Ras activation of a Rac1 exchange factor, Tiam1, mediates neurotrophin-3-induced Schwann cell migration
- *Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305-5125; †Department of Pharmacology, National Research Institute for Child Health and Development, Setagaya, Tokyo 157-8535, Japan; and §Department of Cell and Neurobiology, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089
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Contributed by Eric M. Shooter, August 16, 2005
Abstract
Endogenous neurotrophins positively and negatively regulate migration of premyelinating Schwann cells before the initiation of myelination. Neurotrophin-3 (NT3) acting through the TrkC receptor tyrosine kinase stimulates Schwann cell migration via the Rho GTPases Rac1 and Cdc42. We previously demonstrated that TrkC directly phosphorylates and activates Dbs, the guanine-nucleotide exchange factor (GEF) for Cdc42, to partially mediate Schwann cell migration. Here, we identify T lymphoma invasion and metastasis (Tiam) 1 as the Rac1-specific guanine-nucleotide exchange factor involved in NT3-induced Schwann cell migration. Furthermore, the interaction between the small GTPase Ras and Tiam1 plays an essential role in the activation of Rac1. Taken together, these results suggest that NT3 activation of TrkC stimulates Schwann cell migration through two parallel signaling units, Ras/Tiam1/Rac1 and Dbs/Cdc42, and that Schwann cell migration is uniquely regulated in the case of Ras and Rac1, by two different types of small GTPases.
Continuous glial-neuronal interactions play essential roles in forming the mature nervous system (1). Neurotrophins constitute a critical family of proteins governing the development of neurons (2-4). During the development of the peripheral nervous system, Schwann cells proliferate, migrate along axons, and finally differentiate to form the myelin sheath (1). Previously, we reported that endogenous neurotrophin-3 (NT3) stimulates migration of premyelinating Schwann cells and inhibits myelination through the receptor tyrosine kinase, TrkC, on Schwann cells (5-8). The mechanism by which the NT3 activation of TrkC stimulates Schwann cell migration involves the Rho GTPases Rac1 and Cdc42 and the downstream c-Jun N-terminal kinase (JNK), a subfamily of mitogen-activated protein kinases (MAPKs) (7).
Rho GTPases cycle between inactive (GDP-bound) and active (GTP-bound) conformations, as do small GTPases of the Ras family (9). When active, Rho GTPases Rac1, Cdc42, and RhoA alter the cytoskeleton to mediate various cellular processes, including morphological changes and cell migration (9, 10). The exchange reactions of bound GDP for GTP by Rho GTPases are regulated by ≈70 guanine-nucleotide exchange factors (GEFs) related to the product of the diffusible B cell lymphoma (dbl) gene (9). Rho GTPase-GEFs typically contain a Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain and domains responsible for interactions with other proteins (9). The DH domain is sufficient for catalyzing guanine-nucleotide exchange, and the PH domain helps modulate the activity of the DH domain by binding a phosphoinositide (9). The catalytic activities of Rho GTPase-GEFs are strictly regulated by posttranslational modifications and/or protein-protein interactions upon stimulation with extracellular signals (9).
Despite the recent identification of Dbl's big sister (Dbs) (11, 12) as the Cdc42-GEF in Schwann cell migration induced by NT3 activation of TrkC, there has still been a missing link between TrkC and the other Rho GTPase Rac1 (13). We previously demonstrated that, whereas Dbs is essential for Schwann cell migration via JNK, the inhibition of Dbs is not sufficient to completely inhibit migration, as well as JNK activation. Therefore, the elucidation of the responsible Rac1-GEF(s) and its activation mechanism is required to fully understand the role of NT3 in Schwann cell migration. Here, we show that T lymphoma invasion and metastasis (Tiam) 1 (14-16) is the Rac1-specific GEF involved in Schwann cell migration. Furthermore, the interaction of Tiam1 with Ras·GTP, which is achieved upon stimulation with NT3, is required for the formation of Rac1·GTP. In the present study, we provide an example of a physiological role for the Ras/Tiam1/Rac1/JNK-signaling pathway in the peripheral nervous system. These results suggest that the functional interaction between two different types of small GTPases plays a key role in controlling the early stages of Schwann cell maturation, ultimately leading to myelination.
Methods
For details, see Supporting Materials and Methods, which is published as supporting information on the PNAS web site.
Cell Culture. Primary Schwann cells were prepared from sciatic nerves of Sprague-Dawley rats at postnatal day 2 (5, 6). Schwann cells were cultured on polylysine-coated dishes in DMEM containing 10% heat-inactivated FBS and 10 μg/ml gentamicin at 37°C and were plated for experiments on collagen (type I)-coated dishes. Before performing experiments, we cultured Schwann cells in Sato medium containing 1 mg/ml BSA for 24 h (7, 8, 12). Cos-7 and 293T cells were cultured on cell culture dishes in DMEM containing 10% FBS, 50 units/ml penicillin, and 50 μg/ml streptomycin, and Cos-7 cells were plated for experiments on collagen-coated dishes. Before performing experiments, we cultured Cos-7 cells in DMEM containing 1% FBS and 1 mg/ml BSA for 24 h. Unless otherwise indicated, Schwann cells and Cos-7 cells were pretreated with or without 100 nM K252a (Calbiochem-Novabiochem) for 45 min before stimulation with 10 ng/ml NT3 (Regeneron Pharmaceuticals, Tarrytown, NY) for 30 min. To confirm cell viability under these experimental conditions, Schwann cells and Cos-7 cells were stained with 0.4% trypan blue. Trypan blue-incorporating cells numbered fewer than 1% in each experiment.
Plasmid Transfection. pCMV-FLAG-H-Ras was transfected into Schwann cells by using the Lipofectamine-Plus reagent (Invitrogen), according to the manufacturer's protocol. Cos-7 cells were transfected by the method of calcium phosphate precipitation. In Cos-7 cells, the final amount of the transfected DNA for a 6-cm dish was adjusted to 10 μg by addition of empty vector, pCMV. pCMV-H-Ras (1 μg), pCMV-FLAG-Tiam1 (393-1591) (0.5 μg), pCMV-Rac1 (1 μg), pCMV-Cdc42 (1 μg), or SRα-HA-JNK1 (1 μg) were cotransfected with pCMV-TrkC (0.5 μg), pCMV-FLAG-Tiam1 constructs (0.5-5 μg), or pCMV-H-Ras constructs (1 μg). The medium was replaced 24 h after transfection, and cells were cultured in DMEM containing 1% FBS and 1 mg/ml BSA for 24 h. Transfection efficiency typically exceeded 98% by using pEGFP-C1 as the control.
Small Interfering RNA (siRNA) Transfection. The siRNA was transfected into primary Schwann cells by using the Oligofectamine reagent (Invitrogen), according to the manufacturer's protocol. The medium was replaced 24 h after transfection, and cells were cultured in Sato medium containing 1 mg/ml BSA for 24 h. The efficiencies of Tiam1 depletion were 92 ± 5.5% for the Tiam1 siRNA and 53 ± 8.1% for the H-Ras siRNA.
Boyden Chamber Migration Assay. Cell migration was routinely measured by using a 24-well Boyden chamber (BD Biosciences), as described (7, 8, 12). Briefly, polyethylene terephthalate (8-μm pore size) filters were coated with collagen. Cells (0.5 × 105 cells for Schwann cells or 1 × 105 cells for Cos-7 cells) in 500 μl of medium per well were loaded into the upper chambers, which were inserted into the tissue culture wells containing 25 ng/ml NT3 in 750 μl of medium per well. After incubation at 37°C for 6 h, the filters were stained with Giemsa solution. The number of stained, migrating cells at the bottom surface of the filters was counted at four fields per filter in two to four independent experiments.
Immunoprecipitation and Immunoblotting. Cells were lysed in 200 μl (for 35-mm dishes) or 600 μl (for 60-mm dishes) of lysis buffer A and were centrifuged (7, 8, 12). Aliquots of the supernatants were mixed with protein G resin preadsorbed with various antibodies. The immunoprecipitates or the proteins in the cell lysates were denatured, separated on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, blocked, and immunoblotted.
Pull-Down Assays for Ras, Rac1, Cdc42, and Rac1-GEF Tiam1. See Supporting Materials and Methods.
Assay for JNK. Immunoprecipitated JNK was immunoblotted with anti-phospho-JNK antibody, which recognizes the active form (7, 8, 12). To compare the total amount of JNK, the immunoblotting was also performed with an anti-JNK antibody. Three to five separate experiments were carried out, and a representative experiment is shown in Figs. 6 and 7, which are published as supporting information on the PNAS web site. Levels of the phosphorylated forms were normalized to the amount of total kinase.
Immunofluorescence. Schwann cells on collagen-coated glass coverslips were fixed with 4% paraformaldehyde, blocked with 20% goat serum in PBS-0.1% Tween-20, incubated with primary antibodies, and treated with fluorescently labeled secondary antibodies, as described (8, 12). The primary antibodies used were as follows: anti-Tiam1 and anti-FLAG to identify FLAG-H-Ras.
Statistical Analysis. Values shown represent the mean ± SD from separate experiments. Student's t test was carried out for intergroup comparisons. ANOVA was followed by Fisher's PLSD (protected least significant difference) post hoc comparisons.
Results
The Rac1-Specific GEF Tiam1 Mediates Schwann Cell Migration Induced by NT3 Activation of TrkC. We tested the possibility that Tiam1, the major Rac1-specific GEF in primary Schwann cells, mediates Schwann cell migration induced by stimulation with NT3. We transfected a Tiam1 siRNA oligonucleotide to knock down endogenous Tiam1 expression in Schwann cells. Expression of Tiam1 was markedly down-regulated by transfection with Tiam1 siRNA, whereas expression of Rac1 and β-actin was unaffected, as revealed by immunoblotting (Fig. 1C). Migration was measured by using Boyden chambers. We placed Schwann cells onto the filters in the upper well and allowed them to migrate out onto the lower compartment along a concentration gradient of NT3 for 6 h. The knockdown of Tiam1 inhibited NT3-induced Schwann cell migration by ≈35% (Fig. 1 A and B). To further confirm that NT3 activation of TrkC enhances cell migration through Tiam1, we used a fragment (amino acids 393-841) of Tiam1. Tiam1 (393-841) contains multiple motifs (PH domain and the Ras-binding domain) that interact with various regulatory proteins to elicit dominant-negative effects (14-16). Transfection of Tiam1 (393-841) inhibited NT3-induced migration of Cos-7 cells cotransfected with TrkC by ≈35% (Fig. 1 D and E). Interestingly, amino acids 720-841, which contain only the Ras-binding domain of Tiam1, showed the same dominant-negative effect as Tiam1 (393-841) (Fig. 1G). When transfected, FLAG-tagged constructs of Tiam1 were detectable with an anti-FLAG antibody (Figs. 1 F and H and 8, which is published as supporting information on the PNAS web site). Because the N-terminal region of Tiam1 was difficult to express in cells (14, 15), we could not determine whether the N terminus of Tiam1 had any effect on migration, although it participates in the turnover of Tiam1 protein (16). These results indicate that Tiam1 mediates Schwann cell migration induced by NT3 activation of TrkC and that this signal can be transduced by amino acids 720-841 of Tiam1.
Tiam1 mediates Schwann cell migration induced by NT3 activation of TrkC. (A and B) Schwann cells were transfected with control or Tiam1 siRNA. After incubation with NT3, migration was assayed by using Boyden chambers. (C) To confirm the effect of Tiam1 siRNA, the lysates from transfected cells were immunoblotted with anti-Tiam1, Rac1, or β-actin antibody. (D and E) Cos-7 cells were transfected with TrkC with or without FLAG-Tiam1 (393-841). After incubation with NT3, migration was assayed. (F) To confirm the expression of FLAG-Tiam1 (393-841), the lysates from transfected cells were immunoblotted with anti-FLAG or β-actin antibody. (G) Cos-7 cells were transfected with TrkC with or without FLAG-tagged Tiam1 constructs, and migration was assayed. (H) To confirm the expression of FLAG-tagged Tiam1 constructs, the lysates from transfected cells were immunoblotted with anti-FLAG or β-actin antibody. Data were evaluated by using Student's t test (B and E; *, P < 0.005) and one-way ANOVA (G; *, P < 0.005).
Rac1 and JNK Activation Depends on Tiam1. Because NT3 enhances Schwann cell migration through Rac1 and the downstream JNK (7), we examined whether NT3 activates Rac1 and JNK through Tiam1. We carried out a pull-down assay to detect active GTP-bound Rac1 using the isolated Rac1·GTP and Cdc42-binding domain of αPak. Transfection of Tiam1 siRNA into Schwann cells blocked the NT3-induced activation of endogenous Rac1 (Fig. 2 A and B). Similarly, the amino acids 720-841 of Tiam1, as well as Tiam1 (393-841), blocked the NT3-induced Rac1 activation in Cos-7 cells cotransfected with Rac1 and TrkC (Fig. 2 C-E). The inhibition of Tiam1 had no effect on Cdc42 activation (Fig. 9, which is published as supporting information on the PNAS web site). JNK activity was measured by detecting active, phosphorylated JNK from immunoprecipitated JNK. Knockdown of Tiam1 in Schwann cells inhibited the NT3-induced activation of endogenous JNK by ≈50% (Fig. 6 A and B). Similarly, the amino acids 720-841 of Tiam1, as well as Tiam1 (393-841), inhibited the NT3-induced JNK activation in Cos-7 cells cotransfected with JNK and TrkC (Fig. 8 C-E). Collectively, these results indicate that Tiam1 mediates the signaling pathway linking NT3 activation of TrkC to migration possibly through amino acids 720-841. Although it is clear that amino acids 720-841 of Tiam1 participate in this signal transduction pathway, the amino acids 393-726 of Tiam1 (PH domain) are less effective (Figs. 1G, 2E, and 8E). Because amino acids 393-726 of Tiam1 can associate with various proteins (16), it is still possible that these binding partners may play a partial role in this signal transduction.
Tiam1 is essential for the Rac1 activation induced by NT3. (A and B) Schwann cells were transfected with control or Tiam1 siRNA. After stimulation with NT3, Rac1 activity, which is estimated by the accumulation of Rac1-GTP, was measured with a pull-down assay by using GST-αPak-CRIB in the lysates. The total Rac1 in the cell lysates is shown. (C and D) Cos-7 cells were transfected with Rac1 and TrkC with or without FLAG-Tiam1 (393-841). Rac1 activity was measured with the pull-down assay. The total Rac1 in the cell lysates is also shown. (E) Cos-7 cells were transfected with Rac1 and TrkC with or without FLAG-tagged Tiam1 constructs, and Rac1 activity was measured. Data were evaluated by using Student's t test (B and D; *, P < 0.005) and one-way ANOVA (E; *, P < 0.005).
Next, we performed pull-down assays to detect active Tiam1 by using Rac1 G15A, a guanine-nucleotide-free form of Rac1 (17). Active GEFs preferentially interact with guanine-nucleotide-free forms of the small GTPases (17). A glycine to alanine point mutation of residue 15 in Rac1 decreases its nucleotide binding. After stimulation with NT3, endogenous Tiam1 precipitated with Rac1 G15A was significantly increased in Schwann cells (Fig. 3 A and B) and, in Cos-7 cells, cotransfected with Tiam1 and TrkC (Fig. 3 D and E). K252a, an inhibitor of the Trk receptor tyrosine kinase, blocked these effects (Fig. 3 C and F), suggesting that NT3 acting through TrkC leads to activation of Tiam1.
NT3 activates Tiam1. (A and B) Stimulation with NT3 increased the amount of Tiam1 precipitated with GST-Rac1G15A from the lysates of Schwann cells. The total Tiam1 in the cell lysates is shown. (C) K252a, a Trk tyrosine kinase inhibitor, reduces the amount of Tiam1 precipitated from the lysates of Schwann cells. (D and E) After stimulation with NT3, increased Tiam1 was detected by the pull-down assay in the lysates of Cos-7 cells transfected with FLAG-Tiam1 (393-1591) and TrkC. The total FLAG-Tiam1 (393-1591) in the cell lysates is also shown. (F) K252a reduces the amount of FLAG-Tiam1 (393-1591) precipitated from the lysates of transfected Cos-7 cells. Data were evaluated by using Student's t test (*, P < 0.005).
Ras Is Essential for Tiam1 Activation. Neurotrophins activate Ras in various types of cells (4, 18). We demonstrate that the Ras-binding domain (720-841) of Tiam1 (16, 19) is important in the signaling pathway coupling NT3 activation of TrkC to Schwann cell migration (Figs. 1, 2, and 6). We therefore explored whether Ras regulates NT3-induced migration. Transfection with H-Ras siRNA reduced expression of total Ras protein, whereas expression of Tiam1, Rac1, and β-actin was unaffected (Fig. 4B). Knockdown of H-Ras inhibited NT3-induced migration of Schwann cells by ≈30% (Fig. 4A). Similarly, transfection of dominant-negative H-Ras (H-RasS17N) (Fig. 4D) inhibited NT3-induced migration of Cos-7 cells cotransfected with TrkC (Fig. 4C). Activation of Tiam1, Rac1, and JNK was also inhibited by transfection with H-Ras siRNA (Figs. 4 E and G and 7A) and in Cos-7 cells cotransfected with H-RasS17N (Figs. 4 F and H and 7B). These results indicate that Ras regulates Tiam1 in the signaling pathway coupling NT3 activation of TrkC to migration.
Ras is required for the NT3-induced Tiam1 activation. (A) Schwann cells were transfected with control or H-Ras siRNA. After stimulation with NT3, migration was assayed by using Boyden chambers. (B) To confirm the effect of H-Ras siRNA, the lysates from transfected cells were immunoblotted with anti-Ras, Tiam1, Rac1, or β-actin antibody. (C) Cos-7 cells were transfected with TrkC with or without H-RasS17N, and migration was assayed. (D) To confirm the expression of H-RasS17N, the lysates from transfected cells were immunoblotted with anti-FLAG or β-actin antibody. (E) Schwann cells were transfected with control or H-Ras siRNA, and Rac1 precipitated with GST-αPak-CRIB was measured. (F) Cos-7 cells were transfected with Rac1 and TrkC with or without H-RasS17N, and Rac1 activity was measured. (G) Schwann cells were transfected with control or H-Ras siRNA, and Tiam1 precipitated with GST-Rac1G15A was measured. (H) Cos-7 cells were transfected with FLAG-Tiam1 (393-1591) and TrkC with or without H-RasS17N, and FLAG-Tiam1 (393-1591) precipitated with GST-Rac1G15A was measured. Data were evaluated by using Student's t test (*, P < 0.005; **, P < 0.01).
As shown in Fig. 5A, Tiam1 (720-841) has the ability to specifically associate with RasG12V, which mimics the GTP-bound form, but not to RasS17N, which mimics the GDP-bound form. We thus carried out a pull-down assay using the Ras·GTP-binding domain of Tiam1 to clarify whether Ras is activated after stimulation with NT3. NT3/TrkC led to activation of Ras (Fig. 5 B, C, E, and F) and K252a inhibited this activation (Fig. 5 D and G). Additionally, stimulation with NT3 enhanced the association and colocalization of Tiam1 with H-Ras in Schwann cells (Fig. 5 H and I). Taken together, NT3 activation of TrkC stimulates Ras activity and then induces Tiam1 function to ultimately activate Rac1 and Schwann cell migration.
NT3 activates Ras. (A) Cos-7 cells were transfected with FLAG-H-RasG12V (GTP-bound form) or FLAG-H-RasS17N (GDP-bound form). RasG12V, but not RasS17N, was detected with the pull-down assay by using GST-Tiam1 (720-841). (B and C) Ras activity was measured with the pull-down assay by using GST-Tiam1 (720-841) in the lysates of Schwann cells. (D) The effect of K252a on Ras activation in the lysates of Schwann cells was measured. (E and F) Ras activity was measured a the pull-down assay in the lysates of Cos-7 cells transfected with H-Ras and TrkC. (G) The effect of K252a on Ras activation in the lysates of transfected Cos-7 cells was analyzed. (H) After stimulation with NT3, Tiam1 immunoprecipitates were immunoblotted with antibodies against Ras and Tiam1. Total Ras in the cell lysates is also shown. (I) Schwann cells were transfected with FLAG-H-Ras, incubated with NT3, and stained with antibodies against Tiam1 (red) and the FLAG peptide (green). Stimulation with NT3 increased colocalization. Arrows indicate structures where Tiam1 and H-Ras colocalize. Data were evaluated by using Student's t test (*, P < 0.005).
Discussion
Trk receptors play a crucial role in supporting the survival and differentiation of neurons through the Ras GTPase and the downstream extracellular-regulated protein kinase (ERK), a subfamily of mitogen-activated protein kinases (3, 4). Increasing evidence now suggests important roles for the Trk and p75 receptors in nonneuronal systems. We previously showed that endogenous NT3 acting through TrkC stimulates Schwann cell migration via the signaling cascade coupling Rho GTPases Rac1 and Cdc42 to JNK activation. In the present study, we identify the Rac1-specific GEF Tiam1 as a key mediator of TrkC-induced migration. This conclusion is supported by the finding that knocking down Tiam1 with siRNA or transfection with the dominant-negative form inhibits the downstream signaling pathway from Rac1 to the induction of migration, and that stimulation of TrkC induces the activation of Tiam1. Importantly, Tiam1 activation of Rac1 requires Ras, because knocking down H-Ras with siRNA or transfection with dominant-negative H-Ras suppresses this migratory signaling pathway, and that TrkC stimulation activates Ras significantly. Taken together, these results demonstrate that the interaction between two different types of small GTPases Ras and Rac1 is required for neurotrophin-induced migration of primary Schwann cells. This example is reminiscent of the well-known interaction of Ras with the small GTPase Ral (20). Ras activates Ral through the direct association with the Ral-GEF Ral-GDP dissociation stimulator (GDS) to induce various cellular functions. Interestingly, recent biochemical and bioinformatic research shows that this interaction between Ras and the GEFs for the Rho family of small GTPases (9) is emerging as a common and generalizable signaling pathway. Further experiments concerning the interaction between Ras and the GEFs for the Rho GTPases should allow us to clarify the roles of these GEFs not only in migration but also in many other biological processes.
Tiam1 acts upstream of Rac1 in this signaling pathway of TrkC-induced migration, but it is not the only Rac1-specific GEF that is expressed in primary Schwann cells. Two other Rac1-specific GEFs are the Ras-GRF2 and mSos1/2, which are expressed in Schwann cells. Ras-GRF and mSos contain two different GEF domains, thereby acting as dual GEFs for Rac1 and Ras (21). Specific upstream signals induce the specific GEF activities of Ras-GRF and mSos. Induction of the Rac1-GEF activity of Ras-GRF depends on tyrosine phosphorylation by the Src family (22). It is unlikely that the Rac1 activation by TrkC involves Ras-GRF, because PP1, a specific inhibitor of the Src family, has no effect on the Rac1 activation in Schwann cells (ref. 8 and data not shown). On the other hand, induction of the Rac1-GEF activity of mSos requires the activity of phosphatidylinositol (PI) 3-kinase through two distinct mechanisms (21). The first scenario includes the binding of PI-3,4,5-triphosphate to the PH domain of mSos that is required to induce the Rac1-GEF activity (21). In the second scenario, mSos forms a trimeric complex with the adaptor proteins Abi1 and Eps8, which then induces Rac1-GEF activity (21). Innocenti et al. (23) have also reported that the recruitment of p85, a subunit of PI 3-kinase, to the trimeric complex and the subsequent production of PI-3,4,5-triphosphate are essential for the induction of the Rac1-GEF activity (23). Both pathways are dependent on PI3-kinase for the induction of Rac1 activation. However, because wortmannin, a PI3-kinase inhibitor, has no effect on the Rac1 activation by TrkC in Schwann cells (data not shown), it is unlikely that Rac1 activation by TrkC involves mSos.
Ras-GRF and mSos were originally identified as the conserved GEFs for the Ras family. Although they are not involved in the TrkC-induced Rac1 activation, Ras-GRF and mSos may act upstream of the Ras-GEFs in the Ras/Tiam1/Rac1 cascade. MacDonald et al. (24) identified Ras-GRF as a TrkA-binding partner. TrkA binds and phosphorylates the Ras-GRF through the HIKE motif in the intracellular domain (24). As pointed out by Robinson et al. (25), TrkC also contains the HIKE motif (532 HIKRRDIV-LKRE543). It is conceivable that TrkC interacts with Ras-GRF to stimulate Ras-GEF activity in primary Schwann cells. It has been widely reported that neurotrophin binding to the Trk receptors recruits the adaptor protein Grb2-mSos complex directly or through the Shc family adaptor proteins to tyrosine-phosphorylated intracellular domains of the receptors (26, 27). Like other receptor tyrosine kinases, this step is crucial for the interaction of the Grb2-mSos complex with Ras on the plasma membrane (26, 27). Ras-GRF and/or mSos could be candidates coupling the Ras GEFs to NT3/TrkC and Ras activation in Schwann cells.
In this study, we demonstrate that NT3 activation of TrkC increases the formation of Ras·GTP to stimulate the activity of the Rac1-specific GEF Tiam1, thereby enhancing migration of premyelinating Schwann cells. Concomitant with Tiam1 activation, this signaling pathway requires the Cdc42-GEF Dbs (12) for complete activation of JNK activity and migration. On the basis of these findings, we summarize the proposed signaling pathway in Fig. 10, which is published as supporting information on the PNAS web site. Importantly, NT3 also has the ability to inhibit myelination by Schwann cells in vivo as well as in Schwann cell-neuronal cocultures (5, 6). Further studies on the exact mechanism of Schwann cell migration by the NT3 receptor TrkC should aid in elucidating how NT3 inhibits myelination and in other words the possible relationship between migration and myelination. Recently, the chemical compound NSC23766 has been identified as a first generation Rac1-specific inhibitor that fits into the GTPase-recognition groove of the Rac1-specific GEFs (28). If NSC23766 can be modified to achieve a more specific fit into the Rac1-recognition groove of Tiam1, the small molecule compound as well as the siRNA for Tiam1 should be useful tools for clarifying the roles of Tiam1 in different aspects of the myelination program and also the remyelination process after injury.
Acknowledgments
We thank Drs. W. C. Mobley and Y. Kaziro for insightful discussions and encouragement. We also thank Drs. M. Hoshino, T. Satoh, T. Aoki, H. Maruta, and H. Nakajima for helpful comments. This work was supported by National Institute of Neurological Disorders and Stroke Grant NS 04270, by grants from the Muscular Dystrophy Association and the McGowan Charitable Trust (to E.M.S.), by a National Multiple Sclerosis Society Career Transition Fellowship (to J.R.C.), and by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (to J.Y.).
Footnotes
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↵ ‡ To whom correspondence should be addressed at: Department of Neurobiology, Stanford University School of Medicine, 299 Campus Drive, Stanford, CA 94305-5125. E-mail: eshooter{at}stanford.edu.
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Abbreviations: NT3, neurotrophin-3; JNK, c-Jun N-terminal kinase; GEF, guanine-nucleotide exchange factor; PH, pleckstrin homology; Tiam, T lymphoma invasion and metastasis; siRNA, small interfering RNA; PI, phosphatidylinositol.
- Copyright © 2005, The National Academy of Sciences
