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Phosphorylation of DCC by Fyn mediates Netrin-1 signaling in growth cone guidance.

Meriane M, Tcherkezian J, Webber CA, Danek EI, Triki I, McFarlane S, Bloch-Gallego E, Lamarche-Vane N - J. Cell Biol. (2004)

Bottom Line: Fyn, but not Src, is able to phosphorylate the intracellular domain of DCC in vitro, and we demonstrate that Y1418 is crucial for DCC axon outgrowth function.Both DCC phosphorylation and Netrin-1-induced axon outgrowth are impaired in Fyn(-/-) CN and spinal cord explants.We propose that DCC is regulated by tyrosine phosphorylation and that Fyn is essential for the response of axons to Netrin-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, H3A 2B2, Canada.

ABSTRACT
Netrin-1 acts as a chemoattractant molecule to guide commissural neurons (CN) toward the floor plate by interacting with the receptor deleted in colorectal cancer (DCC). The molecular mechanisms underlying Netrin-1-DCC signaling are still poorly characterized. Here, we show that DCC is phosphorylated in vivo on tyrosine residues in response to Netrin-1 stimulation of CN and that the Src family kinase inhibitors PP2 and SU6656 block both Netrin-1-dependent phosphorylation of DCC and axon outgrowth. PP2 also blocks the reorientation of Xenopus laevis retinal ganglion cells that occurs in response to Netrin-1, which suggests an essential role of the Src kinases in Netrin-1-dependent orientation. Fyn, but not Src, is able to phosphorylate the intracellular domain of DCC in vitro, and we demonstrate that Y1418 is crucial for DCC axon outgrowth function. Both DCC phosphorylation and Netrin-1-induced axon outgrowth are impaired in Fyn(-/-) CN and spinal cord explants. We propose that DCC is regulated by tyrosine phosphorylation and that Fyn is essential for the response of axons to Netrin-1.

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Netrin-1–induced DCC phosphorylation on tyrosine residues and neurite outgrowth are impaired in Fyn−/− mice. (A) E11.5 Fyn+/+ mouse CN were or were not stimulated with Netrin-1 for 10 or 30 min. The neurons were also pretreated with SU6656 before a 10-min stimulation with Netrin-1. DCC was immunoprecipitated from the cell lysates and the products were analyzed by SDS-PAGE. The membrane was immunoblotted with anti-pY and anti-DCC antibodies. (B) E11.5 CN dissected from Fyn−/− mice were or were not stimulated for 10 or 30 min with Netrin-1. DCC immunoprecipitates were analyzed by Western blot using anti-pY and anti-DCC antibodies. (C) Quantitative analysis of the phosphorylation level of DCC on tyrosine residues after Netrin-1 stimulation in E11.5 wild-type or Fyn−/− CN, determined by densitometry (n = 3). Error bars represent SD. (D) E11.5 dorsal spinal cord explants from Fyn+/+ or Fyn−/− mice were cultured for 20 h, alone (control) or in the presence of Netrin-1. Fyn+/+ explants were also treated with both Netrin-1 and SU6656. Bar, 100 μm. (E) Quantification of the total length of axon bundles per explant in micrometers (n = 36). Error bars represent SD.
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fig6: Netrin-1–induced DCC phosphorylation on tyrosine residues and neurite outgrowth are impaired in Fyn−/− mice. (A) E11.5 Fyn+/+ mouse CN were or were not stimulated with Netrin-1 for 10 or 30 min. The neurons were also pretreated with SU6656 before a 10-min stimulation with Netrin-1. DCC was immunoprecipitated from the cell lysates and the products were analyzed by SDS-PAGE. The membrane was immunoblotted with anti-pY and anti-DCC antibodies. (B) E11.5 CN dissected from Fyn−/− mice were or were not stimulated for 10 or 30 min with Netrin-1. DCC immunoprecipitates were analyzed by Western blot using anti-pY and anti-DCC antibodies. (C) Quantitative analysis of the phosphorylation level of DCC on tyrosine residues after Netrin-1 stimulation in E11.5 wild-type or Fyn−/− CN, determined by densitometry (n = 3). Error bars represent SD. (D) E11.5 dorsal spinal cord explants from Fyn+/+ or Fyn−/− mice were cultured for 20 h, alone (control) or in the presence of Netrin-1. Fyn+/+ explants were also treated with both Netrin-1 and SU6656. Bar, 100 μm. (E) Quantification of the total length of axon bundles per explant in micrometers (n = 36). Error bars represent SD.

Mentions: To confirm that Fyn tyrosine kinase is implicated in vivo in the regulation of the phosphorylation of DCC, we examined the phosphorylation level of DCC in embryonic CN from Fyn-deficient (Fyn−/−) mice in response to Netrin-1. As shown in Fig. 6 (A and C), a 10-fold increase in DCC phosphorylation on tyrosine residues is observed after 30 min of Netrin-1 stimulation in wild-type E11.5 CN. However, no tyrosine phosphorylation band is detected when DCC is immunoprecipitated from Fyn−/− CN, after 30 min of Netrin-1 stimulation, or from wild-type CN pretreated with SU6656 before a 10-min Netrin-1 stimulation (Fig. 6, A–C). Next, we examined the ability of dorsal spinal cord explants from Fyn−/− mouse embryos to extend neurites in response to Netrin-1, in comparison with that of Fyn+/+ explants. As shown in Fig. 6 D, explants from Fyn+/+ mice, treated for 20 h with Netrin-1, show maximal axon outgrowth compared with the nontreated control explants. When the Src family kinase inhibitor SU6656 is added with Netrin-1 to the Fyn+/+ explants, the axon outgrowth is completely abolished (Fig. 6, D and E), as observed with rat dorsal spinal cord explants. However, when explants from Fyn−/− dorsal spinal cords are cultured in the presence of Netrin-1 for 20 h, the axon outgrowth is completely abolished in up to 90% of the total number of explants examined (n = 36; Fig. 6, D and E). These results are consistent with the data obtained in vitro, showing the crucial role of Fyn tyrosine kinase in DCC phosphorylation and Netrin-1–induced outgrowth function in vivo.


Phosphorylation of DCC by Fyn mediates Netrin-1 signaling in growth cone guidance.

Meriane M, Tcherkezian J, Webber CA, Danek EI, Triki I, McFarlane S, Bloch-Gallego E, Lamarche-Vane N - J. Cell Biol. (2004)

Netrin-1–induced DCC phosphorylation on tyrosine residues and neurite outgrowth are impaired in Fyn−/− mice. (A) E11.5 Fyn+/+ mouse CN were or were not stimulated with Netrin-1 for 10 or 30 min. The neurons were also pretreated with SU6656 before a 10-min stimulation with Netrin-1. DCC was immunoprecipitated from the cell lysates and the products were analyzed by SDS-PAGE. The membrane was immunoblotted with anti-pY and anti-DCC antibodies. (B) E11.5 CN dissected from Fyn−/− mice were or were not stimulated for 10 or 30 min with Netrin-1. DCC immunoprecipitates were analyzed by Western blot using anti-pY and anti-DCC antibodies. (C) Quantitative analysis of the phosphorylation level of DCC on tyrosine residues after Netrin-1 stimulation in E11.5 wild-type or Fyn−/− CN, determined by densitometry (n = 3). Error bars represent SD. (D) E11.5 dorsal spinal cord explants from Fyn+/+ or Fyn−/− mice were cultured for 20 h, alone (control) or in the presence of Netrin-1. Fyn+/+ explants were also treated with both Netrin-1 and SU6656. Bar, 100 μm. (E) Quantification of the total length of axon bundles per explant in micrometers (n = 36). Error bars represent SD.
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fig6: Netrin-1–induced DCC phosphorylation on tyrosine residues and neurite outgrowth are impaired in Fyn−/− mice. (A) E11.5 Fyn+/+ mouse CN were or were not stimulated with Netrin-1 for 10 or 30 min. The neurons were also pretreated with SU6656 before a 10-min stimulation with Netrin-1. DCC was immunoprecipitated from the cell lysates and the products were analyzed by SDS-PAGE. The membrane was immunoblotted with anti-pY and anti-DCC antibodies. (B) E11.5 CN dissected from Fyn−/− mice were or were not stimulated for 10 or 30 min with Netrin-1. DCC immunoprecipitates were analyzed by Western blot using anti-pY and anti-DCC antibodies. (C) Quantitative analysis of the phosphorylation level of DCC on tyrosine residues after Netrin-1 stimulation in E11.5 wild-type or Fyn−/− CN, determined by densitometry (n = 3). Error bars represent SD. (D) E11.5 dorsal spinal cord explants from Fyn+/+ or Fyn−/− mice were cultured for 20 h, alone (control) or in the presence of Netrin-1. Fyn+/+ explants were also treated with both Netrin-1 and SU6656. Bar, 100 μm. (E) Quantification of the total length of axon bundles per explant in micrometers (n = 36). Error bars represent SD.
Mentions: To confirm that Fyn tyrosine kinase is implicated in vivo in the regulation of the phosphorylation of DCC, we examined the phosphorylation level of DCC in embryonic CN from Fyn-deficient (Fyn−/−) mice in response to Netrin-1. As shown in Fig. 6 (A and C), a 10-fold increase in DCC phosphorylation on tyrosine residues is observed after 30 min of Netrin-1 stimulation in wild-type E11.5 CN. However, no tyrosine phosphorylation band is detected when DCC is immunoprecipitated from Fyn−/− CN, after 30 min of Netrin-1 stimulation, or from wild-type CN pretreated with SU6656 before a 10-min Netrin-1 stimulation (Fig. 6, A–C). Next, we examined the ability of dorsal spinal cord explants from Fyn−/− mouse embryos to extend neurites in response to Netrin-1, in comparison with that of Fyn+/+ explants. As shown in Fig. 6 D, explants from Fyn+/+ mice, treated for 20 h with Netrin-1, show maximal axon outgrowth compared with the nontreated control explants. When the Src family kinase inhibitor SU6656 is added with Netrin-1 to the Fyn+/+ explants, the axon outgrowth is completely abolished (Fig. 6, D and E), as observed with rat dorsal spinal cord explants. However, when explants from Fyn−/− dorsal spinal cords are cultured in the presence of Netrin-1 for 20 h, the axon outgrowth is completely abolished in up to 90% of the total number of explants examined (n = 36; Fig. 6, D and E). These results are consistent with the data obtained in vitro, showing the crucial role of Fyn tyrosine kinase in DCC phosphorylation and Netrin-1–induced outgrowth function in vivo.

Bottom Line: Fyn, but not Src, is able to phosphorylate the intracellular domain of DCC in vitro, and we demonstrate that Y1418 is crucial for DCC axon outgrowth function.Both DCC phosphorylation and Netrin-1-induced axon outgrowth are impaired in Fyn(-/-) CN and spinal cord explants.We propose that DCC is regulated by tyrosine phosphorylation and that Fyn is essential for the response of axons to Netrin-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, H3A 2B2, Canada.

ABSTRACT
Netrin-1 acts as a chemoattractant molecule to guide commissural neurons (CN) toward the floor plate by interacting with the receptor deleted in colorectal cancer (DCC). The molecular mechanisms underlying Netrin-1-DCC signaling are still poorly characterized. Here, we show that DCC is phosphorylated in vivo on tyrosine residues in response to Netrin-1 stimulation of CN and that the Src family kinase inhibitors PP2 and SU6656 block both Netrin-1-dependent phosphorylation of DCC and axon outgrowth. PP2 also blocks the reorientation of Xenopus laevis retinal ganglion cells that occurs in response to Netrin-1, which suggests an essential role of the Src kinases in Netrin-1-dependent orientation. Fyn, but not Src, is able to phosphorylate the intracellular domain of DCC in vitro, and we demonstrate that Y1418 is crucial for DCC axon outgrowth function. Both DCC phosphorylation and Netrin-1-induced axon outgrowth are impaired in Fyn(-/-) CN and spinal cord explants. We propose that DCC is regulated by tyrosine phosphorylation and that Fyn is essential for the response of axons to Netrin-1.

Show MeSH
Related in: MedlinePlus