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Related Collections Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 92 No. 4 (August 15), 1998: pp. 1277-1286 Outside-In Signaling of Soluble and Solid-Phase Fibrinogen Through Integrin IIb3 Is Different and Cooperative With Each Other in a Megakaryoblastic Leukemia Cell Line, CMK By Yumi Tohyama, Kaoru Tohyama, Misao Tsubokawa, Momoyo Asahi, Yataro Yoshida, and Hirohei Yamamura From the Department of Biochemistry, Kobe University School of Medicine, Kobe, Japan; the Department of Laboratory Medicine and Clinical Sciences, and Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto, Japan; the Department of Otorhinolaryngology, Fukui Medical School, Fukui, Japan; and the Department of Biochemistry, Fukui Prefectural University, Fukui, Japan.     ABSTRACT Abstract Introduction Methods Results Discussion References The function and the outside-in signaling pathways of IIb3 were examined in relation to cell adhesion using a megakaryoblastic leukemia cell line, CMK. After 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, the cells adhered to the culture plate and underwent megakaryocytic differentiation with expression of IIb3. Binding of soluble fibrinogen to the cells via IIb3 was dependent on cell adhesion. Cell detaching reduced the affinity of this integrin for soluble fibrinogen, although its surface expression was almost unchanged. In contrast, detached cells became tightly adherent to the fibrinogen-coated plate (solid-phase fibrinogen). The same ligand, fibrinogen, present either in soluble or solid-phase form, triggered differential signaling pathways mediated by IIb3. By the stimulation with soluble fibrinogen, Syk was tyrosine-phosphorylated but FAK was dephosphorylated, whereas solid-phase fibrinogen promptly caused tyrosine phosphorylation of FAK followed by delayed phosphorylation of Syk. In addition, the binding of soluble fibrinogen to the cells adherent to fibrinogen-coated plate resulted in tyrosine phosphorylation of integrin 3 and a complex formation of integrin 3 with Syk. This implies the cooperation of both soluble and solid-phase fibrinogen-mediated signaling pathways. © 1998 by The American Society of Hematology.     INTRODUCTION Abstract Introduction Methods Results Discussion References INTEGRIN IIB3 EXPRESSED on platelet megakaryocyte-lineage cells is one of the integrin family, - and -heterodimeric cell surface receptors and is involved in the interactions between extracellular matrix and the cytoskeleton.1-3 In the process of platelet activation, IIb3 undergoes conformational changes from the resting to the activated form (inside-out signaling). In the activated platelets, soluble fibrinogen binds to IIb3 and triggers outside-in signaling during platelet aggregation, but in the resting form, IIb3 only adheres to fibrinogen-coated plate (binding of the solid-phase fibrinogen).4,5 The function of IIb3 in megakaryocytes has been limitedly characterized as the IIb3-mediated endocytosis of fibrinogen into  granules6 and the modulation of IIb3 activity by thrombopoietin.7 Several studies using cell lines have shown that the cytoplasmic domains of IIb3 are essential for signaling response8-10 and that IIb3-mediated cell adhesion is enhanced by the treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA).11,12 In platelet aggregation, tyrosine phosphorylation of 3 might be important in initiating outside-in signaling through IIb3.13 FAK and Syk are nonreceptor protein tyrosine kinases involved in IIb3-mediated signaling. FAK is found in the focal adhesion and appears to be activated in response to integrin-mediated adhesion. In platelets, FAK is reported to be activated dependently on IIb3-mediated aggregation, whereas the ligation of IIb3 is not sufficient to stimulate FAK phosphorylation.14,15 In fact, a few reports showed that clustering of IIb3 with some anti-IIb3 antibodies does not promote FAK phosphorylation.16,17 Syk is expressed exclusively in hematopoietic cells.18,19 In a number of studies on the activation of Syk, the functional roles of Syk in B-cell receptor (BCR) and some Fc receptors have been extensively examined in association with the immune receptor tyrosine activation motifs (ITAMs).20-22 In platelets, we and others have reported that Syk is activated by a variety of stimulants23-25 and translocates into the cytoskeleton in two steps, one of which is dependent on IIb3-mediated aggregation.26 In addition, Clark et al27 have reported that Syk is activated via engagement of IIb3 by fibrinogen only when the anti-ligand-induced binding site (anti-LIBS) monoclonal antibody (MoAb) that alters IIb3 to the activated form is used. Recently Gao et al28 showed that Syk activation mediated by IIb3 is triggered by the binding of soluble fibrinogen and does not require phosphorylated ITAMs. As for other integrin-mediated signaling, Syk was reported to be activated after the ligation of 1 or 2 integrins.29,30 In this study, we address the possibility of differential signaling pathways mediated by IIb3 when it is engaged via fibrinogen in different forms, either in soluble or solid-phase form, using a megakaryoblastic leukemia cell line, CMK.31     MATERIALS AND METHODS Abstract Introduction Methods Results Discussion References Antibodies.   Anti-Syk MoAb 101 was kindly provided by Wako Pure Chemicals (Tokyo, Japan). Polyclonal antibodies against Syk (sc-573, sc-929) and against FAK (sc-557) were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Antiphosphotyrosine MoAb 4G10 was purchased from Upstate Biotechnology Inc (Lake Placid, NY), antiphosphotyrosine MoAb PY-20 from Transduction Laboratories (Lexington, KY), anti-IIb3 MoAb P2 from Immunotech (Marseille, France),32 anti-IIb3 MoAb for immunophenotyping analysis from DAKO Japan (Kyoto, Japan), fluorescein-labeled anti-IIb3 MoAb specific for the activated form, fluorescein isothiocyanate (FITC)-PAC1, from Becton Dickinson (San Jose, CA),33 anti-integrin 3 MoAb for immunoprecipitation from Southern Biotechnology Associates Inc (Birmingham, AL), and anti-integrin 3 and anti-integrin IIb MoAb for immunoblotting from Affiniti Research Products Ltd (Nottingham, UK). Reagents.   Human fibrinogen was kindly provided by Yoshitomi Pharmaceutical Industries Ltd (Osaka, Japan). TPA, Arg-Gly-Asp-Ser (RGDS), and Arg-Gly-Glu-Ser (RGES) peptides were purchased from Sigma (St Louis, MO). The disulfide-linked peptide, cyclo(S,S)-Mpr.(-phenylimidyl-Lys)GDWPPen-NH2 (cyclic-KGD) was kindly provided by Dr Robert M. Scarborough (COR Therapeutics Inc, South San Francisco, CA).34 Cytochalasin D was purchased from Wako Pure Chemicals (Tokyo, Japan),35 poly-L-lysine from Nacalai Tesque (Kyoto, Japan), and fluorescein-C6-succunimidyl ester (FXS) from PanVera Corp (Madison, WI). Cell culture.   The CMK cell line, provided by Dr T. Sato (Chiba University, Chiba, Japan), was maintained in RPMI 1640 medium with 10% heat-inactivated fetal calf serum (FCS).31 This cell line was derived from a child with megakaryoblastic leukemia and has the property of expressing some characteristics of mature megakaryocytes after TPA treatment.36 The cells were induced to differentiation by the addition of 20 nmol/L TPA and harvested at the indicated times. The stimulation with soluble and solid-phase fibrinogen.   We added the indicated concentrations of fibrinogen dissolved in RPMI 1640 without FCS either to the adherent cells formed after TPA treatment for 3 days (D3-adherent cells) or to the cells detached mechanically by use of a cell scraper after the same TPA treatment (D3-detached cells). To remove the influence of TPA, the medium was exchanged 1 day before the stimulation with fibrinogen. In some cases, the cells were preincubated with cytochalasin D at 37°C or with cyclic-KGD, RGDS or RGES peptides at 4°C for 15 minutes. To examine the cell adhesion to solid-phase fibrinogen or poly-L-lysine, the culture plates were precoated with phosphate-buffered saline (PBS) containing 100 µg/mL fibrinogen or 10 µg/mL poly-L-lysine overnight at 4°C, incubated with 1% bovine serum albumin (BSA) in PBS for 1 hour at 37°C to block nonspecific binding, and washed with PBS before use. The BSA precoated plates were used as a negative control. The detached cells were reincubated on the plates precoated with fibrinogen, poly-L-lysine, or BSA for the indicated times. Immunoprecipitation.   Usually, cells were lysed in a nonionic detergent buffer (1% Triton X-100, 50 mmol/L Tris/HCl, pH 7.5, 150 mmol/L NaCl, 5 mmol/L EDTA, 1 mmol/L sodium vanadate, 1 mmol/L phenylmethylsulfonyl fluoride, and 10 µg/mL leupeptin). In some cases, sodium dodecyl sulfate (SDS)-containing lysis buffer (0.05% SDS, 0.5% sodium deoxycholate, 50 mmol/L Tris/HCl, pH 7.5, 150 mmol/L NaCl, 5 mmol/L EDTA, 1 mmol/L sodium vanadate, 1 mmol/L phenylmethylsulfonyl fluoride, and 10 µg/mL leupeptin) was used. The cell lysates were centrifuged and the supernatants were incubated with the indicated antibodies for 2 hours at 4°C, after which protein A-Sepharose (Pharmacia Biotech, Uppsala, Sweden) was added for 1 hour at 4°C. The precipitates were washed 3 times with the indicated lysis buffer. For immunoblotting, the precipitates were boiled with electrophoresis SDS sample buffer (2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.001% bromophenol blue, and 62.5 mmol/L Tris/HCl, pH 6.8) for 3 minutes. In vitro kinase assay of Syk was performed as previously described.23 Immunoblotting.   For the day course study, whole cell lysates were prepared by boiling with electrophoresis SDS sample buffer for 3 minutes. Whole cell lysates or immunoprecipitates were separated by SDS-PAGE and transferred onto polyvinylidine difluoride membranes (Immobilon; Millipore Corp, Bedford, MA). The membrane blots were blocked with 5% skim milk in T-PBS (PBS containing 0.05% Tween 20) or low-salt Tris buffered-saline containing Tween 20 (low-salt T-TBS; 10 mmol/L Tris/HCl, pH 7.5, 100 mmol/L NaCl containing 0.05% Tween 20; for the detection of tyrosine phosphorylation of integrin 3) for 1 hour at 37°C and then incubated with primary antibodies in T-PBS or low-salt T-TBS for 1 hour at room temperature. After washing, membranes were incubated with horseradish peroxidase-conjugated goat antimouse IgG or antirabbit IgG polyclonal antibodies in T-PBS or low-salt T-TBS for 30 minutes at room temperature. After washing, the enhanced chemiluminescence (ECL) assay was performed and the positive bands were detected using x-ray films. Flow cytometry.   To measure the IIb3-mediated binding of soluble fibrinogen to CMK cells, fibrinogen was labeled with FXS according to the manufacturer's method. Total IIb3 expression was determined using anti-IIb3 MoAb and fluorescein-conjugated antimouse IgG polyclonal antibody. FITC-PAC1, fluorescein-labeled anti-IIb3 MoAb was used to detect the activated form. Each binding assay was performed for 30 minutes at room temperature. The binding assay for adherent cells was performed under the adherent condition and then the cells were detached mechanically for flow cytometric analyses. Flow cytometry was performed on a Becton Dickinson FACScan with Lysys II software. In the case of inhibition assay, anti-IIb3 MoAb was pretreated before the addition of FXS-labeled fibrinogen.     RESULTS Abstract Introduction Methods Results Discussion References TPA-induced differentiation of CMK cells.   When CMK cells were cultured in suspension, the percentage of IIb3-positive cells was less than 10% by immunophenotyping analysis (data not shown). In contrast, on the addition of 20 nmol/L TPA, most of CMK cells adhered to the plate by 3 hours. From day 3, cell spreading became prominent and the IIb3-positivity increased to greater than 90%, but the cell growth was completely arrested (data not shown). The expression of integrin IIb, 3 and protein tyrosine kinase Syk and FAK was examined by immunoblotting analyses using corresponding antibodies before and after TPA treatment (Fig 1). In Fig 1A, the upper band was considered as the precursor of IIb. The bands of IIb and 3 that were hardly detected on day 0 became detectable with time and were constant after day 3 (Fig 1A and B). In contrast, the band of Syk was already detectable on day 0 and increased about fourfold after day 2 (Fig 1C). The expression of FAK was unchanged in this day course study (data not shown). Next, protein tyrosine phosphorylation of whole cell lysates was analyzed. Tyrosine-phosphorylated proteins of 115, 105, 95, 75, and 50 kD were newly detected after the addition of TPA (Fig 1D). The 75-kD band reached a maximal level at 24 hours and then decreased, whereas the other tyrosine-phosphorylated bands were constant throughout the time course study. In the same lysates, tyrosine phosphorylation of FAK was detected at 3 hours after the addition of TPA, and the phosphorylation was maintained up to 72 hours (Fig 1E). View larger version (36K): [in this window] [in a new window]   View larger version (55K): [in this window] [in a new window]   Fig 1. TPA-induced differentiation of CMK cells on day course study. After addition of 20 nmol/L TPA to CMK cells, whole cell lysates were subjected to immunoblotting analyses with (A) anti-integrin IIb MoAb, (B) anti-integrin 3 MoAb, or (C) anti-Syk polyclonal antibody. In each lane, 30 µg protein of cell lysates was loaded. The cell lysates were next immunoprecipitated with (D) antiphosphotyrosine MoAb (PY20) or (E) anti-FAK polyclonal antibody. Immunoblotting analyses were performed with antiphosphotyrosine MoAb (4G10) as described in the Materials and Methods. In (D), arrows indicate the positions of the tyrosine-phosphorylated protein bands. In (E), the blots were stripped and reprobed with the anti-FAK polyclonal antibody. Results are representative of 3 experiments. Binding of anti-IIb3 MoAb and soluble fibrinogen to TPA-induced CMK cells.   The effect of cell adhesion on ligand binding to IIb3 was examined by flow cytometry. Surface expression of IIb3 was almost the same between D3-adherent and D3-detached cells (Fig 2A). In contrast, the binding of FXS-labeled fibrinogen to D3-adherent cells was higher than that to D3-detached cells (Fig 2B). To confirm that the binding is mediated by IIb3, cells were pretreated with anti-IIb3 MoAb or control mouse IgG for 5 minutes at room temperature before the addition of FXS-labeled fibrinogen. As shown in Fig 2C, anti-IIb3 MoAb but not control IgG showed a suppressive effect. Additionally, a larger amount of FITC-PAC1 MoAb specific for the activated form of IIb3 could bind to IIb3 on the surface of D3-adherent than D3-detached cells (Fig 2D). These results suggested that IIb3 on the surface of TPA-induced CMK cells exists in the activated form dependently on cell adhesion. View larger version (23K): [in this window] [in a new window]   Fig 2. Binding of anti-IIb3 MoAb and soluble fibrinogen to TPA-induced CMK cells. D3-adherent, D3-detached, and control CMK cells were incubated with (A) anti-IIb3 complex MoAb or (B) 200 µg/mL FXS-labeled fibrinogen for 30 minutes at room temperature, and flow cytometry was performed. D3-adherent cells were harvested by detaching them mechanically after the binding reaction. (C) D3-adherent and D3-detached cells were pretreated with 40 µg/mL of anti-IIb3 MoAb or control mouse IgG for 5 minutes at room temperature and then treated with 50 µg/mL FXS-labeled fibrinogen, after which flow cytometry was performed. The MFI of FXS-labeled fibrinogen bound to D3-adherent cells was taken as 100%, and results represent the mean ± SD of 3 independent experiments. (D) D3-adherent, D3-detached, and control CMK cells were incubated with the activation-dependent anti-IIb3 MoAb, PAC1 as described above, and flow cytometry was performed. Results of (A), (B), and (D) are representative of 3 experiments. Tyrosine phosphorylation of D3-adherent cells and D3-detached cells after the stimulation with soluble or solid-phase fibrinogen.   Protein tyrosine phosphorylation in D3-adherent and D3-detached cells was analyzed before and after the stimulation with soluble fibrinogen. To remove the direct influence of TPA, the medium was exchanged 1 day before the experiments. In D3-adherent cells, the addition of soluble fibrinogen resulted in gradual shrinking of the cells contrary to cell spreading and increased tyrosine phosphorylation of several proteins, including 160~150-kD, 75-kD, and approximately 72-kD bands, in contrast to the reduction of 115-kD band (Fig 3A). Reprobe assay confirmed that 72-kD band contained Syk (data not shown). As expected from the result of Fig 2, soluble fibrinogen could not trigger any signal in D3-detached cells, whereas 115-kD proteins were tyrosine-dephosphorylated just by detaching (Fig 3A). We then examined whether solid-phase fibrinogen binds to these cells and triggers the outside-in signaling. D3-detached cells were further incubated on the plate precoated with fibrinogen. After 15 minutes, most of the cells adhered to the fibrinogen plate (the cells on fibrinogen plate), but few cells adhered to the BSA-coated plate (negative control). Figure 3B shows that tyrosine phosphorylation of 150-kD, 120-kD, 115-kD, 75~70-kD, 50-kD, and 45-kD proteins was significantly increased by adhesion to the fibrinogen plate. View larger version (43K): [in this window] [in a new window]   View larger version (36K): [in this window] [in a new window]   Fig 3. Tyrosine phosphorylation of D3-adherent cells and D3-detached cells after the stimulation with soluble and solid-phase fibrinogen. (A) D3-adherent and D3-detached cells were stimulated with 1 mg/mL soluble fibrinogen for the indicated times. (B) D3-detached cells were incubated on the fibrinogen plate for the indicated times (0, 15, 30, and 45 minutes). In (A) and (B), the cell lysates were immunoprecipitated with antiphosphotyrosine MoAb (PY20) and immunoblotting analysis was performed with antiphosphotyrosine MoAb (4G10) as described in the Materials and Methods. The positions of the molecular markers are shown to the left in kilodaltons. Solid arrows and a broken arrow indicate the positions of the tyrosine-phosphorylated protein bands and the tyrosine-dephosphorylated protein band, respectively. In (C) and (D), D3-adherent and D3-detached cells were stimulated with 1 mg/mL soluble fibrinogen for the indicated times. The cell lysates were immunoprecipitated with (C) anti-FAK polyclonal antibody or (D) anti-Syk MoAb, and immunoblotting analysis was performed with antiphosphotyrosine MoAb (4G10). The blots were stripped and reprobed with the indicated antibodies. (E) The effects of disintegrin peptides on tyrosine phosphorylation of Syk were examined. D3-adherent cells were treated with 5 mmol/L RGDS, 5 mmol/L RGES, or 40 µmol/L cyclic-KGD peptide for 15 minutes at 4°C and stimulated with 1 mg/mL soluble fibrinogen for 5 minutes, and then tyrosine phosphorylation of Syk was examined as described above. (F) The effect of cytochalasin D on tyrosine phosphorylation of Syk was examined. D3-adherent cells were pretreated with 1 or 5 µmol/L cytochalasin D for 15 minutes at 37°C and then stimulated with 1 mg/mL soluble fibrinogen for 5 minutes, and tyrosine phosphorylation of Syk was examined as described above. Results are representative of 4 experiments. The effects of soluble fibrinogen on tyrosine phosphorylation of FAK and Syk were examined. By the stimulation with soluble fibrinogen, tyrosine phosphorylation of FAK was gradually reduced in D3-adherent cells (Fig 3C). In D3-detached cells, FAK was tyrosine-dephosphorylated just by detaching and soluble fibrinogen showed no effect (Fig 3C). In contrast to FAK, soluble fibrinogen increased tyrosine phosphorylation of Syk, which was readily detected at 1 minute and reached a maximal level at 30 minutes in D3-adherent cells (Fig 3D). As for D3-detached cells, Syk was only faintly tyrosine-phosphorylated, in marked contrast to the results of D3-adherent cells (Fig 3D). In TPA-untreated control CMK cells, soluble fibrinogen did not induce tyrosine phosphorylation of Syk (data not shown). Syk activity assayed in vitro showed a good correlation with tyrosine phosphorylation induced by fibrinogen (data not shown). To further confirm that tyrosine phosphorylation of Syk is specifically mediated by IIb3, the effects of disintegrin peptides were examined. In addition to RGDS and control RGES peptides, cyclic-KGD, a disulfide-linked peptide that has a potent affinity and high specificity for IIb3,34 was used. As shown in Fig 3E, soluble fibrinogen-induced tyrosine phosphorylation of Syk was suppressed by 5 mmol/L RGDS peptide but not by RGES peptide. Cyclic-KGD peptide (40 µmol/L) suppressed the phosphorylation of Syk to almost the level of 5 mmol/L RGDS. To further investigate whether the activation of Syk occurs via direct IIb3-mediated signaling or indirect changes of cytoskeleton triggered by IIb3-mediated signaling, we pretreated the cells with 1 or 5 µmol/L cytochalasin D, an actin polymerization inhibitor. Cytochalasin D failed to inhibit the tyrosine phosphorylation of Syk (Fig 3F). These results indicated that Syk is directly activated by the ligation of soluble fibrinogen and does not require subsequent cytoskeletal reorganization. Binding of soluble fibrinogen to the cells on fibrinogen plate.   We next examined whether soluble fibrinogen has an ability to bind to the cells on fibrinogen plate. Varying concentrations of FXS-labeled fibrinogen were added to D3-adherent cells (plastic plate) and the cells on fibrinogen plate (Fig 4A). The binding was quantitated as the mean fluorescence intensity (MFI). FXS-labeled fibrinogen showed saturation binding at a concentration of about 100 µg/mL in D3-adherent cells and 200 µg/mL in the cells on fibrinogen plate, respectively. At the saturating concentration, MFI of the cells on fibrinogen plate was reduced to about one sixth as compared with that of D3-adherent cells. The decreased binding of fibrinogen to the cells on fibrinogen plate may be due simply to fewer available receptors for binding. Figure 4B shows the binding of FXS-labeled fibrinogen to D3-detached cells, the cells on poly-L-lysine plate, the cells on fibrinogen plate, and D3-adherent cells (plastic plate). The histogram of the cells on fibrinogen plate was shifted to the right as compared with that of the D3-detached cells or the cells on poly-L-lysine plate, which indicated that soluble fibrinogen can bind to the cells on fibrinogen plate. View larger version (15K): [in this window] [in a new window]   View larger version (37K): [in this window] [in a new window]   Fig 4. Binding of soluble fibrinogen to the cells on fibrinogen plate. (A) FXS-labeled fibrinogen was added to D3-adherent cells and the cells on the fibrinogen plate at the indicated concentrations and the binding was characterized by flow cytometry. The MFIs from the histograms for each concentration of FXS-labeled fibrinogen were plotted graphically. (B) FXS-labeled fibrinogen (200 µg/mL) was added either to the D3-detached cells (Detached), the cells on poly-L-lysine plate (broken line), the cells on fibrinogen plate, or D3-adherent cells (Plastic Plate) for 30 minutes at room temperature. Adherent cells were detached mechanically after the binding reaction and subjected to flow cytometry. In (C) and (D), D3-detached cells were incubated on the fibrinogen plate for the indicated times (0, 15, 30, and 45 minutes). The cell lysates were immunoprecipitated with (C) anti-FAK polyclonal antibody or (D) anti-Syk MoAb, and immunoblotting analysis was performed with antiphosphotyrosine MoAb (4G10). The blots were stripped and reprobed with indicated antibodies. +Soluble Fg. indicates the stimulation with 1 mg/mL soluble fibrinogen for 5 minutes. PL shows the result of plating on poly-L-lysine plate for 30 minutes. Ad+Soluble Fg. and De+Soluble Fg. are the results of 1 mg/mL soluble fibrinogen stimulation on D3-adherent cells and D3-detached cells, respectively. Results are representative of 3 experiments. Because we found that binding of solid-phase fibrinogen triggered protein tyrosine phosphorylation of D3-detached cells (Fig 3B), we further examined the effect of solid-phase fibrinogen on tyrosine phosphorylation of FAK and Syk. Tyrosine phosphorylation of FAK was promptly induced by adhesion to fibrinogen plate but not by adhesion to poly-L-lysine plate (Fig 4C) and was suppressed by pretreatment with 5 µmol/L cytochalasin D (data not shown). In contrast, Syk was gradually tyrosine-phosphorylated by adhesion to fibrinogen plate and the degree of phosphorylation was less than that for soluble fibrinogen to D3-adherent cells (Fig 4D, 30 v Ad+Soluble Fg.). Therefore, the patterns of tyrosine phosphorylation triggered by soluble and solid-phase fibrinogen were different. The effect of additional binding of soluble fibrinogen to the cells on fibrinogen plate on tyrosine phosphorylation of FAK and Syk was examined next. Tyrosine phosphorylation of FAK was reduced (Fig 4C, 30 v 30+Soluble Fg.), whereas Syk phosphorylation was even more enhanced (Fig 4D, 30 v 30+Soluble Fg.) and the phosphorylation level was higher than that of D3-adherent cells (Fig 4D, 30+Soluble Fg. v Ad+Soluble Fg.). Association of Syk with IIb3 was detected only after the costimulation with soluble and solid-phase fibrinogen.   To examine the effects of additional stimulation with soluble fibrinogen on the cells on fibrinogen plate, coimmunoprecipitation assays between IIb3 and Syk were performed. Anti-IIb3 complex MoAb P2 coimmunoprecipitated Syk and anti-Syk MoAb 101 also coimmunoprecipitated integrin IIb with Syk after the additional binding of soluble fibrinogen (Fig 5A and B). P2 immunoprecipitated both integrin IIb (Fig 5B) and 3 (data not shown) in 1% Triton X-100 lysis buffer containing EDTA, as previously reported by McGregor et al.32 Because the coimmunoprecipitation between integrin 3 and Syk was not detected using Triton X-100 lysis buffer, we used SDS-containing lysis buffer to obtain nonionic detergent-insoluble fractions. Antiphosphotyrosine MoAb PY20 immunoprecipitated a trace amount of Syk in the cells on fibrinogen plate and then a greater amount on additional stimulation with soluble fibrinogen (Fig 5C). From the same lysates, anti-3 MoAb coimmunoprecipitated Syk (Fig 5C) and, conversely, anti-Syk antibody coimmunoprecipitated integrin 3 after the additional stimulation with soluble fibrinogen (Fig 5D). View larger version (29K): [in this window] [in a new window]   Fig 5. Association of Syk with integrin IIb3 after the costimulation with soluble and solid-phase fibrinogen. D3-detached cells (0), the cells on fibrinogen plate incubated for 30 minutes (30), and the cells on fibrinogen plate after the additional stimulation with 1 mg/mL soluble fibrinogen for 5 minutes (30+Soluble Fg.) were lysed and immunoprecipitated with indicated antibodies. In (A) and (B), the cells were lysed in 1% Triton X-100 lysis buffer. The immunoblotting analysis for each immunoprecipitate was performed with (A) anti-Syk polyclonal antibody or (B) anti-integrin IIb MoAb. In (C) and (D), the cells were lysed in 0.05% SDS-containing buffer. The immunoblotting analysis for each immunoprecipitate was performed with (C) anti-Syk polyclonal antibody or (D) anti-integrin 3 MoAb. Results are representative of 3 experiments. Tyrosine phosphorylation of integrin 3 after the costimulation with soluble and solid-phase fibrinogen.   Between the cytoplasmic tails of integrin IIb and 3, only 3 contains 2 tyrosine residues. Tyrosine phosphorylaton of integrin 3 was detected only on additional stimulation with soluble fibrinogen on the cells on fibrinogen plate by the use of SDS-containing lysis buffer (Fig 6). View larger version (31K): [in this window] [in a new window]   Fig 6. Integrin 3 is tyrosine-phosphorylated after the costimulation with soluble and solid-phase fibrinogen. D3-detached cells (0), the cells on fibrinogen plate incubated for 30 minutes (30), and the cells on fibrinogen plate after the additional stimulation with 1 mg/mL soluble fibrinogen for 5 minutes (30+Soluble Fg.) were lysed in 0.05% SDS-containing buffer and immunoprecipitated with anti-integrin 3 MoAb. Aliquots of each condition were equally divided and electrophoresed on the same gel in duplicate. Immunoblotting analyses were performed as described in the Materials and Methods. Results are representative of 3 experiments.     DISCUSSION Abstract Introduction Methods Results Discussion References CMK cells are known to undergo megakaryocytic differentiation after TPA treatment. During this process, cell growth is arrested, the cells become adherent to the plastic plate, and integrin IIb3 is strongly expressed. In the present study, we examined the function and the outside-in signaling pathways of the newly expressed IIb3 in relation to cell adhesion. CMK cells are reported to express several other integrins such as VLA-4,37 and these molecules might be related to cell adhesion under FCS-containing conditions. In this study, we focused on the relation of IIb3-mediated signaling with cell adhesion and came to the following conclusions: (1) binding of soluble fibrinogen to IIb3 expressed on the surface of TPA-treated CMK cells depends on cell adhesion; (2) the same ligand, fibrinogen, present either in soluble or solid-phase form, triggers differential signaling pathways mediated by IIb3; and (3) as a result of the cooperation of both soluble and solid-phase fibrinogen-mediated signaling pathways, integrin 3 is tyrosine-phosphorylated and forms a complex with Syk. In platelets, agonist-initiated signals convert IIb3 from a resting form to an activated form by increasing its affinity for ligands such as fibrinogen. In CMK cells, soluble fibrinogen binds to IIb3 on the surface of D3-adherent cells without any other stimulations but not on the surface of D3-detached cells. Because TPA is removed, its direct effect might be excluded. One possibility is that newly expressed IIb3 works as an activated form, a functional receptor for soluble fibrinogen, in the adhesion process of megakaryocytic differentiation after TPA treatment. In fact, activation-dependent anti-IIb3 antibody, PAC1, bound preferably to adherent cells. The elevated tyrosine phosphorylation observed in TPA-treated cells may lead to increased IIb3 affinity. Cell detaching resulted in decreased tyrosine phosphorylation of FAK and some other proteins concomitant with the decrease in the affinity of IIb3 for soluble fibrinogen. In contrast, D3-detached cells promptly adhered to fibrinogen plate, indicating binding of solid-phase fibrinogen. These results are consistent with the report that solid-phase but not soluble fibrinogen binds to IIb3 in the resting form in platelets.4 Outside-in signaling triggered by soluble fibrinogen is different in several respects from that of solid-phase fibrinogen, even if the same integrin is mediated. The ligation of soluble fibrinogen to D3-adherent cells resulted in reduced cell spreading and increase in tyrosine phosphorylation of Syk and several proteins, whereas that of some other proteins, including FAK, was decreased. To characterize the mechanism of Syk activation, 2 types of inhibitors, cytochalasin D, an actin polymerization inhibitor and cyclic-KGD peptide, a specific inhibitor of IIb3 were used (Fig 3E and F). Syk appeared to be directly activated and tyrosine-phosphorylated by the ligation of soluble fibrinogen, and the activation occurred at the upstream of the cytoskeletal reorganization. On the other hand, the ligation of solid-phase fibrinogen, namely adhesion to fibrinogen plate, brought about cell spreading and prompt tyrosine phosphorylation of FAK. As for Syk, the ligation of solid-phase fibrinogen caused delayed and faint tyrosine phosphorylation as compared with the result of soluble fibrinogen (Fig 4D). It was also reported that platelet adhesion to fibrinogen triggers only faint Syk tyrosine phosphorylation.38 Postbinding events such as the reorganization of cytoskeletal proteins or phosphorylation of other kinases might be needed for Syk activaton in the case of solid-phase fibrinogen. Thus, there may exist different signaling pathways mediated by IIb3, although it is engaged with the same ligand, fibrinogen, present either in soluble or solid-phase form. This hypothesis is consistent with the results of previous reports by Pelletier et al16 and Defilippi et al.17 They showed that different ligands trigger different actin organization and tyrosine phosphorylation of FAK using a panel of anti-IIb3 antibodies. In the present study, we also propose the presence of different mechanisms involving tyrosine phosphorylation of Syk triggered by soluble or solid-phase fibrinogen. The binding of soluble fibrinogen to the cells adherent to solid-phase fibrinogen gave rise to more enhanced tyrosine phosphorylation of Syk than the binding to D3-adherent cells (Fig 4D). In addition, costimulation of both forms of fibrinogen enabled us to detect coprecipitation of integrin 3 with Syk and tyrosine phosphorylation of 3, which were not detected by the stimulation with a single form of fibrinogen. These results were obtained only with SDS-containing lysis buffer and not with nonionic detergent lysis buffer. The lysis in SDS may have protected the phosphorylated 3 protein from dephosphorylation, as Law et al13 indicated. Furthermore, these molecules may be associated with cytoskeleton, but exactly how integrin 3 and Syk exist in a complex form is unknown. For 1 integrin, Miyamoto et al2 reported that both integrin clustering and occupancy accumulate a number of cytoskeletal proteins and signaling molecules, including tyrosine kinases, in adhesion complexes. We address the possibility that similar accumulation induced by adhesion to solid-phase fibrinogen mediates more effective signaling on additional stimulation with soluble fibrinogen. In platelets, Law et al13 showed that integrin 3 is tyrosine-phosphorylated after aggregation and some Src family kinases and Syk have the ability to phosphorylate 3 in vitro. Although they failed to detect an association of Syk with 3 in vivo or in vitro, Syk might be a candidate for tyrosine kinase phosphorylating 3. In this study, we suggest that 3 is tyrosine-phosphorylated as a result of outside-in signaling triggered by both soluble and solid-phase fibrinogen and that this phosphorylation leads to a stable complex containing Syk and cytoskeletal molecules. Finally, we propose that costimulation of soluble and solid-phase fibrinogen on IIb3 is related to megakaryocytic maturation and function, because this integrin is expressed exclusively in platelet-megakaryocyte lineage cells. Zauli et al7 showed the effect of thrombopoietin on IIb3-dependent adhesion of magakaryocytic cells. Handagama et al6 reported that IIb3 in mature megakaryocytes is the receptor of fibrinogen for endocytosis. In the process of megakaryocytic differentiation, cells come to express IIb3 and adhere to extracellular matrix proteins containing the RGD motif through IIb3 and other integrins. Further studies are needed to evaluate the signaling mechanisms of integrins in the process of cell adhesion and megakaryocytic differentiation.     FOOTNOTES    Submitted October 21, 1997; accepted April 16, 1998.    Supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan for General Scientific Research and Scientific Research on Priority Areas, by the Yamanouchi Foundation for Research on Metabolic Disorders, and in part by Fukui Prefectural Universities Research Foundation for the Promotion of Sciences through Contract.    Address reprint requests to Hirohei Yamamura, MD, Department of Biochemistry, Kobe University School of Medicine, Chuo-ku, Kobe 650, Japan; e-mail: yamamura{at}kobe-u.ac.jp.    The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.     ACKNOWLEDGMENT The authors thank Dr Robert M. Scarborough (COR Therapeutics Inc) for providing a cyclic peptide, Dr Ryukichi Ryo (Kobe University, Kobe, Japan) for discussions, and Yukie Tanaka for technical support.     REFERENCES Abstract Introduction Methods Results Discussion References 1. Clark EA, Brugge JS: Integrins and signal transduction pathways: The road taken. Science 268:233, 1995[Abstract/Free Full Text] 2. Miyamoto S, Teramoto H, Coso OA, Gutkind JS, Burbelo PD, Akiyama SK, Yamada KM: Integrin function: Molecular hierarchies of cytoskeletal and signaling molecules. J Cell Biol 131:791, 1995[Abstract/Free Full Text] 3. Yamada KM, Geiger B: Molecular interactions in cell adhesion complexes. Curr Opin Cell Biol 9:76, 1997[Medline] [Order article via Infotrieve] 4. Savage B, Ruggeri ZM: Selective recognition of adhesive sites in surface-bound fibrinogen by glycoprotein IIb-IIIa on nonactivated platelets. J Biol Chem 266:11227, 1991[Abstract/Free Full Text] 5. Haimovich B, Lipfert L, Brugge JS, Shattil SJ: Tyrosine phosphorylation and cytoskeletal reorganization in platelets are triggered by interacton of integrin receptors with their immobilized ligands. J Biol Chem 268:15868, 1993[Abstract/Free Full Text] 6. Handagama P, Scarborough RM, Shuman MA, Bainton DF: Endocytosis of fibrinogen into megakaryocyte and platelet -granules is mediated by IIb3 (glycoprotein IIb-IIIa). Blood 82:135, 1993[Abstract/Free Full Text] 7. Zauli G, Bassini A, Vitale M, Gibellini D, Celeghini C, Caramelli E, Pierpaoli S, Guidotti L, Capitani S: Thrombopoietin enhances the IIb3-dependent adhesion of megakaryocytic cells to fibrinogen or fibronectin through PI 3 kinase. Blood 89:883, 1997[Abstract/Free Full Text] 8. Ylänne J, Huuskonen J, O'Toole TE, Ginsberg MH, Virtanen I, Gahmberg CG: Mutation of the cytoplasmic domain of the integrin 3 subunit. J Biol Chem 270:9550, 1995[Abstract/Free Full Text] 9. Leong L, Hughes PE, Schwarts MA, Ginsberg MH, Shattil SJ: Integrin signaling: Roles for the cytoplasmic tails of IIb3 in the tyrosine phosphorylation of pp125FAK. J Cell Sci 108:3817, 1995[Abstract] 10. Loh E, Qi W, Vilaire G, Bennett JS: Effects of cytoplasmic domain mutations on the agonist-stimulated ligand binding activity of the platelet integrin IIb3. J Biol Chem 271:30233, 1996[Abstract/Free Full Text] 11. Boudignon-Proudhon C, Patel PM, Parise LV: Phorbol ester enhances integrin IIb3-dependent adhesion of human erythroleukemic cells to activation-dependent monoclonal antibodies. Blood 87:968, 1996[Abstract/Free Full Text] 12. Loh E, Beaverson K, Vilaire G, Qi W, Motimer P, Bennett JS: Agonist-stimulated ligand binding by the platelet integrin IIb3 in a lymphocyte expression system. J Biol Chem 270:18631, 1995[Abstract/Free Full Text] 13. Law DA, Nannizi-Alaimo L, Phillips DR: Outside-in integrin signal transduction. J Biol Chem 271:10811, 1996[Abstract/Free Full Text] 14. Shattil SJ, Haimovich B, Cunningham M, Lipfert L, Parsons JT, Ginsberg MH, Brugge JS: Tyrosine phosphorylation of pp125FAK in platelets requires coordinated signaling through integrin and agonist receptors. J Biol Chem 269:14738, 1994[Abstract/Free Full Text] 15. Huang MM, Lipfert L, Cunningham M, Brugge JS, Ginsberg MH, Shattil SJ: Adhesive ligand binding to integrin IIb3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK. J Cell Biol 122:473, 1993[Abstract/Free Full Text] 16. Pelletier AJ, Kunicki T, Ruggirti ZM, Quaranta V: The activation state of the integrin IIb3 affects outside-in signals leading to cell spreading and focal adhesion kinase phosophorylation. J Biol Chem 270:18133, 1995[Abstract/Free Full Text] 17. Defilippi P, Venturino M, Gulino D, Duperray A, Boquet P, Fiorentini C, Volpe G, Palmieri M, Silengo L, Tarone G: Dissection of pathways implicated in integrin-mediated actin cytoskeleton assembly. J Biol Chem 272:21726, 1997[Abstract/Free Full Text] 18. Taniguchi T, Kobayashi T, Kondo J, Takahashi K, Nakamura H, Suzuki J, Nagai K, Yamada T, Nakamura S, Yamamura H: Molecular cloning of a porcine gene syk that encodes a 72-kDa protein-tyrosine kinase showing high susceptibitily to proteolysis. J Biol Chem 266:15790, 1991[Abstract/Free Full Text] 19. Yanagi S, Kurosaki T, Yamamura H: The structure and function of nonreceptor tyrosine kinase p72syk expressed in hematopoietic cells. Cell Signal 7:185, 1995[Medline] [Order article via Infotrieve] 20. Agarwai A, Salem P, Robbins KC: Involvement of p72syk, a protein-tyrosine kinase, in Fc receptor signaling. J Biol Chem 268:15900, 1993[Abstract/Free Full Text] 21. Hirasawa N, Scharenberg A, Yamamura H, Beaven MA, Kinet JP: A requirement for Syk in the activation of the microtubule-associated protein kinase/phospholipase A2 pathway by FcR1 is not shared by a G protein-coupled receptor. J Biol Chem 270:10960, 1995[Abstract/Free Full Text] 22. Kurosaki T, Johnson SA, Pao L, Sada K, Yamamura H, Cambier JC: Role of the Syk autophosphorylation site and SH2 domains in B cell antigen receptor signaling. J Exp Med 182:1815, 1995[Abstract/Free Full Text] 23. Taniguchi T, Kitagawa H, Yasue T, Yanagi S, Sakai K, Asahi M, Ohta S, Takeuchi F, Nakamura S, Yamamura H: Protein-tyrosine kinase p72sykis activated by thrombin and is negatively regulated through Ca2+ mobilization in platelets. J Biol Chem 268:2277, 1993[Abstract/Free Full Text] 24. Fujii C, Yanagi S, Sada K, Nagai K, Taniguchi T, Yamamura H: Involvement of protein-tyrosine kinase p72syk in collagen-induced signal transduction in platelets. Eur J Biochem 226:243, 1994[Medline] [Order article via Infotrieve] 25. Ichinohe T, Takayama H, Ezumi Y, Yanagi S, Yamamura H, Okuma M: Collagen-stimulated activation of Syk but not c-src is severely comprised in human platelets lacking membrane glycoproteinVI. J Biol Chem 270:28029, 1995[Abstract/Free Full Text] 26. Tohyama Y, Yanagi S, Sada K, Yamamura H: Translocation of p72syk to the cytoskeleton in thrombin-stimulated platelets. J Biol Chem 269:32796, 1994[Abstract/Free Full Text] 27. Clark EA, Shattil SJ, Ginsberg MH, Bolen J, Brugge JS: Regulation of the protein tyrosine kinase p72syk by platelet agonists and the integrin IIb3. J Biol Chem 269:28859, 1994[Abstract/Free Full Text] 28. Gao J, Zoller KE, Ginsberg MH, Brugge JS, Shattil SJ: Regulation of the pp72syk proteintyrosine kinase by platelet integrin IIb3. EMBO J 16:6414, 1997[Medline] [Order article via Infotrieve] 29. Lin TH, Rosales C, Mondal K, Bolen JB, Haskill S, Juliano RL: Integrin-mediated tyrosine phosphorylation and cytokine message induction in monocytic cells. J Biol Chem 270:16189, 1995[Abstract/Free Full Text] 30. Yan SR, Huang M, Berton G: Signaling by adhesion in human neutrophilesActivation of the p72syk tyrosine kinase and formation of protein complexes containing p72syk and Src family kinases in neutrophils spreading over fibrinogen. J Immunol 158:1902, 1997[Abstract] 31. Sato T, Fuse A, Eguchi M, Hayashi Y, Sugita K, Nakazawa S, Minato K, Shima Y, Komori I, Sunami S, Okimoto Y, Nakajima H: Establishment and characterization of a megakaryoblastic cell line (CMK) from a Down's syndrome patient with acute megakaryoblastic leukemia. Exp Haematol 15:495, 1987 32. McGregor JL, Brochier J, Wild F, Follea G, Trzeciak M, James E, Dechavanne M, Mcgregor L, Clemeson KJ: Monoclonal antibodies against platelet membrane glycoproteins. Eur J Biochem 131:427, 1983[Medline] [Order article via Infotrieve] 33. Shattil SJ, Cunningham M, Hoxie JA: Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood 70:307, 1987[Abstract/Free Full Text] 34. Scarborough RM, Naughton MA, Teng W, Rose JW, Philips DR, Nannizzi L, Arfsten A, Campbell AM, Charo IF: Design of potent and specific integrin antagonists. Peptide antagonists with high specificity for glycoprotein IIb-IIIa. J Biol Chem 268:1066, 1993[Abstract/Free Full Text] 35. Casella JF, Flanagan MD, Lin S: Cytochalasin D inhibits actin polymerization and induces depolymerization of actin filaments formed during platelet shape change. Nature 293:302, 1981[Medline] [Order article via Infotrieve] 36. Komatsu N, Suda T, Moroi M, Tokuyama N, Sakaya Y, Okada M, Nishida T, Hirai Y, Sato T, Fuse A, Miura Y: Growth and differentiation of a human megakaryoblastic cell line, CMK. Blood 74:42, 1989[Abstract/Free Full Text] 37. Avraham H, Cowley S, Chi SY, Jiang S, Groopman JE: Characterization of adhesive interactions between human endothelial cells and megakaryocytes. J Clin Invest 91:2378, 1993 38. Haimovich B, Regan C, DiFazio L, Ginalis E, Ji P, Purohit U, Rowley RB, Bolen J, Greco R: The FcRII receptor triggers pp125FAK phosphorylation in platelets. J Biol Chem 271:16332, 1996[Abstract/Free Full Text] © 1998 by the American Society of Hematology.   0006-4971/98/92-0022$3.00/0 CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: Y. Tohyama and H. Yamamura Protein Tyrosine Kinase, Syk: A Key Player in Phagocytic Cells J. Biochem., March 1, 2009; 145(3): 267 - 273. [Abstract] [Full Text] [PDF] Y. Shi, Y. Tohyama, T. Kadono, J. He, S. M. Shahjahan Miah, R. Hazama, C. Tanaka, K. Tohyama, and H. 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