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• W2018233795 abstract "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 αvβ3 (not via the αvβ5 integrin), suggesting that αvβ3 differs from αvβ5 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 αvβ3 as well as the αvβ5 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 αvβ3 integrin on their surface; (ii) that upon adhesion of the β3-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. 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 αvβ3 (not via the αvβ5 integrin), suggesting that αvβ3 differs from αvβ5 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 αvβ3 as well as the αvβ5 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 αvβ3 integrin on their surface; (ii) that upon adhesion of the β3-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. vitronectin extracellular matrix focal adhesion kinase phosphatidylinositol 3-kinase -B, -C, protein kinases A, B, and C mitogen-activated protein kinase extracellular signal-regulated kinase MAPK/ERK kinase bovine aorta endothelial cells c-Jun N-terminal kinase fluorescein isothiocyanate phosphate-buffered saline hemagglutinin fluorescence-activated cell sorting radioimmune precipitation buffer polyacrylamide gel electrophoresis recombinant Vn phosphorylated Vn Vitronectin (Vn)1 is an adhesive glycoprotein found in the extracellular matrix (ECM) of various cells, and in circulating blood (1Preissner K.T. Annu. Rev. Cell Biol. 1991; 7: 275-310Crossref PubMed Scopus (396) Google Scholar, 2Preissner K.T. Jenne D. Thromb. Haemost. 1991; 66: 123-132Crossref PubMed Scopus (85) Google Scholar, 3Tomasini B.R. Mosher D.F. Prog. Hemost. Thromb. 1991; 10: 269-305PubMed Google Scholar). It has been implicated in a large variety of physiological and pathophysiological processes such as hemostasis (4Mohri H. Ohkubo T. Am. J. Clin. Pathol. 1991; 96: 605-609Crossref PubMed Scopus (36) Google Scholar, 5Thiagarajan P. Kelly K.L. J. Biol. Chem. 1988; 263: 3035-3038Abstract Full Text PDF PubMed Google Scholar), tumor cell invasion (6Juliano R.L. Varner J.A. Curr. Opin. Cell Biol. 1993; 5: 812-818Crossref PubMed Scopus (256) Google Scholar, 7Nip J. Shibata H. Loskutoff D.J. Cheresh D.A. Brodt P. J. Clin. Invest. 1992; 90: 1406-1413Crossref PubMed Scopus (146) Google Scholar), angiogenesis (8Varner J.A. Brooks P.C. Cheresh D.A. Cell Adhes. 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Dev. 1990; 32: 287-292Crossref PubMed Scopus (47) Google Scholar, 16Sigurdardottir O. Wiman B. Biochim. Biophys. Acta. 1990; 1035: 56-61Crossref PubMed Scopus (24) Google Scholar, 17Preissner K.T. Biochem. Biophys. Res. Commun. 1990; 168: 966-971Crossref PubMed Scopus (44) Google Scholar, 18Chain D. Kreizman T. Shapira H. Shaltiel S. FEBS Lett. 1991; 285: 251-256Crossref PubMed Scopus (50) Google Scholar). One of the most important properties of Vn is its ability to promote cell attachment, spreading, and migration (19Hayman E.G. Pierschbacher M.D. Ohgren Y. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4003-4007Crossref PubMed Scopus (320) Google Scholar, 20Hayman E.G. Pierschbacher M.D. Suzuki S. Ruoslahti E. Exp. Cell. Res. 1985; 160: 245-258Crossref PubMed Scopus (259) Google Scholar, 21Preissner K.T. Anders E. Grulich H.J. Muller-Berghaus G. Blood. 1988; 71: 1581-1589Crossref PubMed Google Scholar, 22Brown C. Stenn K.S. Falk R.J. Woodley D.T. O'Keefe E.J. J. Invest. Dermatol. 1991; 96: 724-728Crossref PubMed Scopus (37) Google Scholar). In fact, Vn was originally discovered as a “serum spreading factor” (23Holmes R.J. J. Cell Biol. 1967; 32: 297-308Crossref PubMed Scopus (100) Google Scholar). 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 α and β subunits (24Ruoslahti E. Pierschbacher M.D. Cell. 1986; 44: 517-518Abstract Full Text PDF PubMed Scopus (1025) Google Scholar, 25Pierschbacher M.D. Ruoslahti E. Nature. 1984; 309: 30-33Crossref PubMed Scopus (2820) Google Scholar, 26Ruoslahti E. J. Clin. Invest. 1991; 87: 1-5Crossref PubMed Scopus (1477) Google Scholar, 27Ruoslahti E. Noble N.A. Kagami S. Border W.A. Kidney Int. Suppl. 1994; 44: S17-S22PubMed Google Scholar, 28Schwartz M.A. Ingber D.E. Mol. Biol. Cell. 1994; 5: 389-393Crossref PubMed Scopus (170) Google Scholar, 29Hynes R.O. Cell. 1987; 48: 549-554Abstract Full Text PDF PubMed Scopus (3072) Google Scholar, 30Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8941) Google Scholar). There are 17 α and 8 β subunits that heterodimerize to produce 22 different integrins (27Ruoslahti E. Noble N.A. Kagami S. Border W.A. Kidney Int. Suppl. 1994; 44: S17-S22PubMed Google Scholar, 31Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1456) Google Scholar, 32Kumar C.C. Oncogene. 1998; 17: 1365-1373Crossref PubMed Scopus (236) Google Scholar). Several of these integrins, e.g. αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8and the platelet-specific αIIbβ3 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 (33Richardson A. Parsons J.T. Bioessays. 1995; 17: 229-236Crossref PubMed Scopus (256) Google Scholar). 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 Tyr397residue. 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 (31Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1456) Google Scholar, 32Kumar C.C. Oncogene. 1998; 17: 1365-1373Crossref PubMed Scopus (236) Google Scholar, 33Richardson A. Parsons J.T. Bioessays. 1995; 17: 229-236Crossref PubMed Scopus (256) Google Scholar, 34Chen H.C. Appeddu P.A. Isoda H. Guan J.L. J. Biol. Chem. 1996; 271: 26329-26334Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar, 35Schwartz M.A. J. Cell Biol. 1997; 139: 575-578Crossref PubMed Scopus (302) Google Scholar, 36Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2802) Google Scholar, 37Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3757) Google Scholar, 38Schlaepfer D.D. Hanks S.K. Hunter T. Van-der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1426) Google Scholar). 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 (39Gordon J.L. Biochem. J. 1986; 233: 309-319Crossref PubMed Scopus (1393) Google Scholar). For example PKA, released from platelets upon their physiological stimulation with thrombin (40Korc-Grodzicki B. Tauber-Finkelstein M. Shaltiel S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7541-7545Crossref PubMed Scopus (34) Google Scholar, 41Korc-Grodzicki B. Tauber-Finkelstein M. Chain D. Shaltiel S. Biochem. Biophys. Res. Commun. 1988; 157: 1131-1138Crossref PubMed Scopus (39) Google Scholar, 42Shaltiel S. Schvartz I. Korc G.B. Kreizman T. Mol. Cell. Biochem. 1993; 127: 283-287Crossref PubMed Scopus (24) Google Scholar), selectively phosphorylates Vn, and, as a consequence of this phosphorylation, it reduces its grip on plasminogen activator inhibitor-1 (43Shaltiel S. Schvartz I. Gechtman Z. Kreizman T. Preissner K.T. Rosenblatt S. Kost C. Wegerhoff J. Mosher D.F. Biology of Vitronectins and Their Receptors. Elsevier Science, Amsterdam1993: 311-320Google Scholar). Similarly, PKC phosphorylation of Vn was shown to attenuate its cleavage by plasmin (44Gechtman Z. Shaltiel S. Eur. J. Biochem. 1997; 243: 493-501Crossref PubMed Scopus (19) Google Scholar). Several laboratories have shown the occurrence of an extra-cellular CK2 activity on a variety of cells. These include epithelial cells (45Kubler D. Pyerin W. Burow E. Kinzel V. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4021-4025Crossref PubMed Scopus (53) Google Scholar,46Pyerin W. Burow E. Michaely K. Kubler D. Kinzel V. Biol. Chem. Hoppe-Seyler. 1987; 368: 215-227Crossref PubMed Scopus (49) Google Scholar), neutrophils (47Dusenbery K.E. Mendiola J.R. Skubitz K.M. Biochem. Biophys. Res. Commun. 1988; 153: 7-13Crossref PubMed Scopus (24) Google Scholar, 48Skubitz K.M. Ehresmann D.D. Ducker T.P. J. Immunol. 1991; 147: 638-650PubMed Google Scholar), platelets (49Rand M.D. Kalafatis M. Mann K.G. Blood. 1994; 83: 2180-2190Crossref PubMed Google Scholar, 50Kalafatis M. Rand M.D. Jenny R.J. Ehrlich Y.H. Mann K.G. Blood. 1993; 81: 704-719Crossref PubMed Google Scholar), and endothelial cells (51Skubitz K.M. Ehresmann D.D. Cell. Mol. Biol. 1992; 38: 543-560PubMed Google Scholar, 52Hartmann M. Schrader J. Biochim. Biophys. Acta. 1992; 1136: 189-195Crossref PubMed Scopus (13) Google Scholar, 53Eriksson S. Alston S.J. Ekman P. Thromb. Res. 1993; 72: 315-320Abstract Full Text PDF PubMed Scopus (5) Google Scholar). 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 αvβ3 (54Seger D. Gechtman Z. Shaltiel S. J. Biol. Chem. 1998; 273: 24805-24813Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). 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, αvβ5, 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 αvβ3 (but not αvβ5). Furthermore, we show that this enhanced adhesion coincides with a preferential activation of the FAK/PI3K/PKB cascade, rather than the ERK signaling pathway. 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). Monoclonal antibodies against the integrin receptor αvβ5 (P1F6), against αvβ3 (LM609), and against the β3 integrin receptor (MAB 1974) were obtained from Chemicon. Monoclonal antibodies directed against the integrin receptor α3 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. 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.). 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. The monoclonal antibodies used were: P1F6, directed against the integrin receptor αvβ5; LM609, directed against the integrin receptor αvβ3; 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. 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. The cDNA encoding the β3 integrin subunit in pGEM was kindly provided by Dr. P. J. Newman, Blood Research Institute, Milwaukee, WI. The cDNA was digested withDraI 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 β3 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 (54Seger D. Gechtman Z. Shaltiel S. J. Biol. Chem. 1998; 273: 24805-24813Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). 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 (106cells/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). 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). 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, 0.1m Tris-HCl, pH 8.0, and finally twice in 50 mmβ-glycerophosphate, pH 7.3, 1.5 mm EGTA, 1 mmEDTA, 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. We have previously shown (54Seger D. Gechtman Z. Shaltiel S. J. Biol. Chem. 1998; 273: 24805-24813Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) 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 αvβ5 (a characteristic binding receptor for Vn (55Felding H.B. Cheresh D.A. Curr. Opin. Cell Biol. 1993; 5: 864-868Crossref PubMed Scopus (350) Google Scholar)); 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. 1 A) and H1299 cells (Fig. 1 B), whose adhesion to Vn was found to be mediated mainly by αvβ5. In both cases we found an efficient inhibition of cell adhesion by anti-αvβ5 but a minor inhibition by anti-αvβ3. 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 αvβ5. 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 andD). 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 (54Seger D. Gechtman Z. Shaltiel S. J. Biol. Chem. 1998; 273: 24805-24813Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) 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). The results presented above, together with our previous findings with BAEC (54Seger D. Gechtman Z. Shaltiel S. J. Biol. Chem. 1998; 273: 24805-24813Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), imply that the enhanced cell adhesion onto CK2-PVn is mediated by the αvβ3 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 β3subunit. 2The rational of transfecting only with one subunit (β3) rather than co-transfecting both αv and β3 was to use the existing αv pool and make it generate more αvβ3 at the expense of other αv partners (β5). Isolated clones of H1299 cells overexpressing αvβ3 that were identified by immunoblotting with anti-β3, and subsequently characterized by FACS analysis with anti-αvβ3 (Fig.2, A and B), were shown to contain high amounts of the αvβ3integrin on their surface. Quantitation of the FACS analysis indicated that the β3-transfected clones we used contained up to ∼7-fold more αvβ3 than the control vector-transfected clones, whereas the amounts of the αvand of a non-relevant α3 integrin were very similar to the control. In addition, we observed a ∼3-fold reduction of αvβ5 in the β3-transfected clone, presumably due to competition between β5 and the excess of β3 for the limited amount of their common partner, the αv subunit. The involvement of αvβ3 (but not αvβ5) 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-αvβ5 and not by anti-αvβ3 (Fig. 3 A), whereas the adhesion of β3-transfected H1299 cells is blocked by anti-αvβ3 but not by anti-αvβ5 (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 β3 subunit exhibit an ability to enhance cell adhesion on the Vn(T50E,T57E) mutant (compare Fig. 3 C with Fig.3 D). 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 β3-transfected clones (not shown). Following the identification of αvβ3 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 (31Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1456) Google Scholar, 32Kumar C.C. Oncogene. 1998; 17: 1365-1373Crossref PubMed Scopus (236) Google Scholar, 33Richardson A. Parsons J.T. Bioessays. 1995; 17: 229-236Crossref PubMed Scopus (256) Google Scholar, 34Chen H.C. Appeddu P.A. Isoda H. Guan J.L. J. Biol. Chem. 1996; 271: 26329-26334Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar, 35Schwartz M.A. J. Cell Biol. 1997; 139: 575-578Crossref PubMed Scopus (302) Google Scholar, 36Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2802) Google Scholar, 37Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3757) Google Scholar, 38Schlaepfer D.D. Hanks S.K. Hunter T. Van-der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1426) Google Scholar), we first examined the pattern of ERK activation in the stable αvβ3 and αvβ5expressing clones of the H1299 cells mentioned above. In response to cell adhesion to r-Vns, the ERK activation of αvβ5- and αvβ3-containing clones was found to be low and transient (Fig. 4, A andB): 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. 4 C). 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 β3-transfected clone. 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 β3-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. 3This small activation is probably due to the residual cell adhesion through the αvβ3integrin in these cells (about 10%, as detected by the inhibition achieved using specific anti-αvβ3 integrin antibodies, Fig. 3 A). These results suggested to us that the activation of PKB depends on the availability of the αvβ3 integrin. As such, the extent of PKB activation in the β3-transfected cells correlates well with the extent of enhanced cell adhesion onto CK2-PVn. This was demonstrated with β3-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. 5 C). PKB was recently implicated as an important downstream target for PI3K (56Downward J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1178) Google Scholar). To determine whether the PKB activation in our system requires the activation of PI3K (which precedes PKB in several signal transduction processes (cf.Scheme FS1), we treated β3-transfected cells with wortmannin (a PI3K inhibitor) prior to their stimulation by adhesion to Vn(T50E,T57E). Indeed, w" @default.
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• W2018233795 title "The CK2 Phosphorylation of Vitronectin" @default.
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