From the Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel
Received for publication, July 23, 2002, and in revised form, February 3, 2003
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ABSTRACT |
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Galectin-8, a member of the galectin family of
mammalian lectins, is a secreted protein that promotes cell adhesion
and migration upon binding to a subset of integrins through
sugar-protein interactions. Ligation of integrins by galectin-8
triggers a distinct pattern of cytoskeletal organization, including
formation of F-actin-containing microspikes. This is associated with
activation of integrin-mediated signaling cascades (ERK and
phosphatidylinositol 3 kinase (PI3K)) that are much more robust and are
of longer duration than those induced upon cell adhesion to
fibronectin. Indeed, formation of microspikes is enhanced 40% in cells
that overexpress protein kinase B, the downstream effector of PI3K.
Inhibition of PI3K activity induced by wortmannin partially inhibits
cell adhesion and spreading while largely inhibiting microspike
formation in cells adherent to galectin-8. Furthermore, the inhibitory
effects of wortmannin are markedly accentuated in cells overexpressing PKB or p70S6K (CHOPKB and CHOp70S6K
cells), whose adhesion and spreading on galectin-8 (but not on fibronectin) is inhibited ~25-35% in the presence of wortmannin. The above results suggest that galectin-8 is an extracellular matrix protein that triggers a unique repertoire of integrin-mediated signals, which leads to a distinctive cytoskeletal organization and
microspike formation. They further suggest that downstream effectors of PI3K, including PKB and p70 S6 kinase, in part mediate cell adhesion, spreading, and microspike formation induced by galectin-8.
Extracellular matrix
(ECM)1 proteins have
important functions in providing structural integrity to tissues and in
presenting proper environmental cues for cell adhesion, migration,
growth, and differentiation (1-5). These functions rely on
spatio-temporal expression of adhesive as well as anti-adhesive
components of the ECM proteins (6). ECM proteins like fibronectin
(7-9), collagen (10), and laminin (11) are best characterized, though other types of proteins, including mammalian lectins, also function as
modulators of cell adhesion. Selectins mediate cell-cell interactions (12) through calcium-dependent recognition of sialylated
glycans (13, 14), whereas galectins, animal lectins that specifically bind Cellular adhesion to extracellular matrix proteins is mediated by a
diverse class of cell surface We have recently shown that different cell types adhere and spread when
cultured on immobilized galectin-8, a mammalian In the present study we undertook to characterize the signaling
cascades downstream of FAK, which are activated by galectin-8 and
confer upon cells adherent to this lectin a unique cytoskeletal organization. Our results indicate that ligation of integrins by
galectin-8 is associated with GTP loading onto Ras as well as sustained
and potent activation of ERK, PKB, and p70S6K. These downstream
effectors of PI3K modulate cell adhesion and spreading on galectin-8
and account for the extensive F-actin-containing microspikes that are
formed when cells adhere onto galectin-8. Our findings therefore
implicate galectin-8 as an ECM protein that triggers a unique
repertoire of integrin-mediated signals, leading to a distinctive cell
adhesion, spreading, and cytoskeletal organization.
Materials--
Bacterially expressed recombinant galectin-8 was
generated as previously described (25).
Isopropyl- Antibodies--
Cy3-conjugated, affinity-purified
F(ab')2 fragment of goat anti-mouse IgG (H+L) was purchased
from Jackson Immunoresearch Laboratories, Inc. Polyclonal anti-PKB
antibody, monoclonal anti- Cell Cultures--
Chinese hamster ovary (CHO-P) cells were
grown in F12 medium containing 10% FCS. CHOPKB and
CHOp70S6K cells were grown in F12 medium containing 10%
FCS supplemented with 10 µg/ml puromycin for selection. Human
endothelial (HE) ECV304 cells (28) were grown in
Dulbecco's modified Eagle's medium/F12 medium containing 10% FCS.
B16 murine melanoma cells were grown in Dulbecco's modified Eagle's
medium containing 10% FCS. NIH-hIR (NIH) mouse
fibroblasts (overexpressing the insulin receptor (29)) were grown in
Dulbecco's modified Eagle's medium containing 10% FCS.
Generation of Stable Clones of CHOPKB and
CHOp70S6K Cells Overexpressing PKB or p70S6K,
Respectively--
CHO-P cells were stably transfected using
LipofectAMINE as previously described (30). The plasmids used for
overexpression of WT-PKB or WT-p70S6K were pCIS2 or p2B4, respectively
(31, 32). The cells were co-transfected with pBabe-Puro plasmid, which
encodes a puromycin resistance gene. Following transfection, 10 µg/ml
puromycin was added for selection of stable colonies. Puromycin-resistant clones that overexpress WT-PKB or WT-p70S6K were
isolated and further propagated.
Cell Adhesion Assay--
Bacterial or tissue-culture plates were
precoated for 2 h at 22 °C with galectin-8 or fibronectin in
PBS. Cells, grown on tissue-culture plates, were detached from the
plates with 5 mM EDTA, washed with PBS, resuspended in
serum-free medium, and re-seeded on the coated plates. At the indicated
times, cells were washed, and the adherent cells were stained with
0.2% crystal violet and 20% methanol in H2O for 10 min at
22 °C. Excess dye was removed by washes with water, and cells were
solubilized in 1% SDS for 1 h at 22 °C. The amount of the
adherent cells was quantified by measuring the absorbance at 540 nm in
an enzyme-linked immunosorbent assay plate reader-TECAN (Spectra,
Austria). Specific binding was defined as the difference between the
absorbance of cells bound to ligand-coated wells less the absorbance of
cells bound to bovine serum albumin-coated wells. All assays were
performed in quadruplicate.
Binding of Activated Ras to its Effector--
Activation of Ras
was determined by binding assay of the activated (GTP-loaded) forms of
Ras to a minimal RBD of Raf1 (33), coupled to GST (GST·RBD).
GST·RBD, coupled to glutathione-agarose beads, was washed three times
in buffer C (50 mM Tris-HCl, pH 7.5, 10% glycerol, 2.5 mM MgCl2, 200 mM NaCl, 1.0%
Nonidet P-40, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 0.1 µg/ml
trypsin inhibitor, 250 µM phenylmethylsulfonyl
difluoride, 10 mM NaF, 1 mM sodium orthovanadate). CHO-P cells were washed, and extracts were prepared in
buffer C. Insoluble material was removed by 15 min of centrifugation (20,000 × g) at 4 °C. Then, 10-20 µg of fusion
proteins coupled to glutathione-agarose beads were incubated with the
cell extract (0.5 mg) for 45 min at 4 °C. The Ras·GTP-bound
beads were further washed (four times) with buffer F (20 mM
HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% Triton
X-100). The bound proteins were suspended in Laemmli's sample buffer,
resolved by 15% SDS-PAGE, and Western-immunoblotted with the indicated antibodies.
Preparation of Cell Extracts and Immunoblotting--
Cell
extracts were prepared in buffer I (25 mM Tris/HCl, 25 mM NaCl, 0.5 mM EGTA, 2 mM sodium
orthovanadate, 10 mM NaF, 10 mM sodium
pyrophosphate, 80 mM Immunoprecipitation--
Cell extracts (1-2 mg protein) were
incubated for 1 h at 4 °C with monoclonal p130Cas
antibody, followed by additional incubation for 1 h at 4 °C
with 30 µl of protein G-agarose beads. Immunocomplexes were washed twice with buffer J (25 mM Tris/HCl, 25 mM
NaCl, 0.5 mM EGTA, 2 mM sodium orthovanadate,
10 mM NaF, 10 mM sodium pyrophosphate, 80 mM Immunofluorescence Microscopy--
Cultured cells, plated on
glass coverslips, were washed and fixed with paraformaldehyde (3%)
containing 0.5% Triton X-100 (for phalloidin staining) or cold 100%
methanol (for tubulin staining). Following several washes with PBS,
cells were incubated for 1 h at 22 °C with TRITC-labeled
phalloidin or with anti-tubulin monoclonal antibodies. Cells were
washed with PBS and further incubated for 1 h at 22 °C with
secondary Cy3-conjugated goat anti-mouse antibody in PBS. The specimens
were washed, mounted onto glass microscope slides, and examined on
Zeiss fluorescence microscope.
Cell Adhesion onto Galectin-8 Induces a Characteristic Pattern of
Cytoskeletal Organization--
Immobilized galectin-8, similar to
other ECM proteins, promotes cell adhesion and spreading (24). Still,
there is a marked difference in cytoskeletal organization between cells
adherent to galectin-8 versus fibronectin (24).
Time-dependent formation of prominent stress fibers that
traverse the cell body are readily observed
in HE (Fig. 1) and CHO-P cells (Fig.
2) adherent onto fibronectin but are less
abundant in cells adherent to galectin-8. Instead, adhesion and
spreading onto galectin-8 were characterized by initial formation
(within 10 min) of sheet-like projections of the membrane, known as
lamellipodia (34), that were followed by the appearance of short
individual projections (< 2 microns long) at the cellular perimeter,
known as F-actin microspikes (35-37). Microspikes were readily
detected 30-60 min following cell adhesion on galectin-8, and they
continued to develop for at least the first 120 min (Figs. 1 and 2).
Microspike formation induced upon cell adhesion onto galectin-8 is
rather a general phenomenon. It was observed in a number of cell
lines, derived from different tissues and species such as HE cells
(Fig. 1), CHO-P (Fig. 2), and NIH-hIR
fibroblasts. Still, it is not a
ubiquitous phenomenon because certain cell types, such as B16 murine
melanoma cells, fail to produce microspikes upon adhesion to
galectin-8.2 Additional
features characterize cells adherent to galectin-8; whereas vinculin
and paxillin are associated with large focal contacts in cells adherent
to fibronectin, the number and size of vinculin- and
paxillin-containing focal contacts is reduced in cells attached to
galectin-8 (24). The differences in actin-microfilament organization
were not accompanied by differences in microtubules organization, and a
similar microtubular network developed when cells adhered to galectin-8
or fibronectin (Fig. 2B).
Phosphorylation of p130Cas Is Induced upon Cell
Adhesion to Galectin-8--
The differences in cytoskeletal
organization between cells adherent to galectin-8 versus
fibronectin could be attributed to different signaling readouts
elicited upon ligation of cell surface integrins by these two ECM
molecules. We have already demonstrated that FAK phosphorylation, which
occurs at early stages of cell-matrix interactions, is a common signal
emitted upon cell adhesion to fibronectin or galectin-8 (24). Hence,
bifurcation of the signals mediated by fibronectin and galectin-8
presumably takes place downstream of FAK. To address this possibility,
Tyr phosphorylation of p130Cas, a downstream effector of
FAK, was studied. As shown in Fig. 3, the
P-Tyr content of p130Cas increased to a similar extent when
CHO-P cells were allowed to adhere for 60 min onto fibronectin- or
galectin-8-coated plates. The effect was rapid, and maximal levels of
P-Tyr p130Cas were already reached after 15 min of cell
adhesion onto galectin-8 (Fig. 3A). These findings suggest
that galectin-8, similar to fibronectin, triggers Tyr phosphorylation
of p130Cas, presumably through activation of FAK.
Cell Adhesion to Galectin-8 Activates Ras and the MAPK
Cascade--
Next, the effects of galectin-8 on the activation of Ras
and the MAPK cascade were examined. Consistent with previous studies, activation of Ras (Fig. 3B) and phosphorylation of ERK1 and
-2 (Fig. 4) were largely diminished upon
detachment of CHO-P cells from culture plates (e.g. Fig.
3B, time zero and 15-60
min without galectin-8 or fibronectin). Re-adhesion of the
cells onto fibronectin- or galectin-8-coated plates lead to activation
of Ras (Fig. 3B) and phosphorylation of ERK1 and -2 (Fig.
4). Maximal GTP loading onto Ras and phosphorylation of ERK1 and -2 was
achieved by 15 min. However, whereas phosphorylation of ERK1 and -2 in
cells adherent to galectin-8 was sustained for at least 60 min, ERK phosphorylation in cells adherent to fibronectin was transient and
rapidly declined (Fig. 4B). Addition of wortmannin, a PI3K inhibitor, diminished ERK phosphorylation induced either by fibronectin or galectin-8 (Fig. 4), indicating that PI3K is an upstream regulator of ERK1 and -2 phosphorylation, which occurs upon integrin ligation by
galectin-8.
Galectin-8 Induces Robust and Sustained Activation of PKB and
p70S6K--
The above results have indicated that activation of PI3K
triggers the MAPK cascade when cells adhere onto galectin-8. To explore whether other downstream effectors of PI3K are being activated, the
effects of galectin-8 on PKB and p70S6K were studied. Phosphorylation of both proteins was largely diminished upon detachment of CHO-P cells
from culture plates (Fig. 5, A
and B, time zero), whereas re-adhesion of the
cells onto fibronectin or galectin-8 lead to a
time-dependent phosphorylation of PKB and p70S6K (Fig.
5A). Phosphorylation of both proteins was significantly
higher upon cell adhesion to galectin-8 than fibronectin at all time
points tested. For example, at 60 min, the extent of phosphorylation of
PKB and p70S6K was 8- and 3-fold higher, respectively, in cells adherent to galectin-8. Pretreatment with wortmannin largely diminished the phosphorylation of PKB and p70S6K (Fig. 5, A and
B), indicating that these proteins are indeed downstream
effectors of PI3K.
Overexpression of PKB and p70S6K Diverts Adhesion of CHO Cells into
a PI3K-dependent Pathway--
To determine whether
downstream effectors of PI3K affect cell adhesion to galectin-8, PKB
and p70S6K were introduced into CHO-P cells, and stable clones that
overexpress either PKB (CHOPKB) or p70S6K
(CHOp70S6K) were generated. These clones expressed 1.7- and
2.5-fold higher levels of PKB or p70S6K, respectively, than the
endogenous expression levels of these proteins (Fig.
6A). As shown in Fig.
6B, adhesion of CHOPKB and CHOp70S6K
cells to fibronectin or galectin-8 triggered similar signaling responses seen in wild-type CHO-P cells. Phosphorylation of PKB and
p70S6K was largely diminished upon detachment of the cells from culture
plates, whereas re-adhesion stimulated the phosphorylation of the
overexpressed PKB and p70S6K (Fig. 6, B and C).
Again, the signals elicited upon cell adhesion to galectin-8 were
stronger and more sustained when compared with the signals emitted upon cell adhesion onto fibronectin, and pretreatment with wortmannin largely diminished the phosphorylation of PKB and p70S6K (Fig. 6,
B and C). Next, the effects of wortmannin on cell
adhesion were evaluated. As shown in Fig.
7, wortmannin had a slight (~15%) albeit significant inhibitory effect on adhesion of CHO-P cells to
galectin-8. However, this inhibitory effect was markedly accentuated in
CHOPKB and CHOp70S6K cells, whose adhesion onto
galectin-8 was inhibited 35 and 28%, respectively, in the presence of
wortmannin. This inhibitory effect was specific, because cell adhesion
to fibronectin was unaffected by the drug. Thus, overexpression of PKB
and p70S6K generates wortmannin-sensitive signals that promote cell
adhesion onto galectin-8, although the overall adhesion rates (in
the absence of wortmannin) were not affected by the overexpressed PKB
and p70S6K (Fig. 7).
Cellular Spreading over Galectin-8--
An immediate consequence
of cell adhesion is cell spreading. Cells spread much faster on
galectin-8, compared with fibronectin. Although most cells were already
spread on galectin-8 by 10 min, cells adherent to fibronectin were
still round and began to spread only after ~20 min (compare Figs. 1
and 2). Cell spreading (measured as increased cell area) on either
galectin-8 or fibronectin was comparable by 2 h (Fig.
8). This was evident by the similar
distribution of cells areas, with a mean of 875 ± 175 arbitrary
units following 2 h of adhesion on either galectin-8 or
fibronectin, (Fig. 8). Still, the rate of cellular spreading over
galectin-8 was faster, and comparable cell areas were observed in cells
adherent to galectin-8 or fibronectin for 10 and 20 min, respectively.
Interestingly, although the area distribution of cells adherent to
fibronectin by 20 min was rather limited (Fig. 8), a much wider area
distribution (> 700 arbitrary units) characterized cells adherent to
galectin-8 by 10 min (Fig. 8). This phenomenon was not unique to CHO-P
cells (Fig. 8), because a wide area distribution was also observed when B16, HE, or NIH cells adhered and spread over galectin-8 for 10-20 min
(Fig. 9, a-c) as compared
with the limited area distribution of these cells when spread on
fibronectin (Fig. 9, d-f). The reason for this variability
is presently unknown, but as shown in Fig. 10, the variance cannot be attributed
to variable induction of protein synthesis because inclusion of
cycloheximide did not alter the rate or phenotype of cells adherent to
galectin-8. In contrast, cellular attachment on galectin-8 was
inhibited ~50%, whereas spreading hardly took place at 4 °C (Fig.
10), indicating that energy-consuming processes and active protein
trafficking are required to mediate these events.
Wortmannin Inhibits Cell Spreading on Galectin-8--
The faster
initial rate of cell spreading on galectin-8 occurred despite the fact
that the rates of cell adhesion onto galectin-8 or fibronectin were
comparable (compare Fig. 7 and Fig. 8, inset). These results
suggest that the initial affinity of integrins to either galectin-8 or
fibronectin is comparable, although subsequent cytoskeletal
organization associated with cell spreading occurs faster in cells
adherent to galectin-8. To assess the contribution of PKB and p70S6K to
this process, cellular spreading was studied in CHO-P,
CHOPKB, and CHOp70S6K cells. As shown in Fig.
11A, the three cell types
spread to a similar extent following 10 or 20 min of adhesion over
galectin-8 or fibronectin, respectively. Under these conditions about
60% of all populations had areas of < 350 arbitrary units (Fig.
11B). These results indicate that the rate of cell
spreading, as well as cell adhesion (Fig. 7), is independent of the
overexpressed PKB and p70S6K. However, similar to its inhibitory
effects on cell adhesion, wortmannin selectively inhibited cell
spreading on galectin-8 (Fig. 11, A and B), but
not on fibronectin, and its inhibitory effects were more pronounced in
cells overexpressing PKB or p70S6K. Indeed, wortmannin increased the
percentage of CHO-P, CHOPKB, and CHOp70S6K
cells having areas of < 350 arbitrary units (following 10 min of
adhesion) by 20, 30, and 40%, respectively (Fig. 11A).
Hence, overexpression of downstream effectors of PI3K sensitizes the cells to the inhibitory effects of wortmannin, implicating the overexpressed PKB and p70S6K as potential mediators of cell spreading on galectin-8. The inhibitory effects of wortmannin on cell spreading were not restricted to CHO-P cells. As shown in Fig. 9, wortmannin effectively inhibited spreading of HE, NIH, and B16 cells on galectin-8 but had trivial effects on spreading of these cells on fibronectin.
Effects of PI3K and Its Downstream Effectors on Formation of
Microspikes in Cells Adherent to Galectin-8--
Because formation of
microspikes appeared to be a characteristic feature of cells adherent
onto galectin-8, the effects of PI3K and its downstream effectors on
microspike formation were evaluated. Cells were considered as
possessing microspikes when at least 35% of the cellular perimeter
(not in contact with other cells) contained these structures. As shown
in Fig. 12, A and
B, formation of microspikes in cells adherent onto
galectin-8 was significantly (> 60%) inhibited when cells were
treated with wortmannin, implicating PI3K and its downstream effectors
as mediators of microspike formation. This conclusion was supported by
the fact that there was a ~40% increase in formation of microspikes
in CHOPKB cells compared with CHO-P or
CHOp70S6K cells (Fig. 12A), implicating PKB as
being actively involved in signals mediating microspike formation upon
cell adhesion to galectin-8. Microspikes could not be detected in cells
adherent to fibronectin.
In the present study we provide evidence that galectin-8, a
mammalian lectin (24, 27), functions as an ECM protein that triggers a
unique spectrum of signaling events upon ligation of sugar moieties of
integrins. Although cell adhesion onto galectin-8 or fibronectin
activates to the same extent FAK and p130Cas, the signaling
cascades triggered upon adhesion to galectin-8 are characterized by a
robust and sustained activation of ERK-1 and -2, which contrasts with
the transient nature of ERK activation upon cell adhesion to
fibronectin. Similarly, activation of PKB and p70S6K, which serve as
downstream effectors of PI3K, is several -folds more intense and
sustained when cells adhere to galectin-8 than fibronectin. The unique
signaling pattern triggered upon cell adhesion to galectin-8 is
associated with faster cell spreading and with a distinctive
organization of cytoskeletal elements. Prominent stress fibers that
traverse the cell body are readily observed in cells adherent to
fibronectin, but they are less abundant in cells adherent to
galectin-8. Similarly, formation of focal contacts is limited when
cells adhere onto galectin-8 (24). Instead, adhesion to galectin-8
triggers sustained formation of F-actin microspikes. This is rather a
general phenomenon observed in a number of cell lines; still, it is not
a ubiquitous phenomenon because certain cell types, such as B16 murine
melanoma cells, fail to produce microspikes upon adhesion to
galectin-8.
Inhibitors of PI3K impair cell adhesion and spreading on galectin-8 and
the formation of microspikes but have no effects on cells adherent to
fibronectin, indicating that downstream effectors of PI3K selectively
regulate cytoskeletal rearrangements that occur when cells adhere to
and spread on galectin-8. Indeed, overexpression of PKB potentiates the
formation of microspikes, whereas overexpression of PKB or p70S6K
accentuates the sensitivity of cells adherent to and spread on
galectin-8 to inhibitors of PI3K. Hence, the differences in
cytoskeletal organization observed when cells adhere to galectin-8 or
fibronectin can be attributed to differences in the robustness and
duration of the PI3K-mediated signals emitted upon adhesion to the two matrices.
Several lines of evidence support these conclusions. First, we could
demonstrate that cell adhesion onto galectin-8 induces signaling
cascades that are being utilized by integrins upon ligation by other
ECM proteins. Common signaling elements, activated to about the same
extent by galectin-8 and fibronectin, include FAK (24) and
p130Cas (Fig. 3), indicating that upstream elements of
integrin signal transduction, such as FAK and p130Cas, are
activated irrespective of the mode of ligation and clustering of
integrins, which might involve either protein-protein interactions, in
the case of fibronectin, or protein-sugar interactions, in the case of
galectin-8. Hence, the restricted lateral mobility of integrins at the
plane of the membrane upon binding of a bivalent lectin to their
extracellular domains is sufficient to induce a conformational change
that is conveyed to the cytoplasmic domains of integrins and triggers
the recruitment and activation of FAK. However, the overall
ligand-induced conformational change of integrins differs, depending on
whether integrin clustering is induced upon protein-protein or
protein-sugar interactions, because the cytoskeletal organization and
the nature of the distal signals emitted downstream of FAK and
p130Cas differ when cells adhere onto galectin-8 or
fibronectin. Whereas cell adhesion to fibronectin leads to transient
activation of MAPK, engagement of integrins by galectin-8 leads to
sustained activation of ERK-1 and -2. Similarly, ligation of integrins
by galectin-8 results in more robust and sustained activation of PKB
and p70S6K. How is a similar extent of activation of FAK and p130Cas by galectin-8 or fibronectin translated into
differences in the state of activation of their downstream effectors
(Ras, ERK-1,2, PKB, and p70S6K)? One possibility is that a different
set of integrins is ligated by galectin-8 or fibronectin. We have
already shown that galectin-8 preferentially ligates
The robustness and duration of the activation of a given signaling
pathway has far reaching biological consequences. For example, it is
well established that transient activation of the MAPK cascade (e.g. by epidermal growth factor) leads to enhanced
growth of PC-12 cells, whereas stimulation of these cells with nerve
growth factor induces sustained activation of the MAPK cascade, which leads to cellular differentiation (39). Accordingly, the sustained and
robust activation of the MAPK and PI3K signaling pathway upon cell
adhesion to galectin-8 might account for the unique cytoskeletal organization and biological functions of cells adherent to this lectin.
Activation of ERK was inhibited in the presence of wortmannin, suggesting that PI3K is an upstream regulator of ERK signaling, triggered upon cell adhesion to galectin-8 or fibronectin. This conclusion is in accordance with previous studies that have
demonstrated that PI3K may function upstream of Raf-1 but downstream of
Ras upon integrin ligation by fibronectin (40). These results further support the role of integrins as ligands for galectin-8 because, unlike
integrin signaling, activation of the ERK pathway by other means, such
as ligation of growth factor receptors, is most often insensitive to
inhibitors of PI3K (41).
An interesting outcome of the present study is the finding that PKB and
p70S6K are actively involved in mediating cell adhesion and spreading
on galectin-8. Their positive role is particularly evident in cells
overexpressing PKB or p70S6K (CHOPKB and
CHOp70S6K cells), whose adhesion and spreading is inhibited
~30% in the presence of PI3K inhibitors. The role of PKB or p70S6K
as positive regulators of cell adhesion is in accordance with the fact
that ligation of growth factor receptors, which stimulates the activity of PKB or p70S6K (42), potentiates the adhesive process in a number of
cell types (43). At present, the signaling pathways regulating cell
adhesion and spreading are not fully understood. Still, our results
suggest that inhibition of the PI3K activity, induced by wortmannin,
does not directly affect the active conformation of integrins because
wortmannin does not inhibit cell adhesion to fibronectin. Instead, our
results suggest that protein substrates for PKB or p70S6K are
phosphorylated to a higher extent in cells adherent to galectin-8, in
particular CHOPKB and CHOp70S6K cells, and this
enables them to replace other signaling molecules that promote cell
adhesion and spreading on galectin-8. The displacement of the native
signaling elements with downstream effectors of PI3K is an irreversible
process, because inhibition of PI3K activity does not enable the
original participants to resume their positions within the integrin
signaling complex and to turn the adhesive process less
sensitive to PI3K inhibitors, as in non-transfected CHO cells. PKB has
already been implicated as a mediator of cell adhesion (44), but the
role of p70S6K in this process is less obvious. There is little
evidence that p70S6K is required for the processes of cell adhesion,
and activation of p70S6K was shown to be independent of pathways that
regulate formation of focal adhesions (45). Our results suggest that
p70S6K under certain conditions might modulate integrin activation by
selective ECM proteins such as galectin-8, although the direct targets
of this kinase within cell adhesion complexes remain to be determined. Our findings further suggest that cells that overexpress specific signaling elements, such as downstream effectors of PI3K, are bound to
utilize these new elements not only for the promotion of cellular
growth but also for remodeling of their "inside out" signaling
elements and integrins function.
An immediate consequence of cell adhesion is cell spreading. In the
present study we provide evidence that cells spread much faster on
galectin-8, compared with fibronectin. Interestingly, although the
variance of areas of cells adherent to fibronectin at early time points
is rather limited, a much wider variance characterizes cells adherent
to galectin-8. The reason for this variability is presently unknown,
but it cannot be attributed to variable induction of protein synthesis
because inclusion of inhibitors of protein synthesis did not alter the
rate or phenotype of cells adherent to galectin-8. In contrast,
cellular attachment to galectin-8 is inhibited ~50%, whereas
spreading hardly takes place at 4 °C, indicating that
energy-consuming processes and active protein trafficking are required
to mediate these events. In that respect, cell adhesion to galectin-8
resembles cell adhesion to other ECM proteins, which is largely
inhibited at low temperatures (compare Ref. 46).
Cellular attachment and spreading on fibronectin involves an initial
requirement for Cdc42 in the formation of filopodial protrusions and
the subsequent involvement of both Cdc42 and Rac during cell spreading
and organization of the actin cytoskeleton (47). In galectin-8-adherent
cells, focal contacts poorly assemble (24) and microspikes containing
F-actin are formed instead. These structures have been functionally
implicated in cell migration (37), which is readily induced by
galectin-8 (24). Microspikes are readily formed when cells adhere to a
variety of other ECM proteins such as thrombospondin-I (37), laminin-5
(48), and Tenascin-C splice variants (49). On fibronectin, Cdc42- and Rac-dependent formation of microspikes is involved in early
steps of cell adhesion, but these events are transient and microspikes are rapidly replaced by focal contacts (37). In contrast, microspikes are stabilized when cells adhere to galectin-8, and the cells do not
proceed to form highly developed focal contacts (24). Whereas Cdc42
leads to the formation of elongated projections containing F-actin, Rac
leads to the formation of ribbons of short spikes (37). The microspikes
formed when cells adhere to galectin-8 are short and radial and in that
respect resemble microspikes formed when C2C12 cells, overexpressing a
constitutively active Rac, adhere onto thrombospondin-I (36). We can
therefore suggest that formation of microspikes upon cell adhesion to
galectin-8 presumably involves Rac activation. Still, additional
signaling elements, induced by galectin-8, are likely to be involved.
Potential candidates are elements of the PI3K/PKB pathway, which are
activated to a much greater extent by galectin-8 than fibronectin. In
accordance with this idea, addition of wortmannin, a potent inhibitor
of PI3K, effectively inhibits PKB activity and formation of microspikes when cells adhere onto galectin-8. Furthermore, overexpression of PKB
promotes formation of microspikes in cells adherent onto galectin-8.
The possible involvement of PI3K and its downstream effectors in
galectin-8-mediated formation of microspikes is supported by recent
findings implicating the signaling pathway from the insulin-like growth
factor-I receptor through PI3K in the rapid organization of microspikes
at cell-cell junctions (50, 51). PKB, the downstream effector of
PI3K, activates a number of kinases, including the p21-activated kinase
(PAK) (52) that has been implicated as playing a role in actin
organization. PAK inhibits the activity of coffilin (reviewed in
Refs. 53 and 54) and in such a way may inhibit actin depolymerization
and promote formation of F-actin microspikes induced by galectin-8.
Finally, it should be noted that prostate carcinoma tumor antigen-1,
the human isoform of galectin-8, is highly expressed in certain forms
of prostate carcinomas (55) and other tumors (56). In contrast,
galectin-8 expression decreases in human colon cancer when compared
with normal and dysplastic colon tissues (57). This is associated with
reduced migration of the colon cancer cells on immobilized galectin-8.
Because interactions of soluble galectin-8 with cell surface integrins
inhibit cell adhesion (27), whereas immobilized galectin-8 has the
potential to promote cell attachment and spreading (24), galectin-8 may
modulate cell-matrix interactions under a variety of physiological and pathological conditions, depending on the repertoire, duration, and
robustness of signals emitted when cells interact with this lectin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactoside residues (15), were implicated as modulators of
cell-matrix interactions. Although lacking a signal peptide and found
mainly in the cytosol, galectins are externalized by an atypical
secretory mechanism (16) to regulate cell growth, cell transformation,
embryogenesis, and apoptosis (reviewed in Refs. 17 and 18). In
accordance with their proposed functions, galectins enhance or inhibit
cell-matrix interactions (reviewed in Ref. 19).
heterodimeric receptors known as
integrins (2, 20, 21). In addition to mediating cell adhesion,
integrins induce multiple signal transduction pathways that regulate
cytoskeletal rearrangements, cell spreading, migration, differentiation, survival, and cell growth. These processes are associated with activation of a number of signaling elements, (4), most
prominent of which is focal adhesion kinase (FAK), which undergoes
integrin-stimulated autophosphorylation. Tyr-phosphorylated FAK
recruits Grb2-Sos complexes, which activate the Ras-MAPK signaling pathway. FAK also phosphorylates p130Cas, which binds Crk
and generates further signals through c-Jun NH2-terminal
kinase. P-Tyr397 of FAK serves as a binding site for the
SH2 domain of p85
, the regulatory subunit of PI3K that propagates
signals to protein kinase B (PKB) and p70 S6 kinase (p70S6K) (reviewed
in Refs. 4 and 22). Stimulation of integrins also activates the
Rho-family GTPases Rho, Rac, and Cdc42, which mediate the formation of
stress fibers, lamellipodia, and filopodia, respectively (23).
-galactoside-binding protein (24). Galectin-8 (25-27), a member of the galectin family, is
a secreted protein that is widely expressed. It is made of two
homologous carbohydrate-recognition domains linked by a short (~26
amino acids) peptide. Upon secretion, galectin-8 binds to a subset of
cell surface integrins, which include integrin
3
1 or
6
1
but not
4
1 (27). Immobilized galectin-8
is equipotent to fibronectin in promoting cell adhesion and spreading,
effects that involve sugar-protein interactions of integrins with
galectin-8 (24). Accordingly, cell adhesion to galectin-8 is
potentiated in the presence of Mn2+, whereas adhesion is
interrupted in the presence of soluble galectin-8, integrin
1 inhibitory antibodies, EDTA, or thiodigalactoside but
not RGD peptides (24). Whereas immobilized galecin-8 promotes cell
adhesion, soluble galectin-8 interacts both with cell surface integrins
and other soluble ECM proteins and inhibits cell-matrix interactions
(27). These observations suggest that galectin-8 is a matrix protein
that can positively or negatively modulate cell adhesion (24).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside was purchased from
MBI Fermentas (Amherst, NY). Bovine fibronectin, puromycin, wortmannin,
glutathione-agarose beads, cycloheximide, and crystal violet were
purchased from Sigma. Protein G-PLUS-agarose beads were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). LipofectAMINE reagent was
from Invitrogen. A construct of wild-type (WT) PKB in the pCIS2
expression vector was kindly provided by M. Quon (NHLBI, National
Institutes of Health, Bethesda, MD). A construct of WT p70S6K in the
p2B4 expression vector was kindly provided by G. Thomas,
(Friedrich Meischer Institute, Basel, Switzerland). GST·RBD (Ras-binding domain of Raf), coupled to
glutathione-agarose beads was kindly provided by R. Seger (Weizmann
institute, Rehovot, Israel).
-tubulin, and TRITC-labeled phalloidin
were from Sigma. Monoclonal anti-Ras and p130Cas antibodies
were obtained from Transduction Labs (Lexington, KY). Polyclonal
anti-phospho-PKB (Ser473), anti-p70S6K, and
anti-phospho-p70S6K (Thr389) antibodies were obtained from
New England BioLabs, Inc. (Beverly, MA). Polyclonal anti-ERK1 and -2 and monoclonal anti-phospho ERK1 and -2 (Thr183,
Tyr185) antibodies were kindly provided by R. Seger
(Weizmann institute, Rehovot, Israel).
-glycerophosphate, 1% Triton
X-100, 0.5% deoxycholate, 0.05% SDS, 5 µg/ml leupeptin, 10 µg/ml
trypsin inhibitor, and 1 mM phenylmethylsulfonyl
difluoride, pH 7.5). Insoluble material was removed by 15 min of
centrifugation (20,000 × g) at 4 °C. Supernatants
were mixed with 5× concentrated Laemmli's sample buffer,
boiled for 5 min, and resolved on 10% SDS-PAGE under reducing
conditions. Proteins were transferred to nitrocellulose membranes and
Western immunoblotted with the indicated antibodies.
-glycerophosphate, 1% Triton X-100, 5 µg/ml
leupeptin, 10 µg/ml soybean trypsin inhibitor, and 1 mM
phenylmethylsulfonyl difluoride, pH 7.5) and once with PBS. Samples
were mixed with Laemmli's sample buffer, boiled for 5 min, resolved by
means of 10% SDS-PAGE, and immunoblotted with the indicated antibodies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cytoskeletal organization of HE cells
adherent to galectin-8 or fibronectin. Cover glasses were coated
for 2 h at 22 °C with galectin-8 (0.7 µM)
(a-e) or fibronectin (0.04 µM)
(f-j) in PBS. HE cells were incubated for 16 h in
serum-free medium. Then, the cells were detached from the culture
plates with 5 mM EDTA, washed, and incubated in suspension
for 30 min at 37 °C in serum-free medium. Next, cells were seeded on
galectin-8- or fibronectin-coated cover glasses. Following incubation
at 37 °C for the indicated times, cells were fixed and incubated
with TRITC-labeled phalloidin for actin staining.
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Fig. 2.
Cytoskeletal organization of CHO-P cells
adherent to galectin-8 or fibronectin. Cover glasses were coated
for 2 h at 22 °C with galectin-8 (0.7 µM)
(panel A, a-e; panel B, d)
or fibronectin (0.04 µM) (panel A,
f-j; panel B, a, b) in
PBS. CHO-P cells were incubated for 16 h in serum-free medium.
Then the cells were detached from the culture plates with 5 mM EDTA, washed, and incubated in suspension for 30 min at
37 °C in serum-free medium. Next, cells were seeded on galectin-8-
or fibronectin-coated cover glasses. Following incubation at 37 °C
for the indicated times (A) or for 2 h (B) cells
were fixed and incubated with TRITC-labeled phalloidin for actin
staining or were immunostained for tubulin as indicated.
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Fig. 3.
Activation of Ras and phosphorylation of
p130Cas induced upon cell adhesion to galectin-8 or
fibronectin. A, 6-cm bacterial plates were coated with
galectin-8 (0.7 µM) or fibronectin (0.04 µM) for 2 h at 22 °C. CHO-P cells, grown on
tissue culture plates, were incubated for 16 h in serum-free
medium. Cells were detached from the plates with 5 mM EDTA,
washed, kept in suspension for 30 min at 37 °C in serum-free medium,
and seeded in serum-free medium on the coated plates. Following
incubation at 37 °C for the indicated times, cells were washed and
extracted using buffer I. Cell extracts (1.0 mg) were subjected to
immunoprecipitation with anti-p130Cas antibody, resolved by
10% SDS-PAGE, transferred to nitrocellulose membranes, and Western
immunoblotted with anti-P-Tyr antibody (a). Alternatively,
proteins (100 µg) were resolved by 10% SDS-PAGE, transferred to
nitrocellulose membranes, and Western immunoblotted with
anti-p130Cas antibody (b). B, CHO-P
cells were grown and treated as indicated in panel A. Cells
were washed and extracted using buffer C. Cell extracts (0.5 mg) were
incubated with GST·RBD. The bound proteins, corresponding to Ras-GTP,
were resolved by 15% SDS-PAGE, transferred to nitrocellulose
membranes, and Western immunoblotted with anti-Ras antibody
(c). Alternatively, proteins (100 µg) from total cell
extracts were resolved by 15% SDS-PAGE, transferred to nitrocellulose
membranes, and Western immunoblotted with anti-Ras antibody
(d). Quantitation of the intensity of the bands,
corresponding to Ras·GTP is presented as a bar graph. The
results are mean ± S.D. of four experiments.
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Fig. 4.
Activation of ERK-1 and ERK-2 induced upon
cell adhesion to galectin-8 or fibronectin. A, 6-cm
bacterial plates were coated with galectin-8 (0.7 µM) or
fibronectin (0.04 µM) for 2 h at 22 °C. CHO-P
cells grown on tissue culture plates were incubated for 16 h in
serum-free medium. Cells were detached from the plates with 5 mM EDTA, washed, and incubated in suspension for 15 min at
37 °C in serum-free medium. The cells were further incubated for an
additional 15 min in suspension in the presence or absence of 100 nM Wortmannin and were seeded in serum-free medium on the
coated plates. Following incubation at 37 °C for the indicated
times, cells were washed and extracted using buffer I. Proteins (100 µg) were resolved by 10% SDS-PAGE, transferred to nitrocellulose
membranes, and Western immunoblotted with anti-phospho-ERK antibodies
( -pERK1,
-pERK2) (a) or with
anti-ERK antibodies (
-ERK1,
-ERK2)
(b). B, quantitation of the intensity of the
bands, corresponding to the phosphorylation of ERK1 (c) or
ERK2 (d) is presented. Results are the mean ± S.D. of
two independent experiments.
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Fig. 5.
Activation of PKB and p70S6K upon adhesion of
CHO-P cells to galectin-8 or fibronectin. A, CHO-P
cells were incubated in the absence or presence of wortmannin and
seeded on galectin-8- or fibronectin-coated plates as described in Fig.
4A. Cell extracts were made using buffer I. Proteins (100 µg) were resolved by 10% SDS-PAGE, transferred to nitrocellulose
membranes, and Western immunoblotted with (a)
anti-phospho-PKB ( -pPKB); (b) anti-PKB
(
-PKB); (c) anti-phospho-p70S6K
(
-pp70S6K); or (d) anti-p70S6K antibodies
(
-p70S6K). B, quantitation of the intensity of
the bands, corresponding to the phosphorylated PKB (e) or
the phosphorylated p70S6K (f) is presented as a line
graph. Results are the mean ± S.D. of two independent
experiments.
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Fig. 6.
Activation of PKB and p70S6K induced upon
adhesion of CHOPKB and CHOp70S6K cells to
galectin-8 or fibronectin. A, puromycin-resistant cells
that overexpress PKB or p70S6K (CHOPKB or
CHOp70S6K cells, respectively) were isolated and extracted
using buffer I. Proteins (100 µg) were resolved by 10% SDS-PAGE,
transferred to nitrocellulose membranes, and Western immunoblotted with
anti-PKB or anti-p70S6K antibodies. B, CHOPKB
and CHOp70S6K cells were incubated in the absence or
presence of wortmannin and seeded on galectin-8- or fibronectin-coated
plates as described in Fig. 4A. Cells were washed and
extracted using buffer I. Proteins (100 µg) were resolved by 10%
SDS-PAGE, transferred to nitrocellulose membranes, and Western
immunoblotted with (a) anti-phospho-PKB
( -pPKB); (b) anti-PKB (
-PKB);
(c) anti-phospho-p70S6K (
-pp70S6K); or
(d) anti-70S6K antibodies (
-p70S6K).
C, quantitation of the intensity of the bands, corresponding
to the phosphorylated PKB (e) or the phosphorylated p70S6K
(f) is presented.
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Fig. 7.
Effect of wortmannin on cell adhesion.
96-well bacterial plates were coated with galectin-8 (0.7 µM) or fibronectin (0.04 µM) for 2 h
at 22 °C. CHO-P (a and d), CHOPKB
(b and e), and CHOp70S6K
(c and f) cells were grown and incubated for
16 h in serum-free medium. Cells were detached from the plates
with 5 mM EDTA, washed, and incubated in suspension for 15 min at 37 °C in serum-free medium. The cells were further incubated
for an additional 15 min in suspension in the presence or absence of 1 µM wortmannin and seeded in serum-free medium on the
coated wells. Following incubation at 37 °C for the indicated times,
cells were washed, stained with crystal violet, and the number of
adherent cells was determined. Values are the mean ± S.D. of
quadruplicate measurements of a representative experiment. Maximum
adhesion of CHO-P, CHOPKB, and CHOp70S6K onto
galectin-8 refers to 0.423, 0.427, and 0.352 A at 540 nm;
maximum adhesion of CHO-P, CHOPKB, and
CHOp70S6K onto fibronectin refers to 0.429, 0.362, and 0.32 A at 540 nm. p values of non-treated
versus wortmannin-treated CHO-P, CHOPKB, and
CHOp70S6K cells, adherent on galectin-8, were calculated
(*, p < 0.05; ***, p < 0.01).
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Fig. 8.
Adhesion and spreading of CHO-P cells on
galectin-8 or fibronectin. Cover glasses (A) or 96-well
bacterial plates (B) were coated for 2 h at 22 °C
with fibronectin (0.04 µM) or galectin-8 (0.7 µM) in PBS. CHO-P cells (1 × 106 in
section A; 2.5 × 105 in section
B) were seeded on the indicated matrices. Following incubation at
37 °C for the indicated time periods, cells were washed, stained
with crystal violet, and the number of adherent cells was determined.
Values are the mean ± S.D. of quadruplicate measurements of a
representative experiment (B). Alternatively, cells were
fixed, stained with TRITC-labeled phalloidin (A), and
photographed. Quantitation of the cell areas was determined using the
NIH Image program. Values represent the percentage of cells having cell
areas that correspond to 175 ± 175 and up to 2275 ± 175 arbitrary units. The number of cells (n) whose area was
calculated under each experimental condition is indicated.
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Fig. 9.
Effect of wortmannin on adhesion and
spreading of HE, NIH, and B16 cells. Cover glasses were coated for
2 h at 22 °C with fibronectin (0.04 µM) or
galectin-8 (0.7 µM) in PBS. HE (a and
d), NIH (b and e), and B16
(c and f) cells were incubated in the absence or
presence of 1 µM wortmannin and seeded (4 × 105) on galectin-8- or fibronectin-coated cover glasses as
described in Fig. 7. Following incubation at 37 °C for the indicated
time periods, cells were fixed and incubated with TRITC-labeled
phalloidin for actin staining. Quantitation of the cell areas was
determined using the NIH Image program. The number of cells (n)
whose area was calculated under each experimental condition is
indicated.
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Fig. 10.
Effects of low temperature and cycloheximide
on adhesion and spreading of CHO-P cells on galectin-8. Cover
glasses were coated for 2 h at 22 °C with galectin-8 (0.7 µM) in PBS. CHO-P cells were incubated for 16 h in
serum-free medium. Following incubation the cells were further
incubated in the absence or presence of 50 µg/ml cycloheximide for
3 h in serum-free medium. Next, the cells were detached from the
culture plates with 5 mM EDTA, washed, and further
incubated in suspension for 30 min at 37 °C in serum-free medium in
the absence or presence of cycloheximide. The cells were seeded on
galectin-8-coated cover glasses at 37 or 4 °C for the indicated
times. Cells were fixed and incubated with TRITC-labeled phalloidin for
actin staining.
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Fig. 11.
Effect of wortmannin on spreading of CHO
cells overexpressing PKB or p70S6K. Cover glasses were coated for
2 h at 22 °C with fibronectin (0.04 µM) or
galectin-8 (0.7 µM) in PBS. CHO-P (a and
d), CHOPKB (b and e), and
CHOp70S6K (c and f) cells were
incubated in the absence or presence of 1 µM wortmannin
and were seeded (1 × 106) on galectin-8- or
fibronectin-coated cover glasses as described in Fig. 7. Following
incubation at 37 °C for 10 min with galectin-8 or 20 min with
fibronectin, cells were fixed and incubated with TRITC-labeled
phalloidin for actin staining (A). The distribution of the
cell areas was determined using the NIH Image program as described in
the legend to Fig. 8. B, the number of cells (n)
whose area was calculated under each experimental condition is
indicated.
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Fig. 12.
Effect of wortmannin on microspike
formation. Cover glasses were coated for 2 h at 22 °C with
galectin-8 (0.7 µM) (A and B) or
fibronectin (0.04 µM) (A) in PBS. CHO-P,
CHOPKB, and CHOp70S6K cells were incubated in
the absence (panel B, g, i,
k) or presence (panel B, h,
j, i) of 1 µM wortmannin and seeded
on galectin-8- or fibronectin-coated cover glasses as described in Fig.
7. B, following 2 h of incubation at 37 °C, cells
were fixed and incubated with TRITC-labeled phalloidin for actin
staining. Cells having microspikes were counted (A). Values
are the mean ± S.D. of the indicated n
measurements of a representative experiment. The reduction in
the number of cells harboring microspikes upon wortmannin treatment is
highly significant (***, p < 0.01). Similarly, the
number of CHOPKB cells harboring microspikes is
significantly higher (***, p < 0.01) compared with
CHO-P cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
1 or
6
1,
but not
4
1 integrins (27), whereas the
repertoire of integrins ligated by fibronectin is much broader (38). As
a result, the composition of signaling complexes formed between the
different cytoplasmic tails of integrins and their downstream effectors
might differ, depending on whether integrins were clustered by
galectin-8 or fibronectin.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Ronit Sagi-Eisenberg and Benjamin Geiger for helpful discussions and a critical review of this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the CaPCure Israel Foundation, the Moross Center for Cancer Research, and the Israel Cancer Association.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.
Incumbent of the Marte R. Gomez Professorial Chair. To whom
correspondence should be addressed. Tel.: 972-8-9342-380; Fax: 972-8-9344-125; E-mail: yehiel.zick@weizmann.ac.il.
Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M207380200
2 Y. Levy and Y. Zick, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ECM, extracellular matrix; FCS, fetal calf serum; PBS, phosphate-buffered saline; GST, glutathione S-transferase; RBD, Ras binding domain of Raf; FAK, focal adhesion kinase; PKB, protein kinase B; p70S6K, p70 S6 kinase; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; CHO, Chinese hamster ovary; CHOPKB and CHOp70S6K, CHO-P cells overexpressing PKB or p70S6K, respectively; ERK, extracellular-regulated kinase; HE, human endothelial; TRITC, tetramethylrhodamine isothiocyanate; WT, wild-type.
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REFERENCES |
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