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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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Effects of the Tyr-to-Phe mutations in the middle region 2 of Dock7 on NRG1-induced migration of Schwann cells. (A and B) pEGFP, pEGFP-siRNA–-resistant wild-type Dock7, or pEGFP-siRNA–resistant Dock7Y1118F was cotransfected with control or Dock7-1 siRNA into Schwann cells. The number of GFP-fluorescent migrating Schwann cells in Boyden chambers was counted. Bar, 100 μm. (C) Expression of GFP-tagged siRNA-sensitive wild-type Dock7 or Dock7-1 siRNA-resistant Dock7 (wild type, Y1118F, Y1138F, Y1225F, Y1233F, Y1375F, or Y1429F) in Schwann cells are shown in the immunoblots. The cell lysates were immunoblotted with an anti-GFP or -actin antibody. (D and E) Schwann cells were cotransfected with pEGFP-siRNA–resistant wild-type or mutated Dock7 together and Dock7-1 siRNA. The number of GFP-fluorescent migrating Schwann cells was counted. Bar, 100 μm. Error bars show ±SD. Data were evaluated by using one-way ANOVA (n = 16; *, P < 0.01; ***, P < 0.02).
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fig8: Effects of the Tyr-to-Phe mutations in the middle region 2 of Dock7 on NRG1-induced migration of Schwann cells. (A and B) pEGFP, pEGFP-siRNA–-resistant wild-type Dock7, or pEGFP-siRNA–resistant Dock7Y1118F was cotransfected with control or Dock7-1 siRNA into Schwann cells. The number of GFP-fluorescent migrating Schwann cells in Boyden chambers was counted. Bar, 100 μm. (C) Expression of GFP-tagged siRNA-sensitive wild-type Dock7 or Dock7-1 siRNA-resistant Dock7 (wild type, Y1118F, Y1138F, Y1225F, Y1233F, Y1375F, or Y1429F) in Schwann cells are shown in the immunoblots. The cell lysates were immunoblotted with an anti-GFP or -actin antibody. (D and E) Schwann cells were cotransfected with pEGFP-siRNA–resistant wild-type or mutated Dock7 together and Dock7-1 siRNA. The number of GFP-fluorescent migrating Schwann cells was counted. Bar, 100 μm. Error bars show ±SD. Data were evaluated by using one-way ANOVA (n = 16; *, P < 0.01; ***, P < 0.02).

Mentions: Next, we investigated the role of the phosphorylation of Dock7 at the Tyr-1118 position in Schwann cell migration. We cotransfected a plasmid encoding Dock7-1 siRNA-resistant wild-type or Y1118F Dock7 together with a control or Dock7-1 siRNA into Schwann cells. Expression of siRNA-resistant wild-type Dock7 reversed the Dock7-1 siRNA-mediated inhibition of NRG1-induced migration in Boyden chambers, whereas Y1118F Dock7 failed to rescue Dock7-1 siRNA-mediated inhibition of migration (Fig. 8, A and B). Because there is the possibility that the Y1118F mutation has an effect on the protein conformation of Dock7, we tested the effects of the other mutants, Y1138F, Y1225F, Y1233F, Y1375F, and Y1429F, on migration. The Y1138F, Y1225F, Y1233F, or Y1375F mutant rescued siRNA-mediated inhibition of migration at the same level as that of the wild type (Fig. 8, D and E), indicating that the Y1118F mutation mimics the nonphosphorylated form and that the phosphorylation at the Tyr-1118 position is required for migration. The Y1429F mutant could rescue siRNA-mediated inhibition of migration but did not completely. The reason may be that Tyr-1429 interacts functionally with the catalytic DHR-2 because it is adjacent to DHR-2. Alternatively, because the Tyr-1429 position is contained in the canonical phosphatidylinositol-3-kinase binding motif Tyr-X-X-Met (Fig. 5 D; Ponzetto et al., 1993), the binding may partially affect Dock7 activation (Côté et al., 2005). Expression of Dock7-1 siRNA-resistant constructs was not down-regulated by cotransfection with Dock7-1 siRNA, which specifically reduced expression of native siRNA-sensitive nucleotide sequence of Dock7 (Fig. 8 C).


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

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

Effects of the Tyr-to-Phe mutations in the middle region 2 of Dock7 on NRG1-induced migration of Schwann cells. (A and B) pEGFP, pEGFP-siRNA–-resistant wild-type Dock7, or pEGFP-siRNA–resistant Dock7Y1118F was cotransfected with control or Dock7-1 siRNA into Schwann cells. The number of GFP-fluorescent migrating Schwann cells in Boyden chambers was counted. Bar, 100 μm. (C) Expression of GFP-tagged siRNA-sensitive wild-type Dock7 or Dock7-1 siRNA-resistant Dock7 (wild type, Y1118F, Y1138F, Y1225F, Y1233F, Y1375F, or Y1429F) in Schwann cells are shown in the immunoblots. The cell lysates were immunoblotted with an anti-GFP or -actin antibody. (D and E) Schwann cells were cotransfected with pEGFP-siRNA–resistant wild-type or mutated Dock7 together and Dock7-1 siRNA. The number of GFP-fluorescent migrating Schwann cells was counted. Bar, 100 μm. Error bars show ±SD. Data were evaluated by using one-way ANOVA (n = 16; *, P < 0.01; ***, P < 0.02).
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fig8: Effects of the Tyr-to-Phe mutations in the middle region 2 of Dock7 on NRG1-induced migration of Schwann cells. (A and B) pEGFP, pEGFP-siRNA–-resistant wild-type Dock7, or pEGFP-siRNA–resistant Dock7Y1118F was cotransfected with control or Dock7-1 siRNA into Schwann cells. The number of GFP-fluorescent migrating Schwann cells in Boyden chambers was counted. Bar, 100 μm. (C) Expression of GFP-tagged siRNA-sensitive wild-type Dock7 or Dock7-1 siRNA-resistant Dock7 (wild type, Y1118F, Y1138F, Y1225F, Y1233F, Y1375F, or Y1429F) in Schwann cells are shown in the immunoblots. The cell lysates were immunoblotted with an anti-GFP or -actin antibody. (D and E) Schwann cells were cotransfected with pEGFP-siRNA–resistant wild-type or mutated Dock7 together and Dock7-1 siRNA. The number of GFP-fluorescent migrating Schwann cells was counted. Bar, 100 μm. Error bars show ±SD. Data were evaluated by using one-way ANOVA (n = 16; *, P < 0.01; ***, P < 0.02).
Mentions: Next, we investigated the role of the phosphorylation of Dock7 at the Tyr-1118 position in Schwann cell migration. We cotransfected a plasmid encoding Dock7-1 siRNA-resistant wild-type or Y1118F Dock7 together with a control or Dock7-1 siRNA into Schwann cells. Expression of siRNA-resistant wild-type Dock7 reversed the Dock7-1 siRNA-mediated inhibition of NRG1-induced migration in Boyden chambers, whereas Y1118F Dock7 failed to rescue Dock7-1 siRNA-mediated inhibition of migration (Fig. 8, A and B). Because there is the possibility that the Y1118F mutation has an effect on the protein conformation of Dock7, we tested the effects of the other mutants, Y1138F, Y1225F, Y1233F, Y1375F, and Y1429F, on migration. The Y1138F, Y1225F, Y1233F, or Y1375F mutant rescued siRNA-mediated inhibition of migration at the same level as that of the wild type (Fig. 8, D and E), indicating that the Y1118F mutation mimics the nonphosphorylated form and that the phosphorylation at the Tyr-1118 position is required for migration. The Y1429F mutant could rescue siRNA-mediated inhibition of migration but did not completely. The reason may be that Tyr-1429 interacts functionally with the catalytic DHR-2 because it is adjacent to DHR-2. Alternatively, because the Tyr-1429 position is contained in the canonical phosphatidylinositol-3-kinase binding motif Tyr-X-X-Met (Fig. 5 D; Ponzetto et al., 1993), the binding may partially affect Dock7 activation (Côté et al., 2005). Expression of Dock7-1 siRNA-resistant constructs was not down-regulated by cotransfection with Dock7-1 siRNA, which specifically reduced expression of native siRNA-sensitive nucleotide sequence of Dock7 (Fig. 8 C).

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