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ErbB2 directly activates the exchange factor Dock7 to promote Schwann cell migration.

Yamauchi J, Miyamoto Y, Chan JR, Tanoue A - J. Cell Biol. (2008)

Bottom Line: Dock7 knockdown, or expression of Dock7 harboring the Tyr-1118-to-Phe mutation in Schwann cells, attenuates the effects of NRG1.Thus, Dock7 functions as an intracellular substrate for ErbB2 to promote Schwann cell migration.This provides an unanticipated mechanism through which ligand-dependent tyrosine phosphorylation can trigger the activation of Rho GTPase-GEFs of the Dock180 family.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, National Research Institute for Child Health and Development, Setagaya, Tokyo 157-8535, Japan. jyamauchi@nch.go.jp

ABSTRACT
The cellular events that precede myelination in the peripheral nervous system require rapid and dynamic morphological changes in the Schwann cell. These events are thought to be mainly controlled by axonal signals. But how signals on the axons are coordinately organized and transduced to promote proliferation, migration, radial sorting, and myelination is unknown. We describe that the axonal signal neuregulin-1 (NRG1) controls Schwann cell migration via activation of the atypical Dock180-related guanine nucleotide exchange factor (GEF) Dock7 and subsequent activation of the Rho guanine triphosphatases (GTPases) Rac1 and Cdc42 and the downstream c-Jun N-terminal kinase. We show that the NRG1 receptor ErbB2 directly binds and activates Dock7 by phosphorylating Tyr-1118. Dock7 knockdown, or expression of Dock7 harboring the Tyr-1118-to-Phe mutation in Schwann cells, attenuates the effects of NRG1. Thus, Dock7 functions as an intracellular substrate for ErbB2 to promote Schwann cell migration. This provides an unanticipated mechanism through which ligand-dependent tyrosine phosphorylation can trigger the activation of Rho GTPase-GEFs of the Dock180 family.

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NRG1 activation of the ErbB2 and 3 heterodimer stimulates the GEF activity of Dock7. (A–C) 125 ng of immobilized FLAG-Dock7-DHR-2 was incubated with 16 ng/μl GST-Rac1, Cdc42, or RhoA and 3 μM [3H]GDP in 30 μl of reaction buffer for 0–30 min, and the guanine nucleotide binding activities were measured (n = 10). (D–F) The release of [3H]GDP from GST-Rac1-[3H]GDP, Cdc42-[3H]GDP, or GST-RhoA-[3H]GDP by FLAG–Dock7–DHR-2 was measured (n = 10). Immunoprecipitated FLAG-Dbs-DHPH was used as the positive control for the RhoA-GEF. Dock7–DHR-2, closed circle; control, open circle; Dbs-DHPH, closed square. (G–L) 293T cells were transfected with pCMV–FLAG–Dock7–DHR-2 or pCMV-FLAG-Dbs-DHPH. The cell lysates were affinity precipitated with 20 μg each of nucleotide-free GST-Rho GTPase (Rac1G15A, Cdc42G15A, or RhoAG17) or the wild type (Rac1, Cdc42, or RhoA) and immunoblotted with an anti-FLAG antibody. The total FLAG–Dock7–DHR-2 or FLAG-Dbs-DHPH is also shown. Each GST-Rho GTPase was immobilized in the same experimental conditions, subjected to SDS-PAGE, and stained with Coomassie brilliant blue. (M and O) 293T cells were cotransfected with pCMV-FLAG-Dock7, pCMV-ErbB2, and pCMV-ErbB3 and stimulated with or without NRG1 for 30 min. The expression of ErbB2 and 3 in 293T cells was below the detection level of immunoblotting (not depicted). The release of [3H]GDP from GST-Rac1-[3H]GDP or Cdc42-[3H]GDP by immunoprecipitated FLAG-Dock7 was measured (n = 3). (N and P) Cells were cotransfected with pCMV-FLAG-Dock7, pCMV-ErbB2, and pCMV-ErbB3. The affinity precipitation of the cell lysates with GST-Rac1G15A or Cdc42G15A was performed. The total FLAG-Dock7 is also shown. Error bars show ±SD. Data were evaluated by using one-way ANOVA (*, P < 0.01).
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fig5: NRG1 activation of the ErbB2 and 3 heterodimer stimulates the GEF activity of Dock7. (A–C) 125 ng of immobilized FLAG-Dock7-DHR-2 was incubated with 16 ng/μl GST-Rac1, Cdc42, or RhoA and 3 μM [3H]GDP in 30 μl of reaction buffer for 0–30 min, and the guanine nucleotide binding activities were measured (n = 10). (D–F) The release of [3H]GDP from GST-Rac1-[3H]GDP, Cdc42-[3H]GDP, or GST-RhoA-[3H]GDP by FLAG–Dock7–DHR-2 was measured (n = 10). Immunoprecipitated FLAG-Dbs-DHPH was used as the positive control for the RhoA-GEF. Dock7–DHR-2, closed circle; control, open circle; Dbs-DHPH, closed square. (G–L) 293T cells were transfected with pCMV–FLAG–Dock7–DHR-2 or pCMV-FLAG-Dbs-DHPH. The cell lysates were affinity precipitated with 20 μg each of nucleotide-free GST-Rho GTPase (Rac1G15A, Cdc42G15A, or RhoAG17) or the wild type (Rac1, Cdc42, or RhoA) and immunoblotted with an anti-FLAG antibody. The total FLAG–Dock7–DHR-2 or FLAG-Dbs-DHPH is also shown. Each GST-Rho GTPase was immobilized in the same experimental conditions, subjected to SDS-PAGE, and stained with Coomassie brilliant blue. (M and O) 293T cells were cotransfected with pCMV-FLAG-Dock7, pCMV-ErbB2, and pCMV-ErbB3 and stimulated with or without NRG1 for 30 min. The expression of ErbB2 and 3 in 293T cells was below the detection level of immunoblotting (not depicted). The release of [3H]GDP from GST-Rac1-[3H]GDP or Cdc42-[3H]GDP by immunoprecipitated FLAG-Dock7 was measured (n = 3). (N and P) Cells were cotransfected with pCMV-FLAG-Dock7, pCMV-ErbB2, and pCMV-ErbB3. The affinity precipitation of the cell lysates with GST-Rac1G15A or Cdc42G15A was performed. The total FLAG-Dock7 is also shown. Error bars show ±SD. Data were evaluated by using one-way ANOVA (*, P < 0.01).

Mentions: Because the DHR-2 domain of the Dock180-related GEFs shows catalytic activity (Brugnera et al., 2002; Côté and Vuori, 2002; Meller et al., 2002), we tested whether Rho GTPases could be activated by the DHR-2 domain of Dock7. The purified DHR-2 domain (Fig. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200709033/DC1) promoted the exchange, binding, and release of the guanine nucleotide for Rac1 and Cdc42 in a time-dependent manner (Fig. 5, A, B, D, and E), whereas no effect was observed for RhoA (Fig. 5, C and F). Catalytically active GEFs preferentially interact with guanine nucleotide–free forms of the small GTPases (Arthur et al., 2002; Schmidt and Hall, 2002; Rossman et al., 2005). A Gly-to-Ala mutation in the P loop of the small GTPases decreases their guanine nucleotide binding activities (Arthur et al., 2002). We performed an affinity precipitation of the DHR-2 domain of Dock7 with guanine nucleotide–free Rac1G15A, Cdc42G15A, or RhoAG17A as well as wild-type Rac1, Cdc42, or RhoA. DHR-2 specifically coprecipitated with Rac1G15A (Fig. 5 G) and Cdc42G15A (Fig. 5 I) but not with RhoAG17A (Fig. 5 K), which is consistent with the results from the guanine nucleotide exchange assays. In contrast, the DH and pleckstrin homology (PH) domains of Dbs affinity precipitated with Rac1G15A (Fig. 5 H) or Cdc42G15A (Fig. 5 J) as well as with RhoAG17A (Fig. 5 L). Similarly, the affinity precipitation with wild-type Rac1 or Cdc42 also showed binding to DHR-2 but was slightly weaker than the precipitation with each GTPase harboring the Gly-to-Ala mutation. To investigate whether NRG1 activation of the ErbB2 and 3 heterodimer stimulates the GEF activity of Dock7, we cotransfected the plasmids encoding wild-type Dock7, ErbB2, and ErbB3 into 293T cells and measured the exchange of the guanine nucleotide from immunoprecipitated Dock7 for Rac1 and Cdc42. The activity of wild-type Dock7 was significantly increased after stimulation with NRG1 (Fig. 5, M and O). Similarly, NRG1 promoted the affinity-precipitation of Dock7 with Rac1G15A or Cdc42G15A (Fig. 5, N and P).


ErbB2 directly activates the exchange factor Dock7 to promote Schwann cell migration.

Yamauchi J, Miyamoto Y, Chan JR, Tanoue A - J. Cell Biol. (2008)

NRG1 activation of the ErbB2 and 3 heterodimer stimulates the GEF activity of Dock7. (A–C) 125 ng of immobilized FLAG-Dock7-DHR-2 was incubated with 16 ng/μl GST-Rac1, Cdc42, or RhoA and 3 μM [3H]GDP in 30 μl of reaction buffer for 0–30 min, and the guanine nucleotide binding activities were measured (n = 10). (D–F) The release of [3H]GDP from GST-Rac1-[3H]GDP, Cdc42-[3H]GDP, or GST-RhoA-[3H]GDP by FLAG–Dock7–DHR-2 was measured (n = 10). Immunoprecipitated FLAG-Dbs-DHPH was used as the positive control for the RhoA-GEF. Dock7–DHR-2, closed circle; control, open circle; Dbs-DHPH, closed square. (G–L) 293T cells were transfected with pCMV–FLAG–Dock7–DHR-2 or pCMV-FLAG-Dbs-DHPH. The cell lysates were affinity precipitated with 20 μg each of nucleotide-free GST-Rho GTPase (Rac1G15A, Cdc42G15A, or RhoAG17) or the wild type (Rac1, Cdc42, or RhoA) and immunoblotted with an anti-FLAG antibody. The total FLAG–Dock7–DHR-2 or FLAG-Dbs-DHPH is also shown. Each GST-Rho GTPase was immobilized in the same experimental conditions, subjected to SDS-PAGE, and stained with Coomassie brilliant blue. (M and O) 293T cells were cotransfected with pCMV-FLAG-Dock7, pCMV-ErbB2, and pCMV-ErbB3 and stimulated with or without NRG1 for 30 min. The expression of ErbB2 and 3 in 293T cells was below the detection level of immunoblotting (not depicted). The release of [3H]GDP from GST-Rac1-[3H]GDP or Cdc42-[3H]GDP by immunoprecipitated FLAG-Dock7 was measured (n = 3). (N and P) Cells were cotransfected with pCMV-FLAG-Dock7, pCMV-ErbB2, and pCMV-ErbB3. The affinity precipitation of the cell lysates with GST-Rac1G15A or Cdc42G15A was performed. The total FLAG-Dock7 is also shown. Error bars show ±SD. Data were evaluated by using one-way ANOVA (*, P < 0.01).
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fig5: NRG1 activation of the ErbB2 and 3 heterodimer stimulates the GEF activity of Dock7. (A–C) 125 ng of immobilized FLAG-Dock7-DHR-2 was incubated with 16 ng/μl GST-Rac1, Cdc42, or RhoA and 3 μM [3H]GDP in 30 μl of reaction buffer for 0–30 min, and the guanine nucleotide binding activities were measured (n = 10). (D–F) The release of [3H]GDP from GST-Rac1-[3H]GDP, Cdc42-[3H]GDP, or GST-RhoA-[3H]GDP by FLAG–Dock7–DHR-2 was measured (n = 10). Immunoprecipitated FLAG-Dbs-DHPH was used as the positive control for the RhoA-GEF. Dock7–DHR-2, closed circle; control, open circle; Dbs-DHPH, closed square. (G–L) 293T cells were transfected with pCMV–FLAG–Dock7–DHR-2 or pCMV-FLAG-Dbs-DHPH. The cell lysates were affinity precipitated with 20 μg each of nucleotide-free GST-Rho GTPase (Rac1G15A, Cdc42G15A, or RhoAG17) or the wild type (Rac1, Cdc42, or RhoA) and immunoblotted with an anti-FLAG antibody. The total FLAG–Dock7–DHR-2 or FLAG-Dbs-DHPH is also shown. Each GST-Rho GTPase was immobilized in the same experimental conditions, subjected to SDS-PAGE, and stained with Coomassie brilliant blue. (M and O) 293T cells were cotransfected with pCMV-FLAG-Dock7, pCMV-ErbB2, and pCMV-ErbB3 and stimulated with or without NRG1 for 30 min. The expression of ErbB2 and 3 in 293T cells was below the detection level of immunoblotting (not depicted). The release of [3H]GDP from GST-Rac1-[3H]GDP or Cdc42-[3H]GDP by immunoprecipitated FLAG-Dock7 was measured (n = 3). (N and P) Cells were cotransfected with pCMV-FLAG-Dock7, pCMV-ErbB2, and pCMV-ErbB3. The affinity precipitation of the cell lysates with GST-Rac1G15A or Cdc42G15A was performed. The total FLAG-Dock7 is also shown. Error bars show ±SD. Data were evaluated by using one-way ANOVA (*, P < 0.01).
Mentions: Because the DHR-2 domain of the Dock180-related GEFs shows catalytic activity (Brugnera et al., 2002; Côté and Vuori, 2002; Meller et al., 2002), we tested whether Rho GTPases could be activated by the DHR-2 domain of Dock7. The purified DHR-2 domain (Fig. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200709033/DC1) promoted the exchange, binding, and release of the guanine nucleotide for Rac1 and Cdc42 in a time-dependent manner (Fig. 5, A, B, D, and E), whereas no effect was observed for RhoA (Fig. 5, C and F). Catalytically active GEFs preferentially interact with guanine nucleotide–free forms of the small GTPases (Arthur et al., 2002; Schmidt and Hall, 2002; Rossman et al., 2005). A Gly-to-Ala mutation in the P loop of the small GTPases decreases their guanine nucleotide binding activities (Arthur et al., 2002). We performed an affinity precipitation of the DHR-2 domain of Dock7 with guanine nucleotide–free Rac1G15A, Cdc42G15A, or RhoAG17A as well as wild-type Rac1, Cdc42, or RhoA. DHR-2 specifically coprecipitated with Rac1G15A (Fig. 5 G) and Cdc42G15A (Fig. 5 I) but not with RhoAG17A (Fig. 5 K), which is consistent with the results from the guanine nucleotide exchange assays. In contrast, the DH and pleckstrin homology (PH) domains of Dbs affinity precipitated with Rac1G15A (Fig. 5 H) or Cdc42G15A (Fig. 5 J) as well as with RhoAG17A (Fig. 5 L). Similarly, the affinity precipitation with wild-type Rac1 or Cdc42 also showed binding to DHR-2 but was slightly weaker than the precipitation with each GTPase harboring the Gly-to-Ala mutation. To investigate whether NRG1 activation of the ErbB2 and 3 heterodimer stimulates the GEF activity of Dock7, we cotransfected the plasmids encoding wild-type Dock7, ErbB2, and ErbB3 into 293T cells and measured the exchange of the guanine nucleotide from immunoprecipitated Dock7 for Rac1 and Cdc42. The activity of wild-type Dock7 was significantly increased after stimulation with NRG1 (Fig. 5, M and O). Similarly, NRG1 promoted the affinity-precipitation of Dock7 with Rac1G15A or Cdc42G15A (Fig. 5, N and P).

Bottom Line: Dock7 knockdown, or expression of Dock7 harboring the Tyr-1118-to-Phe mutation in Schwann cells, attenuates the effects of NRG1.Thus, Dock7 functions as an intracellular substrate for ErbB2 to promote Schwann cell migration.This provides an unanticipated mechanism through which ligand-dependent tyrosine phosphorylation can trigger the activation of Rho GTPase-GEFs of the Dock180 family.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, National Research Institute for Child Health and Development, Setagaya, Tokyo 157-8535, Japan. jyamauchi@nch.go.jp

ABSTRACT
The cellular events that precede myelination in the peripheral nervous system require rapid and dynamic morphological changes in the Schwann cell. These events are thought to be mainly controlled by axonal signals. But how signals on the axons are coordinately organized and transduced to promote proliferation, migration, radial sorting, and myelination is unknown. We describe that the axonal signal neuregulin-1 (NRG1) controls Schwann cell migration via activation of the atypical Dock180-related guanine nucleotide exchange factor (GEF) Dock7 and subsequent activation of the Rho guanine triphosphatases (GTPases) Rac1 and Cdc42 and the downstream c-Jun N-terminal kinase. We show that the NRG1 receptor ErbB2 directly binds and activates Dock7 by phosphorylating Tyr-1118. Dock7 knockdown, or expression of Dock7 harboring the Tyr-1118-to-Phe mutation in Schwann cells, attenuates the effects of NRG1. Thus, Dock7 functions as an intracellular substrate for ErbB2 to promote Schwann cell migration. This provides an unanticipated mechanism through which ligand-dependent tyrosine phosphorylation can trigger the activation of Rho GTPase-GEFs of the Dock180 family.

Show MeSH
Related in: MedlinePlus