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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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Fyn tyrosine kinase regulates the phosphorylation of DCC and is critical for DCC-induced neurite outgrowth in N1E-115 cells. (A) N1E-115 cells were transfected with empty vector (EV) or pRK5-DCC (DCC). Cells expressing DCC were treated with PP2. DCC was immunoprecipitated from the lysates, and the total amount of DCC was determined using anti-DCC antibodies. Anti-pY antibodies were used to show the level of DCC phosphorylation on tyrosines. (B) The empty vector (EV), DCC, DCC-Y1261F, DCC-Y1272F, DCC-Y1361F, or DCC-Y1418F constructs were transfected either alone or together with Fyn-CA, in N1E-115 cells. Cells expressing DCC were treated with the Src kinase inhibitor PP2. DCC was also cotransfected with Fyn-DN. The expression of these proteins was analyzed by Western blot using anti-DCC and anti-Fyn antibodies, and the phosphorylation levels of these proteins were assessed using anti-pY antibodies. (C) N1E-115 cells were transfected with empty vector (EV) or with DCC, alone or with Src-CA. DCC-transfected cells were also treated with SU6656. After DCC immunoprecipitation, the phosphorylation levels of these proteins were assessed using anti-pY antibodies, and the total amounts of the expressed proteins were determined using anti-DCC and anti-Src antibodies. (D) Quantitative analysis of the tyrosine phosphorylation of DCC and DCC mutant proteins in N1E-115 cells (n = 3). Error bars represent SD. (E) N1E-115 cells were transfected with empty vector (EV), Fyn-CA alone, and DCC either alone or together with Fyn-CA, Fyn-DN, Src-CA, or Src-DN. The cells were costained with anti-DCC antibodies and rhodamine-conjugated phalloidin to visualize the actin filaments. Fyn or Src expression was visualized using anti-p62 antibodies (Santa Cruz Biotechnology, Inc.) (not depicted). Bar, 20 μm. (F) Quantitative analysis of the percent of transfected N1E-115 cells exhibiting neurites shown in E (n = 3) was performed in a blinded fashion. Error bars represent SD.
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fig5: Fyn tyrosine kinase regulates the phosphorylation of DCC and is critical for DCC-induced neurite outgrowth in N1E-115 cells. (A) N1E-115 cells were transfected with empty vector (EV) or pRK5-DCC (DCC). Cells expressing DCC were treated with PP2. DCC was immunoprecipitated from the lysates, and the total amount of DCC was determined using anti-DCC antibodies. Anti-pY antibodies were used to show the level of DCC phosphorylation on tyrosines. (B) The empty vector (EV), DCC, DCC-Y1261F, DCC-Y1272F, DCC-Y1361F, or DCC-Y1418F constructs were transfected either alone or together with Fyn-CA, in N1E-115 cells. Cells expressing DCC were treated with the Src kinase inhibitor PP2. DCC was also cotransfected with Fyn-DN. The expression of these proteins was analyzed by Western blot using anti-DCC and anti-Fyn antibodies, and the phosphorylation levels of these proteins were assessed using anti-pY antibodies. (C) N1E-115 cells were transfected with empty vector (EV) or with DCC, alone or with Src-CA. DCC-transfected cells were also treated with SU6656. After DCC immunoprecipitation, the phosphorylation levels of these proteins were assessed using anti-pY antibodies, and the total amounts of the expressed proteins were determined using anti-DCC and anti-Src antibodies. (D) Quantitative analysis of the tyrosine phosphorylation of DCC and DCC mutant proteins in N1E-115 cells (n = 3). Error bars represent SD. (E) N1E-115 cells were transfected with empty vector (EV), Fyn-CA alone, and DCC either alone or together with Fyn-CA, Fyn-DN, Src-CA, or Src-DN. The cells were costained with anti-DCC antibodies and rhodamine-conjugated phalloidin to visualize the actin filaments. Fyn or Src expression was visualized using anti-p62 antibodies (Santa Cruz Biotechnology, Inc.) (not depicted). Bar, 20 μm. (F) Quantitative analysis of the percent of transfected N1E-115 cells exhibiting neurites shown in E (n = 3) was performed in a blinded fashion. Error bars represent SD.

Mentions: To determine which tyrosine residues of DCC are phosphorylated in vivo, we investigated the phosphorylation status of the various DCC mutant proteins expressed in N1E-115 mouse neuroblastoma cells. We have shown previously that N1E-115 cells constitutively produce Netrin-1 but do not express DCC (Li et al., 2002b). In the presence of serum, N1E-115 cells are round and show lamellipodia formation and multiple filopodia, but they do not extend neurites. The expression of DCC in these cells induces neurite extension (Li et al., 2002b). As shown in Fig. 5 A, DCC is phosphorylated on tyrosine residues in N1E-115 cells, which is consistent with the tyrosine phosphorylation of DCC observed in rat primary CN. To confirm that phosphorylation of DCC is regulated by Fyn tyrosine kinase in N1E-115 cells, we coexpressed constitutively active (CA) Fyn or dominant negative (DN) Fyn with DCC in N1E-115 cells (Fig. 5 B). Indeed, Fyn-CA shows a 30-fold increase in the level of tyrosine phosphorylation of DCC, whereas Fyn-DN inhibits the basal level of tyrosine phosphorylation of DCC, similar to the results observed when cells are treated with PP2 or SU6656 (Fig. 5, A–D). In comparison, the expression of Src-CA in N1E-115 cells did not affect the basal level of DCC phosphorylation (Fig. 5, C and D).


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)

Fyn tyrosine kinase regulates the phosphorylation of DCC and is critical for DCC-induced neurite outgrowth in N1E-115 cells. (A) N1E-115 cells were transfected with empty vector (EV) or pRK5-DCC (DCC). Cells expressing DCC were treated with PP2. DCC was immunoprecipitated from the lysates, and the total amount of DCC was determined using anti-DCC antibodies. Anti-pY antibodies were used to show the level of DCC phosphorylation on tyrosines. (B) The empty vector (EV), DCC, DCC-Y1261F, DCC-Y1272F, DCC-Y1361F, or DCC-Y1418F constructs were transfected either alone or together with Fyn-CA, in N1E-115 cells. Cells expressing DCC were treated with the Src kinase inhibitor PP2. DCC was also cotransfected with Fyn-DN. The expression of these proteins was analyzed by Western blot using anti-DCC and anti-Fyn antibodies, and the phosphorylation levels of these proteins were assessed using anti-pY antibodies. (C) N1E-115 cells were transfected with empty vector (EV) or with DCC, alone or with Src-CA. DCC-transfected cells were also treated with SU6656. After DCC immunoprecipitation, the phosphorylation levels of these proteins were assessed using anti-pY antibodies, and the total amounts of the expressed proteins were determined using anti-DCC and anti-Src antibodies. (D) Quantitative analysis of the tyrosine phosphorylation of DCC and DCC mutant proteins in N1E-115 cells (n = 3). Error bars represent SD. (E) N1E-115 cells were transfected with empty vector (EV), Fyn-CA alone, and DCC either alone or together with Fyn-CA, Fyn-DN, Src-CA, or Src-DN. The cells were costained with anti-DCC antibodies and rhodamine-conjugated phalloidin to visualize the actin filaments. Fyn or Src expression was visualized using anti-p62 antibodies (Santa Cruz Biotechnology, Inc.) (not depicted). Bar, 20 μm. (F) Quantitative analysis of the percent of transfected N1E-115 cells exhibiting neurites shown in E (n = 3) was performed in a blinded fashion. Error bars represent SD.
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fig5: Fyn tyrosine kinase regulates the phosphorylation of DCC and is critical for DCC-induced neurite outgrowth in N1E-115 cells. (A) N1E-115 cells were transfected with empty vector (EV) or pRK5-DCC (DCC). Cells expressing DCC were treated with PP2. DCC was immunoprecipitated from the lysates, and the total amount of DCC was determined using anti-DCC antibodies. Anti-pY antibodies were used to show the level of DCC phosphorylation on tyrosines. (B) The empty vector (EV), DCC, DCC-Y1261F, DCC-Y1272F, DCC-Y1361F, or DCC-Y1418F constructs were transfected either alone or together with Fyn-CA, in N1E-115 cells. Cells expressing DCC were treated with the Src kinase inhibitor PP2. DCC was also cotransfected with Fyn-DN. The expression of these proteins was analyzed by Western blot using anti-DCC and anti-Fyn antibodies, and the phosphorylation levels of these proteins were assessed using anti-pY antibodies. (C) N1E-115 cells were transfected with empty vector (EV) or with DCC, alone or with Src-CA. DCC-transfected cells were also treated with SU6656. After DCC immunoprecipitation, the phosphorylation levels of these proteins were assessed using anti-pY antibodies, and the total amounts of the expressed proteins were determined using anti-DCC and anti-Src antibodies. (D) Quantitative analysis of the tyrosine phosphorylation of DCC and DCC mutant proteins in N1E-115 cells (n = 3). Error bars represent SD. (E) N1E-115 cells were transfected with empty vector (EV), Fyn-CA alone, and DCC either alone or together with Fyn-CA, Fyn-DN, Src-CA, or Src-DN. The cells were costained with anti-DCC antibodies and rhodamine-conjugated phalloidin to visualize the actin filaments. Fyn or Src expression was visualized using anti-p62 antibodies (Santa Cruz Biotechnology, Inc.) (not depicted). Bar, 20 μm. (F) Quantitative analysis of the percent of transfected N1E-115 cells exhibiting neurites shown in E (n = 3) was performed in a blinded fashion. Error bars represent SD.
Mentions: To determine which tyrosine residues of DCC are phosphorylated in vivo, we investigated the phosphorylation status of the various DCC mutant proteins expressed in N1E-115 mouse neuroblastoma cells. We have shown previously that N1E-115 cells constitutively produce Netrin-1 but do not express DCC (Li et al., 2002b). In the presence of serum, N1E-115 cells are round and show lamellipodia formation and multiple filopodia, but they do not extend neurites. The expression of DCC in these cells induces neurite extension (Li et al., 2002b). As shown in Fig. 5 A, DCC is phosphorylated on tyrosine residues in N1E-115 cells, which is consistent with the tyrosine phosphorylation of DCC observed in rat primary CN. To confirm that phosphorylation of DCC is regulated by Fyn tyrosine kinase in N1E-115 cells, we coexpressed constitutively active (CA) Fyn or dominant negative (DN) Fyn with DCC in N1E-115 cells (Fig. 5 B). Indeed, Fyn-CA shows a 30-fold increase in the level of tyrosine phosphorylation of DCC, whereas Fyn-DN inhibits the basal level of tyrosine phosphorylation of DCC, similar to the results observed when cells are treated with PP2 or SU6656 (Fig. 5, A–D). In comparison, the expression of Src-CA in N1E-115 cells did not affect the basal level of DCC phosphorylation (Fig. 5, C and D).

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