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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-induced migration of Schwann cells is dependent on the activation of the Rho GTPases Rac1 and Cdc42. (A) Schwann cells were pretreated with or without 2 ng/ml C. difficile Toxin B or 2 μg/ml C3 exoenzyme, and migration was assayed in Boyden chambers (12 independent fields). (B and C) After the addition of NRG1 for 0–120 min, endogenous Rac1-GTP in the lysates of Schwann cells was affinity precipitated with GST-Pak1-CRIB and immunoblotted with an anti-Rac1 antibody. The levels of Rac1-GTP were normalized to the amount of total Rac1 (n = 3). (D and E) The Rac1 activities were measured at 0–360 min (n = 5). (F–I) Endogenous Cdc42-GTP in the cell lysates was affinity precipitated with GST-Pak1-CRIB. The Cdc42-GTP levels were normalized to the amount of total Cdc42 (n = 3). Error bars show ±SD. Data were evaluated by using one-way ANOVA (*, P < 0.01).
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fig2: NRG1-induced migration of Schwann cells is dependent on the activation of the Rho GTPases Rac1 and Cdc42. (A) Schwann cells were pretreated with or without 2 ng/ml C. difficile Toxin B or 2 μg/ml C3 exoenzyme, and migration was assayed in Boyden chambers (12 independent fields). (B and C) After the addition of NRG1 for 0–120 min, endogenous Rac1-GTP in the lysates of Schwann cells was affinity precipitated with GST-Pak1-CRIB and immunoblotted with an anti-Rac1 antibody. The levels of Rac1-GTP were normalized to the amount of total Rac1 (n = 3). (D and E) The Rac1 activities were measured at 0–360 min (n = 5). (F–I) Endogenous Cdc42-GTP in the cell lysates was affinity precipitated with GST-Pak1-CRIB. The Cdc42-GTP levels were normalized to the amount of total Cdc42 (n = 3). Error bars show ±SD. Data were evaluated by using one-way ANOVA (*, P < 0.01).

Mentions: Next, we tested whether the Rho GTPases Rac1 and Cdc42 are involved in the NRG1-induced migration of Schwann cells, as previously seen in NT3-induced migration (Yamauchi et al., 2004). Pretreatment of Clostridium difficile Toxin B, which glycosylates and blocks the functions of Rho GTPases such as RhoA, Rac1, and Cdc42 (Just et al., 1995), inhibited the NRG1 effect by ∼70% (Fig. 2 A). In contrast, C3 exoenzyme, which ADP ribosylates RhoA and blocks its function (Hirose et al., 1998), did not have any obvious effect. Furthermore, we transfected a siRNA for Rac1 or Cdc42 into Schwann cells. Knockdown of Rac1 inhibited the NRG1-induced migration in Boyden chambers by ∼25% (Fig. S1 D, available at http://www.jcb.org/cgi/content/full/jcb.200709033/DC1) as well as migration from reaggregates on DRG axons (compare videos 1–4 for cells transfected with control siRNA with videos 5 and 6 for cells transfected with Rac1 siRNA, available at http://www.jcb.org/cgi/content/full/jcb.200709033/DC1), which is consistent with recent studies (Benninger et al., 2007; Nodari et al., 2007). Transfection with nonoverlapping siRNA, Cdc42-1 or Cdc42-2, decreased the migration in Boyden chambers by ∼15 and 25%, respectively (Fig. S1, E and F), as well as decreasing the migration from reaggregates on DRG axons (videos 7 and 8 for cells transfected with Cdc42-2 siRNA). However, because the effect after knockdown of Rac1 or Cdc42 is weaker than that of Toxin B, it is possible that Rac1 activity transduces an intracellular signal from NRG1 together with Cdc42 and that Rac1 and Cdc42 may share a common downstream signaling pathway. To examine whether NRG1 directly activates Cdc42 and Rac1, we performed affinity precipitation using the Rac1-GTP and Cdc42-GTP binding domain of Pak1. The activities of Rac1 and Cdc42 reached maximum levels at 60–120 min after stimulation with NRG1 and remained activated for at least 360 min (Fig. 2, B–I). Therefore, NRG1 activation of ErbB2 and 3 can stimulate the increase of Rac1-GTP and Cdc42-GTP to enhance Schwann cell migration.


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-induced migration of Schwann cells is dependent on the activation of the Rho GTPases Rac1 and Cdc42. (A) Schwann cells were pretreated with or without 2 ng/ml C. difficile Toxin B or 2 μg/ml C3 exoenzyme, and migration was assayed in Boyden chambers (12 independent fields). (B and C) After the addition of NRG1 for 0–120 min, endogenous Rac1-GTP in the lysates of Schwann cells was affinity precipitated with GST-Pak1-CRIB and immunoblotted with an anti-Rac1 antibody. The levels of Rac1-GTP were normalized to the amount of total Rac1 (n = 3). (D and E) The Rac1 activities were measured at 0–360 min (n = 5). (F–I) Endogenous Cdc42-GTP in the cell lysates was affinity precipitated with GST-Pak1-CRIB. The Cdc42-GTP levels were normalized to the amount of total Cdc42 (n = 3). Error bars show ±SD. Data were evaluated by using one-way ANOVA (*, P < 0.01).
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Related In: Results  -  Collection

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fig2: NRG1-induced migration of Schwann cells is dependent on the activation of the Rho GTPases Rac1 and Cdc42. (A) Schwann cells were pretreated with or without 2 ng/ml C. difficile Toxin B or 2 μg/ml C3 exoenzyme, and migration was assayed in Boyden chambers (12 independent fields). (B and C) After the addition of NRG1 for 0–120 min, endogenous Rac1-GTP in the lysates of Schwann cells was affinity precipitated with GST-Pak1-CRIB and immunoblotted with an anti-Rac1 antibody. The levels of Rac1-GTP were normalized to the amount of total Rac1 (n = 3). (D and E) The Rac1 activities were measured at 0–360 min (n = 5). (F–I) Endogenous Cdc42-GTP in the cell lysates was affinity precipitated with GST-Pak1-CRIB. The Cdc42-GTP levels were normalized to the amount of total Cdc42 (n = 3). Error bars show ±SD. Data were evaluated by using one-way ANOVA (*, P < 0.01).
Mentions: Next, we tested whether the Rho GTPases Rac1 and Cdc42 are involved in the NRG1-induced migration of Schwann cells, as previously seen in NT3-induced migration (Yamauchi et al., 2004). Pretreatment of Clostridium difficile Toxin B, which glycosylates and blocks the functions of Rho GTPases such as RhoA, Rac1, and Cdc42 (Just et al., 1995), inhibited the NRG1 effect by ∼70% (Fig. 2 A). In contrast, C3 exoenzyme, which ADP ribosylates RhoA and blocks its function (Hirose et al., 1998), did not have any obvious effect. Furthermore, we transfected a siRNA for Rac1 or Cdc42 into Schwann cells. Knockdown of Rac1 inhibited the NRG1-induced migration in Boyden chambers by ∼25% (Fig. S1 D, available at http://www.jcb.org/cgi/content/full/jcb.200709033/DC1) as well as migration from reaggregates on DRG axons (compare videos 1–4 for cells transfected with control siRNA with videos 5 and 6 for cells transfected with Rac1 siRNA, available at http://www.jcb.org/cgi/content/full/jcb.200709033/DC1), which is consistent with recent studies (Benninger et al., 2007; Nodari et al., 2007). Transfection with nonoverlapping siRNA, Cdc42-1 or Cdc42-2, decreased the migration in Boyden chambers by ∼15 and 25%, respectively (Fig. S1, E and F), as well as decreasing the migration from reaggregates on DRG axons (videos 7 and 8 for cells transfected with Cdc42-2 siRNA). However, because the effect after knockdown of Rac1 or Cdc42 is weaker than that of Toxin B, it is possible that Rac1 activity transduces an intracellular signal from NRG1 together with Cdc42 and that Rac1 and Cdc42 may share a common downstream signaling pathway. To examine whether NRG1 directly activates Cdc42 and Rac1, we performed affinity precipitation using the Rac1-GTP and Cdc42-GTP binding domain of Pak1. The activities of Rac1 and Cdc42 reached maximum levels at 60–120 min after stimulation with NRG1 and remained activated for at least 360 min (Fig. 2, B–I). Therefore, NRG1 activation of ErbB2 and 3 can stimulate the increase of Rac1-GTP and Cdc42-GTP to enhance Schwann cell migration.

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