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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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DCC is rapidly phosphorylated in vivo upon Netrin-1 stimulation in embryonic rat CN. (A) Phase-contrast image of E13 rat CN 72 h after plating on laminin. Bar, 40 μm. (B) Immunofluorescence of CN with anti-DCC antibodies showing DCC expression in the cell bodies and along the axons. Bar, 20 μm. (C) E13 rat CN labeled with [32P]orthophosphate for 2 h were either stimulated or not with Netrin-1 for 5, 10, and 30 min. Endogenous DCC was immunoprecipitated (IP) from the cell lysates. The radiolabeled proteins were subjected to SDS-PAGE and identified by autoradiography. The membrane was immunoblotted with anti-pY and anti-DCC antibodies to show the total amount of DCC. (D) Quantitative analysis of the phosphorylation level of DCC after Netrin-1 stimulation of rat CN. Fold increase in total phosphorylation of DCC was determined by densitometry (n = 3). Error bars represent SD. (E and F) The bands corresponding to phosphorylated DCC obtained after 5 min (E) or 30 min (F) of Netrin-1 stimulation were subjected to a phospho–amino acid analysis. (G) Quantitative analysis of DCC phosphorylation on tyrosine residues, using the method described in D (n = 3). Error bars represent SD.
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fig1: DCC is rapidly phosphorylated in vivo upon Netrin-1 stimulation in embryonic rat CN. (A) Phase-contrast image of E13 rat CN 72 h after plating on laminin. Bar, 40 μm. (B) Immunofluorescence of CN with anti-DCC antibodies showing DCC expression in the cell bodies and along the axons. Bar, 20 μm. (C) E13 rat CN labeled with [32P]orthophosphate for 2 h were either stimulated or not with Netrin-1 for 5, 10, and 30 min. Endogenous DCC was immunoprecipitated (IP) from the cell lysates. The radiolabeled proteins were subjected to SDS-PAGE and identified by autoradiography. The membrane was immunoblotted with anti-pY and anti-DCC antibodies to show the total amount of DCC. (D) Quantitative analysis of the phosphorylation level of DCC after Netrin-1 stimulation of rat CN. Fold increase in total phosphorylation of DCC was determined by densitometry (n = 3). Error bars represent SD. (E and F) The bands corresponding to phosphorylated DCC obtained after 5 min (E) or 30 min (F) of Netrin-1 stimulation were subjected to a phospho–amino acid analysis. (G) Quantitative analysis of DCC phosphorylation on tyrosine residues, using the method described in D (n = 3). Error bars represent SD.

Mentions: We first examined whether endogenous DCC is phosphorylated in vivo upon Netrin-1 stimulation in dissociated CN. For this purpose, we dissected the first half of E13 rat dorsal spinal cords that contained the cell bodies of the CN (Tessier-Lavigne et al., 1988). Dissociated CN exhibit long and fasciculated axons enriched in growth cones at the tips of the axons after 72 h in culture (Fig. 1 A). As shown in Fig. 1 B, a high level of DCC expression is observed in the cell bodies and along the neurite extensions as described previously (Li et al., 2002b; Shekarabi and Kennedy, 2002). Thus, we chose this time point to examine the phosphorylation of DCC in vivo in response to Netrin-1. 72 h after plating on laminin-1–coated dishes, the CN were serum-starved and then incubated in phosphate-free medium supplemented with [32P]orthophosphate for 2 h before stimulation with Netrin-1 for different periods of time. Phosphorylation of endogenous DCC is increased 5 min after stimulation with Netrin-1, and a 12-fold increase in the level of DCC phosphorylation is observed after 30 min of stimulation (Fig. 1, C and D). To assess the content of phosphorylated residues on DCC upon Netrin-1 stimulation, we performed a phospho–amino acid analysis of immunoprecipitated DCC after 5 and 30 min of incubation with Netrin-1. Interestingly, DCC is mainly phosphorylated, in vivo, on serine and threonine residues, and to a lesser extent on tyrosine residues (Fig. 1, E and F). The eightfold increase in tyrosine phosphorylation of DCC after 30 min of Netrin-1 stimulation (Fig. 1, F and G) is confirmed by Western blotting using anti-pY antibodies on immunoprecipitated DCC from embryonic commissural neuron lysates (Fig. 1 C). Therefore, Netrin-1 induces DCC phosphorylation in vivo on serine, threonine, and tyrosine residues in embryonic CN.


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)

DCC is rapidly phosphorylated in vivo upon Netrin-1 stimulation in embryonic rat CN. (A) Phase-contrast image of E13 rat CN 72 h after plating on laminin. Bar, 40 μm. (B) Immunofluorescence of CN with anti-DCC antibodies showing DCC expression in the cell bodies and along the axons. Bar, 20 μm. (C) E13 rat CN labeled with [32P]orthophosphate for 2 h were either stimulated or not with Netrin-1 for 5, 10, and 30 min. Endogenous DCC was immunoprecipitated (IP) from the cell lysates. The radiolabeled proteins were subjected to SDS-PAGE and identified by autoradiography. The membrane was immunoblotted with anti-pY and anti-DCC antibodies to show the total amount of DCC. (D) Quantitative analysis of the phosphorylation level of DCC after Netrin-1 stimulation of rat CN. Fold increase in total phosphorylation of DCC was determined by densitometry (n = 3). Error bars represent SD. (E and F) The bands corresponding to phosphorylated DCC obtained after 5 min (E) or 30 min (F) of Netrin-1 stimulation were subjected to a phospho–amino acid analysis. (G) Quantitative analysis of DCC phosphorylation on tyrosine residues, using the method described in D (n = 3). Error bars represent SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172574&req=5

fig1: DCC is rapidly phosphorylated in vivo upon Netrin-1 stimulation in embryonic rat CN. (A) Phase-contrast image of E13 rat CN 72 h after plating on laminin. Bar, 40 μm. (B) Immunofluorescence of CN with anti-DCC antibodies showing DCC expression in the cell bodies and along the axons. Bar, 20 μm. (C) E13 rat CN labeled with [32P]orthophosphate for 2 h were either stimulated or not with Netrin-1 for 5, 10, and 30 min. Endogenous DCC was immunoprecipitated (IP) from the cell lysates. The radiolabeled proteins were subjected to SDS-PAGE and identified by autoradiography. The membrane was immunoblotted with anti-pY and anti-DCC antibodies to show the total amount of DCC. (D) Quantitative analysis of the phosphorylation level of DCC after Netrin-1 stimulation of rat CN. Fold increase in total phosphorylation of DCC was determined by densitometry (n = 3). Error bars represent SD. (E and F) The bands corresponding to phosphorylated DCC obtained after 5 min (E) or 30 min (F) of Netrin-1 stimulation were subjected to a phospho–amino acid analysis. (G) Quantitative analysis of DCC phosphorylation on tyrosine residues, using the method described in D (n = 3). Error bars represent SD.
Mentions: We first examined whether endogenous DCC is phosphorylated in vivo upon Netrin-1 stimulation in dissociated CN. For this purpose, we dissected the first half of E13 rat dorsal spinal cords that contained the cell bodies of the CN (Tessier-Lavigne et al., 1988). Dissociated CN exhibit long and fasciculated axons enriched in growth cones at the tips of the axons after 72 h in culture (Fig. 1 A). As shown in Fig. 1 B, a high level of DCC expression is observed in the cell bodies and along the neurite extensions as described previously (Li et al., 2002b; Shekarabi and Kennedy, 2002). Thus, we chose this time point to examine the phosphorylation of DCC in vivo in response to Netrin-1. 72 h after plating on laminin-1–coated dishes, the CN were serum-starved and then incubated in phosphate-free medium supplemented with [32P]orthophosphate for 2 h before stimulation with Netrin-1 for different periods of time. Phosphorylation of endogenous DCC is increased 5 min after stimulation with Netrin-1, and a 12-fold increase in the level of DCC phosphorylation is observed after 30 min of stimulation (Fig. 1, C and D). To assess the content of phosphorylated residues on DCC upon Netrin-1 stimulation, we performed a phospho–amino acid analysis of immunoprecipitated DCC after 5 and 30 min of incubation with Netrin-1. Interestingly, DCC is mainly phosphorylated, in vivo, on serine and threonine residues, and to a lesser extent on tyrosine residues (Fig. 1, E and F). The eightfold increase in tyrosine phosphorylation of DCC after 30 min of Netrin-1 stimulation (Fig. 1, F and G) is confirmed by Western blotting using anti-pY antibodies on immunoprecipitated DCC from embryonic commissural neuron lysates (Fig. 1 C). Therefore, Netrin-1 induces DCC phosphorylation in vivo on serine, threonine, and tyrosine residues in embryonic CN.

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