Role of the Src Homology 2 Domain-containing Protein Shbin Murine
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Vol. 13, 141–148, March 2002 Cell Growth & Differentiation 141 Role of the Src Homology 2 Domain-containing Protein Shb in Murine Brain Endothelial Cell Proliferation and Differentiation1 Lingge Lu, Kristina Holmqvist, Michael Cross, and which involves proliferation, migration and differentiation of Michael Welsh2 endothelial cells (2, 3). Departments of Medical Cell Biology [L. L., K. H., M. W.], and of Shb is an adaptor protein with proline-rich motifs in its NH2 Genetics and Pathology, Rudbeck Laboratory [M. C.], Uppsala terminus, a central phospho-tyrosine binding domain, and a University, 751 23, Uppsala, Sweden COOH-terminal SH2 domain (4). It has previously been shown that Shb interacts with the PDGF -receptor, FGF Abstract receptor-1, and T-cell receptor via its SH2 domain and par- To study the role of the Src homology 2 (SH2) domain- ticipates in tyrosine kinase-dependent signaling through the containing protein Shb in angiogenesis, wild-type Shb formation of multiprotein complexes (5, 6). Overexpression of and SH2 domain-mutated Shb (R522K Shb) were Shb induces increased apoptosis on serum withdrawal in overexpressed in murine immortalized brain endothelial NIH3T3 cells (7) and growth factor-stimulated differentiation cells. The wild-type Shb cells exhibited an increased in PC12 cells (8). Stimulation with FGF-2 increases Shb ty- rate of apoptosis on serum withdrawal. Both wild-type rosine phosphorylation in bovine adrenal cortex capillary Shb and R522K Shb cells exhibited enhanced endothelial cells (9) and murine IBE cells (10). To further spreading concomitant with cytoskeletal address the role of Shb in angiogenesis, wild-type Shb and rearrangements that occurred independently of SH2 domain-mutated Shb (R522K Shb) were overexpressed fibroblast growth factor (FGF)-2 stimulation. However, in IBE cells. We describe a role for Shb in IBE cell proliferation these effects may partly be caused by altered and differentiation. regulation of Rac1 and Rap1 activation in the Shb cells. The Shb-induced cytoskeletal rearrangements were not Results dependent on phosphatidylinositol 3 kinase activity, Proliferation and Apoptosis of IBE Cells Overexpressing but could be reversed by inhibition of Src family Shb. Overexpression of Shb in the IBE cell clones was con- kinases. FGF-2 failed to further enhance migration of firmed by Western blot analysis (Fig. 1A). The clones dis- wild-type Shb and R522K Shb cells. The R522K Shb played a 2- to 4-fold increase in the Shb content compared cells cultured in collagen gels exhibit diminished with the vector control clones. Probing the same blot for Shc, tubular morphogenesis when treated with FGF-2, Grb2, and ERK revealed no differences in the content of implicating the need for a functional Shb molecule in these proteins. When the cells were maintained in 15% se- this process. These data suggest that Shb plays a role rum at 33°C, no significant differences of the proliferation in the proliferation and differentiation of endothelial rates were observed among the wild-type Shb, R522K Shb, cells and, hence, participates in angiogenesis. and the control IBE cells (Fig. 1B), although the wild-type Shb cells tended to proliferate at a lower rate than the control Introduction cells. The data in Fig. 1B are based on the results obtained Angiogenesis is the process in which new blood capillaries from using two independent clones in each group. The wild- are formed from preexisting vessels, and it occurs in both type Shb cells displayed a significantly lower rate of DNA physiological and pathological situations such as reproduc- synthesis compared with the control cells in the presence of tion, wound healing, rheumatoid arthritis, diabetic retinopa- serum (Fig. 1C). Shb has previously been shown to induce thy, and tumor development (1). Growth factors such as apoptosis in NIH3T3 cells and RINm5F cells when main- FGF-23 and VEGF are major regulators of angiogenesis, tained in low serum (7, 11). We thus, examined the rate of apoptosis in Shb-overexpressing IBE cells after 3 days of serum deprivation by use of flow cytometry after labeling the cells with Annexin-V. As shown in Fig. 1D, wild-type Shb IBE Received 5/29/01; revised 2/6/02; accepted 2/13/02. cells displayed a rate of 34.0 5.5% apoptotic cells, which The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked was significantly higher than the corresponding values in advertisement in accordance with 18 U.S.C. Section 1734 solely to indi- R522K Shb cells (13.4 2.4%) and the control IBE cells cate this fact. (18.6 3.0%). Thus, Shb overexpression also causes ele- 1 Supported by grants from the Juvenile Diabetes Foundation Interna- tional, the Swedish Medical Research Council (31X-10822), the Swedish vated apoptosis in IBE cells when they are deprived of se- Diabetes Association, the Novo-Nordisk Foundation, and the Family Ern- rum. No differences in the rate of apoptosis between the fors Fund. 2 To whom requests for reprints should be addressed, at Department of Medical Cell Biology, Box 571, biomedicum, 751 23, Uppsala, Sweden. Phone: 46-18-4714447; Fax: 46-18-556401; E-mail: michael.welsh@ medcellbiol.uu.se. FBS, fetal bovine serum; IBE, immortalized brain endothelial (cell); PI-3, 3 The abbreviations used are: FGF, fibroblast growth factor; EGF, epider- phosphatidylinositol 3 ; PKB, protein kinase B; FAK, focal adhesion ki- mal growth factor; PDGF, platelet-derived growth factor; VEGF, vascular nase; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d ]pyrimi- EGF; SH2, Src homology 2; ERK, extracellular signal-regulated kinase; dine. 142 Shb and Endothelial Cell Function Fig. 1. Expression levels, proliferation, DNA synthesis, and apoptosis of IBE cells overexpressing Shb. A, overexpression of wild-type and R522K Shb in IBE cells. IBE cells transfected with wild-type or R522K Shb or with empty vector were subjected to Western blot analysis. The blots were probed with anti-Shb antibody, stripped, and reprobed with anti-Grb2, anti-Shc, and anti-ERK. B, IBE cells overexpressing wild-type Shb or R522K Shb were cultured in Ham’s F-12, 15% FBS, and 20 units/ml IFN- at 33°C. Cell numbers were scored on 5 consecutive days. Means SE are given for two observations and are based on two separate clones in each group. C, IBE cells overexpressing both wild-type Shb and R522K Shb were cultured in 15% serum. After 18 h, the cells were labeled with 1 Ci/ml [3H]thymidine for 4 h. Incorporated [3H]thymidine was quantified by scintillation counting. The DNA contents of the samples were measured. , P 0.05 when compared with control. Means SE for six to nine observations are given and are based on three separate clones in each group. D, apoptosis in Shb overexpressing IBE cells. IBE cells overexpressing both wild-type Shb and R522K Shb were starved in serum-free medium, labeled with Annexin-V, and subsequently analyzed by flow cytometry. , P 0.05 compared with the control. Means SE for nine observation are given for three different clones in each group. kDa, Mr in thousands. groups were observed when cultured in the presence of 15% Overexpression of Shb Induces Cell Spreading on Gel- serum (data not shown). atin. Overexpression of wild-type Shb in IBE cells seeded Activation of PKB/Akt. The serine/threonine protein ki- on gelatin lead to an increased cell spreading compared with nase PKB/Akt is known to transmit survival signals, and control cells (Fig. 3). IBE cells expressing R522K Shb also activation of this kinase is paralleled by its phosphorylation showed an increased spreading compared with control cells, (12, 13). To address its putative role for the apoptotic re- although not to the same extent as the wild-type Shb cells. sponse of the wild-type Shb IBE cells, the different cells were IBE Cells Overexpressing Shb Exhibit Cytoskeletal Re- stimulated with FGF-2 for various times, and the activation of arrangements. Growth factors such as PDGF, insulin, and PKB/Akt was then examined. FGF-2 induced activation of EGF have been shown to induce lamellipodia or membrane PKB/Akt to a similar degree in all of the cells at all of the time ruffles in Swiss 3T3 fibroblasts (14). FGF-2 stimulation leads points (Fig. 2). The basal level of PKB/Akt phosphorylation to a slight increase in stress fiber formation rather than for- also appeared to be similar in the different clones, which mation of lamellipodia in the control IBE cells (Fig. 4). The suggests that altered PKB/Akt activity is not responsible for wild-type Shb cells exhibited irregular, densely stained the increased apoptosis of the Shb IBE cells. patches, which were increased when treated with FGF-2. Cell Growth & Differentiation 143 Fig. 2. Activation of PKB/Akt. A, IBE cells were starved as above. After being stimulated with 10 ng/ml FGF-2 for the indicated times, the cells were lysed in SDS sample buffer and subjected to Western blot analysis. The blots were probed with anti-phospho-Akt or anti-total-Akt antibodies. B. relative activation of Akt was quantified by relating densitometric values of phospho-Akt with those of total-Akt. The means SE from two to four independent experiments are given. Fig. 3. Overexpression of Shb induces cell spreading on gelatin. IBE cells were seeded and maintained in Ham’s F-12 supplemented with 15% FBS on gelatin-coated dishes for 2 h. The photographs were taken using The R522K Shb cells displayed the same structure but to a a Nikon TMS microscope. The data are representative for three different lesser extent. These patches were similar to the giant ruffles clones in each group. (irregular multilayered actin structure) that appeared on the dorsal side of PDGF-stimulated PAE cells expressing Y778F mutant PDGF -receptor (15). PDGF induces Rac1 activation and ruffle formation, and this process is inhibited by the PI-3 wild-type Shb cells. Thus, FGF-2 causes both PI-3 kinase/ kinase inhibitor LY294002 (16, 17). To assess a role of PI-3 Rac1 and Src-dependent cytoskeletal changes in control IBE kinase-dependent Rac1 activation in the Shb-dependent cells, whereas Shb induces an altered staining pattern that is changes, cells were pretreated by LY294002 before FGF-2 PI-3-kinase-independent but requires Src activation. addition. In the control cells, FGF-2 failed to cause stress FGF-2 Failed to Promote Chemotaxis in Shb-overex- fiber formation. However, the patchy staining pattern of the pressing IBE Cells. To assess the role of Shb on chemo- wild-type Shb cells remained, which suggests that this is taxis, IBE cells overexpressing wild-type Shb and R522K independent of PI-3 kinase activity. The R522K Shb cells Shb, together with control IBE cells, were examined using displayed a somewhat reduced content of stress fibers. Al- Boyden chamber analysis in the presence or absence of ternatively, the cytoskeletal changes could reflect Src acti- FGF-2. As shown in Fig. 5, FGF-2 could induce distinct vation (18). To address this possibility, cells were treated with migration of control IBE cells as observed previously (21). the Src-family kinase inhibitor PP2 (19, 20). PP2 reverted all FGF-2 stimulation failed to further increase the migration in of the FGF-2 induced changes in the control cells but also both wild-type Shb and R522KShb cells, mainly because of prevented the Shb-dependent patchy staining pattern in the elevated basal cell migration. 144 Shb and Endothelial Cell Function Fig. 4. IBE cells overexpressing Shb ex- hibit FGF-2 independent cytoskeletal rear- rangement. IBE cells on fibronectin-coated culture slides were kept in Ham’s F-12 con- taining 0.1% BSA for 4 h and treated with or without 100 ng/ml FGF-2 for 5 min. To in- hibit PI-3 kinase or Src-family kinase, cells were pretreated with 30 M LY204002 or 2 M PP2 for 30 min before the addition of FGF-2. The cells were fixed in paraformal- dehyde and stained with rhodamine phal- loidin. The slides were examined using a fluorescence microscope. IBE Cells Overexpressing Wild-Type Shb Form Tubular Structures in Response to FGF-2 in the Tube Formation Assay. Shb, with a functional SH2 domain, is required for proper tubular morphogenesis, because IBE cells expressing R522K Shb, form disorganized tubes. Of the four clones repeatedly analyzed, only one gave structures reminiscent of tubes but to a lesser degree than in the control cells (Fig. 6A). These tubular structures were less regular and frequent than those of the control cells. The formation of tubular structures was prominent in wild-type Shb IBE cells (Fig. 6A) and could be detected already after 4 h (Fig. 6B). Rac1 and Rap1 Are Differentially Regulated in Shb- overexpressing IBE Cells. The Rac1-small GTPase func- tions in multiple cellular processes, including lamellipodia formation and membrane ruffling, gene transcription, cell cycle progression, and cell adhesion (22, 23). We assessed the activation of Rac1 in Shb-overexpressing IBE cells. In three independent experiments, FGF-2 stimulation for 2 min slightly activated Rac1 in control IBE cells but not in wild- Fig. 5. FGF-2 fails to promote chemotaxis in Shb overexpressing IBE type Shb cells. FGF-2 induced Rac1 activation in R522K Shb cells. The IBE cells in Ham’s F-12 containing 0.25% BSA were seeded on cells to a degree similar to that in the control cells (Fig. 7A). collagen-coated filter in upper (Boyden) chambers. The lower chambers were filled with Ham’s F-12 containing 0.25% BSA with or without 10 This transient activation was not observed when the cells ng/ml FGF-2. After incubation at 33°C for 4 h, the filters were fixed in were treated with FGF-2 for 5 min (results not shown). Similar ethanol, washed, and stained with Giemsa solution. The cells on the upper results have previously been shown in PDGF-stimulated PhB surface of the filters were wiped away, and the number of the cells on the lower surface of the filters were counted under a microscope. The number fibroblasts overexpressing wild-type Shb (24). We have de- of untreated control cells was set to 100%. Data are shown as mean SE scribed the role of Shb in Rap1 signaling in PC12 cells. Nerve for five independent experiments. , P 0.05. Cell Growth & Differentiation 145 Fig. 6. Functional Shb is required for proper FGF-2-induced tubular morphogenesis. IBE cells were seeded in a collagen matrix for 48 h (A) or 4 h (B) in the absence (untreated) or presence of 5 ng/ml FGF-2. Tube formation was analyzed by a Nikon Eclipse microscope and photographed using a digital camera. The figure is representative for three different clones in each group. growth factor-stimulated-Rap1 activation was observed only Alternatively, IBE cell cycle progression could involve the in PC12 cells overexpressing wild-type Shb (25). In the pres- small GTPase Rac1, which regulates G1 progression through ent study, on FGF-2 stimulation for 5 min, Rap1 was acti- activation of cyclin D1 (30, 31). We observed that overex- vated in both wild-type Shb and R522K Shb IBE cells partly pression of wild-type Shb inhibited growth factor-induced because of decreased basal Rap1 activity, but not in control Rac1 activation in IBE cells and PhB fibroblasts consistent IBE cells (Fig. 7B). These data suggest that Shb may also with the decreased rates of DNA synthesis of the wild-type function in Rap1 signaling in IBE cells. Shb cells. Rac1 is activated in a PI-3 kinase-dependent manner by PDGF (16, 32, 33). Such an activation could be Discussion achieved by direct signaling either from the receptor tyrosine To obtain an understanding of the role of the adaptor protein kinase to PI-3 kinase (34) or via Ras to PI-3 kinase (35). Shb for angiogenesis, IBE cells were transfected with wild- However, FGF-2 stimulation only induces lower activation of type and R522K Shb. The wild-type Shb cells exhibited a PI-3 kinase, and this is thought to occur mainly via Ras (36, higher apoptotic rate under serum deprivation as previously 37). Eps8 mediates the transfer of signals between Ras/PI-3 observed in Shb-overexpressing NIH3T3 cells and RINm5F kinase and Rac by forming a complex with E3b1 and Sos-1 cells (7, 11). This occurred in parallel with a decrease in the (38). It is possible that overexpression of wild-type Shb dis- percentage of cells in the S phase of the cell cycle (results not rupts Rac1 signaling, in part through down-regulation of shown), suggesting that the increase in apoptosis might Eps8, because Eps8 binds Shb via its Src homology 3 do- result from a G1 block of the cell cycle. PKB/Akt is a potential main (5) and Shb may influence the expression level of Eps8 regulator of cell survival and cell cycle progression (26). (39). Shb could also affect Rac1 signaling by regulating Stimulation with FGF-2 induces rapid and transient activa- Rac1-guanine nucleotide exchange factor or Rac1-GTPase- tion of PKB/Akt (12, 13). However, stimulation of PKB/Akt activating protein. Besides mediating effects on the cell cy- was similar in all groups of cells when treated with FGF-2. In cle, the down-regulated Rac1 signaling may have conse- addition, activation of ERK, another putative regulator of quences for the IBE cell cytoskeleton. Shb overexpression apoptosis (27–29), was not affected by Shb overexpression.4 caused a dramatically altered cytoskeletal staining pattern. This was not PI-3 kinase/Rac1 dependent but was reverted by addition of the Src-family inhibitor PP2. Thus, it seems 4 M. J. Cross, L. Lu, P. Magnusson, D. Nyqvist, K. Holmqvist, M. Welsh, that Shb overexpression augments the Src signaling that and L. Claesson-Welsh. The Shb adaptor protein binds to tyrosine 766 in the FGFR-1 and regulates the Ras/MEK/MAPK pathway via FRS2 phos- causes the cytoskeletal changes observed. Indeed, Shb has phorylation in endothelial cells. Submitted for publication. previously been found to associate with Src (5). 146 Shb and Endothelial Cell Function linked as previously noted in the PC12 cells (25, 46) and may, in concert, cause the alterations in the spreading and migration observed. The wild-type Shb cells rapidly formed well-organized tubular structures, compared with the control cells, when cultured in collagen gels in the presence of FGF-2. Because the SH2 domain was required for appropriate tube formation, it seems that FGF-2-dependent tube formation does not correlate with Rac1, Rap1, and FAK activation, nor does it correlate with spreading and migration. However, the addition of a Src-family kinase inhibitor has previously been found to prevent IBE cell tube formation (47). Thus, if Shb augments Src-family kinase signaling, this could have consequences for tubular morpho- genesis. To what extent the observed Shb-dependent cytoskel- etal alterations play a role in IBE cell differentiation remains to be established. It has also been shown that a Mr 125,000 protein binds to the SH2 domain of Shb in IBE cells (10). Stimulation with FGF-2 increased the binding as well as tyro- sine phosphorylation and in vitro kinase activity of this protein. This unknown protein may play a role for Shb-dependent tube formation in IBE cells. Fig. 7. Activation of Rac1 and Rap1 in IBE cells. IBE cells in 10-cm dishes were treated with or without 100 ng/ml FGF-2. In A, for Rac1 assay, Taken together, although Shb induces apoptosis on serum the cells were lysed and incubated with the GST-PAK-CD fusion protein. withdrawal and decreases DNA synthesis, it promotes the dif- In B, for Rap1 assay, the cell lysates were incubated with the RalGDS- ferentiation of IBE cells in the presence of FGF-2 and serum. RBD fusion protein. The bound active Rac1GTP or Rap1GTP was deter- mined by Western blot analysis. Results represent three independent experiments. Units are relative densitometric absorbance when related to lysates. The means SE are given. Materials and Methods Cell Culture and Retrovirus-mediated Overexpression. IBE cells were cultured on gelatin (Sigma Chemical Co.)- Overexpression of Shb in IBE cells also induces FGF-2- coated dishes in Ham’s F-12 (Life Technology), 15% FBS stimulated Rap1 activation independently of the Shb SH2 (Hyclone), and 20 units/ml IFN- (Peprotech, Inc.) at 33°C. domain, unlike in nerve growth factor-stimulated PC12 cells in The wild-type Shb cDNA and R522K Shb mutant (with an which the effect is Shb SH2-domain dependent (25). This sug- inactivation of the SH2 domain) cDNA were inserted into the gests that Shb may use different mechanisms to regulate Rap1 retroviral vector pBABE Puro and transfected into Bosc 23 activation in different cell types. Rap1 has been shown to be- cells by the LipofectAMINE method. Control transfection come activated during cell adhesion, and the Rap1 GTPase- with vector alone was also performed. After 48 h, viral su- activating protein SPA-1 negatively regulates cell adhesion (40, pernatant was collected and virus was added to IBE cells in 41). This is further supported by the fact that Rap1 is involved the presence of Polybrene (4 g/ml). Positive clones were in integrin-mediated cell adhesion (42– 44). Thus, Rap1 activa- selected for puromycin resistance at 48 h after infection. tion could be a significant component in the increased spread- Overexpression of wild-type Shb and R522K Shb was veri- ing in the Shb overexpressing IBE cells. The enhanced migra- fied by Western blot analysis (Fig. 1A). tion in the Shb-overexpressing IBE cells is independent of Cell Proliferation and DNA Synthesis Assay. For the FGF-2 stimulation and Shb SH2 domain. It cannot be attributed proliferation assay, IBE cells overexpressing wild-type Shb, only to the role of Rac1, because the Shb SH2 domain mutation R522K Shb mutant, and control IBE cells (5 104) were seeded did not influence FGF-2-induced Rac1 activation, although in gelatin-coated 24-well plates in Ham’s F-12 supplemented these cells showed a rate of migration similar to that of the wild- with 15% FBS and 20 units/ml IFN- . Cell numbers were type Shb cells. Activated Rap1 also affects the cytoskeleton scored for 4 consecutive days. For DNA synthesis assay, 1.5 through binding to AF-6, which associates with the actin cyto- 104 cells were seeded in 24-well plates coated with 0.1% skeletal regulator profilin (45). Thus, Rap1 may also play a role gelatin and 10 g/ml human fibronectin. After growth in Ham’s in regulating the Shb-induced alterations of IBE cell migration. F-12, 15% FBS, and 20 units/ml IFN- at 33°C for 48 h, the cells FAK is another potential regulator of cell spreading and attach- were starved with Ham’s F-12 and 0.2% BSA for another 48 h. ment. We have, in a separate study,5 observed that Shb over- FGF-2 (5 ng/ml; Roche) was added for 18 h. The cells were expression causes elevated levels of FAK and increased FAK labeled with 1 Ci/ml [3H]thymidine for 4 h, then washed and activity. Indeed, activation of Shb, FAK, and Rap1 may all be sonicated, followed by trichloroacetic acid precipitation before scintillation counting. The DNA contents of the samples were measured in parallel using a fluorometric assay. For all of the 5 K. Holmqvist, M. J. Cross, D. Riley, and M. Welsh. The Shb adaptor assays, two to five different clones from each group were protein causes Src-dependent cell spreading and activation of focal ad- hesion kinase in immortalized brain endothelial cells. Submitted for pub- simultaneously analyzed, and the results were from three inde- lication. pendent experiments. Cell Growth & Differentiation 147 Detection of Apoptosis. Cells (2 104) were seeded on coated tissue culture flasks in Ham’s F-12 supplemented with gelatin-coated 6-cm dishes in Ham’s F-12 supplemented 15% heat-inactivated FBS for 24 h, before the cells were with 15% FBS and 20 units/ml IFN- overnight and starved washed in PBS, and Ham’s F-12 containing 2% FBS was in Ham’s F-12 containing 0.1% BSA for 48 h. Apoptosis was added for 24 h. 24-well plates were coated with a collagen detected by using Annexin-V-Fluos kit (Roche). Cells were solution consisting of 10 Ham’s F-12, collagen Type 1 (Vitro- gently trypsinized and resuspended in Annexin-V-Fluores- gen 100; Collagen Corporation) and 0.1 M NaOH in a ratio of cein and propidium iodide, incubated for 10 min at room 1:8:1, and incubated at 37°C for 1 h, in order for the collagen to temperature, and subsequently analyzed by flow cytometry form a gel. Ham’s F-12 medium was added to prevent cracking with 488 nm excitation and collecting light scatter, green and of the gel. The medium was aspirated before seeding the cells. red fluorescence. Apoptotic cells were defined as cells with Cells (1.5 105/well) in Ham’s F-12 and 0.2% BSA were enhanced Annexin-V fluorescence simultaneously exhibiting seeded in collagen gel-coated 24-well plates and incubated at normal propidium iodide staining. 33°C for 3 h. The cell culture medium was aspirated and a top Determination of PKB/Akt. Cells in 24-well plates were layer of collagen solution was added. The collagen was jellied seeded as described above and treated with FGF-2 (10 ng/ml) for 1 h at 37°C before adding Ham’s F-12 with or without FGF-2 for the indicated times. The cells were lysed with SDS sample at a final concentration of 5 ng/ml. The cells were then incu- buffer containing 2 mM PMSF and were sonicated. After boiling bated at 33°C for 48 h. The cells were fixed in 2.5% glutaral- for 5 min, the samples were subjected to SDS-PAGE. For dehyde/PBS and stored at 4°C. Tube formation was analyzed Western blot analysis, proteins were electrically transferred by a Nikon Eclipse TE 300 Microscope and photographed using onto Immobilon filter (Millipore). The filters were blocked with a SPOT 2 digital camera (Diagnostic Instruments). 5% nonfat milk in PBS, 0.1% Tween 20 and incubated with Determination of Rac1 and Rap1 Activation. Activation either anti-phospho-Akt or anti-total-Akt antibodies (New Eng- of Rac1 and Rap1 were determined as described previously land Biolabs). Immunoreactivity was detected using horserad- (24, 25). Subconfluent cells in 10-cm dishes were maintained ish peroxidase-conjugated secondary antibody and enhanced in Ham’s F-12 containing 0.1% BSA overnight. For the Rac1 chemiluminescence (Amersham Pharmacia Biotech) according assay, cells were stimulated with or without 100 ng/ml FGF-2 to the manufacturer’s instructions. for 2 min. The cells were lysed and incubated with the GST- Cell Spreading and Actin Rearrangement. Cells (3 PAK-CD fusion protein precoupled to glutathione-Sepharose 106) were seeded on gelatin-coated dishes in Ham’s F-12, beads. For the Rap1 assay, cells were stimulated with or supplemented with 15% FBS at 33°C for 2 h. Photographs without 100 ng/ml FGF-2 for 5 min. The cells were lysed and were taken using a Nikon TMS microscope connected to a incubated with the RalGDS-RBD fusion protein precoupled SPOT 2 digital camera (Diagnostic Instruments). For actin re- to glutathione-Sepharose beads. The bound active Rac1GTP arrangement, cells cultured on fibronectin-coated culture slides or Rap1GTP was determined by Western blot analysis. (Becton Dickinson) were kept in Ham’s F-12 containing 0.1% BSA for 4 h and then stimulated with or without 100 ng/ml References FGF-2 for 5 min. To inhibit PI-3 kinase or Src-family kinase, cells 1. Folkman, J. Angiogenesis in cancer, vascular, rheumatoid and other were pretreated with 30 M LY204002 (Sigma Chemical Co.) or diseases. Nat. Med., 1: 27–31, 1995. 2 M PP2 (Calbiochem) for 30 min before the addition of FGF-2. 2. Folkman, J., and Klagsbrun, M. Angiogenic factors. Science (Wash. The cells were fixed in 3% paraformaldehyde and permea- DC), 235: 442– 447, 1987. bilized with acetone. After blocking with 10% FBS, the cells 3. Cross, M. J., and Claesson-Welsh, L. FGF and VEGF function in were incubated with rhodamine phalloidin (Molecular Probes) angiogenesis: signalling pathways, biological responses and therapeutic inhibition. 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