The neurotrophin-3 receptor TrkC directly phosphorylates and activates the nucleotide exchange factor Dbs to enhance Schwann cell migration

Abstract

During the development of the peripheral nervous system, Schwann cells, the myelin-forming glia, migrate along axons before initiating myelination. We previously demonstrated that endogenous neurotrophin-3 (NT3) acting through the TrkC tyrosine kinase receptor enhances migration of premyelinating Schwann cells. This signaling pathway is mediated by the c-Jun N-terminal kinase (JNK) cascade regulated by the Rho GTPases Rac1 and Cdc42. However, missing is the link between TrkC and the GTPases. Here, we show that a guanine-nucleotide exchange factor (GEF), Dbl's big sister (Dbs), couples with TrkC to activate Cdc42 in Schwann cells. Furthermore, TrkC directly phosphorylates Dbs, thereby inducing the Cdc42-GEF activity. Taken together, activation of TrkC triggers Schwann cell migration by regulating Dbs upon direct tyrosine phosphorylation, providing a mechanism whereby a membrane receptor tyrosine kinase can induce the activation of Rho GTPase-GEFs.

Fig. 1.

NT3 activation of TrkC enhances migration of Schwann cells and Cos-7 cells through Dbs. (A, B, and D) Schwann cells were transfected with control or Dbs siRNA. After incubation with NT3, migration was assayed by using Boyden chambers. (C and E) To confirm the effects of the siRNAs, the lysates from transfected cells were immunoblotted with anti-Dbs, JNK, Cdc42, or β-actin antibody. (F and G) Cos-7 cells were transfected with TrkC ± FLAG-DbsΔCat. After incubation with NT3, migration was assayed. (H) To confirm the expression of FLAG-tagged wild-type Dbs or the mutants, the lysates from transfected cells were immunoblotted with anti-FLAG or β-actin antibody. (I and J) Cos-7 cells were transfected with or without FLAG-CA-Dbs, and migration was assayed. Data were evaluated by using Student's t test. *, P < 0.01.

Fig. 2.

Dbs participates in the TrkC-mediated JNK and Cdc42 activation in Schwann cells and Cos-7 cells. (A and B) Schwann cells were transfected with control or Dbs siRNA. (C and D) Cos-7 cells were transfected with HA-JNK and TrkC ± FLAG-DbsΔCat. After stimulation with NT3, JNK was immunoprecipitated (IP) with anti-JNK antibody and immunoblotted with antiphosphorylated JNK or JNK antibody. (E) Cos-7 cells were transfected with HA-JNK ± FLAG-CA-Dbs. The JNK phosphorylation was measured. (F and G) Schwann cells were transfected with control or Dbs siRNA. (H and I) Cos-7 cells were transfected with Cdc42 and TrkC ± FLAG-DbsΔCat. After stimulation with NT3, the Cdc42 activity was measured with the pull-down assay by using GST-αPak-CRIB in the lysates. The total Cdc42 in the cell lysates is also shown. (J) Cos-7 cells were transfected with Cdc42 ± FLAG-CA-Dbs. The Cdc42 activity was measured. Data were evaluated by using Student's t test. *, P < 0.01.

Fig. 3.

Stimulation of the Cdc42-GEF activity of Dbs by NT3 interaction with TrkC. Cos-7 cells transfected with FLAG-Dbs plus TrkC (A–C), HA-Stef (464–1,715) (B), or FLAG-Vav2 (168–379) plus CA-Src (C) were pretreated with or without K252a. After stimulation with NT3, release of [³H]GDP from [³H]GDP-occupied GST-Cdc42, Rac1, and RhoA by the GEF immunoprecipitates was measured. (D and E) After stimulation with NT3, active, tyrosine-phosphorylated FLAG-Dbs was detected by the pull-down assay by using GST-Cdc42G15A from the lysates of Cos-7 cells transfected with FLAG-Dbs and TrkC. The total FLAG-Dbs in the cell lysates is also shown. (F) Effect of K252a on Dbs precipitated from the lysates of Cos-7 cells stimulated with NT3. Active, tyrosine-phosphorylated FLAG-Dbs was detected by the pull-down assay by using GST-Cdc42G15A.

Fig. 4.

TrkC binds, phosphorylates, and activates Dbs in vitro. (A) Purified FLAG-Dbs (2 μg) was applied to SDS/PAGE, stained with Coomassie brilliant blue R-250, and immunoblotted (IB) with antibodies against the Rho GTPases. (B) Purified GST-TrkC kinase domain (2 μg) was resolved by SDS/PAGE, stained, and immunoblotted with antiphosphorylated Trk antibody. (C) Immobilized FLAG-Dbs (250 ng) was incubated with 20 μM cold ATP and 3 ng/μl GST or GST-TrkC kinase domain in 30 μl of reaction buffer, washed, and immunoblotted with anti-GST, pTyr, or FLAG antibody. (D) Release of [³H]GDP from 16 ng/μl GST-Cdc42·[³H]GDP by Dbs preincubated with ATP and GST or GST-TrkC kinase domain (with or without K252a) was measured. Data were evaluated by using Student's t test. *, P < 0.01; **, P < 0.005.

Fig. 5.

NT3 interaction with TrkC mediates phosphorylation and activation of endogenous Dbs in Schwann cells. (A) After stimulation with NT3, the Trk immunoprecipitates were immunoblotted with antibodies against Dbs and pan-Trk. Immunoprecipitated Dbs was quantified and normalized to the total amount of immunoprecipitated Trk, indicated by “1.0” and “2.3” below the Dbs bands. The total Dbs in the cell lysates is also shown. (B) Schwann cells were transfected with FLAG-Dbs and stained with antibodies against FLAG (green) and pan-Trk (red). Structures where Dbs and TrkC colocalize are indicated by arrows. (C and D) Active, tyrosine-phosphorylated Dbs was detected by the pull-down assay by using GST-Cdc42G15A from the cell lysates after incubation with NT3. The total Dbs in the cell lysates is also shown. (E) Effect of K252a on Dbs precipitated from the lysates of Schwann cells stimulated with NT3. Active, tyrosine-phosphorylated Dbs was detected by the pull-down assay by using GST-Cdc42G15A.