electrophoresis and transferred to Immobilon
(Millipore). Expression of protein kinase C In vivo levels of D3
phosphoinositides in response to stimuli were measured as described
previously (8). Briefly, 3T3 cells maintained in Dulbecco's modified
Eagle's medium, 5% fetal calf serum were grown to 80% confluence and
then placed in Dulbecco's modified Eagle's medium, 0.1% fetal calf
serum for 12-16 h. For labeling purposes, monolayers were placed in
phosphate-free Dulbecco's modified Eagle's medium in the absence of
serum for 1 h, followed by 2 mCi/ml of
[32P]orthophosphate for 3 h. Cells were then
stimulated with PDGF (20 ng/ml),
Di-C16-PtdIns-3,4,5-P3, or vehicle control for
10 min. Following stimulation, cells were washed twice with ice-cold phosphate-buffered saline and lysed in 750 µl of methanol, 1 M HCl (1:1). 20 µg of crude brain phosphoinositides
(Sigma) were added as carrier. Lipids were extracted
by the addition of 380 µl of chloroform, and the organic phase was
washed twice with 400 µl of methanol, 0.1 M EDTA.
Phospholipids were then deacylated and prepared for Sepharose A
exchange-high pressure liquid chromatography analysis as described
previously (8).
F-actin was localized in coverslip
adherent cells. Quiescent 3T3-fibroblasts or cells exposed to 40 ng/ml
of PDGF, 5 µM
Di-C16-PtdIns-3,4,5-P3, or 5 µM
PtdIns-4,5-P2 for 10-60 min were fixed by the addition of
an equal volume of 3.7% formaldehyde in phosphate-buffered saline at
37 °C for 30 min. Fixed cells were permeabilized with 0.1 volume of
1% Triton X-100 containing 2 µM
tetramethylrhodamine B isothiocyanate-phalloidin at 37 °C for
60 min (24), washed three times with phosphate-buffered saline for 5 min each, and magnified in a Zeiss IM45 Inverted microscope.
Results were averaged, and statistical
relevance was determined by Student's t test. Data are
presented as mean ± S.E.
Since exogenously added lipid
vesicles and micelles are known to fuse with the plasma membrane of
live cells, we investigated the role of PtdIns-3,4,5-P3 in
cell motility by directly adding this lipid to the cells in a Boyden
chamber. Three independent preparations of PtdIns-3,4,5-P3
were employed to evaluate the motility response of this putative second
messenger (Fig. 1). PtdIns-3,4,5-P3 was
enzymatically generated by adding purified PI 3-kinase to a 1:4 mixture
of PtdIns-4,5-P2 and PtdSer followed by chloroform
extraction of the lipid products. This resulted in conversion of 20%
of the PtdIns-4,5-P2 to PtdIns-3,4,5-P3 to give
a final mixture of 100 µM PtdSer, 25 µM
PtdIns-4,5-P2, 5 µM
PtdIns-3,4,5-P3. The addition of this
PtdSer/PtdIns-4,5-P2/PtdIns-3,4,5-P3 mixture to
the bottom well of the chemotaxis chamber resulted in a 10-fold
increase in motility of NIH 3T3 fibroblasts compared with vehicle
control and a 3-fold increase compared with the
PtdSer/PtdIns-4,5-P2 mixture alone (Fig. 1A).
The small but reproducible motility response to 100 µM
PtdSer, 25 µM PtdIns-4,5-P2 may be due to
either activation of PKC isoforms by the high concentrations of
PtdIns-4,5-P2 (13) or impurities in these lipids not seen
when lower concentrations of lipids were investigated individually (see
"Discussion").
PtdIns-3,4,5-P3 enhances motility
of NIH 3T3 fibroblasts (A and B) and IMCD
epithelial cells (C). Cell motility was evaluated
using a modified Boyden chamber assay with a 48-well microchemotaxis
chamber. A, the lower section of the Boyden chamber was
filled with media alone or media containing PDGF,
PtdSer/PtdIns-4,5-P2 lipid substrate, or enzymatically
generated PtdIns-3,4,5-P3. B and C,
either chemically synthesized (dioctanoyl)-PtdIns-3,4,5-trisphosphate (Di-C8-PI-3,4,5-P3; 5 µM),
chemically synthesized (dipalmitoyl)-PtdIns-3,4,5-trisphosphate (Di-C16-PI-3,4,5-P3; 5 µM),
commercial PtdIns-4,5-bisphosphate (PI-4,5-P2; 5 µM), or PDGF (10 ng/ml) were added to the lower chamber
when indicated. In some experiments, 10 nM wortmannin (wort.) was added. The number of chambers assayed for each
condition is indicated by n. *, p < 0.001 compared with PtdIns-4,5-P2 control.
To more directly examine the isolated effects of
PtdIns-3,4,5-P3, we utilized two synthetically prepared
sources of PtdIns-3,4,5-P3 (Fig. 1, B and
C). 5 µM
Di-C16-PtdIns-3,4,5-P3, which forms micelles when sonicated in the absence of carrier lipids, induced a 7-fold increase in cell motility over base line in 3T3 cells (control, 10.0 ± 1.7 cells/mm2, n = 17;
Di-C16-PtdIns-3,4,5-P3, 70.2 ± 7.9, n = 22; Fig. 1B) and a 4-fold increase in
IMCD cells (control, 7.4.0 ± 1.1 cells/mm2,
n = 27; Di-C16-PtdIns-3,4,5-P3,
34.5.2 ± 2.5, n = 36; Fig. 1C). When
Di-C16-PtdIns-3,4,5-P3 was added to both
compartments of the chemotaxis chamber, a significant but somewhat
smaller number of cells was found to migrate through the pores,
indicating an increase in both chemokinesis and chemotaxis (control,
8.4 ± 0.9 cells/mm2, n = 10;
Di-C16-PtdIns-3,4,5-P3 on the bottom only,
39.0 ± 2.6, n = 11;
Di-C16-PtdIns-3,4,5-P3 on both the top and
bottom, 30.8 ± 1.9, n = 12 (p = 0.016)).
Di-C8-PtdIns-3,4,5-P3, a short chain
PtdIns-3,4,5-P3 that is soluble as a monomer in water, was
also tested in 3T3 cells and found to initiate chemotaxis although to a
lesser extent (control, 10.0 ± 1.7;
Di-C8-PtdIns-3,4,5-P3, 29.4 ± 4.2, n = 27, p = 0.001; Fig. 1B).
There was no chemotactic effect when cells were exposed to 5 µM PtdIns-4,5-P2 (3T3 cells, 11.7 ± 2.8 cells/mm2, n = 23; IMCD cells, 4.5 ± 1.0 cells/mm2, n = 18) or 50 µM PtdSer (3T3 cells, 17.8 ± 3.7 cells/mm2, n = 12; IMCD cells, 10.5 ± 1.5 cells/mm2, n = 6). Concentrations of
Di-C16-PtdIns-3,4,5-P3 from 1 nM to 100 µM were evaluated (Fig. 2). 5 µM was chosen for further experiments, since this
was the lowest dose that consistently resulted in a chemotactic
response.
The polymerization of cytoplasmic actin that follows receptor
stimulation and leads to membrane ruffling and lamellipodia formation
is felt to be downstream of the PI 3-kinase (6, 25). To test this
hypothesis, we evaluated actin filament reorganization and membrane
ruffling following the addition of
Di-C16-PtdIns-3,4,5-P3 (Fig. 3).
The synthetic form of PtdIns-3,4,5-P3 stimulated membrane ruffling in 3T3 fibroblasts to the same extent as PDGF.
PtdIns-4,5-P2 had no effect on quiescent cells.
It was
conceivable that a contaminant or a breakdown product of
PtdIns-3,4,5-P3 might initiate the observed effects via
activation of a cell surface receptor (as has been shown for
lysophosphatidic acid). Although this seemed unlikely, since
PtdIns-3,4,5-P3 made by three different procedures
stimulated cell motility and comparable concentrations of
PtdIns-4,5-P2 and/or PtdSer failed to stimulate cell
motility, we searched for evidence that exogenously added PtdIns-3,4,5-P3 might act via cell surface receptor
activation by examining intracellular production of
PtdIns-3,4-P2 and PtdIns-3,4,5-P3 in
32PO43 The PI 3-kinase inhibitor wortmannin binds irreversibly to the
catalytic subunit of the enzyme and prevents production of the D3
phosphorylated lipid products of the enzyme. 10 nM
wortmannin, the lowest dose that produces reliable inhibition of the PI
3-kinase in vivo in 3T3 fibroblasts and mIMCD-3 cells (19),
caused a 60% inhibition of PDGF- and HGF-dependent cell
motility but had no effect on
Di-C16-PtdIns-3,4,5-P3-stimulated cell movement
in 3T3 cells or in IMCD cells (Fig. 1, B and C).
These results demonstrate that wortmannin at a dose that inhibits
PDGF receptor-mediated activation of the PI 3-kinase does not prevent
PtdIns-3,4,5-P3-initiated motility, further supporting the
hypothesis that these lipids are inserting into the membrane and
directly initiating downstream signaling events.
100 nM wortmannin, a concentration where effects on several
other kinases have been observed, caused essentially complete inhibition of both PDGF and
Di-C16-PtdIns-3,4,5-P3-stimulated cell motility
(data not shown). In light of the observation by Kundra et
al. (1) that selective activation of phospholipase C It was previously shown that activation of PKC by
DAG or TPA can stimulate chemotaxis (31-33). Therefore, we examined
the role of activation of PKC in PtdIns-3,4,5-P3-mediated
chemotaxis. 100 µM DAG produced a consistent increase in
motility of NIH 3T3 cells (control, 1.3 ± 0.2 cells/mm2; DAG, 116.7 ± 13.4, p < 0.001). Of note, 5 µM DAG fails to induce chemotaxis,
while 5 µM Di-C16-PtdIns-3,4,5-P3
does, indicating that the PtdIns-3,4,5-P3 effect is not due
to hydrolysis to DAG. When DAG and
Di-C16-PtdIns-3,4,5-P3 were both present in the
bottom well, the chemotactic rate was similar to that seen with DAG
alone (Di-C16-PtdIns-3,4,5-P3, 55.5 ± 4.5 cells/m2; DAG, 116.7 ± 13.4;
Di-C16-PtdIns-3,4,5-P3 with DAG, 127.3 ± 19.5, n = 12), suggesting that these two stimuli were
acting via the same signaling pathway.
To examine this possibility, the TPA-activable PKC family members were
down-regulated by overnight preincubation of NIH 3T3 cells with 300 nM TPA. Under these conditions, there was a 78% decline in
the concentration of PKC
The PI 3-kinase has been clearly implicated in cell motility by
several laboratories (1, 6, 7, 19), yet the actual mechanism of this
effect is poorly understood. The p85 subunit of the PI 3-kinase has a
BCR homology domain, which is capable of binding GTP-Rac (37, 38) and
may therefore act to recruit activated Rac or associated family members
to the membrane where these signaling proteins have been shown to
initiate motility (39, 40). In addition, PtdIns-3,4,5-P3
has been shown to be capable of activating the actin-severing protein,
gelsolin (28). The present results demonstrate that
PtdIns-3,4,5-P3, the lipid product of the PI 3-kinase, is
capable of directly initiating cell motility and that this effect is
mediated by activation of PKC. The greatest effect on cell motility
occurred as directional movement toward a gradient of
PtdIns-3,4,5-P3 (i.e. chemotaxis), while
nondirectional movement increased as well.
The model we propose to explain the ability of exogenously added
PtdIns-3,4,5-P3 to stimulate cell motility requires that some fraction of the lipid fuse with the plasma membrane and arrive at
the inner leaflet over the 4-h assay period. There it can act similarly
to endogenous PtdIns-3,4,5-P3. This might occur by
different mechanisms for the different PtdIns-3,4,5-P3
preparations. The enzymatically produced PtdIns-3,4,5-P3,
sonicated in the presence of excess PtdSer, is expected to be
distributed in both the inner and outer leaflets of the newly
formed vesicle. Fusion of these vesicles with the plasma membrane
would presumably result in PtdIns-3,4,5-P3 in both
leaflets. The Di-C16-PtdIns-3,4,5-P3 forms
micelles when sonicated2 and is likely to
cause local detergent-like effects when fusing with the plasma
membrane, resulting in distribution of this lipid on both leaflets of
the plasma membrane as well.
Recently, PKC In addition to PKC activation, there are several other targets for the
lipid products of the PI 3-kinase. pp70S6k was the first
target shown to be downstream of PI 3-kinase (42), and recently, Akt
(41, 43) and Rac (38) have also been implicated. The latter is of
particular interest, since microinjection of constitutively active
forms of rac leads to membrane ruffling (39). The recent
availability of synthetic forms of these phosphoinositides should
help identify their targets and determine the pathways that lead to the
motile response.
Previous results from our laboratory and others have shown that
activation of the PI 3-kinase is essential for PDGF and
HGF-dependent cell movement. The present experiments
demonstrate that the lipid products of the PI 3-kinase act directly as
second messengers in cell motility and provide the first indication
that PKC family members are required for the motility effects of this
lipid in vivo.
Nephrology and § Signal Transduction,
Divisions of Experimental Medicine and Hematology-Oncology,
Department of Medicine, Brigham and Women's Hospital, Boston,
Massachusetts 02215, the ** Department of Molecular Genetics, University
of Texas Southwestern Medical Center, Dallas, Texas 75325 and the
Division of Medicinal Chemistry and
Pharmaceutics, College of Pharmacy, University of Kentucky,
Lexington, Kentucky 40506-0286
-phosphatidyl-D-myo-inositol
3,4,5-triphosphate (Di-C16-PtdIns-3,4,5-P3)
also produced actin reorganization and membrane ruffling. Cells
pretreated with 12-O-tetradecanoylphorbol-13-acetate to
cause down-regulation of protein kinase C (PKC) exhibited complete inhibition of cell motility induced by
Di-C16-PtdIns-3,4,5-P3. These results are
consistent with previous observations that PtdIns-3,4,5-P3 activates Ca2+-independent PKC isoforms in
vitro and in vivo and provide the first demonstration
of an in vivo role for the lipid products of the
phosphoinositide 3-kinase. PtdIns-3,4,5-P3 appears to
directly initiate cellular motility via activation of a PKC family
member.
Cell Culture and Reagents
-32P]ATP (3000 Ci/mmol), 5 mM MgCl2, 50 mM Hepes,
pH 7.5, for 60 min. The reaction (200 µl) was stopped by the addition
of 65 µl of 5 N HCl, and lipids were extracted in 400 µl of ChCl3/MeOH (1:1). Lipids were dried and stored at
70 °C until needed. [32P]PtdIns-3,4,5-P3
was quantified by thin layer chromatography (n-propanol, 2 M acetic acid extract (65:35)) and radiation detection on a
Bio-Rad molecular imager. Based on the specific activity of the
[
-32P]ATP, 20% of the PtdIns-4,5-P2 was
converted to PtdIns-3,4,5-P3.
-phosphatidyl-D-myo-inositol
3,4,5-triphosphate (Di-C16-PtdIns-3,4,5-P3)
(21) and
dioctanoyl-L-
-phosphatidyl-D-myo-inositol-3,4,5-trisphosphate were synthesized as described previously (13, 22).
was detected by a
monoclonal antibody specific for this enzyme (Transduction
Laboratories) and quantified using a Molecular Dynamics
PhosphorImager.
Enzymatically Generated and Synthetic PtdIns-3,4,5-P3
Initiate Cell Motility and Ruffling
Fig. 1.
[View Larger Version of this Image (24K GIF file)]
Fig. 2.
Dose response curve for 3T3 fibroblast
chemotaxis to Di-C16-PtdIns-3,4,5-P3 and
PtdIns-4,5-P2. n = 4-6 for each
point.
[View Larger Version of this Image (12K GIF file)]
Fig. 3.
Membrane ruffling by cells exposed to
Di-C16-PtdIns-3,4,5-P3 mimics the response seen
with PDGF. The top panels show that quiescent cells and
cells exposed to 5 µM PtdIns-4,5-P2
(PIP2) are no different, whereas significant
membrane ruffling can be seen in the cells exposed to either 40 ng/ml
PDGF or 5 µM PtdIns-3,4,5-P3 (PIP3) at 10 (middle panels) and 30 min
(bottom panels).
[View Larger Version of this Image (122K GIF file)]
-labeled NIH 3T3
cells following the addition of extracellular Di-C16-PtdIns-3,4,5-P3. This approach was
chosen because the receptors known to initiate chemotaxis (PDGF
receptor, insulin receptor, c-met receptor, lysophosphatidic
acid receptor) have also been found to activate the PI 3-kinase (8,
26-28). While stimulation with PDGF produced a dramatic rise in
intracellular [32P]PtdIns-3,4-P2 and
[32P]PtdIns-3,4,5-P3 (2.3- and 17-fold), no
increase in either of these lipids was seen in cells treated with 5 µM Di-C16-PtdIns-3,4,5-P3.
by the
PDGF receptor resulted in a substantial chemotactic response, even in
the absence of PI 3-kinase activation, this result suggests that other
targets of wortmannin that are likely to be inhibited at the higher
concentration, such as myosin light chain kinase (29) or PtdIns
4-kinase (30), may be critical for cell motility as well.
by Western analysis (Fig. 4), an effect comparable with that seen in human dermal
fibroblasts (34). PKC
was chosen because it shows the greatest
activation to PtdIns-3,4,5-P3 in vitro and
in vivo. The migratory response to
PtdIns-3,4,5-P3 was completely eliminated in NIH 3T3 cells pretreated with TPA (Fig. 5A), while
PDGF-mediated cell movement was inhibited by 70%, a finding
similar to that seen with exposure to 10 nM
wortmannin. In addition, the specific PKC inhibitor calphostin C was
tested (35, 36). NIH 3T3 cells exposed to 100 nM calphostin C (IC50 = 75-100 nM) for 30 min demonstrated a
90% reduction in PtdIns-3,4,5-P3-mediated cell movement
(Fig. 5B). These results suggest that activation of the PI
3-kinase mediates cell motility via the local generation of
PtdIns-3,4,5-P3 and subsequent activation of PKC.
Fig. 4.
TPA down-regulates PKC in 3T3 cells.
NIH 3T3 cells were treated for 12 h with either vehicle control
(
) or 300 nM TPA (+) followed by SDS-polyacrylamide gel
electrophoresis and immunoblotting with an antibody specific for
PKC
. Densitometric analysis of the blot revealed 35.9 ± 4.6 densitometric units for control PKC
versus 8.2 ± 0.9 for cells pretreated with TPA (experiment performed in triplicate;
p = 0.001).
[View Larger Version of this Image (23K GIF file)]
Fig. 5.
Down-regulation of PKC inhibits
Di-C16-PtdIns-3,4,5-P3 stimulated cell
motility. A, 16-h pretreatment with 300 nM TPA caused complete inhibition of the motility response to
Di-C16-PtdIns-3,4,5-P3 and DAG as compared with
vehicle control. PDGF-mediated cell movement was inhibited by 70%.
n = 12. B, 30-min pretreatment of NIH 3T3 cells with 100 nM calphostin C caused a 91% inhibition of
Di-C16-PtdIns-3,4,5-P3-mediated cell motility.
n = 6. *, p < 0.001 versus
control; **, p < 0.01 versus
stimulated.
[View Larger Version of this Image (21K GIF file)]
as well as the atypical PKC
isoform have been found
to be activated downstream of the PtdIns 3-kinase in PDGF- and
epidermal growth factor-stimulated cells (15, 16). In addition, several
researchers have demonstrated that PtdIns-3,4,5-P3 can
directly activate calcium-insensitive PKC family members in vitro (12-14). Our results demonstrate that activation of the
phorbol-sensitive PKC family members enhances cell motility in a
fashion similar to that seen with PtdIns-3,4,5-P3, while
both down-regulation of PKC by overnight treatment with TPA and
inhibition of PKC with calphostin C completely blocked the
PtdIns-3,4,5-P3 response. The present data cannot
distinguish which PKC family member or members are directly involved,
although in vitro data suggest that the PKC
is strongly
up-regulated by these lipids. The observation that high concentrations
of PtdIns-4,5-P2 caused a modest increase in cell motility
(Figs. 1A and 2) is consistent with this hypothesis, since
this polyphosphoinositide has also been found to weakly activate PKC
in vitro (13).
*
This work was supported in part by National Institutes of
Health Grant DK48871.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: Div. of Nephrology,
Dana 517, Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-2147; Fax: 617-667-5276.
1
The abbreviations used are: PDGF,
platelet-derived growth factor; HGF, hepatocyte growth factor; PI,
phosphoinositide; PtdIns, phosphatidylinositol; PtdSer,
phosphatidylserine; DAG, diacylglycerol; TPA,
12-O-tetradecanoylphorbol-13-acetate.
2
P. Janmey, unpublished results.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.