Thrombospondin Signaling of Focal Adhesion Disassembly Requires
Activation of Phosphoinositide 3-Kinase*
Jeffrey A.
Greenwood
,
Manuel A.
Pallero,
Anne B.
Theibert§, and
Joanne E.
Murphy-Ullrich
From the Department of Pathology, Division of Molecular and
Cellular Pathology and the § Department of Neurobiology,
University of Alabama at Birmingham, Birmingham, Alabama 35294
 |
ABSTRACT |
Thrombospondin is an extracellular matrix protein
involved in modulating cell adhesion. Thrombospondin stimulates a rapid loss of focal adhesion plaques and reorganization of the actin cytoskeleton in cultured bovine aortic endothelial cells. The focal
adhesion labilizing activity of thrombospondin is localized to the
amino-terminal domain, specifically amino acids 17-35. Use of a
synthetic peptide (hep I), containing amino acids 17-35 of
thrombospondin, enables us to examine the signaling mechanisms specifically involved in thrombospondin-induced disassembly of focal
adhesions. We tested the hypothesis that activation of phosphoinositide 3-kinase is a necessary step in the thrombospondin-induced signaling pathway regulating focal adhesion disassembly. Both wortmannin and
LY294002, membrane permeable inhibitors of phosphoinositide 3-kinase
activity, blocked hep I-induced disassembly of focal adhesions.
Similarly, wortmannin inhibited hep I-mediated actin microfilament
reorganization and the hep I-induced translocation of
-actinin from
focal adhesion plaques. Hep I also stimulated phosphoinositide 3-kinase
activity approximately 2-3-fold as measured in anti-phosphoinositide
3-kinase and anti-phosphotyrosine immunoprecipitates. Increased
immunoreactivity for the 85-kDa regulatory subunit in anti-phosphotyrosine immunoprecipitates suggests that the p85/p110 form
of phosphoinositide 3-kinase is involved in this pathway. In
32Pi-labeled cells, hep I increased
levels of phosphatidylinositol (3,4,5)-trisphosphate, the major product
of phosphoinositide 3-kinase phosphorylation. These results
suggest that thrombospondin signals the disassembly of focal
adhesions and reorganization of the actin cytoskeleton by a pathway
involving stimulation of phosphoinositide 3-kinase activity.
 |
INTRODUCTION |
Thrombospondin (TSP)1 is
an extracellular matrix protein involved in modulating cell adhesion,
proliferation, and migration (for reviews on TSP, see Refs. 1 and 2).
The structure of TSP1 consists of multiple domains which bind to
various cell surface receptors and interact with other extracellular
molecules. These receptors include heparan sulfate proteoglycans
(3-5), sulfatides (6, 7), CD36 (8, 9), integrins (10), and the 52-kDa integrin-associated protein (11). Differential expression of these
receptors and varying accessibility of the binding domains of TSP1 are
potential mechanisms for regulating the effects of TSP1 on cell
behavior. However, the multiplicity of domains and receptors that exist
for TSP1 has made it difficult to determine the mechanisms by which
TSP1 regulates cellular function.
This laboratory has examined the regulation of cell adhesion by TSP1.
Bovine aortic endothelial (BAE) cells will attach to surfaces coated
with TSP1, but spreading and cytoskeletal organization is not
stimulated (10, 12). Soluble TSP1 induces a rapid disassembly of focal
adhesions and reorganization of the actin cytoskeleton in spread
endothelial cells (12). TSP1-induced disassembly of focal adhesions is
accompanied by a dispersal of vinculin from the focal adhesion plaque,
however, the
v
3 integrin remains clustered (12, 13). The focal adhesion labilizing activity of TSP1 was
localized to the amino-terminal heparin-binding domain, specifically
amino acids 17-35 (14). Using a synthetic peptide, hep I, containing
amino acids 17-35 of TSP1, we can specifically examine mechanisms
involved in the disassembly of focal adhesions induced by TSP1 and
assess the effect of this activity on endothelial cell function.
Previously, we demonstrated that disassembly of focal adhesions in
response to hep I is dependent upon cyclic GMP-dependent
protein kinase (PKG) (15). Cells in which PKG is inhibited or deficient
no longer respond to hep I. However, PKG does not appear to be
activated by hep I and activation of PKG does not stimulate disassembly
of focal adhesions. These results suggest that basal PKG activity is
important for the process of focal adhesion disassembly stimulated by
TSP1. The signaling mechanisms involved in regulating TSP1-induced
disassembly of focal adhesions remain to be elucidated.
A candidate signaling cascade is that involving phosphoinositide
3-kinase (PI 3-kinase). This family of lipid kinases has been shown to
be involved in mitogenesis, vesicle trafficking, integrin activation,
and regulation of the actin cytoskeleton (for reviews on PI 3-kinase,
see Refs. 16-18). PI 3-kinase was first identified as a
phosphatidylinositol kinase activity associated with the virally
encoded protein tyrosine kinase v-Src in cell extracts (19, 20). The
structure of this form of PI 3-kinase was determined to be a
heterodimer consisting of an 85-kDa regulatory subunit and a 110-kDa
catalytic subunit (16, 17). The p85/p110 PI 3-kinase is activated by
interaction of the regulatory subunit, which contains two SH2 domains,
one SH3 domain, and two proline-rich sequences, with receptor and
nonreceptor tyrosine kinases (16, 17). The 110-kDa subunit of PI
3-kinase, which has been shown to be inhibited directly by
wortmannin and LY294002, catalyzes phosphorylation on the
3-position of phosphoinositides leading to increases in
phosphatidylinositol (3,4)-bisphosphate (PtdIns (3,4)-P2) and phosphatidylinositol
(3,4,5)-trisphosphate (PtdIns (3,4,5)-P3) (16,
21-25).
Although PI 3-kinase has been demonstrated to be involved in a
multiplicity of cellular functions, the exact role of its lipid products in cell signaling is not clear. Recent evidence suggests that
the 3-phosphorylated phosphoinositides initiate downstream signaling by
rapidly recruiting and/or activating target proteins. For example, both
PtdIns (3,4)-P2 and PtdIns (3,4,5)-P3 activate calcium-independent protein kinase C types
,
, and
(26, 27)
and PtdIns (3,4)-P2 activates Akt kinase (28). PtdIns (3,4,5)-P3 has been demonstrated to compete with
tyrosine-phosphorylated proteins for binding to SH2 domains, suggesting
that PtdIns (3,4,5)-P3 may recruit SH2-containing proteins
to specific compartments of the cell (29). Evidence is also
accumulating that 3-phosphorylated phosphoinositides bind to and
modulate actin-regulatory proteins such as profilin and gelsolin
(30-32). The diversity of cellular events involving PI 3-kinases
suggest that complex regulatory mechanisms must exist to generate
specific signals.
Implication of PI 3-kinase in the regulation of the actin cytoskeleton
prompted us to propose that PI 3-kinase is involved in the TSP1-induced
disassembly of focal adhesions and reorganization of actin
microfilaments. In current studies, we present evidence that TSP1
stimulates the activation of PI 3-kinase and that PI 3-kinase is a
component of the signaling pathway mediating TSP1-induced disassembly
of focal adhesions and reorganization of the actin cytoskeleton.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Hep I (ELTGAARKGSGRRLVKGPD) and scrambled hep I
(RSKAGTLGERDLKPGARVG) were synthesized, purified, and analyzed by the
UAB Comprehensive Cancer Center/Peptide Synthesis and Analysis shared facility. Phosphatidylinositol was purchased from Avanti Polar Lipids
(Alabaster, AL). Wortmannin, mouse monoclonal antibody to
-actinin
(clone BM752), protein A-Sepharose, crude brain phosphoinositides, and
phosphatidylinositol 4-phosphate were purchased from Sigma. Phosphatidylinositol (4,5)-bisphosphate (PtdIns (4,5)-P2)
was obtained from American Radiolabeled Chemicals (St. Louis, MO), and
LY294002 from Biomol (Plymouth Meeting, PA). Anti-p85-PI 3-kinase (rabbit polyclonal) and anti-phosphotyrosine (mouse monoclonal, clone
PY20 and RC20) antibodies were purchased from Transduction Laboratories
(Lexington, KY). Anti-p85-PI 3-kinase (rabbit polyclonal) agarose beads
and anti-p85-PI 3-kinase (mouse monoclonal) antibodies were purchased
from Upstate Biotechnology Inc. (Lake Placid, NY). 32Pi was from ICN. [32P]ATP was
obtained from Amersham. Bodipy-phallicidin was purchased from Molecular
Probes (Eugene, OR). Horseradish peroxidase-conjugated goat anti-mouse
IgM and fluorescein isothiocyanate-conjugated goat anti-mouse IgM was
purchased from Jackson Laboratories (West Grove, PA). TSP1 was purified
from fresh (24-48 h) human platelets purchased from the Birmingham
American Red Cross as described previously (14).
Cells--
BAE cells were isolated and cultured in DMEM
containing 4.5 g/l glucose, 2 mM glutamine, and 20% fetal
bovine serum (FBS) as described previously (14).
Focal Adhesion Assay--
Focal adhesion assays were performed
as described (12). Briefly, BAE cells were grown on glass coverslips in
DMEM with 20% FBS until near confluence. Cells were rinsed with warm
DMEM to remove serum components and incubated in DMEM for 1 h at
37 °C. Cells were treated with hep I (0.1-1 µM) or
TSP1 (10 µg/ml = 0.078 µM monomer), fixed with 3%
glutaraldehyde, and examined by interference reflection microscopy. A
minimum of 300 cells/condition was evaluated for the presence of focal
adhesions. A cell was scored positive if it contained at least 3 adhesion plaques.
Immunoprecipitation--
Cells were scraped into ice-cold lysis
buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 2 mM
Na3VO4, 1% Triton X-100, 0.5% Nonidet P-40, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 0.2 mM
phenylmethylsulfonyl fluoride) and precleared by centrifugation. Supernatants (1 mg/ml) were incubated with anti-p85-PI 3-kinase (4 µg/ml) or PY20 (10 µg/ml) for 2 h on ice. Protein A-Sepharose was added and incubated for 1 h at 4 °C with shaking.
Immunoprecipitates were washed 3 times in lysis buffer and assayed for
PI 3-kinase activity or immunoblotted.
PI 3-Kinase Activity Assay--
PI 3-kinase activity in
anti-p85-PI 3-kinase and anti-phosphotyrosine (PY20) immunoprecipitates
was assayed as described (36). Immunoprecipitates were washed 2 times
with kinase buffer (10 mM HEPES, pH 7.2, 20 mM
-glycerophosphate, 0.8 mM
Na3VO4, and 30 mM NaCl). A volume
of 30 µl containing the protein A-Sepharose beads and kinase buffer
was left remaining in the tube. Twenty microliters of lipid (see below)
was added to the beads, vortexed, and incubated for 10 min at 37 °C.
The reaction was started by adding 20 µl of kinase buffer containing
17.5 µM ATP (25 µCi of [32P]ATP/sample)
and 17.5 mM MgCl2. The samples were vortexed
and incubated for 10 min at 37 °C. Reactions were stopped by adding 500 µl of chloroform/methanol/water (3:6:1). Lipids were then extracted by adding 175 µl of chloroform and 175 µl of 1 M HCl, vortexing, and centrifuging to separate the phases.
The lower phase was removed, added to 630 µl of methanol, 1 M HCl/chloroform (16:15.8:1), vortexed, and centrifuged.
The lower phase was removed and the lipids separated by TLC on Silica
Gel 60 plates precoated with 1% potassium oxalate. The plates were
developed in methanol/chloroform/water/ammonium hydroxide (8:6:2:1)
when phosphatidylinositol was the substrate and
chloroform/acetone/methanol/acetic acid/water (40:15:13:12:7) when
PtdIns (4,5)-P2 was the substrate (37). The TLC plates were
exposed for autoradiography and quantitated using video-enhanced densitometry. The positions of PtdIns-P, PtdIns-P2, and
PtdIns-P3 as migrated on the TLC plate was visualized by
exposing the plate to I2 vapor and identified using
purified standards.
To prepare the lipid substrates, phosphatidylinositol was dried from
the purchased chloroform solution and then sonicated into kinase buffer
containing 0.2% deoxycholate and 3.5 mM dithiothreitol until clear to achieve a concentration of 3 µg/ml. PtdIns
(4,5)-P2 was prepared for phosphorylation by drying down
with an equal amount of phosphatidylserine and sonicating into kinase
buffer containing 3.5 mM dithiothreitol to get
concentrations of 1.5 µg/ml PtdIns (4,5)-P2 and 1.5 µg/ml phosphatidylserine.
32Pi Labeling of Intact Cells and
Examination of 32P-Labeled Lipids--
Analysis of hep
I-stimulated incorporation of 32P into the lipids in intact
cells was performed as described (38). BAE cells were grown to
approximately 80% confluency in a 100-mm culture dish. Cells were
incubated overnight in 0.2% FBS/DMEM. The media was changed to
phosphate-free DMEM and incubated for 1 h. Cells were then
incubated for 1 h in phosphate-free DMEM containing 1.6 mCi/ml
32Pi. Cells were stimulated for 10 min. The
media was aspirated and the cells scraped and collected in 750 µl of
methanol, 1 M HCl (1:1); 20 µg of crude phosphoinositides
was added as a carrier. The lipids were extracted by adding 380 µl of
chloroform and shaking 15 min at room temperature. Samples were
centrifuged to separate the phases and the upper phase discarded. The
extracted lipids were washed two times with 500 µl of methanol, 0.1 M EDTA (1:0.9), separated by TLC, and analyzed as described
above. These plates were developed in
chloroform/acetone/methanol/acetic acid/water (40:15:13:12:7).
Immunofluorescence--
BAE cells were stained with
Bodipy-phallicidin or
-actinin as described previously (14). Cells
stained for
-actinin were extracted with Triton X-100 buffer (20 mM Tris, pH 7.4, 50 mM NaCl, 1 mM
EGTA, 5 mM EDTA, 50 mM sodium pyrophosphate, 50 µM sodium fluoride, 100 µM sodium
orthovanadate, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 0.5%
Triton X-100) for 5 min on ice prior to fixation.
Immunoblotting--
Soluble proteins were extracted in Triton
X-100 buffer as described above. The extracted cells were rinsed 2 times with ice-cold phosphate-buffered saline on ice and the insoluble
proteins collected in electrophoresis sample buffer. Protein
concentrations were determined by the method of Lowry (39) after acid
precipitation. Samples (30 µg) were resolved by electrophoresis on
SDS-polyacrylamide gels and transferred to nitrocellulose. Blots were
blocked, probed, and developed by enhanced chemiluminescence (NEN Life
Science Products, Boston, MA).
 |
RESULTS |
Inhibitors of PI 3-Kinase Block Focal Adhesion Disassembly Induced
by the Active Sequence of TSP1--
A statistically significant
decrease in focal adhesions can be observed in spread BAE cells 8 min
following administration of hep I, the active sequence from TSP1, with
disassembly complete after 20 min and stable for at least 60 min (Fig.
1). To determine whether PI 3-kinase was
involved in TSP1-induced disassembly of focal adhesions, cells were
treated with hep I in the presence of wortmannin or LY294002, membrane
permeable inhibitors of PI 3-kinase activity. Wortmannin, an
irreversible inhibitor acting at the lipid-binding site (21, 22, 24),
completely blocked the hep I-stimulated disassembly of focal adhesions
at concentrations as low 2.5 nM (Fig.
2A). LY294002, a reversible
inhibitor which competes for the ATP-binding site (21, 23), inhibited
hep I activity at concentrations as low as 5 µM (Fig.
2B). Published data shows that at these low concentrations,
wortmannin and LY294002 do not directly inhibit other lipid kinases or
phospholipase A2 (21-24, 77). Focal adhesion disassembly
stimulated by the intact TSP1 protein was also completely inhibited by
wortmannin and LY294002 (Table I). As
previously reported (14), a synthetic peptide containing the scrambled
amino acid sequence of hep I did not result in the disassembly of focal
adhesions (Table I).

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Fig. 1.
Time course of hep I-stimulated disassembly
of focal adhesions. BAE cells were grown on glass coverslips until
near confluence, rinsed, and then incubated with DMEM for 1 h,
treated with 1 µM hep I for various times, fixed with 3%
glutaraldehyde, and examined for the presence of focal adhesion
positive cells by interference reflection microscopy as described under
"Experimental Procedures." n = 3; error
bars represent S.D.
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Fig. 2.
Wortmannin and LY294002 inhibit the hep
I-induced disassembly of focal adhesions. BAE cells were grown on
glass coverslips until near confluence, rinsed, and then incubated with
DMEM for 1 h, preincubated with various concentrations of
wortmannin (A) or LY294002 (B) for 5-10 min,
treated with DMEM (control) or 1 µM hep I for 30 min,
fixed with 3% glutaraldehyde, and examined for the presence of focal
adhesion positive cells by interference reflection microscopy.
n = 1-8; error bars represent S.D.
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Wortmannin Inhibits Hep I-mediated Actin
Reorganization--
Previous work demonstrated that TSP1 and hep I
stimulate reorganization of the actin cytoskeleton (12, 14). Control
cells display thick stress fibers terminating at the focal adhesion plaques, while the microfilaments in hep I-treated cells appeared thinner, with a more peripheral distribution (12, 14). To determine if
PI 3-kinase was involved in the TSP1-induced rearrangement of the actin
cytoskeleton, the microfilaments were examined in control and hep
I-treated BAE cells pretreated in the absence or presence of 5 nM wortmannin. Consistent with previous observations, phallicidin-stained F-actin microfilaments in hep I-treated cells (Fig.
3b) exhibit a more peripheral
distribution as compared with the more centrally located stress fibers
terminating at focal adhesion plaques in control cells (Fig.
3a). The hep I-stimulated reorganization of the actin
cytoskeleton was completely inhibited by 5 nM wortmannin
(Fig. 3d) with F-actin staining similar to the non-hep
I-treated controls. Treatment with wortmannin alone did not appear to
affect F-actin microfilament organization (Fig. 3c). These
results suggest that PI 3-kinase activity is required for the
rearrangement of actin microfilaments induced by hep I.

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Fig. 3.
Wortmannin inhibits hep I-mediated actin
reorganization. BAE cells were grown on glass coverslips until
near confluence, incubated overnight in 0.2% FBS/DMEM, preincubated
with 5 nM wortmannin for 5 min (c and
d), treated with DMEM (a and c) or 10 µM hep I (b and d) for 30 min,
fixed with 3% formaldehyde, and stained with Bodipy-phallicidin.
F-actin staining was examined by epifluorescence microscopy.
Bar = 10 µM.
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-Actinin is an actin-regulatory protein which binds directly to
actin filaments as well as to other components of focal adhesions forming the protein network anchoring microfilaments to the membrane at
contact sites (40, 41). The localization of
-actinin was examined in
control and hep I-treated cells in the absence or presence of 5 nM wortmannin. Since previous studies have reported that
antibody accessibility to focal adhesion localized
-actinin is
limited (42), cells were extracted with Triton X-100 buffer prior to
fixation and staining. Under these conditions, antibody specific for
-actinin intensely stained focal adhesion plaques (Fig.
4a). Staining for
-actinin
was also observed along actin microfilaments in a beaded pattern. In
hep I-treated cells, a decrease in
-actinin staining of focal
adhesions in comparison to controls was observed (Fig. 4b),
although the beaded pattern of staining was only slightly decreased.
Wortmannin inhibited the hep I-induced decrease in
-actinin staining
of focal adhesion plaques (Fig. 4d) without affecting
-actinin localization in control cells (Fig. 4c). These
results suggest that PI 3-kinase activity is also required for the hep
I-stimulated redistribution of
-actinin from focal adhesions.

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Fig. 4.
Wortmannin inhibits hep I-mediated
redistribution of -actinin. BAE cells were grown on glass
coverslips until near confluence, incubated overnight in 0.2%
FBS/DMEM, preincubated with 5 nM wortmannin for 5 min
(c and d), treated with DMEM (a and
c) or 10 µM hep I (b and
d) for 30 min, extracted with Triton X-100 buffer for 5 min
on ice, and fixed with 3% formaldehyde. -Actinin was localized by
indirect immunofluorescence using a monoclonal antibody specific for
-actinin and examined by epifluorescence microscopy.
Bar = 10 µM.
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To confirm the relocalization of
-actinin in response to hep I
stimulation, immunoblotting was used to examine the distribution of
-actinin in the Triton X-100 soluble and insoluble fractions from
control and hep I-treated cells. Under basal conditions,
-actinin
was detected in the insoluble cytoskeletal fraction almost exclusively.
However, in hep I-treated cells we observed a
time-dependent increase in
-actinin in the soluble
fraction (Fig. 5). These data suggest
that
-actinin translocates to the detergent-soluble fraction as part
of the process of hep I-mediated focal adhesion dissolution.

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Fig. 5.
Hep I stimulates an increase in soluble
-actinin. BAE cells were grown on glass coverslips until near
confluence, rinsed, and then incubated with DMEM for 1 h, treated
with 10 µM hep I for various times, and extracted with
Triton X-100 buffer for 5 min on ice. The soluble fraction (30 µg/lane) was resolved by electrophoresis on a 7.5% polyacrylamide
gel and transferred to a nitrocellulose membrane. Equal protein loading
was confirmed by Ponceau S staining of the membrane prior to
immunoblotting with a monoclonal antibody specific for -actinin
(1:100,000). Results are representative of four separate
experiments.
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Hep I Stimulates PI 3-Kinase Activity--
To further examine the
role of PI 3-kinase in the TSP1-induced disassembly of focal adhesions,
we measured the activation of PI 3-kinase in response to hep I. Complete inhibition of hep I activity by low nanomolar concentrations
of wortmannin suggested that heterodimeric forms of PI 3-kinase,
containing an 85-kDa regulatory subunit, mediate the disassembly of
focal adhesions by hep I (21, 43, 45). Other isoforms of PI 3-kinase
that lack the 85-kDa regulatory subunit require higher concentrations of wortmannin for inhibition (21, 43-45). Previously, researchers have
demonstrated that PI 3-kinase in immunoprecipitates from agonist-stimulated cells show an enhanced activity when assessed in
in vitro kinase assays (16, 17). Therefore soluble lysates from control and hep I-treated cells were immunoprecipitated with antibody recognizing the 85-kDa regulatory subunit of PI 3-kinase. Similar to published studies, the immunoprecipitates were assayed for
PI 3-kinase activity using phosphatidylinositol as a substrate (16,
17). Hep I stimulated the activity of PI 3-kinase 2.1-fold in the
anti-p85-PI 3-kinase immunoprecipitates (Fig.
6). Insulin, which has been demonstrated
to activate PI 3-kinase in various cell types (46-49), stimulated the
activity of PI 3-kinase 4-fold in this assay. These data demonstrate
that PI 3-kinase is activated in response to hep I addition.

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Fig. 6.
Hep I stimulates PI 3-kinase activity in
anti-p85-PI 3-kinase immunoprecipitates. Shown is an
autoradiograph of PI 3-kinase activity in anti-p85-PI 3-kinase
(Transduction Laboratories) immunoprecipitates. BAE cells were
preincubated overnight in 0.2% FBS/DMEM, treated with DMEM, 10 µM hep I, or 10 µg/ml insulin for 10 min, and
soluble lysates obtained for immunoprecipitation as described
under "Experimental Procedures." PI 3-kinase activity was
assayed using phosphatidylinositol as a substrate. The position PtdIns-P migrated on the TLC plate was visualized by exposing the plate to I2 vapor and identified using purified
standards. In comparison with DMEM treated controls, hep I stimulated a
2.1-fold increase in PI 3-kinase activity in the anti-p85-PI
3-kinase immunoprecipitates (n = 3).
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Agonist-stimulated activation of PI 3-kinase often occurs by a pathway
involving activation of tyrosine kinases and is frequently measured in
anti-phosphotyrosine immunoprecipitates (16, 17). Therefore
anti-phosphotyrosine (PY20) immunoprecipitates from control and hep
I-treated cells were assayed for PI 3-kinase activity. Using
phosphatidylinositol as a substrate, a 2.3-fold increase in PI 3-kinase
activity was detected in the anti-phosphotyrosine immunoprecipitates
from hep I-treated cells (data not shown), similar to what was observed
in the anti-p85-PI 3-kinase immunoprecipitates. However, a considerable
level of basal phosphatidylinositol kinase activity was also observed.
To assay specifically for phosphorylation by receptor-stimulated PI
3-kinase, these assays were performed using PtdIns (4,5)-P2
as a substrate. Using PtdIns (4,5)-P2 as a substrate, hep I
stimulated the activation of PI 3-kinase in the anti-phosphotyrosine
immunoprecipitates 2.9-fold (Fig.
7, A and B).
Similar activation of PI 3-kinase was also observed in cells treated
with 10 µg/ml intact TSP1 (Fig. 8).
Scrambled hep I peptide which does not induce focal adhesion
disassembly did not stimulate PI 3-kinase activity (Fig. 8).

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Fig. 7.
Hep I stimulates PI 3-kinase activity in
anti-phosphotyrosine immunoprecipitates. Shown is an
autoradiograph of PI 3-kinase activity in anti-phosphotyrosine
immunoprecipitates. BAE cells were preincubated overnight in 0.2%
FBS/DMEM, stimulated with 10 µM hep I for various times,
and soluble lysates obtained for immunoprecipitation as described under
"Experimental Procedures." PI 3-kinase activity was assayed using
PtdIns (4,5)-P2 (A) as a substrate. The position
PtdIns-P3 migrated on the TLC plate was visualized by
exposing the plate to I2 vapor and identified using
purified standards. Quantitation of PI 3-kinase activity in
A is shown in B (n = 1-4;
error bars represent S.E.). Inset, protein from
the anti-phosphotyrosine immunoprecipitates was immunoblotted with a
monoclonal antibody specific for p85-PI 3-kinase.
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Fig. 8.
TSP1 stimulates PI 3-kinase activity in
anti-phosphotyrosine immunoprecipitates. Autoradiograph of
TSP1-stimulated PI 3-kinase activity in anti-phosphotyrosine
immunoprecipitates compared with controls and hep I. BAE cells were
preincubated overnight in 0.2% FBS/DMEM, stimulated with 0.1 µM hep I, 0.1 µM scrambled hep I, or 10 µg/ml TSP1 for 10 min, and soluble lysates obtained for
immunoprecipitation as described under "Experimental Procedures."
PI 3-kinase activity was assayed using PtdIns (4,5)-P2 as a
substrate. The position PtdIns-P3 migrated on the TLC plate was visualized by exposing the plate to I2 vapor and
identified using purified standards.
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The activation of PI 3-kinase by hep I was observed as early as 2 min
and maintained for the entire 120-min time course. The time course of
PI 3-kinase activation by hep I was nearly identical to the time course
of focal adhesion disassembly induced by hep I (compare Fig. 1 with
Fig. 7B). Hep I-induced (10 min) PI 3-kinase activity was
completely inhibited by the in vitro addition of 50 nM wortmannin, further demonstrating the specificity of the assay (Fig. 7A). To confirm that p85/p110 PI 3-kinase was
involved in the increased PI 3-kinase activity in the
anti-phosphotyrosine immunoprecipitates, immunoblotting with antibodies
specific for the 85-kDa regulatory subunit was performed. A time
dependent increase in p85 immunoreactivity was observed in the
anti-phosphotyrosine immunoprecipitates following treatment with hep I
(Fig. 7B, inset).
To determine if hep I or TSP1 stimulate an increase in the tyrosine
phosphorylation of the 85-kDa regulatory subunit, anti-p85-PI 3-kinase
immunoprecipitates were immunoblotted with anit-phosphotyrosine antibodies. Tyrosine phosphorylation of the 85-kDa regulatory subunit
was not observed in control or treated samples (Fig.
9). However, a tyrosine phosphorylated
protein of ~78 kDa was observed and had increased immunoreactivity in
samples from hep I and TSP1-treated cells compared with controls.
Antibodies specific for p85-PI 3-kinase did not recognize the ~78-kDa
band (Fig. 9).

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Fig. 9.
Hep I and TSP1 do not stimulate tyrosine
phosphorylation of p85-PI 3-kinase. Shown is an immunoblot of
anti-p85-PI 3-kinase polyclonal agarose bead (Upstate Biotechnology
Inc.) immunoprecipitates probed with anti-phosphotyrosine antibodies (RC20, 1:2500). The same blot was stripped and then reprobed with anti-p85-PI 3-kinase monoclonal antibodies (1:1000). BAE cells were
preincubated overnight in 0.2% FBS/DMEM, stimulated with 0.1 µM hep I, 0.1 µM scrambled hep I, or 10 µg/ml TSP1 for 10 min, and soluble lysates obtained for
immunoprecipitation.
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Hep I Stimulates PtdIns (3,4,5)-P3 Production in Intact
Cells--
The primary in vivo products of p85/p110 PI
3-kinase phosphorylation are PtdIns (3,4)-P2 and PtdIns
(3,4,5)-P3 (25). To further establish that TSP1 activates
this type of PI 3-kinase, the levels of PtdIns (3,4,5)-P3
were examined in cells stimulated with hep I. To our knowledge, the
only in vivo mechanism for generating PtdIns
(3,4,5)-P3 is the phosphorylation of PtdIns
(4,5)-P2 by PI 3-kinase. The production of PtdIns
(3,4)-P2 can occur by mechanisms involving phosphorylation
and dephosphorylation (25). Therefore, measurement of PtdIns
(3,4,5)-P3 levels provides clear evidence for the
activation of PI 3-kinase by an agonist. BAE cells were labeled with
32Pi, treated with hep I (10 µM)
for 10 min, and the lipids extracted and separated by TLC. BAE cells
were also stimulated with insulin (10 µg/ml) as a positive control.
Hep I stimulated a 4.1-fold increase in PtdIns (3,4,5)-P3
levels over non-treated controls (Fig.
10). Insulin stimulated a 3.9-fold
increase in PtdIns (3,4,5)-P3. These results confirm the
in vitro observation that the activation of PI 3-kinase is
stimulated by hep I. Together with the inhibitor data, these two
observations support a role for a tyrosine kinase regulated PI 3-kinase
in the TSP1 signaling pathway leading to focal adhesion disassembly.

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|
Fig. 10.
Hep I stimulates an increase in PtdIns
(3,4,5)-P3 levels. Shown is an autoradiograph of
32P-labeled phospholipids from control and stimulated cells
separated by TLC. BAE cells (100-mm dish) were incubated overnight in
0.2% FBS/DMEM, followed by incubation for 60 min in phosphate-free DMEM, and then labeled for 60 min with 1.6 mCi/ml
32Pi. Cells were then treated with DMEM, 10 µM hep I, or 10 µg/ml insulin for 10 min, and lipids
extracted with chloroform. The positions PtdIns-P,
PtdIns-P2, and PtdIns-P3, migrated on the TLC
plate was visualized by exposing the plate to I2 vapor and identified using purified standards. Results are representative of two
separate experiments.
|
|
 |
DISCUSSION |
Cell adhesion involves receptor-mediated cell surface interactions
with the extracellular matrix (40, 41, 50, 51). These interactions play
a central role in the organization of the cytoskeleton, thereby
regulating cell shape and function. Focal adhesions are specialized
structures linking the extracellular matrix to the actin microfilaments
through integrin and syndecan transmembrane receptors. The structure of
the focal adhesion plaque consists of an elaborate network of
interconnecting proteins anchoring the microfilaments to the membrane
at the contact site. Focal adhesions are dynamic and highly regulated
structures which assemble and disassemble during normal cellular
functioning (40, 41, 50, 51). During the formation of focal adhesions,
structural components are recruited and assembled to anchor the
microfilaments to the adhesion site. Several important groups of
proteins have been identified in the assembly process such as protein
kinases, lipid kinases, and small GTP-binding proteins (52-57).
Although, roles for protein kinase C and cyclic
AMP-dependent protein kinase have been identified in
disassembly of focal adhesions (58-65), our understanding of the
disassembly process is limited. Anti-adhesive extracellular matrix
proteins, such as TSP1, have the potential to regulate cell function by
stimulating focal adhesion disassembly and reorganization of the actin
cytoskeleton (13).
Previously, we demonstrated that TSP1-induced disassembly of focal
adhesions in endothelial and smooth muscle cells is dependent on PKG
(15). However, TSP1 does not stimulate the activation of PKG. These
results indicated that basal PKG activity is important for the process
of focal adhesion disassembly. Moreover, previous studies suggest that
the active sequence of TSP1 interacts with a protein receptor at the
cell surface (a receptor candidate has recently been
identified).2 In this paper,
we show that activation of PI 3-kinase is essential for TSP1 signaling
of focal adhesion disassembly and reorganization of the actin
cytoskeleton. This conclusion is based on the following data: 1) two
structurally different inhibitors of PI 3-kinase activity, wortmannin
and LY294002, completely blocked the hep I-induced disassembly of focal
adhesions; 2) wortmannin inhibited the hep I-induced reorganization of
the actin cytoskeleton; 3) hep I stimulated activation of PI 3-kinase
with kinetics nearly identical to that of focal adhesion disassembly;
and 4) hep I stimulated an increase in the cellular levels of PtdIns
(3,4,5)-P3, the major product of PI 3-kinase
phosphorylation. These results are the first direct evidence that
TSP1/hep I initiates intracellular signaling. The relationship between
the activation of PI 3-kinase and basal PKG activity during TSP1
signaling remains to be determined.
Since the initial discovery of PI 3-kinase, several different isoforms
have been identified which have differential sensitivities to
wortmannin inhibition (21, 43-45, 69-72). The observation that low
concentrations of wortmannin (2.5 nM) inhibited the
TSP1-induced disassembly of focal adhesions suggests that a p85/p110 PI
3-kinase mediated this response. In addition, hep I stimulation of PI
3-kinase was detected by immunoprecipitation with antibody
recognizing the 85-kDa regulatory subunit. Hep I also induced an
increase in immunoreactivity for the 85-kDa regulatory subunit in
anti-phosphotyrosine immunoprecipitates correlating to the increase in
PI 3-kinase activity. These observations are consistent with the
involvement of the p85/p110 form of PI 3-kinase. Furthermore, p85/p110
PI 3-kinase has been localized to focal adhesions in endothelial cells
and fibroblasts suggesting a role for this enzyme in focal adhesion
regulation (41, 68). However, these results do not exclude the
possibility that other forms of PI 3-kinase may also play a role in
TSP1 signaling.
Activation of p85/p110 PI 3-kinase has been demonstrated to occur
through the interaction of the 85-kDa regulatory subunit with other
proteins, often tyrosine-phosphorylated proteins. Detection of
TSP1-induced PI 3-kinase activity in anti-phosphotyrosine
immunoprecipitates suggests that the extent of tyrosine phosphorylation
of PI 3-kinase or a tightly associated protein was increased in hep
I-treated cells. Tyrosine phosphorylation of the 85-kDa subunit has
been reported in response to certain agonists (16, 17), however, the
relationship between tyrosine phosphorylation of the regulatory subunit
and catalytic activity is unclear. We were unable to detect tyrosine
phosphorylation of PI 3-kinase, suggesting that immunoprecipitation of
PI 3-kinase activity with anti-phosphotyrosine antibodies most likely
occurred as a result of the association of PI 3-kinase with another
protein which was tyrosine phosphorylated. Consistent with this
conjecture, a tyrosine-phosphorylated protein migrating at ~78
kDa was detected in anti-p85-PI 3-kinase immunoprecipitates. Current
studies are directed at determining the identity of this protein and
its relationship to PI 3-kinase in the TSP1 signaling of focal adhesion
disassembly.
The major products of receptor-stimulated PI 3-kinase activation,
PtdIns (3,4)-P2 and PtdIns (3,4,5)-P3, have
recently become recognized as a second messenger involved in
transducing extracellular signals regulating the actin cytoskeleton
(16, 17), however, the mechanism by which these phosphoinositides
carry out this function is unclear. Recent reports have shown that
certain actin-regulating proteins which bind PtdIns
(4,5)-P2 have equal or greater affinity for PtdIns
(3,4,5)-P3 (37, 73, 74). PtdIns (4,5)-P2 also binds
-actinin, enhancing its ability to promote actin
polymerization. It has been suggested that
-actinin may be important
for maintaining or stabilizing microfilament attachment in mature focal
adhesions (40). Our data show that
-actinin is redistributed from
focal adhesion plaques in response to hep I. Thus, one possibility is that
-actinin is a target for the second messenger action of PtdIns
(3,4,5)-P3 involved in the mechanism of TSP1-induced
disassembly of focal adhesions.
The carboxyl-terminal domain of TSP1 binds to the integrin-associated
protein and enhances spreading of C32 human melanoma cells on
vitronectin (67). The enhanced spreading induced by the
carboxyl-terminal domain was blocked by 10 nM wortmannin
(67), suggesting that at least one other active domain of TSP1 signals through the activation of PI 3-kinase. In fact, there are numerous reports describing a role for PI 3-kinase in agonist-induced activation of specific integrins leading to increased attachment (18). These
studies appear contrary to this report suggesting a role for PI
3-kinase in the disassembly of focal adhesions. There are several
possible explanations. First, integrin-mediated attachment and the
formation of focal adhesions and stress fibers are two distinct and
temporally separated stages of adhesion (33, 34). In fact, Nobes
et al. (35) have demonstrated that PI 3-kinase activity is
not required for the formation of focal adhesions and stress fibers
induced by lysophosphatidic acid, bombesin, or microinjected rho
protein. Furthermore, integrin activation/reorganization does not
appear to be involved in the TSP1-mediated disassembly of focal
adhesions, since
v
3 integrin remains
clustered (12, 13). Second, our experiments were performed on adherent
cells containing focal adhesions and stress fibers, rather than with non-adherent cells lacking these structures. Localization and availability of specific signaling components can differ following adhesion (51). Finally, the work implicating PI 3-kinase in integrin
activation has been performed mostly on immune and hemopoietic cells;
different cell types often respond in different ways.
Focal adhesion kinase (FAK) has been demonstrated to associate with and
activate PI 3-kinase in vitro and in lysates from stimulated
cells (75, 76). To determine if TSP1 stimulates the association of PI
3-kinase with FAK, anti-FAK immunoprecipitates were measured for PI
3-kinase activity. Preliminary results suggested that hep I stimulated
a slight increase in PI 3-kinase activity in anti-FAK
immunoprecipitates (data not shown). However, the PI 3-kinase activity
in the anti-FAK immunoprecipitates represented less than 5% of the PI
3-kinase activity observed in the anti-phosphotyrosine immunoprecipitates. Based on these results, it does not appear that FAK
plays a major role in the activation of PI 3-kinase in response to hep
I.
PI 3-kinase and 3-phosphorylated phosphoinositides have been
demonstrated to play an important role in the reorganization of the
actin cytoskeleton, particularly membrane ruffling (16-18). However,
this is the first demonstration that PI 3-kinase is involved in the
disassembly of focal adhesions. Focal adhesion disassembly stimulated
by TSP1 or hep I does not involve membrane ruffling, suggesting that PI
3-kinase can have multiple, distinct effects on cytoskeletal
organization. Furthermore, insulin, which stimulated PI 3-kinase
activity in these experiments, neither caused membrane ruffling nor
focal adhesion disassembly (data not shown). Therefore, it appears, at
least under these experimental conditions, that PI 3-kinase activity
alone is not sufficient for focal adhesion disassembly or membrane
ruffling. Localization and extent of activity may account for some of
the differences observed in cellular events. Activation of other
signaling pathways in parallel may also be required to elicit certain
responses. Understanding the mechanisms by which specific signaling
pathways result in cellular events is a question fundamental to cell
signaling.
 |
ACKNOWLEDGEMENTS |
We thank Trevor R. Jackson (Cambridge
University) for helpful discussions and Ira J. Blader
(University of Alabama at Birmingham) for assistance with PI 3-kinase
assays.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Training Grant AR07450 from the Multipurpose Arthritis Center at the
University of Alabama at Birmingham and Postdoctoral Fellowship PF-4439
from the American Cancer Society (to J. A. G.), National Institutes of Health Grants R29 MH50102 and DDRC P50HD32901 (to A. B. T.), and National Institutes of Health Grant RO1 HL44575 and
Grant 9640228N from the American Heart Association (to J. E. M.-U.). This work was done during the tenure of an Established Investigator Grant from the American Heart Association and Genentech (to J. E. M.-U.).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 Pathology,
Div. of Molecular and Cellular Pathology, Volker Hall G038, University
of Alabama at Birmingham, Birmingham, AL 35294-0019. E-mail:
jeffrey{at}vh.path.uab.edu; Tel: 205-934-0529; Fax: 205-934-1775.
1
The abbreviations used are: TSP, thrombospondin;
BAE, bovine aortic endothelial; DMEM, Dulbecco's modified Eagle's
medium; PI 3-kinase, phosphoinositide 3-kinase; PtdIns-P,
phosphatidylinositol phosphate; PtdIns (4,5)-P2,
phosphatidylinositol (4,5)-bisphosphate; PtdIns
(3,4)-P2, phosphatidylinositol (3,4)-bisphosphate; PtdIns (3,4,5)-P3, phosphatidylinositol (3,4,5)-trisphosphate;
PKG, cyclic GMP-dependent protein kinase; FBS, fetal bovine
serum; FAK, focal adhesion kinase.
2
J. E. Murphy-Ullrich, M. A. Pallero, and M. Michalak, manuscript in preparation.
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