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Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D.C. 20007, USA; and
* Laboratory of Cellular and Molecular Regulation, NIMH, National Institutes of Health, Bethesda, Maryland 20892, USA
2Correspondence: Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, W226A Research Building, 3970 Reservoir Rd. NW, Washington, DC 20007, USA. E-mail: spiegel{at}bc.georgetown.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: sphingosine-1-phosphate • motility • Src • FAK
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
). These receptors, which include EDG-1, -3, -5, -6, and -8, all bind SPP and dihydro SPP with high affinity but couple to different G-proteins and thus regulate diverse processes. Whereas EDG-1, EDG-6, and EDG-8 couple mainly to Gi, both EDG-3 and EDG-5 activate Gi, Gq, and G12/13 (reviewed in refs 1
2
3
4
). Although these receptors are differentially expressed, they have all been implicated in cell migration (5
6
7
8
9)
. Activation of EDG-1 or EDG-3 by SPP or dihydro SPP in many cell types induces chemotaxis, whereas binding of SPP to EDG-5 abolished directed chemotaxis and membrane ruffling (5
, 10
11
12
13)
. Members of the EDG-1 family differentially regulate the small GTPases of the Rho family, particularly Rho and Rac (14)
, which are downstream of the heterotrimeric G-proteins and are important for cytoskeletal rearrangements (15
, 16)
. Binding of SPP to EDG-1 regulates Rac-coupled cortical actin formation (14)
, whereas binding to EDG-3 and EDG-5 elicits Rho-coupled stress fiber assembly (10)
; EDG-5 also negatively regulates Rac activity (10)
, thereby inhibiting cell migration.
Cell movement is essential throughout life, particularly during development, and is important in many physiological and pathological processes including inflammation, wound healing, tumor growth, metastasis, and angiogenesis. A mutation in the zebrafish homologue of the edg-5 gene, miles apart, was shown to cause defective migration of myocardial cells during vertebrate heart development, revealing a unique role for EDG-5 in regulating cell migration in organogenesis of the heart (9)
. Disruption of the edg-1 gene in mice by Proia and colleagues revealed that SPP/EDG-1 signaling is essential for vascular maturation (17)
. Remarkably, although EDG-1 null embryos died in utero due to massive hemorrhage, they had normal vasculogenesis and a substantially normal blood vessel network, yet were severely impaired in recruitment of smooth muscle cells and pericytes to the vessel walls, presumably due to their defective migration (17)
. Recently, we found that migration of cells from these embryos toward SPP (17)
and platelet-derived growth factor (PDGF) (18)
, which stimulates sphingosine kinase and increases intracellular SPP in many cell types (19)
, was dependent on expression of EDG-1. Moreover, PDGF activated EDG-1, as measured by its phosphorylation and translocation of ß-arrestin, suggesting a new mechanistic concept for cross-communication between a tyrosine kinase receptor, PDGFR, and a GPCR such as EDG-1 (18)
. In this study, we used EDG-1 null fibroblasts to determine the role of EDG-1 in cell growth and survival mediated by SPP and PDGF and examined the molecular mechanisms whereby EDG-1 so dramatically influences directed cell movement.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Embryos were dissected, rinsed with phosphate-buffered saline (PBS), minced, and digested with trypsin (2 min at 37°C in 2.5% trypsin/1 mM EDTA). Trypsin was inactivated by addition of Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS. Cells were subcultured every 3 days for a maximum of 10 passages. Only MEFs from passages 2–9 were used for experiments. Immortalized fibroblasts were obtained from spontaneously transformed MEFs after continuous culture.
DNA synthesis
[3H]Thymidine incorporation into DNA was measured as described (20)
. Values are the means of triplicate determinations and SDs were routinely less than 10% of the mean.
Determination of apoptotic cells
Wild-type and EDG-1-/- embryonic fibroblasts were plated in 24-well clusters at a density of 1 x 104 cells/well in DMEM containing 10% FBS, washed, and incubated in serum-free media containing the indicated agents for 48 h. Cells were then fixed, stained with Hoechst, and apoptosis assessed as described previously (20)
. In some experiments, viable cells were determined by trypan blue exclusion.
Chemotactic motility
Chemotaxis was measured in a modified Boyden chamber as described previously using polycarbonate filters (25x80 mm, 12 µm pore size) coated with collagen type I (50 µg/ml in 5% acetic acid) (5)
. Chemoattractants were added to the lower chamber and cells were added to the upper chamber at 5 x 104 cells per well. After 18 h, nonmigratory cells on the upper membrane surface were mechanically removed and the cells that traversed and spread on the lower surface of the filter were fixed and stained with Diff-Quik. The number of migratory cells per membrane was counted using a microscope with a 20x objective. Each data point is the average number of cells in four random fields, each counted twice, and is the average ± SD of three individual wells.
In vitro wound healing assay
Confluent fibroblast monolayers were wounded by scraping with a pipette tip, washed twice to remove detached cells, and incubated in serum-free DMEM containing 0.1% BSA. After 12 h, cells were fixed with 2% glutaraldehyde in PBS and photographed.
Western blotting
Fibroblasts were scraped in lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, 2 mM sodium orthovanadate, 4 mM sodium pyrophosphate, 100 mM NaF, 1 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml aprotinin). In some experiments, the cytoskeleton-associated, Triton X-100-insoluble fraction was recovered by centrifugation (14,000 g, 10 min, 4°C) and resuspended lysis buffer supplemented with 1% SDS. Equal amounts of proteins were separated by 10% SDS-PAGE and transblotted to nitrocellulose. Anti-paxillin, anti-Cas, and FAK (Transduction Labs, Lexington, KY), c-Src GD11 antibody (Upstate Biotechnology, Lake Placid, NY), and anti-Src [pY418] and FAK [pY577] antibodies (Biosource, Rockville, MD), phospho-p38, p38, and phospho-ERK1/2 antibodies (New England Biolabs, Beverly, MA), anti-PDGF receptor polyclonal antibody, and phosphotyrosine monoclonal antibody 4G10 (Upstate Biotechnology, Lake Placid, NY) were used as primary antibodies. Immunocomplexes were visualized by enhanced chemiluminescence (20)
.
Immunostaining
Cells grown on glass coverslips coated with 50 µg/ml collagen I were incubated overnight in DMEM. Cells were fixed in 3.7% formaldehyde for 30 min at room temperature and permeabilized in 0.5% Triton-X100 for 5 min. Actin filaments were visualized with Alexa 488-conjugated phalloidin (Molecular Probes, Eugene, OR) and focal complexes with antibodies to vinculin (Upstate Biotechnology, Lake Placid, NY), followed by staining with rhodamine-conjugated secondary antibody. After washing three times with PBS, coverslips were mounted on slides using an Anti-Fade kit (Molecular Probes) and cells were examined by confocal microscopy. Where indicated, cells were transfected with 5 µg of green fluorescent protein (GFP)-SPHK1 fusion plasmid (20)
, treated with PDGF for 5 min, fixed, and visualized by confocal fluorescence microscopy.
Src kinase assay
Cells were lysed in RIPA buffer (1% deoxycholate, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 50 mM HEPES (pH 7.4), 4 mM EGTA, 2 mM EDTA, 2 mM NaVO4, 5 µg/ml aprotonin, 5 µg/ml leupeptin, and 1 mM PMSF) and insoluble material was pelleted. Supernatants were precleared by incubation with a 1:1 Protein A/G (Santa Cruz) slurry for 1 h at 4°C, followed by brief centrifugation to remove the beads. Either Src or Yes monoclonal antibodies or Fyn polyclonal antibody were added to lysates and samples incubated for 1 h at 4°C. Antibody complexes were precipitated with protein A/G. Beads were washed in RIPA and then in buffer containing 50 mM PIPES-KOH (pH 7.0), 50 mM KCl, 2.5 mM MgCl2, 1 mM DTT, 1 µg/ml aprotonin, 1 µg/ml leupeptin, 0.5 mM PMSF. Kinase reactions were carried out using a synthetic peptide corresponding to amino acids 6–20 of p34cdc2. Reaction solution (25 µl) containing 500 µM peptide, 200 µM ATP, and 0.4 mCi/ml 
-32P-ATP was added to 25 µl of each sample. In each case, the activity in the absence of substrate peptide was also determined. After 10 min incubations at room temperature, reactions were stopped by centrifugation and spotting an aliquot of the supernatants on Whatman P81 filter paper. Filters were washed in 1% phosphoric acid and radioactivity determined by liquid scintillation counting.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Thus, it was of interest to examine whether this was a PDGF-specific effect or whether migration toward other growth factors and chemoattractants was also EDG-1-dependent. Fibroblasts isolated from EDG-1 knockout mouse embryos (17)
not only did not migrate toward PDGF, but had reduced chemotaxis to IGF-1 and thrombin, whereas migration toward EDG was also reduced, albeit much less (Fig. 1
A). However, migration toward fibronectin was unaffected, suggesting that, in contrast to chemotaxis-driven motility, EDG-1 is not required for haptotactic cell migration.
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In the Boyden chamber cell migration assay, differences in cell shape and size may affect passage through the pores in the membrane. These properties do not affect the wound healing assay, which can also be used to qualitatively compare cell migration. Thus, after making a wound in a monolayer of cells, wild-type mouse embryonic fibroblasts (MEFs) rapidly migrated into the wounded area whereas EDG-1-/- fibroblasts were not able to actively invade the wound (Fig. 1B
). To further examine the role of EDG-1 in cell migration, we established immortalized fibroblast lines from wild-type and EDG-1-/- MEFs. The immortalized EDG-1-/- cell line retained the severe migratory defects previously noted in the MEFs (18
; Fig. 1
) not only toward SPP and PDGF, but also toward IGF-1 (Fig. 1C
). Similar to our previous results with wild-type MEFs (18)
, treatment with pertussis toxin to inactivate Gi-coupled EDG-1 also markedly reduced migration of these EDG-1+/+ immortalized cells toward SPP and PDGF (data not shown). Furthermore, the migratory defect is clearly related to the lack of expression of EDG-1 as enforced expression of EDG-1 was able to reverse the migratory defect of these cells (Fig. 1D
).
SPP- and PDGF-induced cellular proliferation are independent of EDG-1 expression
Recent studies suggest that EDG-1 plays a critical role in SPP-stimulated proliferation of endothelial cells (5
, 14
, 21)
, whereas in other cell types its effect appears to be mediated by intracellular actions (22)
. We used fibroblasts isolated from EDG-1 knockout mouse embryos to definitively determine whether EDG-1 is essential for the mitogenic effects of SPP and PDGF. Unexpectedly, the mitogenic effect of SPP was not abrogated in EDG-1-/- fibroblasts (Fig. 2
A). Moreover, sphingosine, which is readily taken up by cells and converted intracellularly to SPP, was as effective in stimulating proliferation of EDG-1-/- fibroblasts as wild-type fibroblasts. Similarly, the mitogenic effects of PDGF-BB and FBS were not significantly different in wild-type and EDG-1-/- MEFs (Fig. 2A
). In agreement, no significant differences in DNA synthesis in response to SPP, PDGF, or serum could be detected between wild-type and EDG-1-/--immortalized fibroblast cell lines (Fig. 2B
). These results suggest that EDG-1’s function is dispensable for the mitogenic effects of SPP and PDGF.
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Mitogen activated protein (MAP) kinase family, ERK, SAPK/JNK, and p38 play an important role in cell growth, survival, and motility (23)
. Although it is well established that ERK activation is required for PDGF-stimulated DNA synthesis, activation of p38 is required for PDGF-induced cell motility and actin reorganization (24)
. In agreement with previous studies (24)
, in wild-type fibroblasts, PDGF induced sustained activation of ERK1 and ERK2 (Fig. 2C
) and a more transient activation of p38 (Fig. 2C
), whereas SAPK1/JNK was not stimulated at all (data not shown). Although EDG-1 deletion had no significant effect on activation of ERK induced by PDGF, it almost completely eliminated p38 activation (Fig. 2C
).
Survival effects of SPP and PDGF are not compromised by EDG-1 deletion
Activation of EDG-1 by SPP has also been shown to protect endothelial cells from apoptosis (14
, 21)
and this survival effect was markedly attenuated by EDG-1, but not EDG-3 or EDG-5, antisense oligonucleotides (14)
, whereas other studies suggested that suppression of apoptosis was mediated via intracellular actions (20
, 25
26
27
28
29)
. Therefore, it was important to examine the cytoprotective effect of SPP in EDG-1 knockout fibroblasts. In agreement with previous studies (20)
, serum deprivation induced apoptosis in a time-dependent manner, where shrinkage and condensation of nuclei were clearly evident after 48 h (Fig. 3
A, B). Disruption of the edg-1 gene had no significant effect on apoptosis (Fig. 3)
. Moreover, similar to other cell types (25
, 26)
, addition of micromolar concentrations of SPP (Fig. 3A
, 3B
), but not nanomolar concentrations (data not shown), to control or EDG-1-/- fibroblasts markedly suppressed apoptosis induced by serum deprivation or the chemotherapeutic drug doxorubicin. These protective effects were specific and unrelated to EDG-1, because dihydro-SPP, which lacks the trans double bond present in SPP yet binds and activates EDG-1 equally well (5
, 22)
, did not significantly prevent apoptosis in wild-type or mutant fibroblasts. Moreover, there were also no significant differences in either the extent of the cell death responses or the protection by PDGF or SPP of wild-type and EDG-1-/--immortalized fibroblast cell lines to serum starvation or other apoptotic stimuli (Fig. 3C
), including doxorubicin or tumor necrosis factor 
TNF- in the presence of actinomycin D, which sensitizes cells to the toxic effect of TNF- (30)
.
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EDG-1 null fibroblasts display aberrant cytoskeletal architecture and focal contacts in response to PDGF
Cell movement is a complex process orchestrated by the interplay of leading edge formation and the turnover of the focal adhesions that tether the cell to the extracellular matrix. Leading edge formation is under the control of members of the Rho family of small GTPases (Rac, Cdc42, and Rho) (15
, 16)
and involves actin polymerization and the formation of nascent focal adhesion complexes. The turnover of focal adhesions is modulated by tyrosine kinases that reside within these complexes, such as focal adhesion kinase (FAK) (31)
and Src (32
33
34)
. To better understand the migratory defect of EDG-1 null fibroblasts toward PDGF, we first examined the architecture of the cytoskeleton and focal adhesion formation. No obvious differences between quiescent wild-type and EDG-1-/- MEFs were revealed by phalloidin staining of actin filaments or by antibodies to the cytoskeleton protein vinculin, a major component of focal adhesions (Fig. 4
). However, although PDGF, as expected (15
, 16
, 35)
, caused extension of lamellipodia at the cell periphery of wild-type fibroblasts, lamellipodia were almost completely absent in PDGF-treated EDG-1 null fibroblasts (Fig. 4)
. Moreover, vinculin-positive patches were scattered across the ventral surface of EDG-1-/- cells, in contrast to the typical focal adhesions at the cell periphery observed in wild-type cells. Reminiscent of the morphological changes in FAK deleted fibroblasts (31
, 36)
, actin fibers were much more dense around the periphery of EDG-1-deficient cells (Fig. 4G
), rather than organized in long parallel projections equally distributed throughout the cell as in the wild-type fibroblasts.
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Similarly, PDGF did not induce membrane ruffles in immortalized EDG-1 null fibroblasts, but instead triggered the formation of filopodia or microspikes (Figure 5
A, B). To further substantiate the role of EDG-1 in PDGF-induced lamellipodia extension, immortalized EDG-1 null fibroblasts were transfected with GFP-EDG-1 and treated with PDGF (Fig. 5)
. In contrast to untransfected cells, GFP-EDG-1 transfected EDG-1 null fibroblasts exhibited copious ruffling on PDGF treatment (Fig. 5D
, 5F
).
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PDGF induces translocation of sphingosine kinase to ruffles
The acquisition of spatial and functional asymmetry between the front and rear of the cell is a necessary step for directional migration. It has been suggested that components of G-protein receptor systems may accumulate at the front of polarized cells accounting for increased responsiveness to chemoattractants at the anterior (37
38
39)
. Nonetheless, chemoattractant receptors remain evenly distributed on the surface of polarized cells (37
, 39)
and intermediate intracellular signals that are important for directional migration may be produced in a spatial and temporal manner. Thus, it was tempting to speculate that PDGF might elicit this steep signaling gradient by recruitment of sphingosine kinase to the leading edge where local formation of SPP could result in restricted activation of EDG-1. Indeed, although sphingosine kinase is diffusely distributed in the cytosol of unstimulated cells, PDGF rapidly induced translocation to membrane ruffles as visualized with a SPHK-GFP fusion protein (Fig. 6
). Sphingosine, the substrate of SPHK, is a membrane-bound lipid. Hence, recruitment of SPHK to membrane ruffles should generate SPP in a spatially restricted manner.
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Molecular basis for cross-talk between EDG-1 and PDGFR signaling: aberrant FAK phosphorylation and activation
The observation that deletion of edg-1 does not affect proliferative or survival responses to PDGF, yet eliminates PDGF-mediated motility, suggests that the point of signal disruption lies downstream of the PDGFR. Indeed, there were no significant differences in PDGF-induced tyrosine phosphorylation of PDGFR in EDG-1-/- compared to wild-type fibroblasts, reaching a maximum level within 5 min and decreasing thereafter (Fig. 7
A). In wild-type MEFs, PDGF markedly stimulated tyrosine phosphorylation of proteins with molecular masses of 125 and 
60 kDa, which comigrated with FAK and Src, respectively.
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FAK has been implicated in organization and turnover of focal adhesions and is a receptor-proximal sensor that integrates PDGFR, GPCR, and integrin signals required for cell migration (34
, 36
, 40)
. In agreement with other studies (34)
, in wild-type fibroblasts PDGF rapidly increased phosphorylation of cytoskeleton-associated FAK on Y577 (Fig. 7B
), which is located in the kinase catalytic domain and required for maximal activity; in EDG-1-/- MEFs, PDGF had no effect on tyrosine phosphorylation of FAK, which appeared to be constitutively hyperphosphorylated (Fig. 7B
). In agreement with previous studies demonstrating that FAK functions as part of a large cytoskeleton-associated network of signaling proteins, which includes the Src family tyrosine kinases, p130Cas, and paxillin (41)
, PDGF rapidly induced translocation of p130Cas and paxillin to focal adhesions in wild-type MEFs (Fig. 7C
). Similar to FAK-/- cells, levels of the focal adhesion components paxillin and Cas associated with the cytoskeleton appeared to be enhanced in EDG-1-/- cells and not regulated by PDGF (Fig. 7C
).
Edg-1 deletion abrogates PDGF-induced Src activation
Because active recruitment and activation of Src-family protein tyrosine kinases (Src, Yes, and Fyn, hereafter referred to as Src) to FAK at its phosphorylated Y397 site may be the first of several signaling events necessary to promote PDGF-stimulated cell migration (32
, 36
, 42
, 43)
, it was important to determine whether the migration defect might also be related to activation of Src. In wild-type MEFs, similar to previous reports (reviewed in ref 34
), PDGF induced activation of cytoskeleton-associated Src within 5 min (Fig. 8
A), as determined by Western blotting with an antibody specific for phosphotyrosine 418, an autophosphorylation site located in the Src catalytic domain required for full activity. In contrast, basal Src activation was higher and PDGF did not further increase Y418 phosphorylation in the EDG-1 deleted cells, even after 60 min. Identical results were obtained with immortalized fibroblasts (Fig. 8B
). In agreement with previous suggestions that a pertussis toxin sensitive Gi-protein regulates activation of Src by PDGF in airway smooth muscle cells (44
, 45)
, we found that pertussis toxin inhibited PDGF-induced activation of Src, Fyn, and Yes in wild-type cells (Fig. 8C
). Although disruption of EDG-1 abrogated the ability of PDGF to stimulate Src, it only had a small effect on Fyn activation (Fig. 8C
). Taken together, these results suggest that activation of FAK and Src by PDGF is aberrant in the absence of EDG-1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Further support for this notion recently emerged from the demonstration that PDGFR is tethered to EDG-1 providing a platform for integrative signaling by these receptors (46)
. According to this paradigm, stimulation of PDGFR by PDGF activates sphingosine kinase resulting in increased formation of SPP, which in turn stimulates EDG-1 leading to activation of downstream signals critical for cell locomotion (see below). This type of cross-communication seems to be important for microvasculature maturation during development (17)
, when PDGFR-ß positive pericytes are recruited to capillaries from progenitors in the vessel walls through the action of PDGF-BB secreted from endothelial cells (47
, 48)
. EDG-1-, PDGF-BB-, and PDGFR-ß-deficient embryos all lack microvascular pericytes/smooth muscle cells surrounding their vessel walls, causing their rupture and massive hemorrhaging (17
, 47
, 48)
. Dysfunctional migration of EDG-1-/- cells toward PDGF links these phenotypes at the final steps of vasculogenesis, underscoring the importance of endothelial cell–pericyte communication in vascular maturation. Moreover, our findings that EDG-1 plays a critical role in directed cellular motility may provide the underlying mechanism of the newly discovered role of SPP in in vivo angiogenesis (7
, 12
, 14)
. Whether other growth factors such as epidermal growth factor (EGF) and VEGF use similar mechanisms to trans-activate EDG-1 remains to be determined.
How does EDG-1 signaling so profoundly affect directed cell movement?
EDG-1 plays a critical role in integrating the responses of several key elements of directed cell movement, including Rac (18)
, important for lamellipodia formation at the leading edge (15
, 16
, 35)
; FAK and Src, which are necessary for formation and turnover of focal complexes (31
32
33
, 36)
; and stress-activated protein kinase-2 (p38), which is involved in actin reorganization and PDGF-induced cell migration (24)
.
Although the mechanism whereby the Gi-linked receptor EDG-1 regulates Rac activation in response to PDGF is not well understood, both tyrosine kinases of the Src family and PI3K link Gß complexes to activation of Rac via regulation of guanine nucleotide exchange factors (GEFs; reviewed in ref 49
). One of these, T lymphoma invasion and metastasis gene 1 (Tiam1), a specific GEF for Rac (50)
, might be involved as it is activated by both PDGF and SPP/EDG-1 signaling (14
, 51)
. Alternatively, when activated by Gß or recruitment to the membrane mediated by ß-arrestin (52
, 53)
or by binding of Gai to its catalytic domain (54)
, Src can directly phosphorylate Ras-GRF1, thereby inducing GEF activity toward Rac (55)
. Another candidate for a role in Rac activation is the Cas/Crk complex formed downstream of Src and/or FAK activation (56)
, as we previously showed that inhibitors of sphingosine kinase suppressed PDGF-induced Crk phosphorylation but did not affect PDGFR autophosphorylation or phosphorylation of the adaptor protein Shc (57)
. One downstream target of Rac is p38 (58)
. Indeed, binding of SPP to EDG-1 in several cell types has been shown to activate p38 (7)
and inhibitors of this MAP kinase, but not MEK1, the kinase directly upstream of ERK1/2, inhibit motility responses induced by SPP/EDG-1 (59)
. Migration of fibroblasts toward PDGF is inhibited by expression of dominant-negative Rac, whereas blocking the ERK pathway by dominant-negative MEK1 did not inhibit migration toward PDGF (60)
. By contrast, migration toward fibronectin was dependent on the ERK pathway but not on Rac, concordant with our finding that EDG-1 deletion also did not markedly affect migration toward fibronectin, indicating that EDG-1 is not important for haptotaxis. Similar to our results, MEFs from mice with a disruption of the gene encoding G13, which resulted in vascular system defects, also showed greatly impaired migratory responses to thrombin but not to fibronectin (61)
.
Activation of EDG-1 by SPP stimulates tyrosine phosphorylation of FAK (62)
and chemotaxis (5
, 62)
. Recent evidence indicates that PDGF promotes phosphorylation of FAK at Y397, creating an SH2-binding site that acts as a ‘switchable adaptor’ to recruit Src to focal adhesion complexes (36)
. FAK phosphorylation at this indispensable Src binding site functions as part of the cytoskeleton-associated network of signaling molecules downstream not only of PDGFR, but also of integrin and GPCRs, to regulate cell motility (34
, 36)
. We previously showed that autophosphorylation of FAK on Y397 is essential for regulation of cell motility by SPP (63)
.
Migratory deficits were noted in cells lacking Src (64)
or FAK and reintroduction of FAK, but not unphosphorylatable mutant Y397F FAK, in FAK-deficient cells restored their ability to migrate (36)
. Because the tyrosine kinase activity of Src has been shown to promote turnover of focal contacts (33)
, the aberrant cell migration reflects defects in focal adhesion turnover. Notably, PDGF-induced focal adhesion complexes, tyrosine phosphorylation, as well as activation of cytoskeleton-associated Src and FAK, were all dysregulated in the absence of EDG-1. This indicates that recruitment and activation of Src by PDGF depend on activation of EDG-1. These data provide an explanation for the observation that pertussis toxin inhibits activation of Src by PDGF (44
, 45)
. However, it is unlikely that Src is solely responsible for the migratory defects, as it was recently shown that triple null mutations of Src, Yes, and Fyn (64)
, in contrast to the effect of FAK-/- (31
, 36)
, had little effect on PDGF-induced cell migration.
G-protein-coupled receptor signaling at the leading edge
An early event that marks directional responses of cells is the restricted translocation of the pleckstrin homology (PH) domain containing proteins (indicative of local generation of PIP3) in a manner similar to the polarity of distribution of Gß subunits along the leading edge (37
, 38)
. However, the asymmetric redistribution of ß subunits is not sufficiently localized to restrict events to the leading edge (38)
, and it has been suggested that chemoattractant-associated PH recruitment requires an intermediate pathway dependent on the activity of one or more of the small GTPases (39)
. The data presented here identify a new mechanism that impinges on the signaling cascade that brings about this steep signaling gradient. A tantalizing notion is that recruitment of sphingosine kinase to membrane ruffles and local generation of SPP may convert tyrosine kinase receptor signaling into directed migration. Hence, spatially and temporally restricted generation of SPP in response to PDGF results in restricted activation of the GPCR EDG-1, which in turn recruits and activates tyrosine kinases, such as Src and FAK, and the small GTPase Rac at the inner plasma membrane facing the stimulus. This may lead to amplification of signaling at the leading edge of the cell (39)
.
How do cells generate a steep gradient of SPP?
It is reasonable to assume that synthesis and degradation of SPP are differentially regulated. Enhanced formation of SPP by PDGF could be governed by local recruitment and activation of sphingosine kinase at the ruffles, whereas global, rapid, and efficient degradation is catalyzed by several types of lipid phosphate phosphatases. The net result would be an asymmetric buildup of SPP at the site of its formation and localized EDG-1/SPP signaling could play a role in directional responses to chemoattractants.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication July 3, 2001.
Revision received August 22, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 I. D. Jung, J. S. Lee, Y. J. Kim, Y.-I. Jeong, C.-M. Lee, T. Baumruker, A. Billlich, Y. Banno, M. G. Lee, S.-C. Ahn, et al. Sphingosine kinase inhibitor suppresses a Th1 polarization via the inhibition of immunostimulatory activity in murine bone marrow-derived dendritic cells Int. Immunol., April 1, 2007; 19(4): 411 - 426. [Abstract] [Full Text] [PDF]
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 W. T. Gerthoffer Mechanisms of Vascular Smooth Muscle Cell Migration Circ. Res., March 16, 2007; 100(5): 607 - 621. [Abstract] [Full Text] [PDF]
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 D. H. Walter, U. Rochwalsky, J. Reinhold, F. Seeger, A. Aicher, C. Urbich, I. Spyridopoulos, J. Chun, V. Brinkmann, P. Keul, et al. Sphingosine-1-Phosphate Stimulates the Functional Capacity of Progenitor Cells by Activation of the CXCR4-Dependent Signaling Pathway via the S1P3 Receptor Arterioscler Thromb Vasc Biol, February 1, 2007; 27(2): 275 - 282. [Abstract] [Full Text] [PDF]
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 L. Veracini, M. Franco, A. Boureux, V. Simon, S. Roche, and C. Benistant Two distinct pools of Src family tyrosine kinases regulate PDGF-induced DNA synthesis and actin dorsal ruffles J. Cell Sci., July 15, 2006; 119(14): 2921 - 2934. [Abstract] [Full Text] [PDF]
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B. W. Wattenberg, S. M. Pitson, and D. M. Raben The sphingosine and diacylglycerol kinase superfamily of signaling kinases: localization as a key to signaling function J. Lipid Res., June 1, 2006; 47(6): 1128 - 1139. [Abstract] [Full Text] [PDF]
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C. M. Sutherland, P. A. B. Moretti, N. M. Hewitt, C. J. Bagley, M. A. Vadas, and S. M. Pitson The Calmodulin-binding Site of Sphingosine Kinase and Its Role in Agonist-dependent Translocation of Sphingosine Kinase 1 to the Plasma Membrane J. Biol. Chem., April 28, 2006; 281(17): 11693 - 11701. [Abstract] [Full Text] [PDF]
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C. E. Chalfant and S. Spiegel Sphingosine 1-phosphate and ceramide 1-phosphate: expanding roles in cell signaling J. Cell Sci., October 15, 2005; 118(20): 4605 - 4612. [Abstract] [Full Text] [PDF]
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N. C. Hait, S. Sarkar, H. Le Stunff, A. Mikami, M. Maceyka, S. Milstien, and S. Spiegel Role of Sphingosine Kinase 2 in Cell Migration toward Epidermal Growth Factor J. Biol. Chem., August 19, 2005; 280(33): 29462 - 29469. [Abstract] [Full Text] [PDF]
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 Y. Gon, M. R. Wood, W. B. Kiosses, E. Jo, M. G. Sanna, J. Chun, and H. Rosen S1P3 receptor-induced reorganization of epithelial tight junctions compromises lung barrier integrity and is potentiated by TNF PNAS, June 28, 2005; 102(26): 9270 - 9275. [Abstract] [Full Text] [PDF]
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 P. S. Jolly, M. Bektas, K. R. Watterson, H. Sankala, S. G. Payne, S. Milstien, and S. Spiegel Expression of SphK1 impairs degranulation and motility of RBL-2H3 mast cells by desensitizing S1P receptors Blood, June 15, 2005; 105(12): 4736 - 4742. [Abstract] [Full Text] [PDF]
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 Y. Kariya, A. Kihara, M. Ikeda, F. Kikuchi, S. Nakamura, S. Hashimoto, C.-H. Choi, Y.-M. Lee, and Y. Igarashi Products by the sphingosine kinase/sphingosine 1-phosphate (S1P) lyase pathway but not S1P stimulate mitogenesis Genes Cells, June 1, 2005; 10(6): 605 - 615. [Abstract] [Full Text] [PDF]
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S. K. Goparaju, P. S. Jolly, K. R. Watterson, M. Bektas, S. Alvarez, S. Sarkar, L. Mel, I. Ishii, J. Chun, S. Milstien, et al. The S1P2 Receptor Negatively Regulates Platelet-Derived Growth Factor-Induced Motility and Proliferation Mol. Cell. Biol., May 15, 2005; 25(10): 4237 - 4249. [Abstract] [Full Text] [PDF]
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J. H. Finigan, S. M. Dudek, P. A. Singleton, E. T. Chiang, J. R. Jacobson, S. M. Camp, S. Q. Ye, and J. G. N. Garcia Activated Protein C Mediates Novel Lung Endothelial Barrier Enhancement: ROLE OF SPHINGOSINE 1-PHOSPHATE RECEPTOR TRANSACTIVATION J. Biol. Chem., April 29, 2005; 280(17): 17286 - 17293. [Abstract] [Full Text] [PDF]
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 L. Suomalainen, V. Pentikainen, and L. Dunkel Sphingosine-1-Phosphate Inhibits Nuclear Factor {kappa}B Activation and Germ Cell Apoptosis in the Human Testis Independently of Its Receptors Am. J. Pathol., March 1, 2005; 166(3): 773 - 781. [Abstract] [Full Text] [PDF]
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 S. M. Pitson, P. Xia, T. M. Leclercq, P. A.B. Moretti, J. R. Zebol, H. E. Lynn, B. W. Wattenberg, and M. A. Vadas Phosphorylation-dependent translocation of sphingosine kinase to the plasma membrane drives its oncogenic signalling J. Exp. Med., January 3, 2005; 201(1): 49 - 54. [Abstract] [Full Text] [PDF]
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A. Angers-Loustau, R. Hering, T. E. Werbowetski, D. R. Kaplan, and R. F. Del Maestro Src Regulates Actin Dynamics and Invasion of Malignant Glial Cells in Three Dimensions Mol. Cancer Res., November 1, 2004; 2(11): 595 - 605. [Abstract] [Full Text] [PDF]
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B. Sauer, R. Vogler, H. von Wenckstern, M. Fujii, M. B. Anzano, A. B. Glick, M. Schafer-Korting, A. B. Roberts, and B. Kleuser Involvement of Smad Signaling in Sphingosine 1-Phosphate-mediated Biological Responses of Keratinocytes J. Biol. Chem., September 10, 2004; 279(37): 38471 - 38479. [Abstract] [Full Text] [PDF]
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W. Wu, R. D. Mosteller, and D. Broek Sphingosine Kinase Protects Lipopolysaccharide-Activated Macrophages from Apoptosis Mol. Cell. Biol., September 1, 2004; 24(17): 7359 - 7369. [Abstract] [Full Text] [PDF]
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H. Le Stunff, A. Mikami, P. Giussani, J. P Hobson, P. S. Jolly, S. Milstien, and S. Spiegel Role of Sphingosine-1-phosphate Phosphatase 1 in Epidermal Growth Factor-induced Chemotaxis J. Biol. Chem., August 13, 2004; 279(33): 34290 - 34297. [Abstract] [Full Text] [PDF]
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 R. E. Toman, S. G. Payne, K. R. Watterson, M. Maceyka, N. H. Lee, S. Milstien, J. W. Bigbee, and S. Spiegel Differential transactivation of sphingosine-1-phosphate receptors modulates NGF-induced neurite extension J. Cell Biol., August 2, 2004; 166(3): 381 - 392. [Abstract] [Full Text] [PDF]
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A. Olivera, H. M. Rosenfeldt, M. Bektas, F. Wang, I. Ishii, J. Chun, S. Milstien, and S. Spiegel Sphingosine Kinase Type 1 Induces G12/13-mediated Stress Fiber Formation, yet Promotes Growth and Survival Independent of G Protein-coupled Receptors J. Biol. Chem., November 21, 2003; 278(47): 46452 - 46460. [Abstract] [Full Text] [PDF]
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H. Liu, R. E. Toman, S. K. Goparaju, M. Maceyka, V. E. Nava, H. Sankala, S. G. Payne, M. Bektas, I. Ishii, J. Chun, et al. Sphingosine Kinase Type 2 Is a Putative BH3-only Protein That Induces Apoptosis J. Biol. Chem., October 10, 2003; 278(41): 40330 - 40336. [Abstract] [Full Text] [PDF]
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 D. GRATZINGER, S. CANOSA, B. ENGELHARDT, and J. A. MADRI Platelet endothelial cell adhesion molecule-1 modulates endothelial cell motility through the small G-protein Rho FASEB J, August 1, 2003; 17(11): 1458 - 1469. [Abstract] [Full Text] [PDF]
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H. Le Stunff, I. Galve-Roperh, C. Peterson, S. Milstien, and S. Spiegel Sphingosine-1-phosphate phosphohydrolase in regulation of sphingolipid metabolism and apoptosis J. Cell Biol., September 16, 2002; 158(6): 1039 - 1049. [Abstract] [Full Text] [PDF]
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E. Lacana, M. Maceyka, S. Milstien, and S. Spiegel Cloning and Characterization of a Protein Kinase A Anchoring Protein (AKAP)-related Protein That Interacts with and Regulates Sphingosine Kinase 1 Activity J. Biol. Chem., August 30, 2002; 277(36): 32947 - 32953. [Abstract] [Full Text] [PDF]
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S. Spiegel and S. Milstien Sphingosine 1-Phosphate, a Key Cell Signaling Molecule J. Biol. Chem., July 12, 2002; 277(29): 25851 - 25854. [Full Text] [PDF]
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