Plexin-B1 Utilizes RhoA and Rho Kinase to Promote the Integrin-dependent Activation of Akt and ERK and Endothelial Cell Motility*

The semaphorins are a family of proteins originally identified as axon-guiding molecules in the developing nervous system that have been recently shown to regulate many cellular functions, including motility, in a variety of cell types. We have previously shown that in endothelial cells Semaphorin 4D acts through its receptor, Plexin-B1, to elicit a pro-angiogenic phenotype that involves the activation of the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. Here we show through the use of a receptor chimeric approach, Plexin-B1 mutants, and dominant negative and pharmacological inhibitors that this response is dependent upon the activation of RhoA and its downstream target, Rho kinase (ROK). Indeed, we demonstrate that in endothelial cells, Semaphorin 4D promotes the formation of focal adhesion complexes, stress fibers, and the phosphorylation of myosin light chain, a response that was abolished by the use of ROK inhibitors and absent from cells expressing Plexin-B1 mutant constructs incapable of signaling to RhoA. Stress fiber polymerization and contraction are in turn necessary for RhoA-dependent pro-angiogenic signaling through Plexin-B1. Furthermore, we observed that in endothelial cells Plexin-B1 promotes the integrin-mediated activation of Pyk2, resulting in the stimulation of PI3K, Akt, and ERK. These findings provide evidence that Plexin-B1 promotes endothelial cell motility through RhoA and ROK by regulating the integrin-dependent signaling networks that result in the activation of PI3K and Akt.

The semaphorins are a family of transmembrane and secreted proteins originally identified as regulators of axonal growth and neurite extension in the developing nervous system (1,2). Semaphorins are also expressed outside of the nervous system, where they play a role in regulating the morphology, migration, and proliferation in a variety of cell types. For example, semaphorins control the proliferation and activation of lymphocytes (3)(4)(5)(6)(7); development of the lungs (8), vasculature, and heart (9 -12); and tumor growth and metastasis (13,14). Currently, more than 20 semaphorins have been identified, which are grouped into eight classes; classes 1 and 2 are inver-tebrate semaphorins, classes 3-7 are found in vertebrates, and an eighth class, class V, has been identified in some non-neurotropic DNA viruses (1,15).
Plexins, which are receptors for the semaphorins, share homology in their extracellular segment with the scatter factor receptors c-Met and RON but fail to exhibit significant homology with any other known receptor in their intracellular region (16). In humans, at least nine plexins have been identified and grouped into four families, A through D, most of which have been shown to mediate neuronal cell adhesion and contact, nerve fasciculation, and axon guidance (17). Secreted semaphorins, such as those in class 3, require co-receptors, the neuropilins, to bind and initiate intracellular signaling through the plexins (18,19). In the case of the membrane-bound class IV semaphorin Semaphorin 4D (Sema4D), 2 signaling is mediated directly through binding to its single-pass transmembrane receptor, Plexin-B1.
Ligation of plexins by semaphorins initiates a signaling cascade, which in many cases involves small GTPases of the Ras and Rho family and their signaling pathways. For example, Plexin-A1 and Plexin-B1 are known to act as R-Ras GAPs (GTPase-activating proteins) when bound by their respective semaphorins (20,21). Plexin-B1 may also compete for Rac binding with p21-activated kinase; therefore, in addition to inhibiting R-Ras signaling, Plexin-B1 may sequester Rac and inhibit p21-activated kinase activation (22). Plexin-B1 can also inhibit RhoA signaling in certain cell culture conditions through the membrane trapping of p190 RhoGAP (23). In this regard, however, our group and others have found that the Rhospecific GEFs (guanine nucleotide exchange factors), leukemiaassociated RhoGEF (LARG), and PDZ-RhoGEF bind to the C terminus of Plexin-B1 and mediate activation of the small GTPase RhoA in response to Sema4D (24 -29). In line with these findings, we have observed that the robust migratory and pro-angiogenic response elicited by Sema4D in endothelial cells is dependent upon the ability of Plexin-B1 to stimulate RhoA (25). We have also observed that Plexin-B1 stimulation in endothelial cells leads to the activation of an intracellular tyrosine kinase cascade resulting in the stimulation of phosphatidylinositol 3-kinase and Akt (30). Furthermore, a recent study suggests that Plexin-B1 can utilize RhoA to stimulate ERK (31). How Plexin-B1 activates ERK through RhoA and which of the signaling pathways controlled by Plexin-B1 result in phosphatidylinositol 3-kinase and Akt activation are at the present unknown.
In the present study, we used a chimeric receptor approach, Plexin-B1 mutants, and dominant negative and pharmacological inhibitors of RhoA signaling to show that in endothelial cells the Plexin-B1-mediated phosphorylation and activation of Akt, ERK, and Pyk2 are dependent upon its ability to activate RhoA and not on Plexin-B1 residues involved in its RasGAP activity. We show that in those cells capable of signaling to RhoA, Sema4D treatment results in phosphorylation of myosin light chain (MLC), as a consequence of ROK activation, and the downstream polymerization and contraction of actin/myosin stress fibers. We also provide evidence that, in response to Sema4D activation of Plexin-B1, integrin-mediated attachment to the extracellular matrix is required for pro-angiogenic signaling to phosphatidylinositol 3-kinase, Akt, and ERK in endothelial cells downstream from RhoA.
Trk/Plexin Fusion Proteins-Trk-A/Plexin-B1 fusion proteins were made as previously described (28). Briefly, the intracellular portion of Plexin-B1 with and without the PDZ binding motif was cut out of the plasmid pCEFL EGFP Plexin-B1 with NheI/NotI and cloned in-frame with the extracellular and transmembrane portion of the nerve growth factor (NGF) receptor Trk-A in the vector pCEFL-HA. The constructs were then transfected into endothelial cells using Superfect (Qiagen, Valencia, CA) and stable cells selected in G418. A Trk-A/ Plexin-B1 mutant lacking key residues involved in RasGAP activity was generated as previously described (30) using the QuikChange II XL site-directed mutagenesis kit according to the manufacturer's instructions (Stratagene, La Jolla, CA). Mutations were confirmed by sequencing.
Production of Soluble Semaphorin 4D-Semaphorin 4D was produced and purified as described previously (25). Briefly, the extracellular portion of mouse Semaphorin 4D was subjected to PCR and the resulting product cloned into the plasmid pSecTag2B (Invitrogen). This construct was transfected into 293T cells growing in serum-free medium using the calcium chloride (Fluka Chemika, Sigma Aldrich) BES (Fluka Chemika) method (32). Medium containing soluble Semaphorin 4D was collected 65 h post-transfection and purified with TALON metal affinity resin (Clontech Laboratories, Palo Alto, CA) according to the manufacturer's instructions. Concentration and purity of the TALON eluates were determined by SDS-PAGE analysis with silver stain (Amersham Biosciences) and the Bio-Rad assay (Hercules, CA). In all cases, medium collected from parallel transfectants using the empty pSecTag2B vector was used as control.
Measurement of R-Ras Activity-Cells were maintained on plates coated with collagen (10 g/ml; Roche Applied Science) or poly-L-lysine (1 g/ml; Sigma) and treated with 100 ng/ml NGF (Upstate, Lake Placid, NY) for 10 min. The cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% Triton X-100, 20 mM MgCl 2 , and 40 mM ␤-glycero-phosphate) supplemented with protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 l/ml aprotinin and leupeptin; Sigma) and phosphatase inhibitors (2 mM NaF and 0.5 mM sodium orthovanadate; Sigma). Aliquots were reserved for an immunoblot, while the rest was incubated with glutathione-Sepharose 4B beads (Amersham Biosciences) previously bound to glutathione S-transferase fused to the Ras binding domain of c-Raf-1 for 30 min at 4°C to affinity precipitate GTP-bound R-Ras. The beads were washed in ice-cold lysis buffer and subjected to SDS-polyacrylamide gel electrophoresis along with aliquots of total protein and transferred onto a polyvinylidene difluoride membrane (Immobilon P; Millipore Corp., Billerica, MA). The membranes were then incubated with anti-R-Ras antibody (Santa Cruz Biotechnology). Proteins were detected using the ECL chemiluminescence system (Pierce).
Immunoprecipitation-Cells were lysed in buffer (see above), and proteins were immunoprecipitated from the cleared lysates by incubation for 2 h at 4°C with antibody against Pyk2 (BD Biosciences). Immunocomplexes were recovered with ␥-bind-Sepharose beads (GE Healthcare). Lysates and anti-Pyk2 immunoprecipitates were analyzed by Western blotting after SDS-polyacrylamide gel electrophoresis (see above) and immunoblotted with anti-Pyk2, anti-phospho Pyk2, and anti-paxillin antibody. Immunocomplexes were visualized by the ECL chemiluminescence system (Pierce).
Lentiviral Infections-CSCG-based retroviral vectors were prepared as previously reported (33) for EGFP-RGS and EGFP-PDZ expression using 293T cells as the packaging cells. Endothelial cells were infected with viral supernatants for 24 h at 37°C in the presence of 8 g/ml of polybrene (hexadimethrine bromide; Sigma). The plates were washed twice with PBS and returned to normal medium.
Immunofluorescence-Endothelial cells expressing the Trk-A/Plexin-B1 full-length ⌬PBM and RasGAP mutant chimera were grown on sterile glass coverslips in 35-mm six-well plates and treated with 400 ng/ml of purified Sema4D or 100 ng/ml NGF, with and without 25 M HA1077 or 50 M RGDS peptide for 3 min. The cells were processed for phalloidin staining and paxillin immunofluorescence as follows (all steps were carried out at room temperature). The cells were washed in PBS, fixed in 3% paraformaldehyde for 15 min, and permeabilized in 0.5% Triton X-100 for 5 min. Alternatively, 293T cells grown on poly-L-lysine were transfected as indicated and incubated 24 h later with anti-Myc antibodies (1/100 in serum-free Dulbecco's modified Eagle's medium) at 4°C for 1 h to label membraneexposed TrkA chimera before fixation. The cells were blocked in 3% fetal bovine serum for 30 min, followed by a 1-h incubation in a humidity chamber with anti-paxillin antibody (1:200 dilution in PBS with 3% fetal bovine serum; BD Transduction Laboratories). After three more washings with PBS, coverslips were again placed in a humidity chamber for 1 h and covered with a mixture of fluorescein isothiocyanate-conjugated donkey anti-mouse secondary antibody (Jackson Immuno-Research) and phalloidin, (Alexa Fluor 546 phalloidin; Invitrogen), both diluted at 1:400 in PBS with 3% fetal bovine serum. Coverslips were inverted and mounted onto glass slides with Vectashield containing 4Ј,6-diamidino-2-phenylindole (Vector Laboratories) and viewed with fluorescence microscopy. Images were taken using a SPOT digital camera attached to a Zeiss Axiophot microscope (Carl Zeiss, Thornwood, NY). Stress fiber formation was determined as described previously (30). The number of endothelial cells exhibiting stress fiber polymerization were counted and the results expressed as a percentage of the total number of endothelial cells counted.
Migration Assays-Serum-free medium containing 10% fetal bovine serum (positive control), 0.5% bovine serum albumin (negative control), or 400 ng/ml of purified Sema4D was placed in the bottom well of a Boyden chamber and served as a chemoattractant. Serum-starved endothelial cells were added to the top chamber with or without the indicated concentrations of RGDS peptide. The two chambers were separated by a polyvinylpyrrolidone membrane (8-pore size, Osmonics; GE Water Technologies, Trevose, PA) coated with 10 g/ml fibronectin (Invitrogen). The migration assay was then performed as described (30). Briefly, after 7 h, the chamber was disassembled and the membrane stained with Diff-Quick stain (Dade Behring, Deerfield, IL), placed on a glass slide, and scanned. Densitometric quantitation was performed with NIH image software and cell migration expressed as staining intensity relative to the negative control wells. Each experiment was performed in triplicate and average and standard deviation calculated.

Plexin-B1 Signaling Is Dependent upon Its Ability to Activate
RhoA, but Not on Its Ras GAP Activity-We have previously demonstrated that Sema4D induces polymerization of actin stress fibers and chemotaxis in endothelial cells that is dependent upon the ability of Plexin-B1 to activate RhoA (25,30). Our group and others also have observed phosphorylation of Akt, Pyk2, (30), and ERK (31) in response to treatment with Sema4D, although the question remains whether or not this response is specifically dependent upon Rho. To address this question and to examine whether the ability of Plexin-B1 to act as a RasGAP is important in these responses, we generated endothelial cells stably expressing chimeric receptor constructs containing the extracellular portion of the NGF receptor, Trk-A, fused to the full-length intracellular portion of Plexin-B1, the intracellular portion of Plexin-B1 mutated at the arginine residues required for RasGAP activity (20,34,35), and a chimeric receptor where the C-terminal PDZ binding motif of Plexin-B1 necessary for recruiting PDZ-RhoGEF and leukemia-associated RhoGEF and activating RhoA was deleted (28). First, we confirmed the localization of Trk-A/Plexin-B1 wild-type control (WT), Ras GAP mutant (mut), and the C-terminal PDZ binding motif deletion chimera (⌬PBM) in 293T. As shown by confocal analysis after surface staining of living cells, all three chimeric molecules accumulated at the plasma membrane (Fig. 1A). We then examined whether the Trk-A/Plexin-B1 chimera functions as a liganddependent R-RasGAP protein, as shown for the endogenous Plexin-B1 in response to Sema4D (34). We performed a pulldown assay in NGF-treated 293T cells expressing the Trk-A/ Plexin-B1 wild-type receptor (WT) grown on poly-L-lysine or on collagen, which increases endogenous R-Ras-GTP levels ( Fig. 1B), as previously reported (34). Cells transfected with an active mutant of R-Ras were used as an internal control. The level of R-Ras-GTP was clearly diminished in response to NGF in Trk-A/Plexin-B1-expressing cells, consistent with the ligand-activated R-Ras-GAP activity of Plexin-B1 (Fig. 1B) (34). However, the Trk-A/Plexin-B1 R-RasGAP mutant failed to block R-Ras activation, whereas Trk-A/Plexin-B1 lacking its C-terminal PDZ binding motif (⌬PBM) was still able to function as a ligand-dependent R-RasGAP protein (Fig. 1C). We then began investigating how Plexin-B1 signals in endothelial cells, by expressing Trk-A/Plexin-B1 wild-type control (WT) and the R-Ras GAP mutant (mut) and the C-terminal PDZbinding motif deletion (⌬PBM) chimeras (Fig. 1D). Endothelial cells were cultured on fibronectin-coated glass coverslips, treated with NGF to elicit Plexin-B1 signaling, and analyzed under immunofluorescence for development of focal adhesion complexes and polymerization of actin/myosin stress fibers. Wild-type and R-RasGAP mutant chimeras demonstrated robust stress fiber polymerization (Fig. 1, E and F, WT and R-RasGAPmut, respectively), indicating that the ability of Plexin-B1 to act as a R-RasGAP is not necessary for stress fiber polymerization, whereas cells expressing the Trk-A/Plexin-B1 ⌬PBM mutant construct failed to do so (Fig. 1, E and F, ⌬PBM). We also observed phosphorylation and activation of Akt and ERK1/2 in both wild-type (WT) and R-RasGAP mutant (mut)-expressing cells in response to NGF (Fig. 1G, left two columns), but not in cells stably expressing the ⌬PBM mutants incapable of signaling RhoA (Fig. 1G, right two columns).
Activation of RhoA Is Necessary for Plexin-B1 Signaling in Endothelial Cells-The PDZ domain of PDZ-RhoGEF has been shown to disrupt RhoA signaling in response to Plexin-B1 activation in a dominant negative manner by competing for endogenous PDZ binding domains of the Rho GEFs (28). This signaling pathway does not involve the RGS domain of the Rho GEFs, which links G␣12and G␣13-coupled receptors to RhoA activation (36,37). We thus decided to test the effect of PDZ and RGS overexpression on Plexin-B1 signaling. First, confocal analysis showed the correct membrane localization of the Trk-A/Plexin-B1 chimera in 293T cells overexpressing GFP and the PDZ or RGS domains fused to GFP (Fig. 2A). We also confirmed that the overexpression of these domains did not affect the ligand-dependent R-RasGAP activity of the Plexin-B1 intracellular domain (Fig. 2B). Vectors expressing these EGFPtagged RGS and PDZ domains of PDZ-RhoGEF were then engineered and packaged into lentiviruses, which were used to infect endothelial cells. The expression of both EGFP-PDZ and EGFP-RGS constructs in infected cells was confirmed by Western blot for EGFP (Fig. 2C) and visualization of EGFP expression under the fluorescent microscope (Fig. 2D). Endothelial cells treated with Sema4D revealed phosphorylation of Akt, ERK, and Pyk2 when infected by viruses encoding EGFP-RGS, but not with EGFP-PDZ-expressing viruses (Fig. 2E, left two  columns). Phosphorylation of these proteins in control-treated cells, but not in cells treated with the RhoA-specific inhibitor C3 toxin, provided further evidence for the importance of RhoA in Plexin-B1-mediated activation of Akt, ERK, and Pyk2 (Fig. 2E, right two columns).
The phosphorylation of MLC by ROK contributes to actin polymerization and increased myosin light chain contractility downstream from RhoA activation. Thus, the status of phosphorylation of MLC served as a control for monitoring RhoA function (38). As expected, we observed loss of MLC phosphorylation, and therefore actin/myosin stress fiber contraction, in cells infected with the EGFP-PDZ virus but not in EGFP-RGSinfected populations (Fig. 2E, left two columns, P-MLC) and in cells treated with the C3 toxin, but not in the vehicle-treated controls (Fig. 2E, right two columns). Taken together, these results suggested that the phosphorylation and activation of Pyk2, Akt, and ERK1/2, as well as phosphorylation of MLC and hence contraction of the stress fibers that form in Sema4Dtreated endothelial cells, are dependent upon Plexin-B1-mediated activation of RhoA and ROK.
ROK Activity and Actin Tension Are Necessary for Plexin-B1mediated Signaling-To determine the significance of ROK activation in Plexin-B1-mediated phosphorylation of Akt, ERK1/2, and Pyk2, we treated endothelial cells with Sema4D and either vehicle control or the ROK inhibitor fasudil and then analyzed cell lysates for the phosphorylated forms of these pro-FIGURE 1. Plexin-B1 signaling to Akt and ERK is dependent upon its ability to activate RhoA, and not on its R-Ras GAP function. A, 293T cells were grown on poly-L-lysine and transfected for the Myc-tagged chimeric receptors Trk-A/Plexin-B1 (TrkA/PB1) wild-type (WT), Trk-A/Plexin-B1 R-RasGAP mutant (R-RasGAP mut), and Trk-A/Plexin-B1 ⌬PDZ (⌬PBM). Anti-Myc antibodies were incubated at 4°C for 1 h in living cells to allow the binding to plasma membrane-exposed chimeras. Cells were then rinsed and processed for immunostaining and confocal analysis. Scale bar, 10 m. B, 293T were grown on collagen (coll) (10 g/ml) or poly-L-lysine (PLL) (1 g/ml) and transfected with TrkA/Plexin-B1 chimera or R-RasQL constitutively activated mutant. 24 h later, cells were starved in serum-free medium for 2 h (Ϫ), stimulated by NGF 100 ng/ml for 10 min (ϩ), and lysed. R-Ras-GTP was detected upon affinity precipitation using glutathione S-transferase-c-Raf1 binding domain and total R-Ras in the total cell lysates. The chimera expression was detected by anti-Myc immunoblot in the cell extracts. C, similarly, 293T were transfected by the chimeric receptor TrkA/Plexin-B1 WT and its R-RasGAP mutant (R-RasGAP mut) and ⌬PBM and analyzed by Western blots for R-Ras-GTP content. D, immunoblot analysis for Myc shows a band present in cells transfected and selected to express the Myc-tagged chimeric receptors Trk-A/Plexin-B1 wild-type (WT), Trk-A/Plexin-B1 R-RasGAP mutant (R-RasGAP mut), and Trk-A/Plexin-B1 ⌬PBM (⌬PBM). Empty vector-transfected and selected cells fail to exhibit a band (C). Tubulin was used as a loading control. E, endothelial cells stably expressing the chimeric receptor Trk-A/Plexin-B1 wild-type (WT) and its ⌬PBM and R-RasGAP mutant (R-RasGAP mut) were treated for 3 min with 100 ng/ml NGF and analyzed for stress fiber formation. F, the number of endothelial cells exhibiting stress fiber polymerization was counted and the results expressed as a percentage of the total number of endothelial cells counted. G, cells stably expressing the wild-type (WT) and the R-RasGAP mutant (mut) chimeras, but not cells expressing the Trk-A/ Plexin-B1 ⌬PBM (⌬PBM), exhibit phosphorylation of Akt and ERK in response to treatment with 100 ng/ml NGF. Total levels of these proteins were used as a loading control. NOVEMBER 30, 2007 • VOLUME 282 • NUMBER 48 teins. Control cells treated with Sema4D exhibited phosphorylation and activation of Akt, ERK1/2, Pyk2 and phosphorylation of MLC, as before (Fig. 3A, V). However, following treatment with fasudil, this response was lost (Fig. 3A, fasudil). Because the result of activation of ROK is actin/myosin stress fiber polymerization and contraction, we next used blebbistatin, a membrane-permeable molecule that interferes with the phosphate release from non-muscle myosin II (39), to inhibit actinmyosin contraction and examine how it might be linked to activation of Akt, ERK1/2, and Pyk2. Interestingly, although MLC phosphorylation is still observed (Fig. 3B, blebb, P-MLC), Sema4D-treated cells incubated with blebbistatin lost phosphorylation of Pyk2, Akt, Src, and ERK1/2 (Fig. 3B, blebb). This finding suggested that the pathway linking Sema4D/Plexin-B1 to these signaling molecules in endothelial cells is dependent upon the ROK-mediated contraction of actin/myosin stress fibers.

Binding of Integrins to the Extracellular Matrix at Focal Adhesions Is Necessary for Sema4D-induced Cell Responses-
The internal forces generated by stress fiber formation and contraction produce isometric tension in cells if they are adherent to the substrate. This tension can direct the assembly and composition of focal adhesion complexes and trigger adhesion-dependent signaling (40 -44). Thus, based on our results we next explored whether adhesion-dependent signaling contributes to the propagation of the RhoA-dependent, Plexin-B1-mediated signal. For these experiments, endothelial cells were first cultured with and without the RGDS peptide as a competitor for integrin-extracellular matrix interaction. Cells were treated with Sema4D and analyzed under immunofluorescence for development of mature focal adhesion complexes and polymerization of actin filaments. Cells treated with Sema4D exhibited polymerization of actin/myosin stress fibers (Fig. 4A, upper right panel, red, and see below, higher magnification, Fig. 4C, upper right panel, in purple) and localization of paxillin into maturing focal adhesion complexes (Fig. 4A, in  yellow, and Fig. 4C, in white), a phenotype lost in Sema4D-treated cells grown in RGDS peptide (Fig. 4A, middle right panel, and see below, Fig. 4C, lower right panel). Sema4D-treated cells incubated with fasudil also failed to exhibit significant stress fiber/focal adhesion formation (Fig. 4A, fasudil). The quantification of cells exhibiting focal adhesions clearly showed that both fasudil and RGDS blocked the actin polymerization-induced focal adhesions caused by Sema4D (Fig. 4B). These results demonstrated the importance of integrin-mediated adhesion in the RhoA-ROK-dependent assembly of focal adhesion complexes in endothelial cells upon Sema4D treatment.  Fig. 1A. B, 293T cells were grown on collagen (10 g/ml) and transfected for TrkA/PB1 chimera with GFP, RGS-EGFP, or PDZ-EGFP fusion proteins. 24 h later, cells were starved in serum-free medium for 2 h (Ϫ), stimulated by NGF 100 ng/ml for 10 min (ϩ), and lysed. R-Ras-GTP was detected in the glutathione S-transferasec-Raf1 binding domain and total R-Ras and GFP in the total cell lysates. C, immunoblot confirming the expression of both EGFP-PDZ and EGFP-RGS constructs in lentivirus-infected cells. D, EGFP expression in infected cells. E, endothelial cells infected with lentivirus coding for EGFP-RGS (RGS) exhibit phosphorylation of Akt, ERK, Pyk2, and MLC in response to treatment with 400 ng/ml Sema4D, whereas cells infected with lentivirus coding for EGFP-PDZ (PDZ) fail to do so (left panels). Total levels of these proteins and tubulin are used as a loading control. Vehicle control endothelial cells (V) exhibit phosphorylation of Akt, ERK, Pyk2, and MLC in response to treatment with 400 ng/ml Sema4D, whereas cells incubated with the RhoA inhibitor C3 toxin fail to do so (right panels). Total levels of these proteins and tubulin are used as a loading control. The cytoplasmic tyrosine kinase Pyk2 is known to function at a point of convergence of integrins and G-protein-coupled receptor signaling cascades (45). As we have previously observed that Pyk2 is necessary to propagate the signal from Plexin-B1, which is devoid of intrinsic tyrosine kinase activity (30), we therefore examined whether formation of focal adhesion complexes, and hence cell adhesion, is necessary for Pyk2 activation by Sema4D in endothelial cells. As described above, the addition of the RGDS peptide inhibited the formation of stress fibers and paxillin recruitment to focal adhesions upon Sema4D treatment of endothelial cells (Fig. 4C). We performed an immunoprecipitation for Pyk2 in endothelial cells growing on fibronectin, with and without the RGDS peptide. We observed association of phosphorylated Pyk2 with the focal adhesion protein paxillin in response to Sema4D in vehicletreated controls but not in cells grown with RGDS (Fig. 4D, V and RGDS, respectively). We next examined lysates from endothelial cells treated with Sema4D growing on fibronectin-coated plates in the presence and absence of the RGDS peptide for phosphorylated forms of Akt, ERK1/2, MLC, and Pyk2. Not only is Pyk2 activation lost in cells incapable of forming secure attachment to the substrate due to the RGDS peptide but these cells also lose evidence of phosphorylation of paxillin, Akt, and ERK1/2 (Fig. 4E).
To determine the biological significance of the loss of substrate adhesion in Sema4D-mediated endothelial cell migration, chemotaxis toward wells containing Sema4D was measured in cells growing in control medium or medium containing RGDS peptide. Endothelial cells, which normally exhibit robust migration toward Sema4D (30), failed to migrate when integrin-adhesive function was prevented by the RGDS peptide (Fig.  4F). Taken together, these results demonstrate that in response to Sema4D, endothelial cells need to regulate the actomyosin cytoskeleton through RhoA and ROK to promote the integrin-dependent assembly of focal adhesions. In turn, focal adhesion complexes recruit and activate signaling molecules mediating the pro-migratory effects in response to Sema4D treatment. . Binding of integrins to the extracellular matrix at focal adhesions is necessary for Sema4Dinduced, RhoA-and ROK-mediated polymerization and contraction of stress fibers, activation of Akt, ERK1/2, and Pyk2, and endothelial cell chemotaxis. A, endothelial cells growing on fibronectin-coated coverslips were treated with 400 ng/ml Sema4D for 3 min and processed for phalloidin staining of stress fibers (red), an indicator of RhoA activation, and paxillin immunofluorescence (green), demonstrating formation of focal adhesion complexes. 4Ј,6-Diamidino-2-phenylindole staining of the nuclei is also shown (blue). B, endothelial cells incubated with vehicle control (C), 25 M HA1077 (fasudil), or 50 M RGDS peptide were treated for 3 min with 400 ng/ml Sema4D and analyzed for stress fiber and focal adhesions formation. The number of endothelial cells exhibiting focal adhesions was counted and the results expressed as a percentage of the total number of endothelial cells counted. C, endothelial cells were treated as above with 400 ng/ml Sema4D for 3 min and processed for phalloidin staining of stress fibers (purple) and paxillin immunofluorescence (green), demonstrating in these higher magnification pictures the formation of focal adhesion complexes. 4Ј,6-Diamidino-2-phenylindole staining of the nuclei is also shown (blue). D, lysates of endothelial cells similarly treated with vehicle (V) or 50 M RGDS peptide and 400 ng/ml Sema4D for the indicated periods of time were immunoprecipitated for Pyk2 and immunoblotted for the presence of Pyk2, phosphorylated Pyk2, and paxillin. Phosphorylation of Pyk2 in the immunoprecipitated fraction begins to appear at 1 min following treatment, whereas paxillin is found to associate with Pyk2 at detectable levels at 10 min following treatment, but only in vehicle-treated controls. Cells growing in RGDS peptide fail to exhibit Pyk phosphorylation or association of Pyk2 with paxillin. Total Pyk2 was used as a loading control. E, vehicle control endothelial cells (V) exhibit phosphorylation of Akt, ERK, Pyk2, and paxillin in response to treatment with 400 ng/ml Sema4D, whereas cells incubated with 50 M RGDS peptide fail to do so. Total levels of these proteins are used as a loading control. F, endothelial cells in serum-free medium with or without the indicated levels of RGDS peptide were used in a cell migration assay with 400 ng/ml Semaphorin 4D as the chemoattractant. Medium containing 10% fetal bovine serum was used as a positive control for migration (ϩ). Negative control wells contained 0.1% bovine serum albumin (Ϫ). The bars represent migration as determined by densitometry.

DISCUSSION
In the present study, we provide evidence that Plexin-B1 causes the RhoA-ROK-dependent activation of Pyk2, ERK1/2, and Akt and that this process requires integrin function. Indeed, both Plexin-B1 and integrins may act together to mediate endothelial cell migration in response to Sema4D.
Initially, through the use of Plexin-B1 mutants and dominant negative and pharmacological inhibitors of RhoA, we confirmed and extended earlier findings indicating that the proangiogenic phenotype observed in Sema4D-treated endothelial cells is dependent upon RhoA activation. We also observed that phosphorylation and activation of Akt and Pyk2, which are necessary for endothelial cell migration (30), as well as phosphorylation of ERK (31), were blocked by RhoA inhibition. Phosphorylation of MLC, a downstream target of the RhoA effector ROK involved in actin polymerization and stress fiber contraction (38), was also lost upon RhoA inhibition. As might be expected, the ROK inhibitor fasudil prevented phosphorylation of MLC by Sema4D, but it also inhibited the phosphorylation and activation of Akt, ERK, and Pyk2. This strongly supported the fact that Plexin-B1 can activate these downstream cascades through a RhoA-ROK-dependent signaling pathway. How in turn does ROK regulate Akt, ERK1/2, and Pyk2? Surprisingly, we observed that blebbistatin, an inhibitor of actin/myosin cross-linking and contraction (39), abolished the Sema4D-induced phosphorylation of Akt, ERK, and Pyk2, indicating that stress fiber contraction is necessary for the phosphorylation and activation of these signaling molecules downstream from RhoA and ROK.
There is a large body of evidence to suggest that integrins can regulate Rho GTPases (46 -48). Reciprocally, RhoA-mediated contractility of stress fibers leads to the bundling of actin filaments, aggregation of integrins, and assembly of focal adhesion complexes (43,49). These focal adhesion complexes can include the scaffold proteins paxillin and vinculin and cytoskeletal proteins such as talin, tensin, ␣-actinin, and F-actin and induce the recruitment and activation of focal adhesion kinase and Src family kinases (43, 50 -52). When endothelial cells were pretreated with the peptide RGDS, which blocks the ability of integrins to bind to fibronectin (53,54), activation of Plexin-B1 could no longer elicit the assembly of focal adhesions, phosphorylation and activation of Akt, ERK, and Pyk2, and endothelial cell chemotaxis toward Sema4D. Given the broad effects of the RGDS peptide in inhibiting the basal integrin-mediated adhesion, these observations imply that Plexin-B1 signal can only be transmitted and amplified when contracting stress fibers are capable of generating tension within the cell by anchoring to integrins bound to the extracellular matrix. This conclusion is also supported by the loss of Sema4D signaling in blebbistatin-treated cells, where stress fibers fail to reorganize. Overall, we can conclude that Plexin-B1 requires the engagement of integrins through RhoA and ROK and that, in turn, integrins regulate the activation of Pyk2, Akt, and ERK1/2, similarly to the obligatory signaling cross-talk between tyrosine kinase receptors and integrins (50). Thus, we propose that Plexin-B1 can co-opt other cell surface-adhesive receptors, such as integrins, to propagate signal transmission.
A proposed model for Sema4D/Plexin-B1-mediated signaling is shown in Fig. 5, depicting how the sequential engagement of Plexin-B1 and integrins might be necessary for the downstream effects of activated Plexin-B1 in response to Sema4D. In this model, adhesive contacts in the form of clustered integrins are organized and stabilized with the actin cytoskeleton at focal adhesion complexes upon ligation of Plexin-B1 with Sema4D. As stress fibers contract, they generate tension at the focal adhesions, which is converted from a mechanical signal into a biochemical signal, resulting in the tyrosine phosphorylation of Pyk2, the FIGURE 5. A model for Sema4D/Plexin-B1-mediated signaling leading to endothelial cell migration by Plexin-B1. Upon treatment with Sema4D, PDZ RhoGEF and leukemia-associated RhoGEF (LARG) are recruited to Plexin-B1 through association of their PDZ domains with the PDZ binding motif of the receptor. These GEFs then stimulate the dissociation of GDP from Rho through their DH/PH domain, thus activating Rho and eventually its downstream effector Rho kinase (ROK). In turn, ROK signaling stimulates LIM kinase (LIMK), which phosphorylates Cofilin, as well as promotes the phosphorylation of myosin light chain (MLC) by blocking the activity of MLC phosphatase (Myosin PPase), thus enhancing the accumulation of P-MLC caused by MLC kinase (MLCK). This induces polymerization and contraction of actin/myosin stress fibers. If the cells are attached to the extracellular matrix via integrins, the tension generated by contracting stress fibers promotes the assembly of mature focal adhesion complexes and activation of Pyk2. Pyk2 activation results in the phosphorylation of phosphatidylinositol 3-kinase and the activation of Akt and Erk1/2, a response necessary for cell migration and the pro-angiogenic phenotype (30). most apical tyrosine kinase activated upon Plexin-B1 stimulation in endothelial cells (30). Pyk2 then activates a tyrosine kinase-dependent signaling network resulting in the downstream stimulation of phosphatidylinositol 3-kinase, Akt, and ERK1/2, thereby controlling endothelial cell migration.