Limits...
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.

Show MeSH

Related in: MedlinePlus

The intracellular domain of DCC is phosphorylated in vitro by Fyn tyrosine kinase. (A) The extracellular domain of DCC is characterized by four amino-terminal Ig-like domains and six fibronectin type III (FNIII) repeats, the transmembrane domain, and the cytodomain, which contains three regions according to the rat amino acid sequence: P1, P2, and P3. Protein mutants of rat DCC were created by substituting each tyrosine in the cytodomain at 1261, 1272, 1361, and 1418 positions into a phenylalanine residue. (B) Amino acid sequence alignment of DCC proteins from different species and human neogenin. The tyrosine residues corresponding to the rat amino acid sequence of DCC are shown in red. (C) 20 μg of GST fusion proteins containing the cytodomains of the wild-type DCC (DCC-C) and the DCC-C-Y1261F and DCC-C-Y1418F mutants was incubated without or with the active Fyn or Src kinases in the presence of γ-[32P]ATP. As positive controls, Fyn was incubated with 5 μg MBP, and Src with 5 μg of a specific peptide substrate. The products were analyzed by SDS-PAGE followed by autoradiography, and the total amount of GST fusion proteins was determined by Western blot (W.B) with anti-DCC antibodies. Autophosphorylated bands of Fyn and Src are also shown as positive controls of their autoactivation. (D) Quantitative analysis of the percent DCC phosphorylated by Src kinases in vitro. 100% corresponds to the phosphorylation of wild-type DCC-C protein (n = 3). Error bars represent SD.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172574&req=5

fig4: The intracellular domain of DCC is phosphorylated in vitro by Fyn tyrosine kinase. (A) The extracellular domain of DCC is characterized by four amino-terminal Ig-like domains and six fibronectin type III (FNIII) repeats, the transmembrane domain, and the cytodomain, which contains three regions according to the rat amino acid sequence: P1, P2, and P3. Protein mutants of rat DCC were created by substituting each tyrosine in the cytodomain at 1261, 1272, 1361, and 1418 positions into a phenylalanine residue. (B) Amino acid sequence alignment of DCC proteins from different species and human neogenin. The tyrosine residues corresponding to the rat amino acid sequence of DCC are shown in red. (C) 20 μg of GST fusion proteins containing the cytodomains of the wild-type DCC (DCC-C) and the DCC-C-Y1261F and DCC-C-Y1418F mutants was incubated without or with the active Fyn or Src kinases in the presence of γ-[32P]ATP. As positive controls, Fyn was incubated with 5 μg MBP, and Src with 5 μg of a specific peptide substrate. The products were analyzed by SDS-PAGE followed by autoradiography, and the total amount of GST fusion proteins was determined by Western blot (W.B) with anti-DCC antibodies. Autophosphorylated bands of Fyn and Src are also shown as positive controls of their autoactivation. (D) Quantitative analysis of the percent DCC phosphorylated by Src kinases in vitro. 100% corresponds to the phosphorylation of wild-type DCC-C protein (n = 3). Error bars represent SD.

Mentions: DCC comprises a large extracellular domain of four immunoglobulin repeats followed by six fibronectin type III repeats, a single transmembrane domain, and a cytoplasmic tail with three conserved motifs (P1, P2, and P3) (Grunwald and Klein, 2002; Fig. 4 A). The P3 region is involved in the ligand-gated multimerization of DCC that is required to mediate the Netrin-1–induced attraction response (Stein and Tessier-Lavigne, 2001). The P1 region interacts with the intracellular domain of the UNC-5 receptor family, inducing the heterodimerization between DCC and UNC-5 receptors that has been shown to be involved in some, but not all, repulsive events induced by Netrin-1 (Merz et al., 2001). The cytoplasmic tail of DCC contains four tyrosine residues highly conserved across rat, mouse, human, and X. laevis species (Fig. 4 B). Only tyrosine 1272 is conserved in Frazzled, the Drosophila melanogaster orthologue of mammalian DCC, and only Y1418 is conserved in neogenin, a member of the DCC family of proteins. Surprisingly, none of the four tyrosine residues is conserved in UNC-40, the C. elegans orthologue of DCC. The four tyrosine residues in the intracellular domain of the rat DCC are located within the limits of the P1, P2, and P3 regions, which suggests that they may play a critical role in DCC function (Fig. 4 A). Interestingly, only the motif pYEQD containing Y1418 is a likely phosphorylation target site of Src tyrosine kinases, as it resembles the known consensus sequence pYEEI for Src kinases (Songyang et al., 1993). Thus, we examined whether purified Src or Fyn directly phosphorylates in vitro the cytoplasmic domain of DCC (DCC-C). Truncated DCC lacking the majority of the extracellular domain was expressed as a GST fusion protein and purified as described in Materials and methods. As shown in Fig. 4 C, purified Fyn, but not Src, phosphorylates DCC-C after a 10-min incubation. Using a small range of DCC-C concentrations, we roughly estimated a Km value of 70 nM, which suggests that DCC is a good substrate for Fyn. To determine which tyrosine residues are phosphorylated by Fyn, each tyrosine of the intracellular domain of DCC was substituted with a phenylalanine residue. Three DCC mutant proteins containing either the Y1261F, Y1272F, or Y1361F amino acid substitutions are phosphorylated by Fyn at a similar level as DCC-C (Fig. 4, C and D; and not depicted). However, phosphorylation of DCC-C-Y1418F by Fyn is significantly reduced compared with that of DCC-C (Fig. 4, C and D), which indicates that this Y1418 is a phosphorylation target site of Fyn in vitro.


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)

The intracellular domain of DCC is phosphorylated in vitro by Fyn tyrosine kinase. (A) The extracellular domain of DCC is characterized by four amino-terminal Ig-like domains and six fibronectin type III (FNIII) repeats, the transmembrane domain, and the cytodomain, which contains three regions according to the rat amino acid sequence: P1, P2, and P3. Protein mutants of rat DCC were created by substituting each tyrosine in the cytodomain at 1261, 1272, 1361, and 1418 positions into a phenylalanine residue. (B) Amino acid sequence alignment of DCC proteins from different species and human neogenin. The tyrosine residues corresponding to the rat amino acid sequence of DCC are shown in red. (C) 20 μg of GST fusion proteins containing the cytodomains of the wild-type DCC (DCC-C) and the DCC-C-Y1261F and DCC-C-Y1418F mutants was incubated without or with the active Fyn or Src kinases in the presence of γ-[32P]ATP. As positive controls, Fyn was incubated with 5 μg MBP, and Src with 5 μg of a specific peptide substrate. The products were analyzed by SDS-PAGE followed by autoradiography, and the total amount of GST fusion proteins was determined by Western blot (W.B) with anti-DCC antibodies. Autophosphorylated bands of Fyn and Src are also shown as positive controls of their autoactivation. (D) Quantitative analysis of the percent DCC phosphorylated by Src kinases in vitro. 100% corresponds to the phosphorylation of wild-type DCC-C protein (n = 3). Error bars represent SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172574&req=5

fig4: The intracellular domain of DCC is phosphorylated in vitro by Fyn tyrosine kinase. (A) The extracellular domain of DCC is characterized by four amino-terminal Ig-like domains and six fibronectin type III (FNIII) repeats, the transmembrane domain, and the cytodomain, which contains three regions according to the rat amino acid sequence: P1, P2, and P3. Protein mutants of rat DCC were created by substituting each tyrosine in the cytodomain at 1261, 1272, 1361, and 1418 positions into a phenylalanine residue. (B) Amino acid sequence alignment of DCC proteins from different species and human neogenin. The tyrosine residues corresponding to the rat amino acid sequence of DCC are shown in red. (C) 20 μg of GST fusion proteins containing the cytodomains of the wild-type DCC (DCC-C) and the DCC-C-Y1261F and DCC-C-Y1418F mutants was incubated without or with the active Fyn or Src kinases in the presence of γ-[32P]ATP. As positive controls, Fyn was incubated with 5 μg MBP, and Src with 5 μg of a specific peptide substrate. The products were analyzed by SDS-PAGE followed by autoradiography, and the total amount of GST fusion proteins was determined by Western blot (W.B) with anti-DCC antibodies. Autophosphorylated bands of Fyn and Src are also shown as positive controls of their autoactivation. (D) Quantitative analysis of the percent DCC phosphorylated by Src kinases in vitro. 100% corresponds to the phosphorylation of wild-type DCC-C protein (n = 3). Error bars represent SD.
Mentions: DCC comprises a large extracellular domain of four immunoglobulin repeats followed by six fibronectin type III repeats, a single transmembrane domain, and a cytoplasmic tail with three conserved motifs (P1, P2, and P3) (Grunwald and Klein, 2002; Fig. 4 A). The P3 region is involved in the ligand-gated multimerization of DCC that is required to mediate the Netrin-1–induced attraction response (Stein and Tessier-Lavigne, 2001). The P1 region interacts with the intracellular domain of the UNC-5 receptor family, inducing the heterodimerization between DCC and UNC-5 receptors that has been shown to be involved in some, but not all, repulsive events induced by Netrin-1 (Merz et al., 2001). The cytoplasmic tail of DCC contains four tyrosine residues highly conserved across rat, mouse, human, and X. laevis species (Fig. 4 B). Only tyrosine 1272 is conserved in Frazzled, the Drosophila melanogaster orthologue of mammalian DCC, and only Y1418 is conserved in neogenin, a member of the DCC family of proteins. Surprisingly, none of the four tyrosine residues is conserved in UNC-40, the C. elegans orthologue of DCC. The four tyrosine residues in the intracellular domain of the rat DCC are located within the limits of the P1, P2, and P3 regions, which suggests that they may play a critical role in DCC function (Fig. 4 A). Interestingly, only the motif pYEQD containing Y1418 is a likely phosphorylation target site of Src tyrosine kinases, as it resembles the known consensus sequence pYEEI for Src kinases (Songyang et al., 1993). Thus, we examined whether purified Src or Fyn directly phosphorylates in vitro the cytoplasmic domain of DCC (DCC-C). Truncated DCC lacking the majority of the extracellular domain was expressed as a GST fusion protein and purified as described in Materials and methods. As shown in Fig. 4 C, purified Fyn, but not Src, phosphorylates DCC-C after a 10-min incubation. Using a small range of DCC-C concentrations, we roughly estimated a Km value of 70 nM, which suggests that DCC is a good substrate for Fyn. To determine which tyrosine residues are phosphorylated by Fyn, each tyrosine of the intracellular domain of DCC was substituted with a phenylalanine residue. Three DCC mutant proteins containing either the Y1261F, Y1272F, or Y1361F amino acid substitutions are phosphorylated by Fyn at a similar level as DCC-C (Fig. 4, C and D; and not depicted). However, phosphorylation of DCC-C-Y1418F by Fyn is significantly reduced compared with that of DCC-C (Fig. 4, C and D), which indicates that this Y1418 is a phosphorylation target site of Fyn in vitro.

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