From the Department of Biological Regulation, The Weizmann Institute of Science, Rehovot IL-76100, Israel
Received for publication, May 3, 2000, and in revised form, February 16, 2001
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ABSTRACT |
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Phosphorylation of vitronectin (Vn) by casein
kinase II was previously shown to occur at Thr50 and
Thr57 and to augment a major physiological function of
vitronectin-cell adhesion and spreading. Here we show that this
phosphorylation increases cell adhesion via the
Vitronectin (Vn)1 is an
adhesive glycoprotein found in the extracellular matrix (ECM) of
various cells, and in circulating blood (1-3). It has been implicated
in a large variety of physiological and pathophysiological processes
such as hemostasis (4, 5), tumor cell invasion (6, 7), angiogenesis
(8-11), and in the control of plasminogen activation (12-18).
One of the most important properties of Vn is its ability to promote
cell attachment, spreading, and migration (19-22). In fact, Vn was
originally discovered as a "serum spreading factor" (23). The cell
adhesion, spreading, and migration activities of Vn are associated with
its RGD sequence located near the N terminus of the protein (positions
45-47). This sequence is recognized by the family of receptors known
as the integrins: heterodimers composed of It is well known that cell adhesion is a complex process that was shown
to involve an activation of several Vn receptors and a variety of
intra-cellular signaling pathways. For example, the focal adhesion
kinase (FAK) was shown to play a central role in mediating the signal
from integrins (33). It does so by its autophosphorylation on
Tyr397 upon integrin stimulation. This autophosphorylation
leads to the recruitment and activation of intra-cellular mediators
such as PI3K, as well as the Src family kinases, by an interaction of
their SH2 domain with the autophosphorylated Tyr397
residue. The PI3K binding to Tyr397 leads to activation of
PKB, whereas the Src family of kinases further phosphorylates FAK on
Tyr925 leading to the recruitment of additional signaling
molecules that bring about an activation of the ERK pathway
(31-38).
We have previously shown that Vn can be functionally modulated by
extra-cellular phosphorylation, making use of the kinase co-substrate
ATP found at micromolar levels in the exterior of cells (39). For
example PKA, released from platelets upon their physiological
stimulation with thrombin (40-42), selectively phosphorylates Vn, and,
as a consequence of this phosphorylation, it reduces its grip on
plasminogen activator inhibitor-1 (43). Similarly, PKC phosphorylation
of Vn was shown to attenuate its cleavage by plasmin (44). Several
laboratories have shown the occurrence of an extra-cellular CK2
activity on a variety of cells. These include epithelial cells (45,
46), neutrophils (47, 48), platelets (49, 50), and endothelial cells
(51-53). Subsequently, we showed that Vn is a substrate for CK2, which
phosphorylates Vn at Thr50 and Thr57.
Furthermore, we found that this phosphorylation significantly enhances
the adhesion and spreading of bovine aorta endothelial cells (BAEC),
presumably because the phosphorylated Vn has a higher affinity for
One of the major obstacles in revealing the mechanism of action of
CK2-phosphorylated Vn originates from the well known fact that Vn (like
other adhesion proteins) can bind to several integrins, including the
specific Vn-binding integrin, Chemicals, Materials, and Enzymes--
The following
materials were purchased from the commercial sources:
[35S]methionine (Amersham Pharmacia Biotech);
nitrocellulose membranes (Schleicher & Schuell); restriction enzymes
(Roche Molecular Biochemicals or Life Technologies, Inc.);
Taq DNA polymerase (Promega).
Antibodies--
Monoclonal antibodies against the integrin
receptor Tissue Cultures--
HeLa cells were grown in Dulbecco's
modified Eagle's medium supplemented with 10% (v/v) heat-inactivated
fetal calf serum and glutamine (0.5 mg/ml). H1299 cells were grown in
RPMI supplemented with 10% (v/v) heat-inactivated fetal calf serum and
glutamine (0.5 mg/ml). The cells were grown in an incubator (37 °C)
with an atmosphere containing 5% CO2. The Sf-9 and High-5
insect cells were maintained in Grace's insect medium (Life
Technologies, Inc.) supplemented with 10% (v/v) heat-inactivated fetal
bovine serum and grown in an incubator (27 °C). For the expression
of recombinant Vns, a serum-free medium (Sf-900 II, Life Technologies,
Inc.) was used. All media for insect cells were supplemented with 50 µg/ml Gentamicin and 12.5 µg/ml Fungizone (Life Technologies, Inc.).
Cell Adhesion Assay--
Serial dilutions of r-Vns were added to
24-well plates (250 µl) for 1.5 h at 22 °C to allow coating
of the plates. Thereafter the solutions were aspirated, and 0.5 ml of
serum free medium containing 1 mg/ml hemoglobin was added for 30 min at
37 °C. Confluent cells plated on 10-cm plates were labeled with 30 µCi of [35S]methionine for 3-4 h at 37 °C. The
cells were collected (using 5 mM EDTA) into serum free
medium, centrifuged (5 min at 1200 × g), and
resuspended into a serum free medium adjusting their concentration to
106 cells/ml. Cell suspensions (250 µl) were added to
each coated well for 30 min at 37 °C. The cells were washed three
times with 0.5 ml of PBS, and the adhered cells were treated with 0.5 ml of 1% Triton X-100 in PBS for 5 min. Samples of 0.4 ml were
transferred into scintillation vials for counting. The quantitation of
cell adhesion is reported as the residual radioactivity (a mean of triplicates in cpm) of the cells tested, after their extensive washing
(three times with 0.5 ml of PBS). This comparison was convenient and
valid, because each assay was carried out with an identical volume of
cell suspension, and an identical number of cells. When cell adhesion
assays were performed in 48-well plates, all the components and
treatments of the assay were scaled down accordingly.
Inhibition of Cell Adhesion by Function-inhibiting Monoclonal
Antibodies--
The monoclonal antibodies used were: P1F6, directed
against the integrin receptor FACS Analysis--
Confluent cells grown on 10-cm plates were
collected as described under cell adhesion and brought to a
concentration of 5 × 105 cells in 100 µl of PBS
containing 1% bovine serum albumin and 0.02% sodium azide. The cells
were incubated with monoclonal antibodies (final concentration, 4 µg/100 µl) for 1 h on ice with occasional agitation. They were
then washed three times with 1 ml of PBS containing 1% bovine serum
albumin, and 0.02% sodium azide using a cooled microcentrifuge
(4 °C). After the last wash, the cells were resuspended in 100 µl
of the above-mentioned buffer, supplemented with FITC-conjugated goat
anti-mouse IgG (final concentration of 5 µg/100 µl). The cells were
allowed to bind the antibodies during 1 h (on ice) with occasional
agitation, then washed as above and resuspended in 0.5 ml of PBS
(containing the above constituents) for FACS analysis in a FACScan
Becton Dickinson (530 filter). For each antibody, 5000 cells were
analyzed. Control cells were incubated with the secondary antibody only.
Expression of the Preparation of Cell Lysates for the Detection of Activated
Kinases (ERK, JNK, p38 MAPK, PKB, and FAK)--
Plates (10 cm) were
coated with the r-Vns for 1.5 h at 22 °C. Thereafter the
solutions were aspirated and 3 ml of serum free medium containing 1 mg/ml hemoglobin was added and incubated for 30 min at 37 °C.
Serum-starved cells were collected (using 5 mM EDTA) into
serum free medium containing 1 mg/ml hemoglobin (106
cells/ml). The cells were plated on top of the r-Vns and incubated for
various time periods at 37 °C then washed three times with PBS
(ice-cold) and scraped (on ice) into 500 µl of a RIPA buffer. The
lysates were collected and centrifuged (20,000 × g 15 min at 4 °C), and aliquots of the resulting supernatants were
assayed for their protein concentration (Pierce protein assay).
Detection of Kinase Activation--
Equal amounts of proteins
obtained from the cell lysates described above were loaded onto
SDS-PAGE, transferred to nitrocellulose paper, and immunoblotted with
antibodies exclusively recognizing the active form of the kinase in
question (anti-activated ERK, JNK, p38 MAPK, or PKB antibodies). The
same samples were also analyzed using anti-total kinase antibodies,
which detect the total amount of the kinase in question (activated and
non-activated).
Detection of FAK Phosphorylation--
Protein samples (600 µg)
obtained from the cell lysates described above were immunoprecipitated
using anti-FAK antibodies immobilized on agarose beads (mixing end to
end for 2 h at 4 °C). The immunoprecipitated samples were
washed once with RIPA buffer, twice with 0.5 M LiCl, 0.1 M Tris-HCl, pH 8.0, and finally twice in 50 mM
Comparing the Adhesion of
In the course of our studies we found that BAEC cells do not express
Cells Containing
The involvement of An ERK Activation Cannot Account for the Enhanced Cell Adhesion
Observed with CK2-PVn--
Following the identification of
The Increased Activation of the PKB Pathway Can Account for the
Enhanced Cell Adhesion Mediated by
PI3K Is Essential for the Promotion of Cell Adhesion and for the
Activation of PKB--
PKB was recently implicated as an important
downstream target for PI3K (56). To determine whether the PKB
activation in our system requires the activation of PI3K (which
precedes PKB in several signal transduction processes (cf.
Scheme 1), we treated
In conclusion, it is evident from our results (i) that the PKB
activation (which occurs upon exposure of cells to Vn/CK2-PVn) depends
on the availability of the Cells Containing Intra-cellular protein phosphorylation is now well
established as a central regulatory mechanism. In the last few years,
several reports provided evidence for the occurrence of protein kinases outside the cell, raising the possibility that protein phosphorylation may also regulate extra-cellular processes (40, 41, 45-48). This possibility was supported by the identification of specific target
substrates for the kinases in the cell exterior. Some reports further
indicated that the physiological function of such specific substrates
is modulated upon their phosphorylation (for a review see Ref. 42). For
example, it was shown that Vn is functionally modulated by PKA, a
kinase released from platelets upon their physiological stimulation
with thrombin (40-42). Similarly, a PKC phosphorylation of Vn was
shown to attenuate its cleavage by plasmin (44).
In addition to PKA and PKC, Vn was recently shown to be a substrate for
CK2, which was found to single out and selectively phosphorylate Vn at
Thr50 and Thr57 to bring about a significant
enhancement of one of Vn's well known physiological functions: cell
adhesion and spreading (54). The clinical importance of this modulation
is evident in view of the fact that invasive metastasis involves an
enhanced adhesion of tumor cells to the ECM (6) by binding to
integrins, in particular A major implication of the findings presented in this report is that
the CK2 phosphorylation of Vn enhances cell adhesion via
In view of our finding that the enhanced cell adhesion onto
Vn(T50E,T57E) is mediated by Based on the results presented here, we suggest that
although both One of the important messages reported here lies in the
fact that it identifies two intracellular signaling pathways that are
unequally activated upon binding of Vn and CK2-PVn, at least to the
cells we tested in this study. Both pathways (one functioning via
Taken together, the results presented here together with our results
reported earlier (54) indicate the occurrence of a cell surface
receptor (v
3 (not via the
v
5 integrin), suggesting that
v
3 differs from
v
5 in its biorecognition profile. Although both the phospho (CK2-PVn) and non-phospho (Vn) analogs of
vitronectin (simulated by mutants Vn(T50E,T57E), and
Vn(T50A,T57A), respectively) trigger the
v
3 as well as the
v
5 integrins, and equally activate the
ERK pathway, these two forms are different in their activation of the
focal adhesion kinase/phosphatidylinositol 3-kinase (PI3K)/protein
kinase B (PKB) pathway. Specifically, we show (i) that, upon exposure
of cells to Vn/CK2-PVn, their PKB activation depends on the
availability of the
v
3 integrin on their
surface; (ii) that upon adhesion of the
3-transfected cells onto the CK2-PVn, the extent of PKB activation coincides with the
enhanced adhesion of these cells, and (iii) that both the PKB
activation and the elevation in the adhesion of these cells is
PI3K-dependent. The occurrence of a cell surface receptor that specifically distinguishes between a phosphorylated and a non-phosphorylated analog of Vn, together with the fact that it preferentially activates a distinct intra-cellular signaling pathway, suggest that extra-cellular CK2 phosphorylation may play an important role in the regulation of cell adhesion and migration.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits
(24-30). There are 17
and 8
subunits that heterodimerize to
produce 22 different integrins (27, 31, 32). Several of these
integrins, e.g.
v
1,
v
3,
v
5,
v
6, and
v
8
and the platelet-specific
IIb
3 integrin,
are known to recognize and bind Vn.
v
3 (54).
v
5, and
that this family of integrins can, in turn, activate different
intra-cellular pathways. Here we extend our studies on the consequences
of the CK2 phosphorylation of Vn and show that the enhanced cell
adhesion involves
v
3 (but not
v
5). Furthermore, we show that this
enhanced adhesion coincides with a preferential activation of the
FAK/PI3K/PKB cascade, rather than the ERK signaling pathway.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v
5 (P1F6), against
v
3 (LM609), and against the
3 integrin receptor (MAB 1974) were obtained from
Chemicon. Monoclonal antibodies directed against the integrin receptor
3 were from Serotec. Monoclonal antibodies against active ERK, JNK,
and p38 MAPK were from Sigma Chemical Co. Monoclonal antibodies
against phospho-tyrosine (PY99) were from Santa Cruz Biotechnology.
Polyclonal antibodies against total ERK, JNK, p38 MAPK, FAK, and goat
anti-mouse IgG FITC-conjugated antibodies were purchased from Sigma;
anti-active PKB (polyclonal antibodies) were from New England BioLabs.
v
5; LM609,
directed against the integrin receptor
v
3; and HA, directed against
hemagglutinin as control. Plates (24 wells) were coated with 5 µg/ml
of the Vn to be assayed (250 µl) for 1.5 h 22 °C, then the
nonspecific adsorption sites were blocked with 0.5 ml of serum free
medium containing 1 mg/ml hemoglobin (30 min at 37 °C). The cells
were treated as described above to yield a concentration of
105 cells/ml. Before starting the cell adhesion assay, the
cells were preincubated with increasing concentrations of monoclonal antibodies (gentle shaking, for 30 min at 22 °C). Thereafter, the
cells were washed once with 10 ml of serum free medium containing 1 mg/ml hemoglobin and resuspended to yield a concentration of 105 cells/ml. An aliquot of this cell suspension (250 µl)
was added to the Vn-coated wells, and the adhesion assay was allowed to proceed as described above.
3 Integrin Subunit and the r-Vn
Mutants--
The cDNA encoding the
3 integrin
subunit in pGEM was kindly provided by Dr. P. J. Newman, Blood
Research Institute, Milwaukee, WI. The cDNA was digested with
DraI and XbaI then treated with Klenow and
subcloned into an EcoRV-digested pcDNA3 vector.
Transfections of H1299 cells were done using LipofectAMINE according to
the manufacturer's instructions (Life Technologies, Inc.). The cells were transfected with the
3 subunit cDNA in
pcDNA3 or, for control, with the empty vector of pcDNA3.
Transfected cells were grown on 0.6 mg/ml Geneticin (G418), and single
stable clones were isolated. Preparation of the r-Vn mutants and their
expression in insect cells was carried as described previously
(54).
-glycerophosphate, pH 7.3, 1.5 mM EGTA, 1 mM
EDTA, 1 mM dithiothreitol, and 0.1 sodium vanadate. After
the last wash, the samples were boiled in Laemmli's sample buffer and
subjected on SDS-PAGE. The gels were transferred to nitrocellulose
membranes and blotted either with antibodies against phosphotyrosine
(PY99, to detect phosphorylated FAK), or with antibodies against FAK (to determine the total FAK as a reference value) in each lane.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v
3- and of
v
5-bearing Cells in Their Response to Vn
and to CK2-phosphorylated Vn--
We have previously shown (54) that
the CK2 phosphorylation of Vn results in a significant enhancement of
BAEC cell adhesion (~2.5-fold, average of three experiments), as
indicated by the number of cells that adhere to increasing
concentrations of immobilized Vn. We also showed that the effect of the
CK2 phosphorylation could be reproduced with a mutant Vn(T50E,T57E) (a
close analog of CK2-PVn representing the phospho form of Vn), when
compared with Vn(T50A,T57A) (a close analog of Vn representing the
non-phospho form of Vn).
v
5 (a characteristic binding receptor for
Vn (55)); therefore, we considered the possibility that this integrin
might be involved in a response to CK2-PVn by cells that do express this integrin. To find out whether this is the case, we used HeLa cells
(Fig. 1A) and H1299 cells
(Fig. 1B), whose adhesion to Vn was found to be mediated
mainly by
v
5. In both cases we found an
efficient inhibition of cell adhesion by
anti-
v
5 but a minor inhibition by
anti-
v
3. A similar inhibition of cell
adhesion by both antibodies was also obtained with Vn(T50A,T57A) (not
shown), raising the possibility that the adhesion of these cells to
both forms of Vn is mediated by
v
5. In
line with this finding, the adhesion profile of HeLa as well as H1299
cells to immobilized Vn(T50E,T57E) was found to be essentially
identical to their adhesion to Vn(T50A,T57A) (Fig. 1, C and
D). In this context it should be noted that (i) the same
adsorption profile of the cells was obtained whether Vn(T50E,T57E) or
Vn(T50A,T57A) was used as a substratum (54) and (ii) in all experiments
comparing Vn(T50A,T57A) with Vn(T50E,T57E) we ran a similar
experiment with wild type r-Vn and showed that, within
experimental error, it was identical to Vn(T50A,T57A).
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Fig. 1.
The adhesion of HeLa or H1299 cells to r-Vn
is mediated mainly by
v
5,
which does not discriminate between the phospho and non-phospho Vn
analogs. A, inhibition of HeLa cell adhesion to r-Vns
by antibodies raised against
v
3 or
v
5. B, inhibition of H1299
cell adhesion to r-Vns by antibodies raised against
v
3 or
v
5.
Polystyrene plates were coated with 5 µg/ml Vn(T50E,T57E).
[35S]Met-labeled HeLa/H1299 cells were preincubated (with
gentle shaking) for 30 min at 22 °C, in the presence of increasing
amounts of monoclonal antibodies against
v
3 or against
v
5 (as indicated in the figure), before
the adhesion assay was performed. The cell adhesion was determined by
the residual radioactivity on the plates after extensive washing (see
"Experimental Procedures"). The percent of adhered cells was
calculated in comparison to a control of non-relevant
anti-hemagglutinin monoclonal antibodies. Identical results (not shown)
were obtained when plates were coated with Vn(T50A,T57A). C,
HeLa cells adhesion on the phospho and non-phospho Vn analogs.
D, H1299 cells adhesion on the phospho and non-phospho Vn
analogs. Polystyrene plates were coated with increasing concentrations
of the two r-Vns. [35S]Met-labeled HeLa/H1299 cells were
plated on top of the r-Vns for 30 min. Cell adhesion was determined by
counting the residual radioactivity after washing, as described under
"Experimental Procedures."
v
3 Exhibit an
Enhanced Cell Adhesion upon Exposure to CK2-PVn--
The results
presented above, together with our previous findings with BAEC (54),
imply that the enhanced cell adhesion onto CK2-PVn is mediated by the
v
3 receptor. To confirm this suggestion we endowed H1299 cells (which do not exhibit an enhanced cell adhesion
in response to CK2-PVn) with a capability to exhibit an enhanced cell
adhesion onto Vn(T50E,T57E) and thus to "discriminate" between the
phospho- and non-phospho forms of Vn. This was achieved by transfecting
H1299 cells with the
3
subunit.2 Isolated clones of
H1299 cells overexpressing
v
3 that were identified by immunoblotting with anti-
3, and
subsequently characterized by FACS analysis with
anti-
v
3 (Fig.
2, A and B), were
shown to contain high amounts of the
v
3
integrin on their surface. Quantitation of the FACS analysis indicated
that the
3-transfected clones we used contained up to
~7-fold more
v
3 than the control vector-transfected clones, whereas the amounts of the
v
and of a non-relevant
3 integrin were very similar to
the control. In addition, we observed a ~3-fold reduction of
v
5 in the
3-transfected clone, presumably due to competition between
5 and the
excess of
3 for the limited amount of their common
partner, the
v subunit.
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Fig. 2.
Characterization of the integrins expressed
in the stable clones of H1299 cells. A, overexpression
of integrin 3 subunit in H1299 cells as determined by
immunoblot. Soluble fractions obtained from extracts of H1299 stably
transfected with the cDNA of
3 subunit, or with
vector alone (pcDNA3), were analyzed by immunoblotting with
anti-
3 monoclonal antibodies. Lane 1,
non-transfected cells; lanes 2 and 3, two
different vector-transfected clones; lanes 4 and
5, two different
3-transfected clones.
B, quantitation of the integrins expressed in the stable
clones of H1299 cells by FACS analysis. Cells were incubated with
monoclonal antibodies directed against the indicated integrin, followed
by incubation with the secondary antibodies, FITC-conjugated goat
anti-mouse IgG (heavy line). Control cells were incubated
only with the secondary antibody (light line).
v
3 (but not
v
5) in the enhanced cell adhesion is best
illustrated in Fig. 3, which shows
that the adhesion of vector-transfected H1299 cells is blocked by
anti-
v
5 and not by
anti-
v
3 (Fig. 3A), whereas the
adhesion of
3-transfected H1299 cells is blocked by
anti-
v
3 but not by
anti-
v
5 (B). In line with
these findings, the vector-transfected H1299 cells do not discern
Vn(T50E,T57E) from Vn(T50A,T57A), whereas cells overexpressing the
3 subunit exhibit an ability to enhance cell adhesion on
the Vn(T50E,T57E) mutant (compare Fig. 3C with Fig. 3D). It should be noted that the occurrence of a
relationship between the integrin content of cells, their adhesion, and
the ensuing intracellular signaling triggered by Vn were also observed with two additional
3-transfected clones (not
shown).
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Fig. 3.
Transfection with
3 endows H1299 cells with the
capability to discriminate between the CK2-PVn analog Vn(T50E,T57E) and
the non-phosphorylated Vn analog (T50A,T57A). A,
inhibition of vector-transfected H1299 cell adhesion to r-Vns by
antibodies raised against
v
3 or
v
5. B, inhibition of
3-transfected H1299 cell adhesion to r-Vns by antibodies
raised against
v
3 or
v
5. C, adhesion of
vector-transfected H1299 cells to r-Vns. D, adhesion of
3-transfected H1299 cells to r-Vns. The assay of cell
adhesion and its inhibition were carried out as described in the legend
to Fig. 1.
v
3 as a CK2-PVn-specific mediator of the
enhanced adhesion obtained with this phosphorylation, we attempted to
identify an intra-cellular signaling pathway that might be responsible
for this enhancement. Because the activation of ERKs in response to the
stimulation of cells by ECM proteins was already established (31-38),
we first examined the pattern of ERK activation in the stable
v
3 and
v
5
expressing clones of the H1299 cells mentioned above. In response to
cell adhesion to r-Vns, the ERK activation of
v
5- and
v
3-containing clones was found to be low
and transient (Fig. 4, A and
B): It was found to peak within 10 min after plating and to
decline thereafter. No significant change in the pattern of ERK
activation that could correlate with the enhancement of cell adhesion
was observed (Fig. 4C). These results raised the possibility
that an alternative signaling pathway(s) (other than the ERK pathway),
might be involved in the enhanced adhesion observed with the
3-transfected clone.
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Fig. 4.
ERK activation triggered by r-Vns in the
stably transfected H1299 cells. Plates were coated with the two
r-Vns or with the non-integrin adhesive molecule,
poly-D-lysine (PDL). Cells were plated on top of
the coated plates for the time indicated, or kept in suspension
(Sus). Thereafter, cells were harvested in RIPA buffer and
the soluble fractions were collected after centrifugation. Protein
concentration was determined and equal amounts of protein were loaded
on SDS-PAGE. The gels were transferred to nitrocellulose membranes and
blotted with antibodies against active ERK or with antibodies against
total ERK (whether phosphorylated or not). Extracts were obtained from
the vector-transfected cells (A), or from the
3-transfected cells (B). The ERK activation
was quantitated by densitometry. The data illustrated represent the
average of three separate experiments (C; open
symbols are for cells transfected with the vector, and
filled symbols are for cells transfected with
3).
v
3--
Because we found that the
activation of ERK cannot account for the enhanced cell adhesion, we
looked into other signaling pathways such as the JNK, p38 MAPK, and PKB
pathways that were previously shown to be activated by Vn-binding
integrins. Although no adhesion-triggered activation of JNK and p38
MAPK was detected in the various clones we used (data not shown), we
found that the activation of PKB in the
3-transfected
cells (Fig. 5) led to a significantly
enhanced activation of this kinase, in comparison to the very low PKB
activation in the vector-transfected
cells.3 These results
suggested to us that the activation of PKB depends on the availability
of the
v
3 integrin. As such, the extent of PKB activation in the
3-transfected cells correlates
well with the extent of enhanced cell adhesion onto CK2-PVn. This was demonstrated with
3-transfected cells that were plated
on Vn(T50E,T57E), whose enhanced adhesion resulted in an increased PKB
activation (~30-fold over the PDL control), whereas the PKB
activation obtained in cells plated onto Vn(T50A,T57A) was found to be
only 18-fold over the control (Fig. 5C).
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Fig. 5.
PKB activation triggered by r-Vns in the
stably transfected H1299 cells. Plates were coated with the two
r-Vns or with PDL. Cells were plated on top of the coated plates for
the time indicated, or kept in suspension (Sus). Thereafter,
cells were harvested in RIPA buffer and the soluble fractions were
collected after centrifugation. Protein concentration was determined
and equal amounts of protein were loaded on SDS-PAGE. The gels were
transferred to nitrocellulose membranes and blotted with antibodies
against active PKB or with antibodies against total PKB (whether
phosphorylated or not). Extracts were obtained from the
vector-transfected cells (A), or from the
3-transfected cells (B). The PKB activation
was quantitated by densitometry. The data illustrated represent the
average of three separate experiments (C; open
symbols are for cells transfected with the vector, and
filled symbols are for cells transfected with
3).
3-transfected cells with wortmannin (a PI3K inhibitor)
prior to their stimulation by adhesion to Vn(T50E,T57E). Indeed,
wortmannin prevents PKB activation (Fig.
6A), presumably through a PI3K
inhibition, indicating that the enhanced adhesion mediated by
v
3 transmits the signal to PKB via PI3K.
In line with this result, the enhanced adhesion of the
3-transfected cells was reduced by preincubation with
wortmannin (before allowing the cells to adhere) (Fig. 6B) or with another PI3K inhibitor LY294002 (Fig. 6C). As
expected, these two inhibitors blocked cell adhesion onto both the
phospho- and the non-phospho forms of Vn, because PKB is activated by
both forms of Vn. However, the reduction in the elevation in cell
adhesion onto CK2-PVn over Vn, illustrated by using PI3K inhibitors,
clearly indicates the involvement of this pathway in elevating cell
adhesion on CK2-PVn. The specific involvement of the PI3K-PKB pathway
in the adhesion of cells onto the phospho and the non-phospho forms of
Vn was supported by our finding that the MEK inhibitor PD98059 does not
inhibit the cell adhesion onto these two Vns (Fig. 6D). In
that context it should be noted that, in our experiments, the PKB
activation occurs within 5-10 min after plating the cells (the cells
are attached but not spread), whereas the adhesion of the cells
proceeds for 30 min when the cells are already adhered and spread. This
observation sets the stage for a detailed study aimed at the
identification of the sequence of events that lead from cell adhesion
to cell spreading, namely at the elucidation of the mechanism by which
downstream mediators of PKB influence the cell-spreading process.
View larger version (31K):
[in a new window]
Scheme 1.
Schematic presentation of the ERK and PKB
signaling pathways in response to integrin stimulation. The
enhanced cell adhesion onto CK2-PVn depends on the availability of
v
3 but not of
v
5 on the cell surface. This enhanced
adhesion coincides with the increased activation of the FAK-PI3K-PKB
(rather than the ERK) signaling pathway (Scheme modified from C. C. Kumar (32)).
View larger version (19K):
[in a new window]
Fig. 6.
Inhibition of PKB activation by inhibitors of
PI3K also inhibits cell adhesion. A, the PKB activation
triggered by Vn(T50E,T57E) in the 3-transfected H1299
cells is inhibited by wortmannin. Plates were coated with Vn(T50E,T57E)
(A, lanes 2) or with PDL (A,
lanes 1). The cells were preincubated with dimethyl
sulfoxide (DMSO) (0.1%), or with wortmannin (100 nM/0.1% DMSO), for 15 min (37 °C) with gentle shaking,
then were plated on top of the coated plates for 10 min. Adhered cells
were harvested in RIPA buffer, and the soluble fractions were collected
after centrifugation. The protein concentration was determined and
equal amounts of protein were loaded on SDS-PAGE. The gels were
transferred to nitrocellulose membranes and blotted with anti-active
PKB or with anti-total PKB. B, the adhesion of
3-transfected H1299 cells to Vn(T50E,T57E) is inhibited
by wortmannin. Polystyrene plates were coated with increasing
concentrations of the two r-Vns: Vn(T50A,T57A) (
,
) and
Vn(T50E,T57E) (
,
). H1299 cells stably transfected with the
3 subunit were labeled with [35S]Met. The
labeled cells were preincubated with wortmannin (100 nM/0.1% DMSO; filled symbols) or with DMSO
(0.1%; empty symbols) for 15 min (37 °C) with gentle
shaking, then plated on top of the two r-Vns. The cell adhesion after
30 min was determined by counting the residual radioactivity after
extensive washing as described under "Experimental Procedures."
C, the adhesion of
3-transfected H1299 cells
to Vn(T50E,T57E) is inhibited by LY294002. Polystyrene plates were
coated with increasing concentrations of the two r-Vns: Vn(T50A,T57A)
(
,
); and Vn(T50E,T57E) (
,
). H1299 cells stably
transfected with the
3 subunit were labeled with
[35S]Met. The labeled cells were preincubated with
LY294002 (25 µM/0.1% DMSO; filled symbols) or
with DMSO (0.1%; empty symbols) for 15 min (37 °C) with
gentle shaking then plated on top of the two r-Vns. The cell adhesion
after 30 min was determined by counting the residual radioactivity
after extensive washing as described under "Experimental
Procedures." D, the adhesion of
3-transfected H1299 cells to Vn(T50E,T57E) is not
inhibited by PD98059. Polystyrene plates were coated with increasing
concentrations of the two r-Vns: Vn(T50A,T57A) (
,
) and
Vn(T50E,T57E) (
,
). H1299 cells stably transfected with the
3 subunit were labeled with [35S]Met. The
labeled cells were preincubated with PD98059 (25 µM/0.1%
DMSO; filled symbols) or with DMSO (0.1%; empty
symbols) for 15 min (37 °C) with gentle shaking then plated on
top of the two r-Vns. The cell adhesion after 30 min was determined by
counting the residual radioactivity after extensive washing as
described under "Experimental Procedures."
v
3 integrin on
the surface of the cells; (ii) that the extent of PKB activation (that
takes place upon exposure of the
3-transfected cells to
CK2-PVn) coincides with the specific enhanced adhesion of these cells
upon their binding to CK2-PVn; and (iii) that both the PKB activation
and the subsequent enhanced adhesion of the cells are
PI3K-dependent, because the inhibition of PI3K (upstream of
PKB) prevents the PKB activation and reduces cell adhesion (Scheme
1).
v
3 and
v
5 Differ in Their FAK Phosphorylation
Pattern upon Their Adhesion onto Phospho and Non-phospho Forms of
Vn--
As mentioned above, there is a significant difference in the
intensity of the PKB activation upon exposure of
3-transfected cells to the phospho and the non-phospho
forms of Vn (Fig. 5). To account for this difference in intensity
(shown here to be PI3K-dependent (Fig. 6A)), we
compared their FAK phosphorylation pattern, i.e. the
possible activation of an upstream kinase in this pathway. It is well
known that the phosphorylation of FAK is an early event detected in
response to integrin stimulation (33). Upon this stimulation, FAK is
autophosphorylated on Tyr397, creating a high affinity
binding site for a variety of kinases containing an SH2 domain,
including the PI3K and Src family kinases. Src further phosphorylates
FAK on Tyr925, leading to the recruitment of GRB2, which is
known to activate the ERK pathway (38). Therefore, we monitored the FAK
phosphorylation upon attachment of vector/
3-transfected
cells onto the r-Vns mentioned above. As seen in Fig.
7, the FAK phosphorylation is different
in these two cell lines. Although there is a gradual activation of FAK
that peaks after 20 min in the vector-transfected clone (Fig.
7A), the FAK phosphorylation in
3-transfected
cells is weaker and transient. It peaks after 5-10 min and declines thereafter (Fig. 7B). The time course of FAK phosphorylation
in the
3-transfected cells coincides with that of PKB
activation (Fig. 5B). Moreover, although no differences in
FAK phosphorylation were observed when vector-transfected cells were
plated either on Vn(T50A,T57A) or on Vn(T50E,T57E) (Fig. 7,
B and C), a preferential increase in FAK
phosphorylation was observed when
3-transfected cells
were plated on the Vn(T50E,T57E) mutant (5 min, Fig. 7B). Although a small increase, this signal is amplified, and a better reflection of it is viewed in the differences observed in the downstream kinase PKB (Fig. 5). The FAK phosphorylation in the vector-transfected cells is significantly more intense at the peak of
the response (20-30 min). This may suggest that in the vector-transfected cells (
v
5) another
kinase may further phosphorylate FAK, whereas in the
3-transfected cells (
v
3)
FAK autophosphorylation brings about the association with PI3K, which
does not further phosphorylate FAK but, rather, specifically activates
the PKB pathway. We conclude that the extra-cellular stimulation by
CK2-PVn (as represented by Vn(T50E,T57E)) is transmitted via
v
3 and that the PI3K pathway is involved
in the enhanced cell adhesion.
View larger version (27K):
[in a new window]
Fig. 7.
FAK phosphorylation triggered by r-Vns in the
stably transfected H1299 cells. Plates were coated with the two
r-Vns or with the non-integrin adhesive molecule,
poly-D-lysine (PDL). Cells were plated on top of
the coated plates for the time indicated, or kept in suspension
(Sus). Thereafter, cells were harvested in RIPA buffer and
the soluble fractions were collected after centrifugation. Protein
samples (600 µg) obtained from the cell lysates described above were
immunoprecipitated using anti-FAK antibodies immobilized on agarose
beads as described under "Experimental Procedures." The samples
containing the immunoprecipitated FAK were boiled in Laemmli's sample
buffer and subjected on SDS-PAGE. The gels were transferred to
nitrocellulose membranes and blotted with antibodies against
phosphotyrosine (PY99) or with antibodies against FAK. Extracts
obtained from the vector-transfected cells (A), or from the
3-transfected cells (B). The FAK
phosphorylation was quantitated by densitometry. The data illustrated
represent the average of three separate experiments (C;
open symbols are for cells transfected with the vector, and
filled symbols are for cells transfected with
3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v
3. In fact,
this integrin has been implicated in the acquisition of metastatic
invasiveness (57). In melanoma, for example, the expression of
v
3 was shown to correlate with
invasiveness (58) and with tumorigenic capacity (57, 59). In the case
of Vn, the specificity in the recognition of its CK2-phosphorylated
form may have a special importance in cancer, because Vn seems to be an
important ligand in the
v
3-mediated
adhesion of tumor cells. In line with this fact, human melanoma cells
derived from lymphatic metastases were shown to use
v
3 to adhere to lymph node Vn (7), in a
Vn-mediated manner, as indicated by the fact that the replacement of Vn
by fibronectin had no effect on invasion (60).
v
3 and not via
v
5. This can be deduced from our finding that cells that adhere mostly via
v
5,
(e.g. HeLa cells, or the H1299 lung carcinoma cells) do not
distinguish between the mutant Vn(T50E,T57E) and Vn(T50A,T57A).
Furthermore, we report here that the enhanced cell adhesion can be
quantitatively accounted for by assuming that the integrin
v
3 alone is involved in the preferential recognition of the Vn analog Vn(T50E,T57E), i.e. in the
specific response to CK2-PVn. In line with this conclusion the H1299
lung carcinoma cells (whose adhesion to Vn is mediated by
v
5) are not able to discriminate between
Vn(T50E,T57E) and Vn(T50A,T57A) but gain the ability to discriminate
between these two mutants upon their stable transfection with the
3 integrin subunit.
v
3 and our
previous observation that this enhancement is due to an increased
affinity toward this integrin, we undertook to identify the signaling
pathway that is involved in this increased affinity. Several signaling
cascades were previously shown to be activated by the integrin family
of receptors (31-34, 36-38, 61, 62). Having in our hands cells that
use
v
3 to adhere onto Vn, and essentially
identical companion cells that use
v
5 to
adhere to this integrin ligand, enabled us to identify a signaling
pathway, which is differentially activated upon adhesion of these cells
to CK2-phosphorylated and non-phosphorylated Vn analogs. Specifically,
we were able to show that the phospho and non-phospho forms of Vn
trigger both
v
3 and
v
5, leading to a similar activation of
ERK. These results suggest that the activation of ERK occurs via the
subunit (61), which has not been modified in these cells. The fact
that the activation of ERK is not influenced by the introduction to the
3 subunit supports this suggestion. In contrast, the PKB
activation seems to depend on the availability of the
3
subunit, and therefore, is preferentially activated by the phospho form
of Vn. We presume that this enhanced activation of PKB, which is
v
3- and PI3K-dependent,
results in the enhanced cell adhesion by the CK2-PVn analog
(Vn(T50E,T57E)).
v
3 and
v
5 share common structural elements that
recognize and bind equally well the core protein shared by Vn and PVn,
v
3 contains additional recognition
elements that bind the two phosphate groups specifically introduced in
Vn by its CK2 phosphorylation. Specifically, this result raises the
possibility that the ligand binding site of
v
3 possesses recognition elements to
CK2-PVn that are not present in
v
5. This
suggestion, which is supported by additional experimental evidence
using a set of RGD-containing peptides as
inhibitors,4 can account for
the distinct behavior of the
v
3 and
v
5 integrins and specifically for the
v
3-mediated enhanced activation of the
PI3K/PKB pathway that correlates with the increased cell adhesion.
v
3 and
v
5
and one via
v
3 (Scheme 1)) are activated
upon adhesion of the cells onto Vns. However, although the activation of ERK (triggered by both
v
3 and
v
5) was not modified upon cell adhesion
onto CK2-PVn, the activation of PKB (triggered by
v
3 but not by
v
5) is elevated upon adhesion to CK2-PVn.
It is this elevation that is correlated with the enhanced cell
adhesion. Therefore, we propose that the PI3K/PKB pathway (and not the
ERK pathway) reflects the
v
3-mediated
enhanced cell adhesion (Scheme 1). In line with this proposal, we found
that blocking the activation of ERK by an MEK inhibitor did not have an
effect on cell adhesion. In contrast, the blocking of PKB by PI3K
inhibitors reduced cell adhesion.
v
3) and an intracellular
signaling pathway that distinguish between a CK2-phosphorylated and a
non-phosphorylated form of Vn. These results are based on a CK2-phospho
and a non-phospho form of Vn, two mutant analogs of Vn, three different
cell lines, and four independent cell clones. We believe that these
findings indicate that the extra-cellular phosphorylation of
Vn by CK2 may well be a physiological process with a distinct
regulatory role in the control of cell adhesion and spreading.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Iris Schvartz for stimulating discussions and Shoshana Lichter and Tamar Hanoch for technical assistance.
![]() |
FOOTNOTES |
---|
* This research was supported in part by the Israel Science Foundation.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.
The Incumbent of the Kleeman Chair in Biochemistry. To whom
correspondence should be addressed: Dept. of Biological Regulation, The
Weizmann Institute of Science, Rehovot IL-76100, Israel. Tel.: 972-8-934-4016 or 972-8-934-4526; Fax: 972-8-9342804; E-mail: shmuel.shaltiel@weizmann.ac.il.
Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.M003766200
2
The rational of transfecting only with one
subunit (3) rather than co-transfecting both
v and
3 was to use the existing
v pool and make it generate more
v
3 at the expense of other
v partners (
5).
3
This small activation is probably due to
the residual cell adhesion through the v
3
integrin in these cells (about 10%, as detected by the inhibition
achieved using specific anti-
v
3 integrin antibodies, Fig. 3A).
4 M. Garazi, I. Schvartz, D. Seger, and S. Shaltiel, in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Vn, vitronectin; ECM, extracellular matrix; FAK, focal adhesion kinase; PI3K, phosphatidylinositol 3-kinase; PKA, -B, -C, protein kinases A, B, and C; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; BAEC, bovine aorta endothelial cells; JNK, c-Jun N-terminal kinase; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; HA, hemagglutinin; FACS, fluorescence-activated cell sorting; RIPA, radioimmune precipitation buffer; PAGE, polyacrylamide gel electrophoresis; r-Vn, recombinant Vn; PVn, phosphorylated Vn.
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