The CK2 Phosphorylation of Vitronectin

PROMOTION OF CELL ADHESION VIA THE alpha vbeta 3-PHOSPHATIDYLINOSITOL 3-KINASE PATHWAY*

Dalia Seger, Rony Seger, and Shmuel ShaltielDagger

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha vbeta 3 (not via the alpha vbeta 5 integrin), suggesting that alpha vbeta 3 differs from alpha vbeta 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 alpha vbeta 3 as well as the alpha vbeta 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 alpha vbeta 3 integrin on their surface; (ii) that upon adhesion of the beta 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

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 alpha  and beta  subunits (24-30). There are 17 alpha  and 8 beta  subunits that heterodimerize to produce 22 different integrins (27, 31, 32). Several of these integrins, e.g. alpha vbeta 1, alpha vbeta 3, alpha vbeta 5, alpha vbeta 6, and alpha vbeta 8 and the platelet-specific alpha IIbbeta 3 integrin, are known to recognize and bind Vn.

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 alpha vbeta 3 (54).

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, alpha vbeta 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 alpha vbeta 3 (but not alpha vbeta 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

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 alpha vbeta 5 (P1F6), against alpha vbeta 3 (LM609), and against the beta 3 integrin receptor (MAB 1974) were obtained from Chemicon. Monoclonal antibodies directed against the integrin receptor alpha 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.

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 alpha vbeta 5; LM609, directed against the integrin receptor alpha vbeta 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.

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 beta 3 Integrin Subunit and the r-Vn Mutants-- The cDNA encoding the beta 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 beta 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).

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 beta -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

Comparing the Adhesion of alpha vbeta 3- and of alpha vbeta 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).

In the course of our studies we found that BAEC cells do not express alpha vbeta 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 alpha vbeta 5. In both cases we found an efficient inhibition of cell adhesion by anti-alpha vbeta 5 but a minor inhibition by anti-alpha vbeta 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 alpha vbeta 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).


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 1.   The adhesion of HeLa or H1299 cells to r-Vn is mediated mainly by alpha vbeta 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 alpha vbeta 3 or alpha vbeta 5. B, inhibition of H1299 cell adhesion to r-Vns by antibodies raised against alpha vbeta 3 or alpha vbeta 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 alpha vbeta 3 or against alpha vbeta 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."

Cells Containing alpha vbeta 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 alpha vbeta 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 beta 3 subunit.2 Isolated clones of H1299 cells overexpressing alpha vbeta 3 that were identified by immunoblotting with anti-beta 3, and subsequently characterized by FACS analysis with anti-alpha vbeta 3 (Fig. 2, A and B), were shown to contain high amounts of the alpha vbeta 3 integrin on their surface. Quantitation of the FACS analysis indicated that the beta 3-transfected clones we used contained up to ~7-fold more alpha vbeta 3 than the control vector-transfected clones, whereas the amounts of the alpha v and of a non-relevant alpha 3 integrin were very similar to the control. In addition, we observed a ~3-fold reduction of alpha vbeta 5 in the beta 3-transfected clone, presumably due to competition between beta 5 and the excess of beta 3 for the limited amount of their common partner, the alpha v subunit.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 2.   Characterization of the integrins expressed in the stable clones of H1299 cells. A, overexpression of integrin beta 3 subunit in H1299 cells as determined by immunoblot. Soluble fractions obtained from extracts of H1299 stably transfected with the cDNA of beta 3 subunit, or with vector alone (pcDNA3), were analyzed by immunoblotting with anti-beta 3 monoclonal antibodies. Lane 1, non-transfected cells; lanes 2 and 3, two different vector-transfected clones; lanes 4 and 5, two different beta 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).

The involvement of alpha vbeta 3 (but not alpha vbeta 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-alpha vbeta 5 and not by anti-alpha vbeta 3 (Fig. 3A), whereas the adhesion of beta 3-transfected H1299 cells is blocked by anti-alpha vbeta 3 but not by anti-alpha vbeta 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 beta 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 beta 3-transfected clones (not shown).


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 3.   Transfection with beta 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 alpha vbeta 3 or alpha vbeta 5. B, inhibition of beta 3-transfected H1299 cell adhesion to r-Vns by antibodies raised against alpha vbeta 3 or alpha vbeta 5. C, adhesion of vector-transfected H1299 cells to r-Vns. D, adhesion of beta 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.

An ERK Activation Cannot Account for the Enhanced Cell Adhesion Observed with CK2-PVn-- Following the identification of alpha vbeta 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 alpha vbeta 3 and alpha vbeta 5 expressing clones of the H1299 cells mentioned above. In response to cell adhesion to r-Vns, the ERK activation of alpha vbeta 5- and alpha vbeta 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 beta 3-transfected clone.


View larger version (29K):
[in this window]
[in a new window]
 
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 beta 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 beta 3).

The Increased Activation of the PKB Pathway Can Account for the Enhanced Cell Adhesion Mediated by alpha vbeta 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 beta 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 alpha vbeta 3 integrin. As such, the extent of PKB activation in the beta 3-transfected cells correlates well with the extent of enhanced cell adhesion onto CK2-PVn. This was demonstrated with beta 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).


View larger version (31K):
[in this window]
[in a new window]
 
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 beta 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 beta 3).

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 beta 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 alpha vbeta 3 transmits the signal to PKB via PI3K. In line with this result, the enhanced adhesion of the beta 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 this window]
[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 alpha vbeta 3 but not of alpha vbeta 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 this window]
[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 beta 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 beta 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) (triangle , black-triangle) and Vn(T50E,T57E) (open circle , ). H1299 cells stably transfected with the beta 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 beta 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) (triangle , black-triangle); and Vn(T50E,T57E) (open circle , ). H1299 cells stably transfected with the beta 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 beta 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) (triangle , black-triangle) and Vn(T50E,T57E) (open circle , ). H1299 cells stably transfected with the beta 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."

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 alpha vbeta 3 integrin on the surface of the cells; (ii) that the extent of PKB activation (that takes place upon exposure of the beta 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).

Cells Containing alpha vbeta 3 and alpha vbeta 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 beta 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/beta 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 beta 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 beta 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 beta 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 (alpha vbeta 5) another kinase may further phosphorylate FAK, whereas in the beta 3-transfected cells (alpha vbeta 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 alpha vbeta 3 and that the PI3K pathway is involved in the enhanced cell adhesion.


View larger version (27K):
[in this window]
[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 beta 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 beta 3).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha vbeta 3. In fact, this integrin has been implicated in the acquisition of metastatic invasiveness (57). In melanoma, for example, the expression of alpha vbeta 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 alpha vbeta 3-mediated adhesion of tumor cells. In line with this fact, human melanoma cells derived from lymphatic metastases were shown to use alpha vbeta 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).

A major implication of the findings presented in this report is that the CK2 phosphorylation of Vn enhances cell adhesion via alpha vbeta 3 and not via alpha vbeta 5. This can be deduced from our finding that cells that adhere mostly via alpha vbeta 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 alpha vbeta 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 alpha vbeta 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 beta 3 integrin subunit.

In view of our finding that the enhanced cell adhesion onto Vn(T50E,T57E) is mediated by alpha vbeta 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 alpha vbeta 3 to adhere onto Vn, and essentially identical companion cells that use alpha vbeta 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 alpha vbeta 3 and alpha vbeta 5, leading to a similar activation of ERK. These results suggest that the activation of ERK occurs via the alpha  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 beta 3 subunit supports this suggestion. In contrast, the PKB activation seems to depend on the availability of the beta 3 subunit, and therefore, is preferentially activated by the phospho form of Vn. We presume that this enhanced activation of PKB, which is alpha vbeta 3- and PI3K-dependent, results in the enhanced cell adhesion by the CK2-PVn analog (Vn(T50E,T57E)).

Based on the results presented here, we suggest that although both alpha vbeta 3 and alpha vbeta 5 share common structural elements that recognize and bind equally well the core protein shared by Vn and PVn, alpha vbeta 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 alpha vbeta 3 possesses recognition elements to CK2-PVn that are not present in alpha vbeta 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 alpha vbeta 3 and alpha vbeta 5 integrins and specifically for the alpha vbeta 3-mediated enhanced activation of the PI3K/PKB pathway that correlates with the increased cell adhesion.

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 alpha vbeta 3 and alpha vbeta 5 and one via alpha vbeta 3 (Scheme 1)) are activated upon adhesion of the cells onto Vns. However, although the activation of ERK (triggered by both alpha vbeta 3 and alpha vbeta 5) was not modified upon cell adhesion onto CK2-PVn, the activation of PKB (triggered by alpha vbeta 3 but not by alpha vbeta 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 alpha vbeta 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.

Taken together, the results presented here together with our results reported earlier (54) indicate the occurrence of a cell surface receptor (alpha vbeta 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.

Dagger 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 (beta 3) rather than co-transfecting both alpha v and beta 3 was to use the existing alpha v pool and make it generate more alpha vbeta 3 at the expense of other alpha v partners (beta 5).

3 This small activation is probably due to the residual cell adhesion through the alpha vbeta 3 integrin in these cells (about 10%, as detected by the inhibition achieved using specific anti-alpha vbeta 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Preissner, K. T. (1991) Annu. Rev. Cell Biol. 7, 275-310[CrossRef]
2. Preissner, K. T., and Jenne, D. (1991) Thromb. Haemost. 66, 123-132[Medline] [Order article via Infotrieve]
3. Tomasini, B. R., and Mosher, D. F. (1991) Prog. Hemost. Thromb. 10, 269-305[Medline] [Order article via Infotrieve]
4. Mohri, H., and Ohkubo, T. (1991) Am. J. Clin. Pathol. 96, 605-609[Medline] [Order article via Infotrieve]
5. Thiagarajan, P., and Kelly, K. L. (1988) J. Biol. Chem. 263, 3035-3038[Abstract/Free Full Text]
6. Juliano, R. L., and Varner, J. A. (1993) Curr. Opin. Cell Biol. 5, 812-818[Medline] [Order article via Infotrieve]
7. Nip, J., Shibata, H., Loskutoff, D. J., Cheresh, D. A., and Brodt, P. (1992) J. Clin. Invest. 90, 1406-1413[Medline] [Order article via Infotrieve]
8. Varner, J. A., Brooks, P. C., and Cheresh, D. A. (1995) Cell Adhes. Commun. 3, 367-374[Medline] [Order article via Infotrieve]
9. Brooks, P. C., Clark, R. A., and Cheresh, D. A. (1994) Science 264, 569-571[Medline] [Order article via Infotrieve]
10. Brooks, P. C., Montgomery, A. M., Rosenfeld, M., Reisfeld, R. A., Hu, T., Klier, G., and Cheresh, D. A. (1994) Cell 79, 1157-1164[Medline] [Order article via Infotrieve]
11. Brooks, P. C., Stromblad, S., Klemke, R., Visscher, D., Sarkar, F. H., and Cheresh, D. A. (1995) J. Clin. Invest. 96, 1815-1822[Medline] [Order article via Infotrieve]
12. Lindahl, T. L., Sigurdardottir, O., and Wiman, B. (1989) Thromb. Haemost. 62, 748-751[Medline] [Order article via Infotrieve]
13. Mimuro, J., and Loskutoff, D. J. (1989) J. Biol. Chem. 264, 5058-5063[Abstract/Free Full Text]
14. Owensby, D. A., Morton, P. A., Wun, T. C., and Schwartz, A. L. (1991) J. Biol. Chem. 266, 4334-4340[Abstract/Free Full Text]
15. Seiffert, D., Mimuro, J., Schleef, R. R., and Loskutoff, D. J. (1990) Cell Differ. Dev. 32, 287-92[Medline] [Order article via Infotrieve]
16. Sigurdardottir, O., and Wiman, B. (1990) Biochim. Biophys. Acta 1035, 56-61[Medline] [Order article via Infotrieve]
17. Preissner, K. T. (1990) Biochem. Biophys. Res. Commun. 168, 966-971[Medline] [Order article via Infotrieve]
18. Chain, D., Kreizman, T., Shapira, H., and Shaltiel, S. (1991) FEBS Lett. 285, 251-256[CrossRef][Medline] [Order article via Infotrieve]
19. Hayman, E. G., Pierschbacher, M. D., Ohgren, Y., and Ruoslahti, E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4003-4007[Abstract]
20. Hayman, E. G., Pierschbacher, M. D., Suzuki, S., and Ruoslahti, E. (1985) Exp. Cell. Res. 160, 245-258[Medline] [Order article via Infotrieve]
21. Preissner, K. T., Anders, E., Grulich, H. J., and Muller-Berghaus, G. (1988) Blood 71, 1581-1589[Abstract]
22. Brown, C., Stenn, K. S., Falk, R. J., Woodley, D. T., and O'Keefe, E. J. (1991) J. Invest. Dermatol. 96, 724-728[Abstract]
23. Holmes, R. J. (1967) J. Cell Biol. 32, 297-308[Abstract/Free Full Text]
24. Ruoslahti, E., and Pierschbacher, M. D. (1986) Cell 44, 517-518[Medline] [Order article via Infotrieve]
25. Pierschbacher, M. D., and Ruoslahti, E. (1984) Nature 309, 30-33[Medline] [Order article via Infotrieve]
26. Ruoslahti, E. (1991) J. Clin. Invest. 87, 1-5[Medline] [Order article via Infotrieve]
27. Ruoslahti, E., Noble, N. A., Kagami, S., and Border, W. A. (1994) Kidney Int. Suppl. 44, S17-S22[Medline] [Order article via Infotrieve]
28. Schwartz, M. A., and Ingber, D. E. (1994) Mol. Biol. Cell 5, 389-393[Abstract]
29. Hynes, R. O. (1987) Cell 48, 549-554[Medline] [Order article via Infotrieve]
30. Hynes, R. O. (1992) Cell 69, 11-25[Medline] [Order article via Infotrieve]
31. Schwartz, M. A., Schaller, M. D., and Ginsberg, M. H. (1995) Annu. Rev. Cell Dev. Biol. 11, 549-599[CrossRef][Medline] [Order article via Infotrieve]
32. Kumar, C. C. (1998) Oncogene 17, 1365-1373[CrossRef][Medline] [Order article via Infotrieve]
33. Richardson, A., and Parsons, J. T. (1995) Bioessays 17, 229-236[Medline] [Order article via Infotrieve]
34. Chen, H. C., Appeddu, P. A., Isoda, H., and Guan, J. L. (1996) J. Biol. Chem. 271, 26329-26334[Abstract/Free Full Text]
35. Schwartz, M. A. (1997) J. Cell Biol. 139, 575-578[Free Full Text]
36. Clark, E. A., and Brugge, J. S. (1995) Science 268, 233-239[Medline] [Order article via Infotrieve]
37. Giancotti, F. G., and Ruoslahti, E. (1999) Science 285, 1028-1032[Abstract/Free Full Text]
38. Schlaepfer, D. D., Hanks, S. K., Hunter, T., and Van-der Geer, P. (1994) Nature 372, 786-791[Medline] [Order article via Infotrieve]
39. Gordon, J. L. (1986) Biochem. J. 233, 309-319[Medline] [Order article via Infotrieve]
40. Korc-Grodzicki, B., Tauber-Finkelstein, M., and Shaltiel, S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7541-7545[Abstract]
41. Korc-Grodzicki, B., Tauber-Finkelstein, M., Chain, D., and Shaltiel, S. (1988) Biochem. Biophys. Res. Commun. 157, 1131-1138[Medline] [Order article via Infotrieve]
42. Shaltiel, S., Schvartz, I., Korc, G. B., and Kreizman, T. (1993) Mol. Cell. Biochem. 127, 283-287
43. Shaltiel, S., Schvartz, I., Gechtman, Z., and Kreizman, T. (1993) in Biology of Vitronectins and Their Receptors (Preissner, K. T. , Rosenblatt, S. , Kost, C. , Wegerhoff, J. , and Mosher, D. F., eds) , pp. 311-320, Elsevier Science, Amsterdam
44. Gechtman, Z., and Shaltiel, S. (1997) Eur. J. Biochem. 243, 493-501[Abstract]
45. Kubler, D., Pyerin, W., Burow, E., and Kinzel, V. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4021-4025[Abstract]
46. Pyerin, W., Burow, E., Michaely, K., Kubler, D., and Kinzel, V. (1987) Biol. Chem. Hoppe-Seyler 368, 215-227[Medline] [Order article via Infotrieve]
47. Dusenbery, K. E., Mendiola, J. R., and Skubitz, K. M. (1988) Biochem. Biophys. Res. Commun. 153, 7-13[Medline] [Order article via Infotrieve]
48. Skubitz, K. M., Ehresmann, D. D., and Ducker, T. P. (1991) J. Immunol. 147, 638-650[Abstract/Free Full Text]
49. Rand, M. D., Kalafatis, M., and Mann, K. G. (1994) Blood 83, 2180-2190[Abstract/Free Full Text]
50. Kalafatis, M., Rand, M. D., Jenny, R. J., Ehrlich, Y. H., and Mann, K. G. (1993) Blood 81, 704-719[Abstract]
51. Skubitz, K. M., and Ehresmann, D. D. (1992) Cell. Mol. Biol. 38, 543-560[Medline] [Order article via Infotrieve]
52. Hartmann, M., and Schrader, J. (1992) Biochim. Biophys. Acta 1136, 189-195[Medline] [Order article via Infotrieve]
53. Eriksson, S., Alston, S. J., and Ekman, P. (1993) Thromb. Res. 72, 315-320[Medline] [Order article via Infotrieve]
54. Seger, D., Gechtman, Z., and Shaltiel, S. (1998) J. Biol. Chem. 273, 24805-24813[Abstract/Free Full Text]
55. Felding, H. B., and Cheresh, D. A. (1993) Curr. Opin. Cell Biol. 5, 864-868[Medline] [Order article via Infotrieve]
56. Downward, J. (1998) Curr. Opin. Cell Biol. 10, 262-267[CrossRef][Medline] [Order article via Infotrieve]
57. Marshall, J. F., and Hart, I. R. (1996) Semin Cancer Biol 7, 129-138[CrossRef][Medline] [Order article via Infotrieve]
58. Gehlsen, K. R., Davis, G. E., and Sriramarao, P. (1992) Clin. Exp. Metastasis 10, 111-120[Medline] [Order article via Infotrieve]
59. Marshall, J. F., Nesbitt, S. A., Helfrich, M. H., Horton, M. A., Polakova, K., and Hart, I. R. (1991) Int. J. Cancer 49, 924-931[Medline] [Order article via Infotrieve]
60. Seftor, R. E., Seftor, E. A., Gehlsen, K. R., Stetler, S. W., Brown, P. D., Ruoslahti, E., and Hendrix, M. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1557-1561[Abstract]
61. Wary, K. K., Mariotti, A., Zurzolo, C., and Giancotti, F. G. (1998) Cell 94, 625-634[Medline] [Order article via Infotrieve]
62. Defilippi, P., Olivo, C., Venturino, M., Dolce, L., Silengo, L., and Tarone, G. (1999) Microsc. Res. Tech. 47, 67-78[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.