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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 phosphorylation on tyrosine residues is Src family kinase dependent and is critical for Netrin-1–mediated axon outgrowth. (A) Labeling of CN was or was not followed by stimulation with Netrin-1 for 10 or 30 min. The neurons were also treated or not, for 2 h, with either genistein or PP2 before a 10-min Netrin-1 stimulation. (C) Nonlabeled rat CN were also treated for 2 h with different concentrations of SU6656 before a 10-min Netrin-1 stimulation. (A and C) DCC was immunoprecipitated (IP) from the cell lysates and the products were analyzed by SDS-PAGE and autoradiography. The membrane was immunoblotted with anti-pY and anti-DCC antibodies to analyze the total amount of DCC. (B) Quantitative analysis of the total DCC phosphorylation after genistein or PP2 treatments (n = 4). (D) Quantitative analysis of DCC phosphorylation on tyrosine residues after genistein, PP2, or SU6656 treatments. (B and D) Error bars represent SD. (E) E13 rat dorsal spinal cord explants were cultured for 36 h, alone (control), in the presence of Netrin-1, or with both Netrin-1 and either PP2 or SU6656. Bar, 100 μm. (F) Quantification of the total length of axon bundles per explant in micrometers. Error bars represent SD.
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fig2: DCC phosphorylation on tyrosine residues is Src family kinase dependent and is critical for Netrin-1–mediated axon outgrowth. (A) Labeling of CN was or was not followed by stimulation with Netrin-1 for 10 or 30 min. The neurons were also treated or not, for 2 h, with either genistein or PP2 before a 10-min Netrin-1 stimulation. (C) Nonlabeled rat CN were also treated for 2 h with different concentrations of SU6656 before a 10-min Netrin-1 stimulation. (A and C) DCC was immunoprecipitated (IP) from the cell lysates and the products were analyzed by SDS-PAGE and autoradiography. The membrane was immunoblotted with anti-pY and anti-DCC antibodies to analyze the total amount of DCC. (B) Quantitative analysis of the total DCC phosphorylation after genistein or PP2 treatments (n = 4). (D) Quantitative analysis of DCC phosphorylation on tyrosine residues after genistein, PP2, or SU6656 treatments. (B and D) Error bars represent SD. (E) E13 rat dorsal spinal cord explants were cultured for 36 h, alone (control), in the presence of Netrin-1, or with both Netrin-1 and either PP2 or SU6656. Bar, 100 μm. (F) Quantification of the total length of axon bundles per explant in micrometers. Error bars represent SD.

Mentions: Because the Src family kinases, particularly the Src and Fyn members, play pivotal roles in neuronal signaling cascades (Sperber et al., 2001), we examined the implication of the Src family in the phosphorylation of DCC. In vivo, [32P]orthophosphate-labeled CN were left untreated or treated with either the wide-spectrum tyrosine kinase inhibitor genistein (Akiyama et al., 1987) or one of the two different Src family kinase specific inhibitors PP2 (Hanke et al., 1996) and SU6656 (Blake et al., 2000) before a 10-min stimulation with Netrin-1. By Western blotting using anti-pY antibodies, we show that tyrosine phosphorylation of DCC is completely inhibited when the cells have been treated with genistein (Fig. 2, A and B). PP2 as well as SU6656 treatments of the CN also lead to the inhibition of DCC tyrosine phosphorylation, which indicates that the Src kinases are implicated in this phosphorylation event (Fig. 2, A, C, and D). Interestingly, immunoprecipitation of 32P-radiolabeled DCC reveals that both genistein and PP2 significantly decreased the total phosphorylation of DCC in response to Netrin-1 (Fig. 2, A and B). Because serine residues account for most of the phosphorylation sites in DCC (Fig. 1, E and F), these data suggest that Src family kinase activity is critical for initiation of the molecular events leading to Netrin-1–dependent phosphorylation of DCC.


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 phosphorylation on tyrosine residues is Src family kinase dependent and is critical for Netrin-1–mediated axon outgrowth. (A) Labeling of CN was or was not followed by stimulation with Netrin-1 for 10 or 30 min. The neurons were also treated or not, for 2 h, with either genistein or PP2 before a 10-min Netrin-1 stimulation. (C) Nonlabeled rat CN were also treated for 2 h with different concentrations of SU6656 before a 10-min Netrin-1 stimulation. (A and C) DCC was immunoprecipitated (IP) from the cell lysates and the products were analyzed by SDS-PAGE and autoradiography. The membrane was immunoblotted with anti-pY and anti-DCC antibodies to analyze the total amount of DCC. (B) Quantitative analysis of the total DCC phosphorylation after genistein or PP2 treatments (n = 4). (D) Quantitative analysis of DCC phosphorylation on tyrosine residues after genistein, PP2, or SU6656 treatments. (B and D) Error bars represent SD. (E) E13 rat dorsal spinal cord explants were cultured for 36 h, alone (control), in the presence of Netrin-1, or with both Netrin-1 and either PP2 or SU6656. Bar, 100 μm. (F) Quantification of the total length of axon bundles per explant in micrometers. Error bars represent SD.
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Related In: Results  -  Collection

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fig2: DCC phosphorylation on tyrosine residues is Src family kinase dependent and is critical for Netrin-1–mediated axon outgrowth. (A) Labeling of CN was or was not followed by stimulation with Netrin-1 for 10 or 30 min. The neurons were also treated or not, for 2 h, with either genistein or PP2 before a 10-min Netrin-1 stimulation. (C) Nonlabeled rat CN were also treated for 2 h with different concentrations of SU6656 before a 10-min Netrin-1 stimulation. (A and C) DCC was immunoprecipitated (IP) from the cell lysates and the products were analyzed by SDS-PAGE and autoradiography. The membrane was immunoblotted with anti-pY and anti-DCC antibodies to analyze the total amount of DCC. (B) Quantitative analysis of the total DCC phosphorylation after genistein or PP2 treatments (n = 4). (D) Quantitative analysis of DCC phosphorylation on tyrosine residues after genistein, PP2, or SU6656 treatments. (B and D) Error bars represent SD. (E) E13 rat dorsal spinal cord explants were cultured for 36 h, alone (control), in the presence of Netrin-1, or with both Netrin-1 and either PP2 or SU6656. Bar, 100 μm. (F) Quantification of the total length of axon bundles per explant in micrometers. Error bars represent SD.
Mentions: Because the Src family kinases, particularly the Src and Fyn members, play pivotal roles in neuronal signaling cascades (Sperber et al., 2001), we examined the implication of the Src family in the phosphorylation of DCC. In vivo, [32P]orthophosphate-labeled CN were left untreated or treated with either the wide-spectrum tyrosine kinase inhibitor genistein (Akiyama et al., 1987) or one of the two different Src family kinase specific inhibitors PP2 (Hanke et al., 1996) and SU6656 (Blake et al., 2000) before a 10-min stimulation with Netrin-1. By Western blotting using anti-pY antibodies, we show that tyrosine phosphorylation of DCC is completely inhibited when the cells have been treated with genistein (Fig. 2, A and B). PP2 as well as SU6656 treatments of the CN also lead to the inhibition of DCC tyrosine phosphorylation, which indicates that the Src kinases are implicated in this phosphorylation event (Fig. 2, A, C, and D). Interestingly, immunoprecipitation of 32P-radiolabeled DCC reveals that both genistein and PP2 significantly decreased the total phosphorylation of DCC in response to Netrin-1 (Fig. 2, A and B). Because serine residues account for most of the phosphorylation sites in DCC (Fig. 1, E and F), these data suggest that Src family kinase activity is critical for initiation of the molecular events leading to Netrin-1–dependent phosphorylation of DCC.

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