Gi-mediated Cas Tyrosine Phosphorylation in Vascular Endothelial Cells Stimulated with Sphingosine 1-Phosphate

Since blood platelets release sphingosine 1-phosphate (Sph-1-P) upon activation, it is important to examine the effects of this bioactive lipid on vascular endothelial cell functions from the viewpoint of platelet-endothelial cell interactions. In the present study, we examined Sph-1-P-stimulated signaling pathways related to human umbilical vein endothelial cell (HUVEC) motility, with a special emphasis on the cytoskeletal docking protein Crk-associated substrate (Cas). Sph-1-P stimulated tyrosine phosphorylation of Cas, which was inhibited by the Giinactivator pertussis toxin but not by the Rho inactivator C3 exoenzyme or the Rho kinase inhibitor Y-27632. Fyn constitutively associated with and phosphorylated Cas, suggesting that Cas tyrosine phosphorylation may be catalyzed by Fyn. Furthermore, upon HUVEC stimulation with Sph-1-P, Crk, through its SH2 domain, interacted with tyrosine-phosphorylated Cas, and the Cas-Crk complex translocated to the cell periphery (membrane ruffles), through mediation of Gi (Fyn) but not Rho. In contrast, tyrosine phosphorylation of focal adhesion kinase, and formation of stress fibers and focal adhesion were mediated by Rho but not Gi(Fyn). Finally, Sph-1-P-enhanced HUVEC motility, assessed by a phagokinetic assay using gold sol-coated plates and a Boyden's chamber assay, was markedly inhibited not only by pertussis toxin (or the Fyn kinase inhibitor PP2) but also by C3 exoenzyme (or Y-27632). In HUVECs stimulated with Sph-1-P, these data suggest the following: (i) cytoskeletal signalings may be separable into Gi-mediated signaling pathways (involving Cas) and Rho-mediated ones (involving FAK), and (ii) coordinated signalings from both pathways are required for Sph-1-P-enhanced HUVEC motility. Since HUVECs reportedly express the Sph-1-P receptors EDG-1 (coupled with Gi) and EDG-3 (coupled with G13 and Gq) and the EDG-3 antagonist suramin was found to block specifically Rho-mediated responses, it is likely that Cas-related responses following Gi activation originate from EDG-1, whereas Rho-related responses originate from EDG-3.

Sphingosine 1-phosphate (Sph-1-P) 1 has been shown to elicit a great variety of responses, including stimulation of cell proliferation and survival, regulation of cell motility, and cytoskeletal reorganization, in numerous cell types (1)(2)(3)(4). Blood platelets are unique in that they store Sph-1-P abundantly (possibly due to the presence of highly active Sph kinase and lack of Sph-1-P lyase) and release this bioactive lipid extracellularly upon stimulation (5,6). Hence, it is important to examine the effects of Sph-1-P on vascular endothelial cell functions from the viewpoint of platelet-endothelial cell interactions that may be involved in such diverse activities as thrombosis, hemostasis, angiogenesis, and atherosclerosis. We previously showed that Sph-1-P prevents apoptosis induced by withdrawal of growth factors and stimulates DNA synthesis in human umbilical endothelial cells (HUVECs) (7). Recently, Sph-1-P has been reported to induce endothelial cell migration and morphogenesis into capillary-like networks and act as an angiogenic molecule (8 -10).
Although originally postulated to function as an intracellular second messenger (11), it is now established that Sph-1-P exerts many of its effects as an extracellular mediator (1)(2)(3)(4). Recently, plasma membrane receptors for Sph-1-P have been identified; the endothelial differentiation gene (EDG)-1, EDG-3, and EDG-5 (earlier known as AGR16 or H218) exhibit overlapping, as well as distinct, patterns of expression in various tissues as Sph-1-P receptors (12)(13)(14)(15)(16). In HUVECs, EDG-1 was shown to be abundantly expressed, whereas EDG-3 was expressed at a lower level (8). Consistent with the presence of its high affinity cell surface receptors on vascular endothelial cells, nanomolar Sph-1-P reportedly induces a variety of responses in these cells (7)(8)(9)(10). As for intracellular signalings, Sph-1-P induces Ca 2ϩ mobilization, mitogen-activated protein kinase activation, and Rho family small G protein activation (8 -10). Functionally, Sph-1-P stimulates endothelial cell proliferation through a G i -coupled receptor, probably EDG-1 (8,9). Furthermore, Sph-1-P induces adherens junction assembly, migration, capillary tube formation, and promotion of angiogenesis (8 -10). In this case, the signals mediated via EDG-1 (coupled with G i ) and EDG-3 (coupled with G 13 and G q ) are both necessary, and Rho-and Rac-mediated signalings are involved (8). Sph-1-P induction of angiogenesis through these EDG receptors may be related to involvement of activated platelets in this important process in vivo. However, the overall signaling pathways leading to these cytoskeletal events have not been elucidated. In the present study, we examined Sph-1-P-stimulated signaling pathways related to HUVEC motility with a special emphasis on Crk-associated substrate (Cas), a docking protein involved in cytoskeletal reorganization (17).
Cell Culture and Preparation-HUVECs were prepared as described previously (7). They were grown and maintained in Dulbecco's modified Eagle's medium supplemented with 10 ng/ml of recombinant human basic fibroblast growth factor (PeproTech EC, London, UK), 20% fetal calf serum (ICN Biomedicals, Aurora, OH), penicillin G (100 units/ml), and streptomycin sulfate (100 mg/ml), at 37°C under an atmosphere of 5% CO 2 and 95% room air. HUVECs were not used after the sixth passage. The cells were replated in either 10-or 3.5-cm dishes coated with 0.2% gelatin (Sigma) and were used when confluent. When HU-VECs were serum-starved, serum plus basic fibroblast growth factor were removed from the medium 2 h before stimulation. Human platelets were prepared as described previously (20).
Immunoprecipitation and in Vitro Kinase Assay-HUVECs were lysed in a 1ϫ ice-cold lysis buffer (50 mM Tris (pH 7.4), 1% Triton X-100, 1% Nonidet P-40, 20 mM NaF, 100 mM NaCl, 6 mM EDTA, 1 mM Na 3 VO 4 , 0.5 mM phenylmethylsulfonyl fluoride, and 25 g/ml of leupeptin) with the aid of a cell scraper. The lysates were stood on ice for 1 h with occasional mixing, followed by centrifugation at 15,000 ϫ g for 10 min. The protein concentrations of the resultant supernatant were determined by dye binding (21), and 0.5 mg of protein was used for immunoprecipitation; some of the lysate supernatant was used as the whole cell lysate. All subsequent immunoprecipitation steps were carried out at 4°C. The samples were precleared with protein A-Sepharose 4B, and the resultant supernatants were incubated for 4 h with the antibody indicated. Protein A-Sepharose 4B was then added and further incubated for 2 h. The resulting Sepharose beads were washed with a 1ϫ lysis buffer three times. The samples were then separated into 2 aliquots. One was solubilized with an SDS sample buffer and used for immunoblotting. When indicated, the other was processed further for an in vitro kinase assay as follows. The beads were washed once with a low salt buffer (10 mM Tris (pH 7.2), 100 mM NaCl, and 5 mM MnCl 2 ) and then incubated with 35 l of 20 mM Hepes (pH 7.4) containing 5 mM MgCl 2 and 12.5 g of acid-treated enolase. The reaction was initiated by the addition of 15 l of kinase reaction buffer (300 mM Hepes/NaOH (pH 8.0), 30 M Na 3 VO 4 , 150 mM MgCl 2 , 15 mM MnCl 2 , and 5 M [␥-32 P]ATP (0.75 Ci)). After 8 min at room temperature, the reaction was terminated by the addition of 25 l of 3ϫ SDS sample buffer, and then the contents were boiled for 3 min. The proteins were separated by an SDS-PAGE and electroblotted onto a PVDF membrane. The membrane was treated with 1 M KOH at 60°C for 30 min and then 10% acetic acid at 20°C for 15 min and dried. The phosphorylated protein was quantified using a BAS-2000 PhosphorImager analyzer (Fuji Film, Tokyo, Japan).
GST Fusion Protein Production and Binding Studies-The plasmid that encoded the GST fusion protein containing the SH2 domain of Crk was kindly provided by Dr. M. Matsuda (International Medical Center, Tokyo, Japan). The fusion protein construct was transformed into Escherichia coli for protein production. The protein produced was purified on a glutathione-Sepharose column by affinity chromatography, according to the manufacturer's recommendations (Amersham Pharmacia Biotech). For the binding experiments, the HUVEC lysates were precleared with glutathione-Sepharose 4B, mixed with 20 g of GST fusion protein, and incubated for 1 h at 4°C on a rotary shaker. Glutathione-Sepharose 4B beads were added to preabsorb the protein complex. Following incubation for 1 h, the beads were centrifuged and washed three times with a 1ϫ lysis buffer. The bound proteins were eluted with a 1ϫ SDS sample buffer and boiled for 3 min. The proteins were separated on an SDS-PAGE and immunoblotted, as described above.
Actin Staining-For actin staining, cells were fixed with 3% paraformaldehyde in PBS for 40 min and then permeabilized with 0.2% Triton X-100 for 8 min. Actin filaments were detected by staining with 0.1 g/ml TRITC-conjugated phalloidin. Actin staining was observed and photographed using a confocal microscope.
Immunofluorescent Staining-Fixed and permeabilized cells were incubated with anti-paxillin mAb (2.5 g/ml), anti-Cas mAb (25 g/ml), or anti-Crk mAb (2.5 g/ml) for 2 h at room temperature, washed in PBS, and then incubated for 1 h with FluoroLink TM Cy3 TM -labeled goat anti-mouse IgG (Amersham Pharmacia Biotech). After incubation, the cells were washed three times with PBS. Immunofluorescent staining was observed and photographed using a confocal microscope.
Phagokinetic Assay Using Gold Sol-coated Plates-Random cell motility and phagocytotic activity were jointly estimated as the area of phagokinetic tracks on gold sol particle-coated plates, as described previously (22). Briefly, 3.5-cm dishes coated with 0.2% gelatin were incubated with colloidal gold for 45 min and then washed two times with PBS. HUVECs (2000 cells) in Dulbecco's modified Eagle's medium containing 2% fetal calf serum were added to each dish. After 16 h at 37°C, phagokinetic tracks were visualized using dark field illumination with a confocal microscope. The area cleared of gold particles was measured after photography, and the mean value for 20 cells was calculated in each experiment.
Migration Assay-HUVEC migration was assessed by a modified Boyden's chamber assay, i.e. in Transwell cell culture chambers (Costar, Cambridge, MA) basically as described previously (23). Polycarbonate filters with 8 m pores, used to separate the upper and lower chamber, were coated with 500 g/ml of Matrigel (Becton Dickinson, Bedford, MA). The coated filters were washed with a serum-free medium and dried immediately. Then HUVECs were added to the upper compartment of the chamber at a density of 1 ϫ 10 5 /100 l of medium containing 0.1% bovine serum albumin and incubated for 4 h at 37°C. HUVECs were allowed to migrate toward an indicated chemoattractant in the lower chamber. After the reaction, the filters were fixed and stained with trypan blue. After removal of nonmigrated cells by wiping with cotton swabs, cells that had migrated through the filter to the lower surface were counted manually under a microscope in five predetermined fields at a magnification of ϫ 200.

Expression of Cas in HUVECs and its Tyrosine Phosphorylation upon Stimulation with
Sph-1-P-We first checked the expression of cytoskeletal proteins such as Cas, FAK, talin, and vinculin in HUVECs. The same amount of HUVEC lysate protein was resolved on an SDS-PAGE and probed with each antibody. As expected, these cytoskeletal proteins were confirmed to be expressed in HUVECs (Fig. 1A). One notable finding was the abundant expression of Cas in HUVECs, al-though this was evaluated only in comparison with platelets ( Fig. 1A). To investigate the involvement of Cas in Sph-1-P signaling pathways, we examined the effect of Sph-1-P on its tyrosine phosphorylation in HUVECs. As shown in Fig. 1B, Sph-1-P induced tyrosine phosphorylation of this cytoskeletal docking protein, reaching a maximal level 15-30 min after stimulation. The time course of Cas tyrosine phosphorylation induced by Sph-1-P was similar to that of FAK (Fig. 1B).
Effects of Pertussis Toxin and C3 Exoenzyme on Sph-1-Pinduced Cas Tyrosine Phosphorylation in HUVECs-Bacterial toxins have been shown to be useful in the elucidation of cellular responses elicited by Sph-1-P. Sph-1-P-induced mitogen-activated protein kinase activation, inhibition of adenylyl cyclase, and growth stimulation are reportedly inhibited by pertussis toxin (8,13,24), which ADP-ribosylates and inactivates G i (25). In some systems, it was reported that Sph-1-Pinduced phospholipase C activation (and the resultant intracellular Ca 2ϩ mobilization) was sensitive to pertussis toxin (8,13,24). On the other hand, cytoskeletal reorganization and morphological changes induced by Sph-1-P are inhibited not by pertussis toxin but by C3 exoenzyme (8,16,26), which ADPribosylates and inactivates Rho (18).
In HUVECs, Cas tyrosine phosphorylation induced by Sph-1-P was abolished by pretreatment with pertussis toxin ( Fig.  2A, upper). On the other hand, pretreatment with C3 exoenzyme failed to affect Sph-1-P-induced Cas tyrosine phosphorylation (Fig. 2B, upper). In contrast, Sph-1-P-induced FAK tyrosine phosphorylation was only marginally inhibited by pertussis toxin (Fig. 2A, lower) but completely inhibited by C3 exoenzyme (Fig. 2B, lower). We further examined the effects of Y-27632, a specific Rho kinase inhibitor (19). This compound did not inhibit Cas tyrosine phosphorylation induced by Sph-1-P; it rather induced a slight Cas phosphorylation by itself and enhanced the Sph-1-P effect (Fig. 2C, upper) for unknown reason(s). Sph-1-P-induced FAK tyrosine phosphorylation was abolished by Y-27632 (Fig. 2C, lower) as well as by C3 exoenzyme. These findings were unexpected in view of the previous findings that Cas plays important role(s) in cytoskeletal reorganization through C3 exoenzyme-inhibitable Rho activation (27). Our data suggest that, in HUVECs stimulated with Sph-1-P, cytoskeletal signalings may be separable into G i -mediated signaling pathways (involving Cas) and Rho-mediated ones (involving FAK).

Involvement of Fyn in Cas Tyrosine Phosphorylation in HU-VECs Stimulated with Sph-1-P-Since
Src family tyrosine kinases reportedly phosphorylate Cas in several systems (28 -30), we tested this possibility in Sph-1-P-stimulated HUVECs. In immunoprecipitation studies using each Src family tyrosine kinase, Fyn, but not Src or Lyn, was found to be constitutively associated with Cas in HUVECs (Fig. 3A). Fyn/Cas association was also observed when immunoprecipitates with anti-Cas antibody were probed with anti-Fyn antibody (data not shown). Furthermore, the kinase activity of Fyn immunoprecipitates increased upon Sph-1-P stimulation; in vitro kinase assays of Fyn immunoprecipitates revealed stimulation-dependent 32 P i incorporation into Fyn, enolase, and a 130-kDa protein (confirmed as Cas with immunoblotting) (Fig. 3B). Fyn auto-phosphorylation clearly occurred before Cas phosphorylation (Fig.  3B). These data suggest that HUVEC Cas is constitutively associated with Fyn, and tyrosine-phosphorylated through Fyn activation, following Sph-1-P stimulation. Since there were few bands other than Cas, Fyn, and enolase (added as an exogenous substrate), in the in vitro Fyn kinase assay (Fig. 3B), the Cas/Fyn interaction may be highly specific in HUVECs stimulated with Sph-1-P.
We further examined the involvement of Fyn activation in Cas tyrosine phosphorylation with the use of PP2, a specific inhibitor of Src family tyrosine kinase(s) (31). As expected, pretreatment with PP2 abolished tyrosine phosphorylation of Cas (Fig. 4A), but not FAK (Fig. 4B), in Sph-1-P-stimulated HUVECs. Under these conditions, auto-phosphorylation of Fyn was completely inhibited by PP2 (data not shown). These data confirm that tyrosine phosphorylation of Cas, but not FAK, is a downstream target of Fyn tyrosine kinase in HUVECs stimulated with Sph-1-P. As described above, Cas tyrosine phosphorylation was inhibited by pertussis toxin (but not by C3 exoenzyme or Y-27632), and most probably catalyzed by Fyn. Hence, we next investigated whether Fyn auto-phosphorylation was regulated by G i activation. In HUVECs stimulated with Sph-1-P, Fyn (but not Src) auto-phosphorylation was observed in an in vitro kinase assay (Fig. 5). Pretreatment with pertussis toxin inhibited the Sph-1-P-induced Fyn auto-phosphorylation (Fig. 5B). Furthermore, Fyn auto-phosphorylation was not affected by pretreatment with C3 exoenzyme or Y-27632 (data not shown). These results further strengthen the idea that Sph-1-P induces Cas tyrosine phosphorylation via G i -mediated Fyn activation in HUVECs.
Association of Cas with Crk in HUVECs Stimulated with Sph-1-P-Cas has a cluster of 15 potential SH2-binding motifs that are thought to have a preferential affinity for the Crk-SH2 domain (17). In addition, the Crk-Cas complex reportedly has a role in integrin-mediated cell migration in several systems (32,33). Accordingly, we examined whether Sph-1-P-induced phosphorylation of Cas led to the formation of a complex between endogenous Crk and Cas in HUVECs. When immunoprecipitates with anti-Crk mAb were immunoblotted with anti-phosphotyrosine antibody, a 130-kDa tyrosine-phosphorylated protein was specifically detected in a manner dependent on Sph-1-P stimulation (Fig. 6A). Immunoblotting with anti-Cas mAb revealed that this 130-kDa protein was Cas; Cas weakly associated with Crk in resting HUVECs, and this association was augmented after Sph-1-P stimulation (Fig. 6A). This stimulationdependent Cas/Crk interaction was also detected using immunoprecipitates with anti-Cas polyclonal antibody, followed by anti-Crk immunoblotting (data not shown). Furthermore, the SH2 domain of Crk was shown to bind to Cas in HUVECs stimulated with Sph-1-P, and this interaction was disrupted by pretreatment with pertussis toxin but not with C3 exoenzyme (Fig. 6B), as was the case with Cas tyrosine phosphorylation (Fig. 2). These data suggest that Crk, through its SH2 domain, interacts with tyrosine-phosphorylated Cas in HUVECs stimulated with Sph-1-P.
Localization of Both Cas and Crk to Membrane Ruffles, but Not to Focal Adhesion, in Sph-1-P-stimulated HUVECs-Targeting of signaling proteins to specific or appropriate regions of the cell is important for regulation of various cellular functions, especially cytoskeletal reorganization (34). Previous studies demonstrated that most Cas exists in the cytosol in resting states and shifts to membrane ruffles (32) or focal adhesions (35) during the initial attachment of cells to matrix proteins. To analyze the correlation between the intracellular localization of Cas/Crk and the formation of stress fibers or focal adhesions in HUVECs stimulated with Sph-1-P, the cells were examined using (immuno)fluorescent staining and confocal microscopy. Sph-1-P induced HUVEC stress fiber formation (F-actin bundling) (Fig. 7A) and an increase in paxillin staining, indicative of focal adhesion assembly (Fig. 7B). These cytoskeletal responses induced by Sph-1-P were completely inhibited by Y-27632 but not by PP2 (Fig. 7, A and B). On the other hand, Cas was mainly localized in the cytoplasm in resting states and translocated to the cell periphery after Sph-1-P stimulation (Fig. 7C). Simultaneous phalloidin staining studies showed that Cas translocation was observed in the cells assembling a mesh work of actin filaments at the cell periphery (Fig. 7A). In addition, not only Cas but also Crk was found to translocate to cortical actin in a manner dependent on Sph-1-P stimulation (Fig. 7D). Furthermore, Sph-1-P-elicited translocation of Cas and Crk was completely abolished by PP2, but not by Y-27632 (Fig. 7, C and D). These data suggest that HUVEC stimulation with Sph-1-P results in translocation of Cas and Crk (which form a complex upon stimulation) to the cell periphery (membrane ruffles) through G i (Fyn) mediation and that these responses are independent of stress fiber and focal adhesion formation, which are mediated by Rho.
Requirement of Both Rho-and G i -mediated Pathways for Sph-1-P-enhanced HUVEC Motility-As described above, in HUVECs, Cas is likely to be tyrosine-phosphorylated by Fyn (which undergoes activation through a G i -mediated pathway), whereas FAK tyrosine phosphorylation (and the resultant cytoskeletal reorganization) is mediated mainly by a Rho-mediated pathway. Since Cas and FAK have been implicated in the regulation of cell motility in various systems (30,32), we investigated the involvement of these pathways in Sph-1-P-enhanced HUVEC motility, assessed by a phagokinetic assay using gold sol-coated plates (for chemokinesis) and a Boyden's chamber assay (for migration). Consistent with recent studies (9, 10), Sph-1-P potently stimulated HUVEC chemokinesis (Fig. 8A) and migration (Fig. 8B). Pertussis toxin or PP2 completely inhibited these Sph-1-P-elicited responses (Fig. 8, A and  B). Furthermore, treatment with C3 exoenzyme (Fig. 8, A and  B) or Y-27632 (data not shown) also resulted in inhibition of Sph-1-P-enhanced cell motility. These data suggest that Sph-1-P-enhanced HUVEC motility requires coordinated signalings from G i and Rho activation.
Effects of Suramin on Sph-1-P-induced Cytoskeletal Responses-To discriminate definitively between the signaling pathways mediated by the HUVEC Sph-1-P receptors EDG-1 and EDG-3, we used the polycyclic anionic compound suramin, which selectively antagonizes EDG-3 (36). Of Sph-1-P-elicited cytoskeletal responses, the Rho-mediated formation of stress fibers and focal adhesions were specifically inhibited by pretreatment of the cells with suramin (Fig. 9, A and B). On the other hand, the G i -mediated Cas/Crk translocation to the cell periphery was not affected by this EDG-3 antagonist (Fig. 9, A  and D). These data suggest that EDG-3 plays an important role in cytoskeletal responses mediated by Rho but not G i . (17). Cas can be phosphorylated through a variety of pathways, including integrin, G protein-coupled receptors, and receptor-type tyrosine kinases (28,35,37). Cas is thought to be a component of focal adhesion and contributes to cytoskeletal reorganization through assembly of actin filaments (30). Cas becomes tyrosine-phosphorylated with kinetics similar to FAK in response to integrin-mediated cell adhesion (30) and, in some cells, is found to be actually phosphorylated by FAK (38). However, the mechanism by which Cas is phosphorylated and how this docking protein is functionally involved remain to be clarified in vascular endothelial cells, especially when they are stimulated with the ligand for G protein-coupled receptors.

Sph-1-P-induced HUVEC Cas Tyrosine Phosphorylation through Mediation of G i and Fyn but Not Rho-Cas was originally identified as a tyrosine-phosphorylated protein of 130 kDa in cells transformed by v-Src or v-Crk
In this study, we first confirmed the abundant expression of Cas in HUVECs, and then we examined the mechanism by which Cas is phosphorylated and its possible involvement in cytoskeletal reorganization and motility upon stimulation with Sph-1-P; Sph-1-P acts on its G protein-coupled receptors expressed on HUVECs (see Introduction). Consistent with previous studies (26,39), we confirmed that inactivation of Rho by C3 exoenzyme or inhibition of Rho kinase by Y-27632 resulted in inhibition of Sph-1-P-induced FAK tyrosine phosphorylation, which was examined in parallel with Cas phosphorylation. The inhibition of FAK phosphorylation was accompanied by elimination of stress fiber formation and focal adhesion assembly. It is likely that Sph-1-P-induced activation of Rho/ Rho kinase (through G 12/13 ), leading to phosphorylation (inactivation) of myosin light chain phosphatase (39,40), and the resultant myosin light chain phosphorylation (39,40) were inhibited by treatment with C3 exoenzyme or Y-27632. Contrary to our initial expectations, however, Cas was phosphorylated in a manner distinct from FAK in HUVECs; the association of FAK with Cas was barely observed in immunoprecipitation studies (data not shown). Sph-1-P-induced Cas tyrosine phosphorylation was accompanied by Cas interaction with Crk and localization to membrane ruffles (but not focal adhesion). Importantly, all these Cas reactions elicited by Sph-1-P were abolished by pertussis toxin but not C3 exoenzyme or Y27632. Although Cas has been shown to be localized to focal adhesions and dependent on Rho signaling pathways in several cell types (27,39), Cas functional responses independent of these have also been reported (32,41). Cas is markedly concentrated in membrane ruffles and has an important role during cell migration (32,41). Cas accumulates at the leading edge of the lamellipodia, and assembly of a Cas-Crk complex and Rac activation are necessary for membrane ruffling, independent of actinmyosin contraction (32,41). It is likely that Cas tyrosine phosphorylation and Cas-Crk complex formation in membrane ruffles, following G i activation, are related to Rac (rather than Rho) activation in Sph-1-P-stimulated HUVECs.
Another important finding in our study was that Cas may be phosphorylated by Fyn in HUVECs stimulated with Sph-1-P. It has been reported that Src family tyrosine kinase(s) phosphorylate Cas in several systems (28 -30) and that the C-terminal region of Cas can associate with several Src kinases in vitro (28 -30). We found that in HUVECs, Fyn, but not Src or Lyn, associated with Cas, and Cas tyrosine phosphorylation was inhibited by the specific Src family tyrosine kinase inhibitor PP2. In addition, we confirmed that Fyn actually phosphorylated Cas in an in vitro kinase assay. Recently, Okuda et al. (42) reported that in HUVECs exposed to shear stress, Src phosphorylates Cas. They used the clone 28 mAb, which recognizes the activated form of Src family kinases (43), to examine en- dogenous Src activity, but this mAb has also been found to recognize activated Fyn (as well as Src) (44).

Involvement of G i -mediated Cas Pathways in HUVEC Motility Enhanced by Sph-1-P in Cooperation with Rho-mediated
Pathways-In this study, we found that Sph-1-P-enhanced HUVEC motility was inhibited by pertussis toxin or PP2, as is the case with Cas tyrosine phosphorylation and the resultant formation and translocation (to membrane ruffles) of the Cas-Crk complex. Recent observation of Cas(Ϫ/Ϫ) fibroblasts suggested that Cas plays a key role in mediating cell migration (45). It has also been reported that formation of a Cas-Crk adapter protein complex serves as a molecular switch facilitating a Rac-dependent cell migration response in the extracellular matrix (32). By taking these recent findings into consideration, it is likely that G i -mediated Cas tyrosine phosphorylation and its related responses are necessary for enhanced cell motility in HUVECs stimulated with Sph-1-P.
On the other hand, Sph-1-P-induced HUVEC motility was also abolished by C3 exoenzyme or Y-27632, suggesting the requirement of Rho/Rho kinase pathway. Although a rapid reorganization of actin to the cell edge is important for initiation of migration for a cell to move, it must also organize actin HUVECs were pretreated without or with pertussis toxin (PTX), C3 exoenzyme, or PP2 as described in the legend for Fig. 2. A, HUVEC chemokinesis was measured with a phagokinetic assay (using gold sol-coated plates). HUVECs were incubated in the absence or presence of 1 M Sph-1-P, and after 16 h, the area cleared of gold particles for each cell was measured. Columns and error bars represent the mean Ϯ S.D. (n ϭ 3). B, HUVEC migration was assessed with a modified Boyden's chamber assay. Sph-1-P (100 nM) was placed in the lower chamber (or not added), and HUVECs were allowed to migrate. Columns and error bars represent the mean Ϯ S.D. (n ϭ 3).

FIG. 9. Effects of suramin on stress fiber and focal adhesion formation and
Cas/Crk translocation in Sph-1-P-stimulated HUVECs. Serum-starved HUVECs were pretreated without or with 500 M suramin for 5 min. The cells were then challenged without or with 1 M Sph-1-P for 30 min, fixed, and incubated with TRITC-phalloidin (A), anti-paxillin mAb (B), anti-Cas mAb (C), or anti-Crk mAb (D). Actin staining and immunofluorescent staining were performed as described under "Experimental Procedures." The data shown are representative of three separate experiments. into a functional actin-myosin motor unit capable of generating contractile force resulting from Rho activation (46). Indeed, Rho activation is required for the crawling movements of NIH3T3 cells on a flat surface (47), for hepatocyte growth factor-induced motility of mouse keratinocytes (48), and for lysophosphatidic acid-induced invasion of hepatoma cells (49). Migratory defects have also been reported in cells lacking FAK (50); FAK appears to modulate focal adhesion turnover (46,50). Also in endothelial cell motility enhanced by Sph-1-P (which we examined in this study), the C3 exoenzyme-sensitive Rho mediation and accompanying FAK activation seem to play important role(s) in cooperation with G i -mediated Cas/Crk pathways.
HUVECs reportedly express the Sph-1-P receptors EDG-1 and EDG-3, the former being coupled predominantly with G i and the latter with G 13 and G q (8,51,52). It has been postulated that stimulation of both the Rac pathway via EDG-1 and the Rho pathway via EDG-3 are necessary for cell morphogenesis and adherens junction assembly (8). In this context, Casrelated responses following G i (Fyn) activation may originate from EDG-1, whereas Rho (and FAK)-related responses originate from EDG-3. To discriminate definitively between these pathways, we employed the selective EDG-3 antagonist suramin (36). As expected, suramin was found to inhibit the Rhomediated formation of stress fibers and focal adhesion but not the G i -mediated response. Synergism between the two independent pathways from EDG-1 and EDG-3 seems crucial in Sph-1-P-elicited signalings in vascular endothelial cells, especially those related to cytoskeletal reorganization. Since Sph-1-P, released from activated platelets and interacting with endothelial cells, is likely to play important roles under conditions in which critical platelet-endothelial cell interactions (including thrombosis, hemostasis, angiogenesis, and atherosclerosis) occur, thorough elucidation of EDG signaling pathways in vascular endothelial cells will lead to a greater understanding of vascular events or diseases.