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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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Inhibition of Src family kinases abolishes Netrin-1–induced turning response of RGC growth cones. (A, B, D, and E) X. laevis RGC growth cones extending from a 24-h stage 24 explant culture. Pulsatile ejection of Netrin-1 from a pipette (*) with a 0.5–1.0-μm opening was used to set up a concentration gradient next to an extending RGC growth cone (arrowheads). (A and D) Trajectories of growth cones before applying the pipette solution (arrows). (B and E) Growth cones (arrows) 45 min after continuous exposure to the Netrin-1 concentration gradient. The growth cone in the control bath (A and B) turned toward the Netrin-1 source, whereas the growth cone bathed in 0.25 μM of Src kinase inhibitor PP2 (D and E) ignored the Netrin-1 source. (C and F) Superimposed neurite trajectories of growth cones exposed to Netrin-1 source in a control medium (C) and growth cones exposed to Netrin-1 in the presence of PP2 (F). (G) Representation of the mean turning angles of the growth cones in C and F (P < 0.05; unpaired two-tailed t test). Error bars represent SD.
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fig3: Inhibition of Src family kinases abolishes Netrin-1–induced turning response of RGC growth cones. (A, B, D, and E) X. laevis RGC growth cones extending from a 24-h stage 24 explant culture. Pulsatile ejection of Netrin-1 from a pipette (*) with a 0.5–1.0-μm opening was used to set up a concentration gradient next to an extending RGC growth cone (arrowheads). (A and D) Trajectories of growth cones before applying the pipette solution (arrows). (B and E) Growth cones (arrows) 45 min after continuous exposure to the Netrin-1 concentration gradient. The growth cone in the control bath (A and B) turned toward the Netrin-1 source, whereas the growth cone bathed in 0.25 μM of Src kinase inhibitor PP2 (D and E) ignored the Netrin-1 source. (C and F) Superimposed neurite trajectories of growth cones exposed to Netrin-1 source in a control medium (C) and growth cones exposed to Netrin-1 in the presence of PP2 (F). (G) Representation of the mean turning angles of the growth cones in C and F (P < 0.05; unpaired two-tailed t test). Error bars represent SD.

Mentions: To address the physiological significance of phosphorylation of DCC, we examined the question of whether Src family kinases are involved in mediating the axon outgrowth–promoting effects of Netrin-1. Explants of E13 rat dorsal spinal cord cultured in a three-dimensional collagen gel in the presence of 160 ng/ml of Netrin-1 show maximal commissural axon outgrowth (Fig. 2, E and F), as reported previously (Serafini et al., 1996). When PP2 or SU6656 were added in the presence of Netrin-1 to the explants, the increase in axon outgrowth was completely abolished, similar to the results in the control (Fig. 2, E and F). These results demonstrate that inhibition of the Src kinases interferes with the effect of Netrin-1 to mediate commissural axon outgrowth, suggesting a critical role for the Src kinases in Netrin-1–DCC signaling. To determine whether Src kinases are required for Netrin-1 to mediate axon attraction, we used the in vitro turning assay in which Xenopus laevis retinal ganglion cells (RGC) turn toward a source of Netrin-1 (de la Torre et al., 1997). As shown in Fig. 3 B, a retinal growth cone extending from a 24-h, stage 24 explant culture turned toward the source of Netrin-1 in the control bath. In contrast, the retinal growth cone ignored the source of Netrin-1 in the presence of PP2 (Fig. 3 E). A trace of the paths taken by all growth cones is represented in Fig. 3 (C and F). The turning angles taken by the retinal growth cones were quantified and are shown in Fig. 3 G. These results reveal the implication of Src family kinases in the turning response of growth cones to Netrin-1.


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)

Inhibition of Src family kinases abolishes Netrin-1–induced turning response of RGC growth cones. (A, B, D, and E) X. laevis RGC growth cones extending from a 24-h stage 24 explant culture. Pulsatile ejection of Netrin-1 from a pipette (*) with a 0.5–1.0-μm opening was used to set up a concentration gradient next to an extending RGC growth cone (arrowheads). (A and D) Trajectories of growth cones before applying the pipette solution (arrows). (B and E) Growth cones (arrows) 45 min after continuous exposure to the Netrin-1 concentration gradient. The growth cone in the control bath (A and B) turned toward the Netrin-1 source, whereas the growth cone bathed in 0.25 μM of Src kinase inhibitor PP2 (D and E) ignored the Netrin-1 source. (C and F) Superimposed neurite trajectories of growth cones exposed to Netrin-1 source in a control medium (C) and growth cones exposed to Netrin-1 in the presence of PP2 (F). (G) Representation of the mean turning angles of the growth cones in C and F (P < 0.05; unpaired two-tailed t test). Error bars represent SD.
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fig3: Inhibition of Src family kinases abolishes Netrin-1–induced turning response of RGC growth cones. (A, B, D, and E) X. laevis RGC growth cones extending from a 24-h stage 24 explant culture. Pulsatile ejection of Netrin-1 from a pipette (*) with a 0.5–1.0-μm opening was used to set up a concentration gradient next to an extending RGC growth cone (arrowheads). (A and D) Trajectories of growth cones before applying the pipette solution (arrows). (B and E) Growth cones (arrows) 45 min after continuous exposure to the Netrin-1 concentration gradient. The growth cone in the control bath (A and B) turned toward the Netrin-1 source, whereas the growth cone bathed in 0.25 μM of Src kinase inhibitor PP2 (D and E) ignored the Netrin-1 source. (C and F) Superimposed neurite trajectories of growth cones exposed to Netrin-1 source in a control medium (C) and growth cones exposed to Netrin-1 in the presence of PP2 (F). (G) Representation of the mean turning angles of the growth cones in C and F (P < 0.05; unpaired two-tailed t test). Error bars represent SD.
Mentions: To address the physiological significance of phosphorylation of DCC, we examined the question of whether Src family kinases are involved in mediating the axon outgrowth–promoting effects of Netrin-1. Explants of E13 rat dorsal spinal cord cultured in a three-dimensional collagen gel in the presence of 160 ng/ml of Netrin-1 show maximal commissural axon outgrowth (Fig. 2, E and F), as reported previously (Serafini et al., 1996). When PP2 or SU6656 were added in the presence of Netrin-1 to the explants, the increase in axon outgrowth was completely abolished, similar to the results in the control (Fig. 2, E and F). These results demonstrate that inhibition of the Src kinases interferes with the effect of Netrin-1 to mediate commissural axon outgrowth, suggesting a critical role for the Src kinases in Netrin-1–DCC signaling. To determine whether Src kinases are required for Netrin-1 to mediate axon attraction, we used the in vitro turning assay in which Xenopus laevis retinal ganglion cells (RGC) turn toward a source of Netrin-1 (de la Torre et al., 1997). As shown in Fig. 3 B, a retinal growth cone extending from a 24-h, stage 24 explant culture turned toward the source of Netrin-1 in the control bath. In contrast, the retinal growth cone ignored the source of Netrin-1 in the presence of PP2 (Fig. 3 E). A trace of the paths taken by all growth cones is represented in Fig. 3 (C and F). The turning angles taken by the retinal growth cones were quantified and are shown in Fig. 3 G. These results reveal the implication of Src family kinases in the turning response of growth cones to Netrin-1.

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