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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 1418 is critical for Netrin-1–induced Rac1 activation. (A) COS-7 cells coexpressing DCC, Y1261F-, or Y1418F-DCC mutants together with Myc-Rac1 protein were or were not treated with Netrin-1 for 5 min. The GTP-loaded Rac1 were pulled down from the cell lysates using GST-Cdc42/Rac interactive binding domain-Pak1 fusion protein. The proteins from the pull-down and from the total cell lysate were analyzed by Western blot using anti-DCC antibodies and anti-Myc antibodies to detect both Rac1-GTP and the total Rac1. (B) Quantitative analysis of Rac1 activation by DCC and DCC tyrosine mutants after Netrin-1 treatment (n = 5). Error bars represent SD.
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fig8: DCC phosphorylation on tyrosine 1418 is critical for Netrin-1–induced Rac1 activation. (A) COS-7 cells coexpressing DCC, Y1261F-, or Y1418F-DCC mutants together with Myc-Rac1 protein were or were not treated with Netrin-1 for 5 min. The GTP-loaded Rac1 were pulled down from the cell lysates using GST-Cdc42/Rac interactive binding domain-Pak1 fusion protein. The proteins from the pull-down and from the total cell lysate were analyzed by Western blot using anti-DCC antibodies and anti-Myc antibodies to detect both Rac1-GTP and the total Rac1. (B) Quantitative analysis of Rac1 activation by DCC and DCC tyrosine mutants after Netrin-1 treatment (n = 5). Error bars represent SD.

Mentions: We have previously demonstrated that expression of DCC in fibroblasts specifically activates the small GTPase Rac1 in a Netrin-1–dependent manner and that Rac1 activity is essential for DCC-induced neurite outgrowth in N1E-115 cells (Li et al., 2002b). To determine whether substitution of Y1418 by a phenylalanine residue interferes with the signaling pathways leading to Rac1 activation after Netrin-1 stimulation, we performed a pull-down assay in which GTP-loaded Rac1 was trapped by specific binding to the Cdc42/Rac interactive binding domain of p65PAK fused to GST (GST-PAK). DCC or the various DCC mutants were coexpressed with Myc-tagged Rac1 in COS-7 cells for 24 h and serum-starved overnight. After 5 min of Netrin-1 stimulation, protein lysates were prepared and incubated with GST-PAK, and the amount of Rac1-GTP precipitated by GST-PAK was determined by Western blot analysis. As shown in Fig. 8 (A and B), Netrin-1 stimulates an eightfold increase in the level of activated Rac1. The expression of Y1261F-, Y1272F-, or Y1361F-DCC mutant proteins shows a slight decrease in the level of activated Rac1 induced by Netrin-1 compared with that when DCC is expressed (Fig. 8, A and B; and not depicted). In contrast, activation of Rac1 by Netrin-1 is completely abolished when Y1418F mutant protein is expressed in COS-7 cells (Fig. 8, A and B). Thus, phosphorylation of Y1418 seems to be crucial for the activation of Rac1 by DCC. Together with the results obtained in N1E-115 cells, these data provide evidence for an important role of the phosphorylation of Y1418 in the signaling pathways mediated by the Netrin-1 receptor DCC, leading to neurite outgrowth.


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 1418 is critical for Netrin-1–induced Rac1 activation. (A) COS-7 cells coexpressing DCC, Y1261F-, or Y1418F-DCC mutants together with Myc-Rac1 protein were or were not treated with Netrin-1 for 5 min. The GTP-loaded Rac1 were pulled down from the cell lysates using GST-Cdc42/Rac interactive binding domain-Pak1 fusion protein. The proteins from the pull-down and from the total cell lysate were analyzed by Western blot using anti-DCC antibodies and anti-Myc antibodies to detect both Rac1-GTP and the total Rac1. (B) Quantitative analysis of Rac1 activation by DCC and DCC tyrosine mutants after Netrin-1 treatment (n = 5). Error bars represent SD.
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Related In: Results  -  Collection

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fig8: DCC phosphorylation on tyrosine 1418 is critical for Netrin-1–induced Rac1 activation. (A) COS-7 cells coexpressing DCC, Y1261F-, or Y1418F-DCC mutants together with Myc-Rac1 protein were or were not treated with Netrin-1 for 5 min. The GTP-loaded Rac1 were pulled down from the cell lysates using GST-Cdc42/Rac interactive binding domain-Pak1 fusion protein. The proteins from the pull-down and from the total cell lysate were analyzed by Western blot using anti-DCC antibodies and anti-Myc antibodies to detect both Rac1-GTP and the total Rac1. (B) Quantitative analysis of Rac1 activation by DCC and DCC tyrosine mutants after Netrin-1 treatment (n = 5). Error bars represent SD.
Mentions: We have previously demonstrated that expression of DCC in fibroblasts specifically activates the small GTPase Rac1 in a Netrin-1–dependent manner and that Rac1 activity is essential for DCC-induced neurite outgrowth in N1E-115 cells (Li et al., 2002b). To determine whether substitution of Y1418 by a phenylalanine residue interferes with the signaling pathways leading to Rac1 activation after Netrin-1 stimulation, we performed a pull-down assay in which GTP-loaded Rac1 was trapped by specific binding to the Cdc42/Rac interactive binding domain of p65PAK fused to GST (GST-PAK). DCC or the various DCC mutants were coexpressed with Myc-tagged Rac1 in COS-7 cells for 24 h and serum-starved overnight. After 5 min of Netrin-1 stimulation, protein lysates were prepared and incubated with GST-PAK, and the amount of Rac1-GTP precipitated by GST-PAK was determined by Western blot analysis. As shown in Fig. 8 (A and B), Netrin-1 stimulates an eightfold increase in the level of activated Rac1. The expression of Y1261F-, Y1272F-, or Y1361F-DCC mutant proteins shows a slight decrease in the level of activated Rac1 induced by Netrin-1 compared with that when DCC is expressed (Fig. 8, A and B; and not depicted). In contrast, activation of Rac1 by Netrin-1 is completely abolished when Y1418F mutant protein is expressed in COS-7 cells (Fig. 8, A and B). Thus, phosphorylation of Y1418 seems to be crucial for the activation of Rac1 by DCC. Together with the results obtained in N1E-115 cells, these data provide evidence for an important role of the phosphorylation of Y1418 in the signaling pathways mediated by the Netrin-1 receptor DCC, leading to neurite outgrowth.

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